Fourier transform infrared spectroscopy to quantify collagen and elastin in an in vitro model of extracellular matrix degradation in aorta.

PubMed

Cheheltani, Rabee; McGoverin, Cushla M; Rao, Jayashree; Vorp, David A; Kiani, Mohammad F; Pleshko, Nancy

2014-06-21

Extracellular matrix (ECM) is a key component and regulator of many biological tissues including aorta. Several aortic pathologies are associated with significant changes in the composition of the matrix, especially in the content, quality and type of aortic structural proteins, collagen and elastin. The purpose of this study was to develop an infrared spectroscopic methodology that is comparable to biochemical assays to quantify collagen and elastin in aorta. Enzymatically degraded porcine aorta samples were used as a model of ECM degradation in abdominal aortic aneurysm (AAA). After enzymatic treatment, Fourier transform infrared (FTIR) spectra of the aortic tissue were acquired by an infrared fiber optic probe (IFOP) and FTIR imaging spectroscopy (FT-IRIS). Collagen and elastin content were quantified biochemically and partial least squares (PLS) models were developed to predict collagen and elastin content in aorta based on FTIR spectra. PLS models developed from FT-IRIS spectra were able to predict elastin and collagen content of the samples with strong correlations (RMSE of validation = 8.4% and 11.1% of the range respectively), and IFOP spectra were successfully used to predict elastin content (RMSE = 11.3% of the range). The PLS regression coefficients from the FT-IRIS models were used to map collagen and elastin in tissue sections of degraded porcine aortic tissue as well as a human AAA biopsy tissue, creating a similar map of each component compared to histology. These results support further application of FTIR spectroscopic techniques for evaluation of AAA tissues.

Long-term type 1 diabetes alters the deposition of collagens and proteoglycans in the early pregnant myometrium of mice.

PubMed

Favaro, Rodolfo R; Raspantini, Priscila R; Salgado, Renato M; Fortes, Zuleica B; Zorn, Telma M T

2015-04-01

We have previously shown that long-term type 1 diabetes affects the structural organization, contractile apparatus and extracellular matrix (ECM) of the myometrium during early pregnancy in mice. This study aimed to identify which myometrial ECM components are affected by diabetes, including fibril-forming collagen types I, III and V, as well as proteoglycans, decorin, lumican, fibromodulin and biglycan. Alloxan-induced type 1 diabetic female mice were divided into subgroups D1 and D2, formed by females that bred 90-100 and 100-110 days after diabetes induction, respectively. The deposition of ECM components in the myometrium was evaluated by immunohistochemistry/immunofluorescence. The subgroup D1 showed decreased deposition of collagen types I and III in the external muscle layer (EML) and decreased collagen types III and V in the internal muscle layer (IML). Collagen types I and III were decreased in both muscle layers of the subgroup D2. In addition, increased deposition of collagen types I and III and lumican as well as decreased collagen type V were observed in the connective tissue between muscle layers of D2. Lumican was decreased in the EML of the subgroups D1 and D2. Fibromodulin was repressed in the IML and EML of both D1 and D2. In contrast, decorin deposition diminished only in muscle layers of D2. No changes were noticed for biglycan. Subgroups D1 and D2 showed distinct stages of progression of diabetic complications in the myometrium, characterized by both common and specific sets of changes in the ECM composition.

Fourier Transform Infrared Spectroscopy to Quantify Collagen and Elastin in an In Vitro Model of Extracellular Matrix Degradation in Aorta

PubMed Central

Cheheltani, Rabee; McGoverin, Cushla M.; Rao, Jayashree; Vorp, David A.; Kiani, Mohammad F.; Pleshko, N.

2014-01-01

Extracellular matrix (ECM) is a key component and regulator of many biological tissues including aorta. Several aortic pathologies are associated with significant changes in the composition of the matrix, especially in the content, quality and type of aortic structural proteins, collagen and elastin. The purpose of this study was to develop an infrared spectroscopic methodology that is comparable to biochemical assays to quantify collagen and elastin in aorta. Enzymatically degraded porcine aorta samples were used as a model of ECM degradation in abdominal aortic aneurysm (AAA). After enzymatic treatment, Fourier transform infrared (FTIR) spectra of the aortic tissue were acquired by an infrared fiber optic probe (IFOP) and FTIR imaging spectroscopy (FT-IRIS). Collagen and elastin content were quantified biochemically and partial least squares (PLS) models were developed to predict collagen and elastin content in aorta based on FTIR spectra. PLS models developed from FT-IRIS spectra were able to predict elastin and collagen content of the samples with strong correlations (RMSE of validation = 8.4% and 11.1% of the range respectively), and IFOP spectra were successfully used to predict elastin content (RMSE = 11.3% of the range). The PLS regression coefficients from the FT-IRIS models were used to map collagen and elastin in tissue sections of degraded porcine aortic tissue as well as a human AAA biopsy tissue, creating a similar map of each component compared to histology. These results support further application of FTIR spectroscopic techniques for evaluation of AAA tissues. PMID:24761431

Global Analysis Reveals the Complexity of the Human Glomerular Extracellular Matrix

PubMed Central

Byron, Adam; Humphries, Jonathan D.; Randles, Michael J.; Carisey, Alex; Murphy, Stephanie; Knight, David; Brenchley, Paul E.; Zent, Roy; Humphries, Martin J.

2014-01-01

The glomerulus contains unique cellular and extracellular matrix (ECM) components, which are required for intact barrier function. Studies of the cellular components have helped to build understanding of glomerular disease; however, the full composition and regulation of glomerular ECM remains poorly understood. We used mass spectrometry-based proteomics of enriched ECM extracts for a global analysis of human glomerular ECM in vivo and identified a tissue-specific proteome of 144 structural and regulatory ECM proteins. This catalog includes all previously identified glomerular components plus many new and abundant components. Relative protein quantification showed a dominance of collagen IV, collagen I, and laminin isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein network analysis enabled the creation of a glomerular ECM interactome, which revealed a core of highly connected structural components. More than one half of the glomerular ECM proteome was validated using colocalization studies and data from the Human Protein Atlas. This study yields the greatest number of ECM proteins relative to previous investigations of whole glomerular extracts, highlighting the importance of sample enrichment. It also shows that the composition of glomerular ECM is far more complex than previously appreciated and suggests that many more ECM components may contribute to glomerular development and disease processes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000456. PMID:24436468

3D biomaterial matrix to support long term, full thickness, immuno-competent human skin equivalents with nervous system components.

PubMed

Vidal, Sarah E Lightfoot; Tamamoto, Kasey A; Nguyen, Hanh; Abbott, Rosalyn D; Cairns, Dana M; Kaplan, David L

2018-04-24

Current commercially available human skin equivalents (HSEs) are used for relatively short term studies (∼1 week) due in part to the time-dependent contraction of the collagen gel-based matrix and the limited cell types and skin tissue components utilized. In contrast, here we describe a new matrix consisting of a silk-collagen composite system that provides long term, stable cultivation with reduced contraction and degradation over time. This matrix supports full thickness skin equivalents which include nerves. The unique silk-collagen composite system preserves cell-binding domains of collagen while maintaining the stability and mechanics of the skin system for long-term culture with silk. The utility of this new composite protein-based biomaterial was demonstrated by bioengineering full thickness human skin systems using primary cells, including nerves and immune cells to establish an HSE with a neuro-immuno-cutaneous system. The HSEs with neurons and hypodermis, compared to in vitro skin-only HSEs controls, demonstrated higher secretion of pro-inflammatory cytokines. Proteomics analysis confirmed the presence of several proteins associated with inflammation across all sample groups, but HSEs with neurons had the highest amount of detected protein due to the complexity of the model. This improved, in vitro full thickness HSE model system utilizes cross-linked silk-collagen as the biomaterial and allows reduced reliance on animal models and provides a new in vitro tissue system for the assessment of chronic responses related to skin diseases and drug discovery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Accelerated collagen turnover in women with angina pectoris without obstructive coronary artery disease: An iPOWER substudy.

PubMed

Nielsen, Signe H; Mygind, Naja D; Michelsen, Marie M; Bechsgaard, Daria F; Suhrs, Hannah E; Genovese, Federica; Nielsen, Henning B; Brix, Susanne; Karsdal, Morten; Prescott, Eva; Kastrup, Jens

2018-05-01

Aim Collagens are major cardiac extracellular matrix components, known to be actively remodelled and accumulated during diffuse myocardial fibrosis. We evaluated whether accelerated collagen turnover described by neo-epitope biomarkers reflecting collagen formation and degradation separates patients with diffuse myocardial fibrosis from asymptomatic controls. Methods and results Seventy-one women with angina pectoris without significant coronary artery disease assessed by invasive coronary angiogram were included. Competitive enzyme-linked immunosorbent assays (ELISAs) measuring circulating protein fragments in serum assessed the formation and degradation of collagen type III (Pro-C3, C3M and C3C), IV (P4NP7S and C4M), V (Pro-C5 and C5M) and VI (Pro-C6 and C6M), and degradation of collagen type I (C1M). Serum samples from 32 age-matched asymptomatic women were included as controls. Symptomatic women presented significantly elevated levels of Pro-C6, C3C, C3M, C4M and C8-C ( p < 0.0001-0.0058) and significantly decreased levels of Pro-C3, C5M and C6M ( p < 0.0001-0.041), reflecting accelerated collagen turnover and an imbalanced collagen formation and degradation compared to controls. Cardiac magnetic resonance T1 mapping was performed to determine extracellular volume fraction and thus diffuse myocardial fibrosis. A significant association was identified between C5M and extracellular volume fraction by cardiac magnetic resonance ( p = 0.01). Conclusion Women with angina pectoris, but without significant obstructive coronary artery disease, showed an imbalanced collagen turnover compared to asymptomatic controls. The examined biomarkers are tools to monitor active collagen remodelling in patients with angina pectoris, in risk of developing myocardial fibrosis.

Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding.

PubMed

Barnes, Aileen M; Carter, Erin M; Cabral, Wayne A; Weis, MaryAnn; Chang, Weizhong; Makareeva, Elena; Leikin, Sergey; Rotimi, Charles N; Eyre, David R; Raggio, Cathleen L; Marini, Joan C

2010-02-11

Osteogenesis imperfecta is a heritable disorder that causes bone fragility. Mutations in type I collagen result in autosomal dominant osteogenesis imperfecta, whereas mutations in either of two components of the collagen prolyl 3-hydroxylation complex (cartilage-associated protein [CRTAP] and prolyl 3-hydroxylase 1 [P3H1]) cause autosomal recessive osteogenesis imperfecta with rhizomelia (shortening of proximal segments of upper and lower limbs) and delayed collagen folding. We identified two siblings who had recessive osteogenesis imperfecta without rhizomelia. They had a homozygous start-codon mutation in the peptidyl-prolyl isomerase B gene (PPIB), which results in a lack of cyclophilin B (CyPB), the third component of the complex. The proband's collagen had normal collagen folding and normal prolyl 3-hydroxylation, suggesting that CyPB is not the exclusive peptidyl-prolyl cis-trans isomerase that catalyzes the rate-limiting step in collagen folding, as is currently thought. 2010 Massachusetts Medical Society

Lack of Cyclophilin B in Osteogenesis Imperfecta with Normal Collagen Folding

PubMed Central

Barnes, Aileen M.; Carter, Erin M.; Cabral, Wayne A.; Weis, MaryAnn; Chang, Weizhong; Makareeva, Elena; Leikin, Sergey; Rotimi, Charles N.; Eyre, David R.; Raggio, Cathleen L.; Marini, Joan C.

2011-01-01

SUMMARY Osteogenesis imperfecta is a heritable disorder that causes bone fragility. Mutations in type I collagen result in autosomal dominant osteogenesis imperfecta, whereas mutations in either of two components of the collagen prolyl 3-hydroxylation complex (cartilage-associated protein [CRTAP] and prolyl 3-hydroxylase 1 [P3H1]) cause autosomal recessive osteogenesis imperfecta with rhizomelia (shortening of proximal segments of upper and lower limbs) and delayed collagen folding. We identified two siblings who had recessive osteogenesis imperfecta without rhizomelia. They had a homozygous start-codon mutation in the peptidyl-prolyl isomerase B gene (PPIB), which results in a lack of cyclophilin B (CyPB), the third component of the complex. The proband’s collagen had normal collagen folding and normal prolyl 3-hydroxylation, suggesting that CyPB is not the exclusive peptidyl-prolyl cis–trans isomerase that catalyzes the rate-limiting step in collagen folding, as is currently thought. PMID:20089953

Chondrogenic properties of collagen type XI, a component of cartilage extracellular matrix.

PubMed

Li, Ang; Wei, Yiyong; Hung, Clark; Vunjak-Novakovic, Gordana

2018-08-01

Cartilage extracellular matrix (ECM) has been used for promoting tissue engineering. However, the exact effects of ECM on chondrogenesis and the acting mechanisms are not well understood. In this study, we investigated the chondrogenic effects of cartilage ECM on human mesenchymal stem cells (MSCs) and identified the contributing molecular components. To this end, a preparation of articular cartilage ECM was supplemented to pellets of chondrogenically differentiating MSCs, pellets of human chondrocytes, and bovine articular cartilage explants to evaluate the effects on cell proliferation and the production of cartilaginous matrix. Selective enzymatic digestion and screening of ECM components were conducted to identify matrix molecules with chondrogenic properties. Cartilage ECM promoted MSC proliferation, production of cartilaginous matrix, and maturity of chondrogenic differentiation, and inhibited the hypertrophic differentiation of MSC-derived chondrocytes. Selective digestion of ECM components revealed a contributory role of collagens in promoting chondrogenesis. The screening of various collagen subtypes revealed strong chondrogenic effect of collagen type XI. Finally, collagen XI was found to promote production and inhibit degradation of cartilage matrix in human articular chondrocyte pellets and bovine articular cartilage explants. Our results indicate that cartilage ECM promotes chondrogenesis and inhibits hypertrophic differentiation in MSCs. Collagen type XI is the ECM component that has the strongest effects on enhancing the production and inhibiting the degradation of cartilage matrix. Copyright © 2018 Elsevier Ltd. All rights reserved.

Extracellular Matrix and Dermal Fibroblast Function in the Healing Wound

PubMed Central

Tracy, Lauren E.; Minasian, Raquel A.; Caterson, E.J.

2016-01-01

Significance: Fibroblasts play a critical role in normal wound healing. Various extracellular matrix (ECM) components, including collagens, fibrin, fibronectin, proteoglycans, glycosaminoglycans, and matricellular proteins, can be considered potent protagonists of fibroblast survival, migration, and metabolism. Recent Advances: Advances in tissue culture, tissue engineering, and ex vivo models have made the examination and precise measurements of ECM components in wound healing possible. Likewise, the development of specific transgenic animal models has created the opportunity to characterize the role of various ECM molecules in healing wounds. In addition, the recent characterization of new ECM molecules, including matricellular proteins, dermatopontin, and FACIT collagens (Fibril-Associated Collagens with Interrupted Triple helices), further demonstrates our cursory knowledge of the ECM in coordinated wound healing. Critical Issues: The manipulation and augmentation of ECM components in the healing wound is emerging in patient care, as demonstrated by the use of acellular dermal matrices, tissue scaffolds, and wound dressings or topical products bearing ECM proteins such as collagen, hyaluronan (HA), or elastin. Once thought of as neutral structural proteins, these molecules are now known to directly influence many aspects of cellular wound healing. Future Directions: The role that ECM molecules, such as CCN2, osteopontin, and secreted protein, acidic and rich in cysteine, play in signaling homing of fibroblast progenitor cells to sites of injury invites future research as we continue investigating the heterotopic origin of certain populations of fibroblasts in a healing wound. Likewise, research into differently sized fragments of the same polymeric ECM molecule is warranted as we learn that fragments of molecules such as HA and tenascin-C can have opposing effects on dermal fibroblasts. PMID:26989578

Attachment of Actinobacillus suis H91-0380 and Its Isogenic Adhesin Mutants to Extracellular Matrix Components of the Tonsils of the Soft Palate of Swine

PubMed Central

Bujold, Adina R.

2016-01-01

Tonsils conduct immune surveillance of antigens entering the upper respiratory tract. Despite their immunological function, they are also sites of persistence and invasion of bacterial pathogens. Actinobacillus suis is a common resident of the tonsils of the soft palate in pigs, but under certain circumstances it can invade, causing septicemia and related sequelae. Twenty-four putative adhesins are predicted in the A. suis genome, but to date, little is known about how they might participate in colonization or invasion. To better understand these processes, swine tonsil lysates were characterized by mass spectrometry. Fifty-nine extracellular matrix (ECM) proteins were identified, including small leucine-rich proteoglycans, integrins, and other cell surface receptors. Additionally, attachment of the wild type and 3 adhesin mutants to 5 ECM components was evaluated. Exponential cultures of wild-type A. suis adhered significantly more than stationary cultures to all ECM components studied except collagen I. During exponential growth, the A. suis Δflp1 mutant attached less to collagen IV while the ΔompA mutant attached less to all ECMs. The ΔcomE1 strain attached less to collagen IV, fibronectin, and vitronectin during exponential growth and exhibited differential attachment to collagen I over short adherence time points. These results suggest that Flp1, OmpA, and ComE1 are important during early stages of attachment to ECM components found in tonsils, which supports the notion that other adhesins have compensatory effects during later stages of attachment. PMID:27481253

Components, structure, biogenesis and function of the Hydra extracellular matrix in regeneration, pattern formation and cell differentiation.

PubMed

Sarras, Michael P

2012-01-01

The body wall of Hydra is organized as an epithelial bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM), termed mesoglea by early biologists. Morphological studies have determined that Hydra ECM is composed of two basal lamina layers positioned at the base of each epithelial layer with an intervening interstitial matrix. Molecular and biochemical analyses of Hydra ECM have established that it contains components similar to those seen in more complicated vertebrate species. These components include such macromolecules as laminin, type IV collagen, and various fibrillar collagens. These components are synthesized in a complicated manner involving cross-talk between the epithelial bilayer. Any perturbation to ECM biogenesis leads to a blockage in Hydra morphogenesis. Blockage in ECM/cell interactions in the adult polyp also leads to problems in epithelial transdifferentiation processes. In terms of biophysical parameters, Hydra ECM is highly flexible; a property that facilitates continuous movements along the organism's longitudinal and radial axis. This is in contrast to the more rigid matrices often found in vertebrates. The flexible nature of Hydra ECM can in part now be explained by the unique structure of the organism's type IV collagen and fibrillar collagens. This review will focus on Hydra ECM in regard to: 1) its general structure, 2) its molecular composition, 3) the biophysical basis for the flexible nature of Hydra's ECM, 4) the relationship of the biogenesis of Hydra ECM to regeneration of body form, and 5) the functional role of Hydra ECM during pattern formation and cell differentiation.

Genetics Home Reference: Stickler syndrome

MedlinePlus

... Stickler syndrome provide instructions for making components of collagens, which are complex molecules that give structure and ... genes impair the production, processing, or assembly of collagen molecules. Defective collagen molecules or reduced amounts of ...

Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation

PubMed Central

Weis, MaryAnn; Rai, Jyoti; Hudson, David M.; Dimori, Milena; Zimmerman, Sarah M.; Hogue, William R.; Swain, Frances L.; Burdine, Marie S.; Mackintosh, Samuel G.; Tackett, Alan J.; Suva, Larry J.; Eyre, David R.

2016-01-01

Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis. PMID:27119146

Global Gene Expression Analysis in PKCα-/- Mouse Skin Reveals Structural Changes in the Dermis and Defective Wound Granulation Tissue.

PubMed

Cooper, Nichola H; Balachandra, Jeya P; Hardman, Matthew J

2015-12-01

The skin's mechanical integrity is maintained by an organized and robust dermal extracellular matrix (ECM). Resistance to mechanical disruption hinges primarily on homeostasis of the dermal collagen fibril architecture, which is regulated, at least in part, by members of the small leucine-rich proteoglycan (SLRP) family. Here we present data linking protein kinase C alpha (PKCα) to the regulated expression of multiple ECM components including SLRPs. Global microarray profiling reveals deficiencies in ECM gene expression in PKCα-/- skin correlating with abnormal collagen fibril morphology, disorganized dermal architecture, and reduced skin strength. Detailed analysis of the skin and wounds from wild-type and PKCα-/- mice reveals a failure to upregulate collagen and other ECM components in response to injury, resulting in delayed granulation tissue deposition in PKCα-/- wounds. Thus, our data reveal a previously unappreciated role for PKCα in the regulation of ECM structure and deposition during skin wound healing.

Genetics Home Reference: Fuchs endothelial dystrophy

MedlinePlus

... a protein that is part of type VIII collagen. Type VIII collagen is largely found within the cornea, surrounding the endothelial cells. Specifically, type VIII collagen is a major component of a tissue at ...

Hyaluronan in aged collagen matrix increases prostate epithelial cell proliferation

PubMed Central

Damodarasamy, Mamatha; Vernon, Robert B.; Chan, Christina K.; Plymate, Stephen R.; Wight, Thomas N.

2015-01-01

The extracellular matrix (ECM) of the prostate, which is comprised primarily of collagen, becomes increasingly disorganized with age, a property that may influence the development of hyperplasia and cancer. Collageous ECM extracted from the tails of aged mice exhibits many characteristics of collagen in aged tissues, including the prostate. When polymerized into a 3-dimensional (3D) gel, these collagen extracts can serve as models for the study of specific cell-ECM interactions. In the present study, we examined the behaviors of human prostatic epithelial cell lines representing normal prostate epithelial cells (PEC), benign prostatic hyperplasia (BPH-1), and adenocarcinoma (LNCaP) cultured in contact with 3D gels made from collagen extracts of young and aged mice. We found that proliferation of PEC, BPH-1, and LNCaP cells were all increased by culture on aged collagen gels relative to young collagen gels. In examining age-associated differences in the composition of the collagen extracts, we found that aged and young collagen had a similar amount of several collagen-associated ECM components, but aged collagen had a much greater content of the glycosaminoglycan hyaluronan (HA) than young collagen. The addition of HA (of similar size and concentration to that found in aged collagen extracts) to cells placed in young collagen elicited significantly increased proliferation in BPH-1 cells, but not in PEC or LNCaP cells, relative to controls not exposed to HA. Of note, histochemical analyses of human prostatic tissues showed significantly higher expression of HA in BPH and prostate cancer stroma relative to stroma of normal prostate. Collectively, these results suggest that changes in ECM involving increased levels of HA contribute to the growth of prostatic epithelium with aging. PMID:25124870

A Novel Functional Role of Collagen Glycosylation

PubMed Central

Jürgensen, Henrik J.; Madsen, Daniel H.; Ingvarsen, Signe; Melander, Maria C.; Gårdsvoll, Henrik; Patthy, Laszlo; Engelholm, Lars H.; Behrendt, Niels

2011-01-01

Collagens make up the most abundant component of interstitial extracellular matrices and basement membranes. Collagen remodeling is a crucial process in many normal physiological events and in several pathological conditions. Some collagen subtypes contain specific carbohydrate side chains, the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens. The molecular basis for this interaction is known to involve the fibronectin type II domain but nothing is known about the function of the lectin domains in this respect. In this study, we have investigated a possible role of the single active lectin domain of uPARAP/Endo180 in the interaction with collagens. By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate the endocytic efficiency of the receptor toward highly glycosylated collagens such as basement membrane collagen IV. Surprisingly, this property was not shared by the mannose receptor, which internalized glycosylated collagens independently of its lectin function. This role of modulating its uptake efficiency by a specific receptor is a previously unrecognized function of collagen glycosylation. PMID:21768090

Phenotypic Screening Identifies Synergistically Acting Natural Product Enhancing the Performance of Biomaterial Based Wound Healing.

PubMed

Sivasubramanian, Srinivasan; Chandrasekar, Gayathri; Svensson Akusjärvi, Sara; Thangam, Ramar; Sathuvan, Malairaj; Kumar, R B S; Hussein, Hawraa; Vincent, Savariar; Madhan, Balaraman; Gunasekaran, Palani; Kitambi, Satish S

2017-01-01

The potential of multifunctional wound heal biomaterial relies on the optimal content of therapeutic constituents as well as the desirable physical, chemical, and biological properties to accelerate the healing process. Formulating biomaterials such as amnion or collagen based scaffolds with natural products offer an affordable strategy to develop dressing material with high efficiency in healing wounds. Using image based phenotyping and quantification, we screened natural product derived bioactive compounds for modulators of types I and III collagen production from human foreskin derived fibroblast cells. The identified hit was then formulated with amnion to develop a biomaterial, and its biophysical properties, in vitro and in vivo effects were characterized. In addition, we performed functional profiling analyses by PCR array to understand the effect of individual components of these materials on various genes such as inflammatory mediators including chemokines and cytokines, growth factors, fibroblast stimulating markers for collagen secretion, matrix metalloproteinases, etc., associated with wound healing. FACS based cell cycle analyses were carried out to evaluate the potential of biomaterials for induction of proliferation of fibroblasts. Western blot analyses was done to examine the effect of biomaterial on collagen synthesis by cells and compared to cells grown in the presence of growth factors. This work demonstrated an uncomplicated way of identifying components that synergistically promote healing. Besides, we demonstrated that modulating local wound environment using biomaterials with bioactive compounds could enhance healing. This study finds that the developed biomaterials offer immense scope for healing wounds by means of their skin regenerative features such as anti-inflammatory, fibroblast stimulation for collagen secretion as well as inhibition of enzymes and markers impeding the healing, hydrodynamic properties complemented with other features including non-toxicity, biocompatibility, and safety.

Phenotypic Screening Identifies Synergistically Acting Natural Product Enhancing the Performance of Biomaterial Based Wound Healing

PubMed Central

Sivasubramanian, Srinivasan; Chandrasekar, Gayathri; Svensson Akusjärvi, Sara; Thangam, Ramar; Sathuvan, Malairaj; Kumar, R. B. S.; Hussein, Hawraa; Vincent, Savariar; Madhan, Balaraman; Gunasekaran, Palani; Kitambi, Satish S.

2017-01-01

The potential of multifunctional wound heal biomaterial relies on the optimal content of therapeutic constituents as well as the desirable physical, chemical, and biological properties to accelerate the healing process. Formulating biomaterials such as amnion or collagen based scaffolds with natural products offer an affordable strategy to develop dressing material with high efficiency in healing wounds. Using image based phenotyping and quantification, we screened natural product derived bioactive compounds for modulators of types I and III collagen production from human foreskin derived fibroblast cells. The identified hit was then formulated with amnion to develop a biomaterial, and its biophysical properties, in vitro and in vivo effects were characterized. In addition, we performed functional profiling analyses by PCR array to understand the effect of individual components of these materials on various genes such as inflammatory mediators including chemokines and cytokines, growth factors, fibroblast stimulating markers for collagen secretion, matrix metalloproteinases, etc., associated with wound healing. FACS based cell cycle analyses were carried out to evaluate the potential of biomaterials for induction of proliferation of fibroblasts. Western blot analyses was done to examine the effect of biomaterial on collagen synthesis by cells and compared to cells grown in the presence of growth factors. This work demonstrated an uncomplicated way of identifying components that synergistically promote healing. Besides, we demonstrated that modulating local wound environment using biomaterials with bioactive compounds could enhance healing. This study finds that the developed biomaterials offer immense scope for healing wounds by means of their skin regenerative features such as anti-inflammatory, fibroblast stimulation for collagen secretion as well as inhibition of enzymes and markers impeding the healing, hydrodynamic properties complemented with other features including non-toxicity, biocompatibility, and safety. PMID:28769790

Genetics Home Reference: familial porencephaly

MedlinePlus

... one component of a protein called type IV collagen. Type IV collagen molecules attach to each other to form complex ... and support cells in many tissues. Type IV collagen networks play an important role in the basement ...

Fibromodulin deficiency reduces collagen structural network but not glycosaminoglycan content in a syngeneic model of colon carcinoma.

PubMed

Olsson, P Olof; Kalamajski, Sebastian; Maccarana, Marco; Oldberg, Åke; Rubin, Kristofer

2017-01-01

Tumor barrier function in carcinoma represents a major challenge to treatment and is therefore an attractive target for increasing drug delivery. Variables related to tumor barrier include aberrant blood vessels, high interstitial fluid pressure, and the composition and structure of the extracellular matrix. One of the proteins associated with dense extracellular matrices is fibromodulin, a collagen fibrillogenesis modulator expressed in tumor stroma but scarce in normal loose connective tissues. Here, we investigated the effects of fibromodulin on stroma ECM in a syngeneic murine colon carcinoma model. We show that fibromodulin deficiency decreased collagen fibril thickness but glycosaminoglycan content and composition were unchanged. Furthermore, vascular density, pericyte coverage and macrophage amount were unaffected. Fibromodulin can therefore be a unique effector of dense collagen matrix assembly in tumor stroma and, without affecting other major matrix components or the cellular composition, can function as a main agent in tumor barrier function.

Collagen IV and basement membrane at the evolutionary dawn of metazoan tissues

PubMed Central

Fidler, Aaron L; Darris, Carl E; Chetyrkin, Sergei V; Pedchenko, Vadim K; Boudko, Sergei P; Brown, Kyle L; Gray Jerome, W; Hudson, Julie K; Rokas, Antonis; Hudson, Billy G

2017-01-01

The role of the cellular microenvironment in enabling metazoan tissue genesis remains obscure. Ctenophora has recently emerged as one of the earliest-branching extant animal phyla, providing a unique opportunity to explore the evolutionary role of the cellular microenvironment in tissue genesis. Here, we characterized the extracellular matrix (ECM), with a focus on collagen IV and its variant, spongin short-chain collagens, of non-bilaterian animal phyla. We identified basement membrane (BM) and collagen IV in Ctenophora, and show that the structural and genomic features of collagen IV are homologous to those of non-bilaterian animal phyla and Bilateria. Yet, ctenophore features are more diverse and distinct, expressing up to twenty genes compared to six in vertebrates. Moreover, collagen IV is absent in unicellular sister-groups. Collectively, we conclude that collagen IV and its variant, spongin, are primordial components of the extracellular microenvironment, and as a component of BM, collagen IV enabled the assembly of a fundamental architectural unit for multicellular tissue genesis. DOI: http://dx.doi.org/10.7554/eLife.24176.001 PMID:28418331

Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane type IV collagens and form a novel family widely distributed in invertebrates.

PubMed

Aouacheria, Abdel; Geourjon, Christophe; Aghajari, Nushin; Navratil, Vincent; Deléage, Gilbert; Lethias, Claire; Exposito, Jean-Yves

2006-12-01

Collagens are thought to represent one of the most important molecular innovations in the metazoan line. Basement membrane type IV collagen is present in all Eumetazoa and was found in Homoscleromorpha, a sponge group with a well-organized epithelium, which may represent the first stage of tissue differentiation during animal evolution. In contrast, spongin seems to be a demosponge-specific collagenous protein, which can totally substitute an inorganic skeleton, such as in the well-known bath sponge. In the freshwater sponge Ephydatia mülleri, we previously characterized a family of short-chain collagens that are likely to be main components of spongins. Using a combination of sequence- and structure-based methods, we present evidence of remote homology between the carboxyl-terminal noncollagenous NC1 domain of spongin short-chain collagens and type IV collagen. Unexpectedly, spongin short-chain collagen-related proteins were retrieved in nonsponge animals, suggesting that a family related to spongin constitutes an evolutionary sister to the type IV collagen family. Formation of the ancestral NC1 domain and divergence of the spongin short-chain collagen-related and type IV collagen families may have occurred before the parazoan-eumetazoan split, the earliest divergence among extant animal phyla. Molecular phylogenetics based on NC1 domain sequences suggest distinct evolutionary histories for spongin short-chain collagen-related and type IV collagen families that include spongin short-chain collagen-related gene loss in the ancestors of Ecdyzosoa and of vertebrates. The fact that a majority of invertebrates encodes spongin short-chain collagen-related proteins raises the important question to the possible function of its members. Considering the importance of collagens for animal structure and substratum attachment, both families may have played crucial roles in animal diversification.

Peripheral Blood Mononuclear Cells Enhance the Anabolic Effects of Platelet-Rich Plasma on Anterior Cruciate Ligament Fibroblasts

PubMed Central

Yoshida, Ryu; Murray, Martha M.

2012-01-01

Use of platelet-rich plasma (PRP) has shown promise in various orthopaedic applications, including treatment of anterior cruciate ligament (ACL) injuries. However, various components of blood, including peripheral blood mononuclear cells (PBMCs), are removed in the process of making PRP. It is yet unknown whether these PBMCs have a positive or negative effect on fibroblast behavior. To begin to define the effect of PBMCs on ACL fibroblasts, ACL fibroblasts were cultured on three-dimensional collagen scaffolds for 14 days with and without PBMCs. ACL fibroblasts exposed to PBMCs showed increased type I and type III procollagen gene expression, collagen protein expression, and cell proliferation when the cells were cultured in the presence of platelets and plasma. However, addition of PBMCs to cells cultured without the presence of platelets had no effect. The increase in collagen gene and protein expression was accompanied by an increase in IL-6 expression by the PBMCs with exposure to the platelets. Our results suggest that the interaction between platelets and PBMCs leads to an IL-6 mediated increase in collagen expression by ACL fibroblasts. PMID:22767425

Genetics Home Reference: multicentric osteolysis, nodulosis, and arthropathy

MedlinePlus

... to cut (cleave) a protein called type IV collagen. Type IV collagen is a major structural component of basement membranes, ... enzyme, preventing the normal cleavage of type IV collagen. It is unclear how a loss of enzyme ...

Characterization of collagen II fibrils containing biglycan and their effect as a coating on osteoblast adhesion and proliferation.

PubMed

Douglas, Timothy; Heinemann, Sascha; Hempel, Ute; Mietrach, Carolin; Knieb, Christiane; Bierbaum, Susanne; Scharnweber, Dieter; Worch, Hartmut

2008-04-01

Collagen has been used as a coating material for titanium-based implants for bone contact and as a component of scaffolds for bone tissue engineering. In general collagen type I has been used, however very little attention has been focussed on collagen type II. Collagen-based coatings and scaffolds have been enhanced by the incorporation of the glycosaminoglycan chondroitin sulphate (CS), however the proteglycan biglycan, which is found in bone and contains glycosaminoglycan chains consisting of CS, has not been used as a biomaterial component. The study had the following aims: firstly, five different collagen II preparations were compared with regard to their ability to bind CS and biglycan and the changes in fibril morphology thereby induced. Secondly, the effects of biglycan on the adhesion of primary rat osteoblasts (rO) as well as the proliferation of rO, primary human osteoblasts (hO) and the osteoblast-like cell line 7F2 were studied by culturing the cells on surfaces coated with collagen II fibrils containing biglycan. Fibrils of the collagen II preparation which bound the most biglycan were used to coat titanium surfaces. Bare titanium, titanium coated with collagen II fibrils and titanium coated with collagen II fibrils containing biglycan were compared. It was found that different collagen II preparations showed different affinities for CS and biglycan. In four of the five preparations tested, biglycan reduced fibril diameter, however the ability of a preparation to bind more biglycan did not appear to lead to a greater reduction in fibril diameter. Fibrils containing biglycan promoted the formation of focal adhesions by rO and significantly enhanced the proliferation of hO but not of rO or 7F2 cells. These results should encourage further investigation of biglycan as a component of collagen-based scaffolds and/or coatings.

Thermal and infrared-diode laser effects on indocyanine-green-treated corneal collagen

NASA Astrophysics Data System (ADS)

Timberlake, George T.; Patmore, Ann; Shallal, Assaad; McHugh, Dominic; Marshall, John

1993-07-01

It has been suggested that laser welds of collagenous tissues form by interdigitation and chemical bonding of thermally 'unraveled' collagen fibrils. We investigated this proposal by attempting to weld highly collagenous, avascular corneal tissue with an infrared (IR) diode laser as follows. First, the temperature at which corneal collagen shrinks and collagen fibrils 'split' into subfibrillary components was determined. Second, since use of a near-IR laser wavelength necessitated addition of an absorbing dye (indocyanine green (ICG) to the cornea, we measured absorption spectra of ICG-treated tissue to ensure that peak ICG absorbance did not change markedly when ICG was present in the cornea. Third, using gel electrophoresis of thermally altered corneal collagen, we searched for covalently crosslinked compounds predicted by the proposed welding mechanism. Finally, we attempted to weld partial thickness corneal incisions infused with ICG. Principal experimental findings were as follows: (1) Human corneal (type I) collagen splits into subfibrillary components at approximately 63 degree(s)C, the same temperature that produces collagen shrinkage. (2) Peak ICG absorption does not change significantly in corneal stroma or with laser heating. (3) No evidence was found for the formation of novel compounds or the loss of proteins as a result of tissue heating. All tissue treated with ICG, however, exhibited a novel 244 kD protein band indicating chemical activity between collagen and corneal stromal components. (4) Laser welding corneal incisions was unsuccessful possibly due to shrinkage of the sides of the incision, lack of incision compression during heating, or a less than optimal combination of ICG concentration and radiant exposure. In summary, these experiments demonstrate the biochemical and morphological complexity of ICG-enhanced IR laser-tissue welding and the need for further investigation of laser welding mechanisms.

Distribution of Collagen I, III, and IV and Laminin in the Human Liver during Prenatal Development.

PubMed

Jović, Marko; Nikolić, Ivan; Todorović, Vera; Petrović, Aleksandar; Petrović, Vladimir; Mojsilović, Marijola; Denčić, Tijana

2018-06-27

In the absence of systematized data on the extracellular matrix components during prenatal liver development, the present study aimed to investigate the time of appearance and distribution of collagen types I, III, and IV and laminin. The study material included embryonic and fetal livers, aged 7-37 weeks, categorized into 3 trimesters. The material was stained using hematoxylin-eosin and immunohistochemistry methods for the identification of collagen I, III, and IV and laminin. Collagen I was detected near the end of the first trimester in the capsules and walls of interlobular veins. As the liver matures, collagen I is increasingly abundant in the capsules, portal area connective tissues, arterial walls, interlobular veins, sinusoids, and central veins. Collagen III and collagen IV appear in the middle of the first trimester in the capsules, portal areas, and walls of central veins, as well as the sinusoids particularly. In trimesters 2 and 3, these collagens are increasingly present in all the structures, but collagen IV is also present in nerve fibers. Laminin is sporadically present adjacent to the sinusoids in trimester 1, while in trimesters 2 and 3 this protein commonly appears in the walls of arteries and interlobular veins, in the basal membrane of bile ducts, and in nerve fibers. The contents of collagen I, III, and IV increase during prenatal development in the liver capsule, arterial and vein walls, sinusoids, and portal area. Laminin expression is consistent with that of the collagens with the exception that, within lobules, laminin disappears with liver maturation. © 2018 S. Karger AG, Basel.

In vitro 3D full thickness skin equivalent tissue model using silk and collagen biomaterials

PubMed Central

Bellas, Evangelia; Seiberg, Miri; Garlick, Jonathan; Kaplan, David L.

2013-01-01

Current approaches to develop skin equivalents often only include the epidermal and dermal components. Yet, full thickness skin includes the hypodermis, a layer below the dermis of adipose tissue containing vasculature, nerves and fibroblasts, necessary to support the epidermis and dermis. In the present study, we developed a full thickness skin equivalent including an epidermis, dermis and hypodermis that could serve as an in vitro model for studying skin development, disease or as a platform for consumer product testing as a means to avoid animal testing. The full thickness skin equivalent was easy to handle and was maintained in culture for greater than 14 days while expressing physiologically relevant morphologies of both the epidermis and dermis, as seen by keratin 10, collagen I and collagen IV expression. The skin equivalent produced glycerol and leptin, markers of adipose tissue metabolism. This work serves as a foundation for our understanding of some of the necessary factors needed to develop a stable, functional model of full-thickness skin. PMID:23161763

Collagenous Extracellular Matrix Biomaterials for Tissue Engineering: Lessons from the Common Sea Urchin Tissue.

PubMed

Goh, Kheng Lim; Holmes, David F

2017-04-25

Scaffolds for tissue engineering application may be made from a collagenous extracellular matrix (ECM) of connective tissues because the ECM can mimic the functions of the target tissue. The primary sources of collagenous ECM material are calf skin and bone. However, these sources are associated with the risk of having bovine spongiform encephalopathy or transmissible spongiform encephalopathy. Alternative sources for collagenous ECM materials may be derived from livestock, e.g., pigs, and from marine animals, e.g., sea urchins. Collagenous ECM of the sea urchin possesses structural features and mechanical properties that are similar to those of mammalian ones. However, even more intriguing is that some tissues such as the ligamentous catch apparatus can exhibit mutability, namely rapid reversible changes in the tissue mechanical properties. These tissues are known as mutable collagenous tissues (MCTs). The mutability of these tissues has been the subject of on-going investigations, covering the biochemistry, structural biology and mechanical properties of the collagenous components. Recent studies point to a nerve-control system for regulating the ECM macromolecules that are involved in the sliding action of collagen fibrils in the MCT. This review discusses the key attributes of the structure and function of the ECM of the sea urchin ligaments that are related to the fibril-fibril sliding action-the focus is on the respective components within the hierarchical architecture of the tissue. In this context, structure refers to size, shape and separation distance of the ECM components while function is associated with mechanical properties e.g., strength and stiffness. For simplicity, the components that address the different length scale from the largest to the smallest are as follows: collagen fibres, collagen fibrils, interfibrillar matrix and collagen molecules. Application of recent theories of stress transfer and fracture mechanisms in fibre reinforced composites to a wide variety of collagen reinforcing (non-mutable) connective tissue, has allowed us to draw general conclusions concerning the mechanical response of the MCT at specific mechanical states, namely the stiff and complaint states. The intent of this review is to provide the latest insights, as well as identify technical challenges and opportunities, that may be useful for developing methods for effective mechanical support when adapting decellularised connective tissues from the sea urchin for tissue engineering or for the design of a synthetic analogue.

Collagenous Extracellular Matrix Biomaterials for Tissue Engineering: Lessons from the Common Sea Urchin Tissue

PubMed Central

Goh, Kheng Lim; Holmes, David F.

2017-01-01

Scaffolds for tissue engineering application may be made from a collagenous extracellular matrix (ECM) of connective tissues because the ECM can mimic the functions of the target tissue. The primary sources of collagenous ECM material are calf skin and bone. However, these sources are associated with the risk of having bovine spongiform encephalopathy or transmissible spongiform encephalopathy. Alternative sources for collagenous ECM materials may be derived from livestock, e.g., pigs, and from marine animals, e.g., sea urchins. Collagenous ECM of the sea urchin possesses structural features and mechanical properties that are similar to those of mammalian ones. However, even more intriguing is that some tissues such as the ligamentous catch apparatus can exhibit mutability, namely rapid reversible changes in the tissue mechanical properties. These tissues are known as mutable collagenous tissues (MCTs). The mutability of these tissues has been the subject of on-going investigations, covering the biochemistry, structural biology and mechanical properties of the collagenous components. Recent studies point to a nerve-control system for regulating the ECM macromolecules that are involved in the sliding action of collagen fibrils in the MCT. This review discusses the key attributes of the structure and function of the ECM of the sea urchin ligaments that are related to the fibril-fibril sliding action—the focus is on the respective components within the hierarchical architecture of the tissue. In this context, structure refers to size, shape and separation distance of the ECM components while function is associated with mechanical properties e.g., strength and stiffness. For simplicity, the components that address the different length scale from the largest to the smallest are as follows: collagen fibres, collagen fibrils, interfibrillar matrix and collagen molecules. Application of recent theories of stress transfer and fracture mechanisms in fibre reinforced composites to a wide variety of collagen reinforcing (non-mutable) connective tissue, has allowed us to draw general conclusions concerning the mechanical response of the MCT at specific mechanical states, namely the stiff and complaint states. The intent of this review is to provide the latest insights, as well as identify technical challenges and opportunities, that may be useful for developing methods for effective mechanical support when adapting decellularised connective tissues from the sea urchin for tissue engineering or for the design of a synthetic analogue. PMID:28441344

Quantification of collagen contraction in three-dimensional cell culture.

PubMed

Kopanska, Katarzyna S; Bussonnier, Matthias; Geraldo, Sara; Simon, Anthony; Vignjevic, Danijela; Betz, Timo

2015-01-01

Many different cell types including fibroblasts, smooth muscle cells, endothelial cells, and cancer cells exert traction forces on the fibrous components of the extracellular matrix. This can be observed as matrix contraction both macro- and microscopically in three-dimensional (3D) tissues models such as collagen type I gels. The quantification of local contraction at the micron scale, including its directionality and speed, in correlation with other parameters such as cell invasion, local protein or gene expression, can provide useful information to study wound healing, organism development, and cancer metastasis. In this article, we present a set of tools to quantify the flow dynamics of collagen contraction, induced by cells migrating out of a multicellular cancer spheroid into a three-dimensional (3D) collagen matrix. We adapted a pseudo-speckle technique that can be applied to bright-field and fluorescent microscopy time series. The image analysis presented here is based on an in-house written software developed in the Matlab (Mathworks) programming environment. The analysis program is freely available from GitHub following the link: http://dx.doi.org/10.5281/zenodo.10116. This tool provides an automatized technique to measure collagen contraction that can be utilized in different 3D cellular systems. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of a collagen type I adhesin of Bacteroides fragilis.

PubMed

Galvão, Bruna P G V; Weber, Brandon W; Rafudeen, Mohamed S; Ferreira, Eliane O; Patrick, Sheila; Abratt, Valerie R

2014-01-01

Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼ 31 and ∼ 34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼ 31 kDa and the ∼ 34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼ 31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein.

Identification of a Collagen Type I Adhesin of Bacteroides fragilis

PubMed Central

Galvão, Bruna P. G. V.; Weber, Brandon W.; Rafudeen, Mohamed S.; Ferreira, Eliane O.; Patrick, Sheila; Abratt, Valerie R.

2014-01-01

Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼31 and ∼34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼31 kDa and the ∼34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein. PMID:24618940

In vivo study of dermal collagen of striae distensae by confocal Raman spectroscopy.

PubMed

Lung, Pam Wen; Tippavajhala, Vamshi Krishna; de Oliveira Mendes, Thiago; Téllez-Soto, Claudio A; Schuck, Desirée Cigaran; Brohem, Carla Abdo; Lorencini, Marcio; Martin, Airton Abrahão

2018-04-01

This research work mainly deals with studying qualitatively the changes in the dermal collagen of two forms of striae distensae (SD) namely striae rubrae (SR) and striae albae (SA) when compared to normal skin (NS) using confocal Raman spectroscopy. The methodology includes an in vivo human skin study for the comparison of confocal Raman spectra of dermis region of SR, SA, and NS by supervised multivariate analysis using partial least squares discriminant analysis (PLS-DA) to determine qualitatively the changes in dermal collagen. These groups are further analyzed for the extent of hydration of dermal collagen by studying the changes in the water content bound to it. PLS-DA score plot showed good separation of the confocal Raman spectra of dermis region into SR, SA, and NS data groups. Further analysis using loading plot and S-plot indicated the participation of various components of dermal collagen in the separation of these groups. Bound water content analysis showed that the extent of hydration of collagen is more in SD when compared to NS. Based on the results obtained, this study confirms the active involvement of dermal collagen in the formation of SD. It also emphasizes the need to study quantitatively the role of these various biochemical changes in the dermal collagen responsible for the variance between SR, SA, and NS.

Tumor tissue characterization using polarization-sensitive second harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Tokarz, Danielle; Cisek, Richard; Golaraei, Ahmad; Krouglov, Serguei; Navab, Roya; Niu, Carolyn; Sakashita, Shingo; Yasufuku, Kazuhiro; Tsao, Ming-Sound; Asa, Sylvia L.; Barzda, Virginijus; Wilson, Brian C.

2015-06-01

Changes in the ultrastructure of collagen in various tumor and non-tumor human tissues including lung, pancreas and thyroid were investigated ex vivo by a polarization-sensitive second harmonic generation (SHG) microscopy technique referred to as polarization-in, polarization-out (PIPO) SHG. This involves measuring the orientation of the linear polarization of outgoing SHG as a function of the linear polarization orientation of incident laser radiation. From the PIPO SHG data, the second-order nonlinear optical susceptibility tensor component ratio, χ(2) ZZZ'/χ(2) ZXX', for each pixel of the SHG image was obtained and presented as color-coded maps. Further, the orientation of collagen fibers in the tissue was deduced. Since the χ(2) ZZZ'/χ(2) ZXX' values represent the organization of collagen in the tissue, theses maps revealed areas of altered collagen structure (not simply concentration) within tissue sections. Statistically-significant differences in χ(2) ZZZ'/χ(2) ZXX' were found between tumor and non-tumor tissues, which varied from organ to organ. Hence, PIPO SHG microscopy could potentially be used to aid pathologists in diagnosing cancer. Additionally, PIPO SHG microscopy could aid in characterizing the structure of collagen in other collagen-related biological processes such as wound repair.

Myocardin-Related Transcription Factor A Epigenetically Regulates Renal Fibrosis in Diabetic Nephropathy

PubMed Central

Xu, Huihui; Wu, Xiaoyan; Qin, Hao; Tian, Wenfang; Chen, Junliang; Sun, Lina; Fang, Mingming

2015-01-01

Diabetic nephropathy (DN) is one of the most common complications associated with diabetes and characterized by renal microvascular injury along with accelerated synthesis of extracellular matrix proteins causing tubulointerstitial fibrosis. Production of type I collagen, the major component of extracellular matrix, is augmented during renal fibrosis after chronic exposure to hyperglycemia. However, the transcriptional modulator responsible for the epigenetic manipulation leading to induction of type I collagen genes is not clearly defined. We show here that tubulointerstitial fibrosis as a result of DN was diminished in myocardin-related transcription factor A (MRTF-A) -deficient mice. In cultured renal tubular epithelial cells and the kidneys of mice with DN, MRTF-A was induced by glucose and synergized with glucose to activate collagen transcription. Notably, MRTF-A silencing led to the disappearance of prominent histone modifications indicative of transcriptional activation, including acetylated histone H3K18/K27 and trimethylated histone H3K4. Detailed analysis revealed that MRTF-A recruited p300, a histone acetyltransferase, and WD repeat-containing protein 5 (WDR5), a key component of the histone H3K4 methyltransferase complex, to the collagen promoters and engaged these proteins in transcriptional activation. Estradiol suppressed collagen production by dampening the expression and binding activity of MRTF-A and interfering with the interaction between p300 and WDR5 in renal epithelial cells. Therefore, targeting the MRTF-A–associated epigenetic machinery might yield interventional strategies against DN-associated renal fibrosis. PMID:25349198

Genetics Home Reference: hereditary angiopathy with nephropathy, aneurysms, and muscle cramps syndrome

MedlinePlus

... one component of a protein called type IV collagen . Type IV collagen molecules attach to each other to form complex ... and support cells in many tissues. Type IV collagen networks play an important role in the basement ...

Hydration and distance dependence of intermolecular shearing between collagen molecules in a model microfibril.

PubMed

Gautieri, Alfonso; Pate, Monica I; Vesentini, Simone; Redaelli, Alberto; Buehler, Markus J

2012-08-09

In vertebrates, collagen tissues are the main component responsible for force transmission. In spite of the physiological importance of these phenomena, force transmission mechanisms are still not fully understood, especially at smaller scales, including in particular collagen molecules and fibrils. Here we investigate the mechanism of molecular sliding between collagen molecules within a fibril, by shearing a central molecule in a hexagonally packed bundle mimicking the collagen microfibril environment, using varied lateral distance between the molecules in both dry and solvated conditions. In vacuum, the central molecule slides under a stick-slip mechanism that is due to the characteristic surface profile of collagen molecules, enhanced by the breaking and reformation of H-bonds between neighboring collagen molecules. This mechanism is consistently observed for varied lateral separations between molecules. The high shearing force (>7 nN) found for the experimentally observed intermolecular distance (≈1.1 nm) suggests that in dry samples the fibril elongation mechanism relies almost exclusively on molecular stretching, which may explain the higher stiffnesses found in dry fibrils. When hydrated, the slip-stick behavior is observed only below 1.3 nm of lateral distance, whereas above 1.3 nm the molecule shears smoothly, showing that the water layer has a strong lubricating effect. Moreover, the average force required to shear is approximately the same in solvated as in dry conditions (≈2.5 nN), which suggests that the role of water at the intermolecular level includes the transfer of load between molecules. Copyright © 2012 Elsevier Ltd. All rights reserved.

Analysis of human knee osteoarthritic cartilage using polarization sensitive second harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Kumar, Rajesh; Grønhaug, Kirsten M.; Romijn, Elisabeth I.; Drogset, Jon O.; Lilledahl, Magnus B.

2014-05-01

Osteoarthritis is one of the most prevalent joint diseases in the world. Although the cause of osteoarthritis is not exactly clear, the disease results in a degradation of the quality of the articular cartilage including collagen and other extracellular matrix components. We have investigated alterations in the structure of collagen fibers in the cartilage tissue of the human knee using mulitphoton microscopy. Due to inherent high nonlinear susceptibility, ordered collagen fibers present in the cartilage tissue matrix produces strong second harmonic generation (SHG) signals. Significant morphological differences are found in different Osteoarthritic grades of cartilage by SHG microscopy. Based on the polarization analysis of the SHG signal, we find that a few locations of hyaline cartilage (mainly type II collagen) is being replaced by fibrocartilage (mainly type I cartilage), in agreement with earlier literature. To locate the different types and quantify the alteration in the structure of collagen fiber, we employ polarization-SHG microscopic analysis, also referred to as _-tensor imaging. The image analysis of p-SHG image obtained by excitation polarization measurements would represent different tissue constituents with different numerical values at pixel level resolution.

Fluorescent Labeling of Collagen Production by Cells for Noninvasive Imaging of Extracellular Matrix Deposition.

PubMed

Bardsley, Katie; Yang, Ying; El Haj, Alicia J

2017-04-01

Extracellular matrix (ECM) is an essential component of tissues and provides both integrity and biological cues for cells. Collagen is one of the major proteins found within the ECM and therefore is an essential component of all engineered tissues. Therefore, in this article, we present a method for the online real-time monitoring of collagen deposition in three-dimensional engineered constructs. This method revolves around modification of collagen through the addition of azide-L-proline to cell culture media. The incorporation of azide-L-proline into the neocollagen produced by cells can then be detected by reaction with 10 mM of a Click-IT Alexa Fluor 488 DIBO Alkyne. The reaction was shown as being specific to the collagen as little background staining was observed in cultures, which did not contain the modified proline, and the staining was also depleted after treatment with collagenase and colocalization of collagen type I staining by immunochemistry assay. Real-time online staining of collagen deposition was observed under different culture conditions without affecting proliferation. Collagen deposition was observed to be increased under mechanical stimulation; however, the localization varied across stimulation regimes. This is a new technique for real-time monitoring of cell-produced collagen and will be a valuable addition to the tissue engineering field.

In Situ D-periodic Molecular Structure of Type II Collagen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antipova, Olga; Orgel, Joseph P.R.O.

Collagens are essential components of extracellular matrices in multicellular animals. Fibrillar type II collagen is the most prominent component of articular cartilage and other cartilage-like tissues such as notochord. Its in situ macromolecular and packing structures have not been fully characterized, but an understanding of these attributes may help reveal mechanisms of tissue assembly and degradation (as in osteo- and rheumatoid arthritis). In some tissues such as lamprey notochord, the collagen fibrillar organization is naturally crystalline and may be studied by x-ray diffraction. We used diffraction data from native and derivative notochord tissue samples to solve the axial, D-periodic structuremore » of type II collagen via multiple isomorphous replacement. The electron density maps and heavy atom data revealed the conformation of the nonhelical telopeptides and the overall D-periodic structure of collagen type II in native tissues, data that were further supported by structure prediction and transmission electron microscopy. These results help to explain the observed differences in collagen type I and type II fibrillar architecture and indicate the collagen type II cross-link organization, which is crucial for fibrillogenesis. Transmission electron microscopy data show the close relationship between lamprey and mammalian collagen fibrils, even though the respective larger scale tissue architecture differs.« less

Studies on collagen-tannic acid-collagenase ternary system: Inhibition of collagenase against collagenolytic degradation of extracellular matrix component of collagen.

PubMed

Krishnamoorthy, Ganesan; Sehgal, Praveen Kumar; Mandal, Asit Baran; Sadulla, Sayeed

2012-06-01

We report the detailed studies on the inhibitory effect of tannic acid (TA) on Clostridium histolyticum collagenase (ChC) activity against degradation of extracellular matrix component of collagen. The TA treated collagen exhibited 64% resistance against collagenolytic hydrolysis by ChC, whereas direct interaction of TA with ChC exhibited 99% inhibition against degradation of collagen and the inhibition was found to be concentration dependant. The kinetic inhibition of ChC has been deduced from the extent of hydrolysis of N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA). This data provides a selective competitive mode of inhibition on ChC activity seems to be influenced strongly by the nature and structure of TA. TA showed inhibitor activity against the ChC by molecular docking method. This result demonstrated that TA containing digalloyl radical possess the ability to inhibit the ChC. The inhibition of ChC in gaining new insight into the mechanism of stabilization of collagen by TA is discussed.

Epidermolysis Bullosa Acquisita: Autoimmunity to Anchoring Fibril Collagen

PubMed Central

Chen, Mei; Kim, Gene H.; Prakash, Lori; Woodley, David T.

2012-01-01

Epidermolysis bullosa acquisita (EBA) is a rare and acquired autoimmune subepidermal bullous disease of the skin and mucosa. EBA includes various distinct clinical manifestations resembling Bullous Pemphigus, Brunsting-Perry pemphigoid, or cicatricial pemphigoid. These patients have autoantibodies against type VII collagen, an integral component of anchoring fibrils, which are responsible for attaching the dermis to the epidermis. Destruction or perturbation of the normally functioning anchoring fibrils clinically results in skin fragility, blisters, erosions, scars, milia and nail loss, all features reminiscent of genetic dystrophic epidermolysis bullosa. These anti-type VII collagen antibodies are “pathogenic” because when injected into a mouse, the mouse develops an EBA-like blistering disease. Currently treatment is often unsatisfactory, however some success has been achieved with colchichine, dapsone, photopheresis, plasmaphresis, infliximab, rituximab and IVIG. PMID:21955050

Fibrous nonlinear elasticity enables positive mechanical feedback between cells and ECMs

PubMed Central

Hall, Matthew S.; Alisafaei, Farid; Ban, Ehsan; Feng, Xinzeng; Hui, Chung-Yuen; Shenoy, Vivek B.; Wu, Mingming

2016-01-01

In native states, animal cells of many types are supported by a fibrous network that forms the main structural component of the ECM. Mechanical interactions between cells and the 3D ECM critically regulate cell function, including growth and migration. However, the physical mechanism that governs the cell interaction with fibrous 3D ECM is still not known. In this article, we present single-cell traction force measurements using breast tumor cells embedded within 3D collagen matrices. We recreate the breast tumor mechanical environment by controlling the microstructure and density of type I collagen matrices. Our results reveal a positive mechanical feedback loop: cells pulling on collagen locally align and stiffen the matrix, and stiffer matrices, in return, promote greater cell force generation and a stiffer cell body. Furthermore, cell force transmission distance increases with the degree of strain-induced fiber alignment and stiffening of the collagen matrices. These findings highlight the importance of the nonlinear elasticity of fibrous matrices in regulating cell–ECM interactions within a 3D context, and the cell force regulation principle that we uncover may contribute to the rapid mechanical tissue stiffening occurring in many diseases, including cancer and fibrosis. PMID:27872289

Verification of echo amplitude envelope analysis method in skin tissues for quantitative follow-up of healing ulcers

NASA Astrophysics Data System (ADS)

Omura, Masaaki; Yoshida, Kenji; Akita, Shinsuke; Yamaguchi, Tadashi

2018-07-01

We aim to develop an ultrasonic tissue characterization method for the follow-up of healing ulcers by diagnosing collagen fibers properties. In this paper, we demonstrated a computer simulation with simulation phantoms reflecting irregularly distributed collagen fibers to evaluate the relationship between physical properties, such as number density and periodicity, and the estimated characteristics of the echo amplitude envelope using the homodyned-K distribution. Moreover, the consistency between echo signal characteristics and the structures of ex vivo human tissues was verified from the measured data of normal skin and nonhealed ulcers. In the simulation study, speckle or coherent signal characteristics are identified as periodically or uniformly distributed collagen fibers with high number density and high periodicity. This result shows the effectiveness of the analysis using the homodyned-K distribution for tissues with complicated structures. Normal skin analysis results are characterized as including speckle or low-coherence signal components, and a nonhealed ulcer is different from normal skin with respect to the physical properties of collagen fibers.

Loss of Interneuron-Derived Collagen XIX Leads to a Reduction in Perineuronal Nets in the Mammalian Telencephalon.

PubMed

Su, Jianmin; Cole, James; Fox, Michael A

2017-02-01

Perineuronal nets (PNNs) are lattice-like supramolecular assemblies of extracellular glycoproteins that surround subsets of neuronal cell bodies in the mammalian telencephalon. PNNs emerge at the end of the critical period of brain development, limit neuronal plasticity in the adult brain, and are lost in a variety of complex brain disorders diseases, including schizophrenia. The link between PNNs and schizophrenia led us to question whether neuronally expressed extracellular matrix (ECM) molecules associated with schizophrenia contribute to the assembly of these specialized supramolecular ECM assemblies. We focused on collagen XIX-a minor, nonfibrillar collagen expressed by subsets of telencephalic interneurons. Genetic alterations in the region encoding collagen XIX have been associated with familial schizophrenia, and loss of this collagen in mice results in altered inhibitory synapses, seizures, and the acquisition of schizophrenia-related behaviors. Here, we demonstrate that loss of collagen XIX also results in a reduction of telencephalic PNNs. Loss of PNNs was accompanied with reduced levels of aggrecan (Acan), a major component of PNNs. Despite reduced levels of PNN constituents in collagen XIX-deficient mice ( col19a1 - / - ), we failed to detect reduced expression of genes encoding these ECM molecules. Instead, we discovered a widespread upregulation of extracellular proteases capable of cleaving Acan and other PNN constituents in col19a1 - / - brains. Taken together, these results suggest a mechanism by which the loss of collagen XIX speeds PNN degradation and they identify a novel mechanism by which the loss of collagen XIX may contribute to complex brain disorders.

Collagen type I and III synthesis by Tenon's capsule fibroblasts in culture: individual patient characteristics and response to mitomycin C, 5-fluorouracil, and ascorbic acid.

PubMed Central

Gross, R L

1999-01-01

PURPOSE: This study was performed to better understand the differences between patients in specific components of wound healing as it may pertain to glaucoma filtration surgery, including the use of antimetabolites. METHODS: Human Tenon's capsule fibroblasts were obtained at the time of glaucoma filtering surgery and established in individual cell cultures from 35 glaucoma patients. The dose-response to 5-fluorouracil (5FU) and mitomycin C (MMC) was determined. The individual cell lines were exposed to the antimetabolites and ascorbic acid with measurement of collagen type I and III production by an ELISA-type dot blot assay. These results were then statistically compared to the individual patient characteristics including age, race, previous surgery and medications, and type of glaucoma. RESULTS: 5-FU had little effect on collagen type I and III production or protein synthesis. MMC had an inhibitory effect on collagen secretion and total protein synthesis with increasing concentration. Photomicrographs of the cells after each treatment condition revealed characteristic morphologic changes when compared to controls. There was a large range of collagen type I and III production with correlation between the amounts of each collagen type secreted in response to the antimetabolites. However, there was no correlation with accepted risk factors for filtration failure. CONCLUSION: These antimetabolites act similarly on different cell lines in a nonspecific manner. The results suggest that the increased risk of filtration failure due to age, race, diagnosis, and previous conjunctival surgery is not due to differences in secretion of collagen types I and III by Tenon's capsule fibroblasts. Images FIGURE 3 PMID:10703140

Collagen XII and XIV, new partners of cartilage oligomeric matrix protein in the skin extracellular matrix suprastructure.

PubMed

Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

2012-06-29

The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.

Collagen XII and XIV, New Partners of Cartilage Oligomeric Matrix Protein in the Skin Extracellular Matrix Suprastructure*

PubMed Central

Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R.; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

2012-01-01

The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone. PMID:22573329

Mechanical properties of a collagen fibril under simulated degradation.

PubMed

Malaspina, David C; Szleifer, Igal; Dhaher, Yasin

2017-11-01

Collagen fibrils are a very important component in most of the connective tissue in humans. An important process associated with several physiological and pathological states is the degradation of collagen. Collagen degradation is usually mediated by enzymatic and non-enzymatic processes. In this work we use molecular dynamics simulations to study the influence of simulated degradation on the mechanical properties of the collagen fibril. We applied tensile stress to the collagen fiber at different stages of degradation. We compared the difference in the fibril mechanical priorities due the removal of enzymatic crosslink, surface degradation and volumetric degradation. As anticipated, our results indicated that, regardless of the degradation scenario, fibril mechanical properties is reduced. The type of degradation mechanism (crosslink, surface or volumetric) expressed differential effect on the change in the fibril stiffness. Our simulation results showed dramatic change in the fibril stiffness with a small amount of degradation. This suggests that the hierarchical structure of the fibril is a key component for the toughness and is very sensitive to changes in the organization of the fibril. The overall results are intended to provide a theoretical framework for the understanding the mechanical behavior of collagen fibrils under degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fibrinogen inhibits fibroblast-mediated contraction of collagen

PubMed Central

Nien, Yih-Dar; Han, Yuan-Ping; Tawil, Bill; Chan, Linda S.; Tuan, Tai-Lan; Garner, Warren L.

2008-01-01

Extracellular matrix changes in composition and organization as it transitions from the provisional matrix of the fibrin/platelet plug to collagen scar in healed wounds. The manner in which individual matrix proteins affect these activities is not well established. In this article we describe the interactions of two important extracellular matrix components, fibrin and collagen, using an in vitro model of wound contraction, the fibroblast-populated collagen lattice. We utilized different fibrinogen sources and measured tissue reorganization in floating and tensioned collagen lattices. Our results showed that both fibrin and fibrinogen decreased the contraction of fibroblast populated collagen lattices in a dose-dependent manner. Polymerization of fibrinogen to fibrin using thrombin had no effect on this inhibition. Further, there was no effect due to changes in protein concentration, alternate components of the fibrin sealant, or the enzymatic action of thrombin. These results suggest that the initial stability of the fibrin provisional matrix is due to the fibrin, because this protein appears to inhibit contraction of the matrix. This may be important in the early phases of wound healing when clot stability is vital for hemostasis. Later, as fibrin is replaced by collagen, wound contraction can occur. PMID:12950643

Evaluation of epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes and concerns on osteoblasts.

PubMed

Chu, Chenyu; Deng, Jia; Xiang, Lin; Wu, Yingying; Wei, Xiawei; Qu, Yili; Man, Yi

2016-10-01

Collagen membranes have ideal biological and mechanical properties for supporting infiltration and proliferation of osteoblasts and play a vital role in guided bone regeneration (GBR). However, pure collagen can lead to inflammation, resulting in progressive bone resorption. Therefore, a method for regulating the level of inflammatory cytokines at surgical sites is paramount for the healing process. Epigallocatechin-3-gallate (EGCG) is a component extracted from green tea with numerous biological activities including an anti-inflammatory effect. Herein, we present a novel cross-linked collagen membrane containing different concentrations of EGCG (0.0064%, 0.064%, and 0.64%) to regulate the level of inflammatory factors secreted by pre-osteoblast cells; improve cell proliferation; and increase the tensile strength, wettability, and thermal stability of collagen membranes. Scanning electron microscope images show that the surfaces of collagen membranes became smoother and the collagen fiber diameters became larger with EGCG treatment. Measurement of the water contact angle demonstrated that introducing EGCG improved membrane wettability. Fourier transform infrared spectroscopy analyses indicated that the backbone of collagen was intact, and the thermal stability was significant improved in differential scanning calorimetry. The mechanical properties of 0.064% and 0.64% EGCG-treated collagen membranes were 1.5-fold greater than those of the control. The extent of cross-linking was significantly increased, as determined by a 2,4,6-trinitrobenzenesulfonic acid solution assay. The Cell Counting Kit-8 (CCK-8) and live/dead assays revealed that collagen membrane cross-linked by 0.0064% EGCG induced greater cell proliferation than pure collagen membranes. Additionally, real-time polymerase chain reaction and enzyme-linked immunosorbent assay results showed that EGCG significantly affected the production of inflammatory factors secreted by MC3T3-E1 cells. Taken together, our results indicate that treatment of collagen membranes with appropriate concentrations of EGCG has an anti-inflammatory effect and shows promise for GBR applications. Copyright © 2016. Published by Elsevier B.V.

The mesangial matrix in the normal and sclerotic glomerulus.

PubMed

Rosenblum, N D

1994-02-01

Mesangial sclerosis is a final common pathway to glomerular destruction in a variety of glomerular diseases. The expression of several classes of extracellular matrix (ECM) molecules has been defined in the normal and diseased mesangial matrix (MM). However, the manner in which these ECM components determine the three dimensional structure and function of the MM remains to be defined. Structural studies of the MM suggest that its constituent molecules are regionally organized into subcompartments with different three dimensional structures. The diversity of matrix molecules expressed within the MM as well as the organization of these components in nonrenal ECM's, such as the cornea, provides further support for this organizational model. The study of the cornea has also revealed that novel short chain collagenous proteins partially determine the three dimensional structure of the matrix. Recently, a novel collagen, type VIII collagen, has been described in mesangial cells and in the intact glomerulus. It is hypothesized that type VIII collagen is expressed both as a polymer and as a monomer within the glomerulus, and depending on its conformation, may serve unique functions. In the chronically diseased MM, normal MM components are overexpressed and fibrillar collagens are expressed de novo in a delayed fashion. Enhanced proteoglycan expression, observed early in disease, may determine increased volume of the mesangium. This, in turn, may stimulate the production of fibrillar collagens by mesangial cells resulting in a fibrillar noncompliant mesangial matrix.

The Rationale for Joint Mobilization.

ERIC Educational Resources Information Center

Burkhardt, Sandy

This paper presents an overview of the functions of connective tissue and the mechanisms of joint injury and contracture formation in relation to therapeutic exercise. The components of connective tissue operation are explained, including fibroblasts, macrophages, plasma cells, and collagen. An examination of the histology of connective tissue as…

Concentration determination of collagen and proteoglycan in bovine nasal cartilage by Fourier transform infrared imaging and PLS

NASA Astrophysics Data System (ADS)

Zhang, Xuexi; Xiao, Zhi-Yan; Yin, Jianhua; Xia, Yang

2014-09-01

Fourier transform infrared imaging (FTIRI) combined with chemometrics can be used to detect the structure of bio-macromolecule, measure the concentrations of some components, and so on. In this study, FTIRI with Partial Least-Squares (PLS) regression was applied to study the concentration of two main components in bovine nasal cartilage (BNC), collagen and proteoglycan. An infrared spectrum library was built by mixing the collagen and chondroitin 6-sulfate (main of proteoglycan) at different ratios. Some pretreatments are needed for building PLS model. FTIR images were collected from BNC sections at 6.25μm and 25μm pixel size. The spectra extracted from BNC-FTIR images were imported into the PLS regression program to predict the concentrations of collagen and proteoglycan. These PLS-determined concentrations are agreed with the result in our previous work and biochemical analytical results. The prediction shows that the concentrations of collagen and proteoglycan in BNC are comparative on the whole. However, the concentration of proteoglycan is a litter higher than that of collagen, to some extent.

Matrix metalloproteinase-2 and its correlation with basal membrane components laminin-5 and collagen type IV in paediatric burn patients measured with Surface Plasmon Resonance Imaging (SPRI) biosensors.

PubMed

Weremijewicz, Artur; Matuszczak, Ewa; Sankiewicz, Anna; Tylicka, Marzena; Komarowska, Marta; Tokarzewicz, Anna; Debek, Wojciech; Gorodkiewicz, Ewa; Hermanowicz, Adam

2018-06-01

The purpose of this study was the determination of matrix metalloproteinase-2 and its correlation with basal membrane components laminin-5 and collagen type IV in the blood plasma of burn patients measured with Surface Plasmon Resonance Imaging (SPRI) biosensors. 31 children scalded by hot water who were managed at the Department of Paediatric Surgery between 2014-2015, after primarily presenting with burns in 4-20% TBSA were included into the study (age 9 months up to 14 years, mean age 2,5+1 years). There were 10 girls and 21 boys. Venous blood samples were drawn 2-6h, and 12-16h after the thermal injury, and on the subsequent days 3, 5 and 7. The matrix metalloproteinase-2, collagen type IV and laminin-5 concentrations were assessed using Surface Plasmon Resonance Imaging by the investigators blinded to the other data. The MMP-2, laminin-5 and collagen type IV concentrations in the blood plasma of patients with burns, were highest 12-16h after thermal injury, the difference was statistically significant. The MMP-2, laminin-5 and collagen type IV concentrations measured 3 days, 5 days and 7 days after the thermal injury, slowly decreased over time, and on the 7th day reached the normal range, when compared with the concentration measured in controls. Current work is the first follow-up study regarding MMP-2 in burns. MMP-2, laminin-5 and collagen type IV levels were elevated early after burn injury in the plasma of studied patients, and were highest 12-16h after the injury. MMP-2, laminin-5 and collagen type IV levels were not proportional to the severity of the burn. We believe in the possibility that the gradual decrease of MMP-2, collagen type IV and laminin-5 concentrations could be connected with the process of healing, but to prove it, more investigation is needed in this area. The SPR imaging biosensor is a good diagnostic tool for determination of MMP-2, laminin-5 and collagen type IV in blood plasma of patients with burns. Copyright © 2017 Elsevier Ltd and ISBI. All rights reserved.

Arterial extracellular matrix: a mechanobiological study of the contributions and interactions of elastin and collagen.

PubMed

Chow, Ming-Jay; Turcotte, Raphaël; Lin, Charles P; Zhang, Yanhang

2014-06-17

The complex network structure of elastin and collagen extracellular matrix (ECM) forms the primary load bearing components in the arterial wall. The structural and mechanobiological interactions between elastin and collagen are important for properly functioning arteries. Here, we examined the elastin and collagen organization, realignment, and recruitment by coupling mechanical loading and multiphoton imaging. Two-photon excitation fluorescence and second harmonic generation methods were performed with a multiphoton video-rate microscope to capture real time changes to the elastin and collagen structure during biaxial deformation. Enzymatic removal of elastin was performed to assess the structural changes of the remaining collagen structure. Quantitative analysis of the structural changes to elastin and collagen was made using a combination of two-dimensional fast Fourier transform and fractal analysis, which allows for a more complete understanding of structural changes. Our study provides new quantitative evidence, to our knowledge on the sequential engagement of different arterial ECM components in response to mechanical loading. The adventitial collagen exists as large wavy bundles of fibers that exhibit fiber engagement after 20% strain. The medial collagen is engaged throughout the stretching process, and prominent elastic fiber engagement is observed up to 20% strain after which the engagement plateaus. The fiber orientation distribution functions show remarkably different changes in the ECM structure in response to mechanical loading. The medial collagen shows an evident preferred circumferential distribution, however the fiber families of adventitial collagen are obscured by their waviness at no or low mechanical strains. Collagen fibers in both layers exhibit significant realignment in response to unequal biaxial loading. The elastic fibers are much more uniformly distributed and remained relatively unchanged due to loading. Removal of elastin produces similar structural changes in collagen as mechanical loading. Our study suggests that the elastic fibers are under tension and impart an intrinsic compressive stress on the collagen. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Genetics Home Reference: fibrochondrogenesis

MedlinePlus

... provide instructions for making components of type XI collagen, which is a complex molecule that gives structure ... the body's joints and organs. Specifically, type XI collagen is found in cartilage, a tough but flexible ...

Optimally designed collagen/polycaprolactone biocomposites supplemented with controlled release of HA/TCP/rhBMP-2 and HA/TCP/PRP for hard tissue regeneration.

PubMed

Kim, WonJin; Jang, Chul Ho; Kim, GeunHyung

2017-09-01

Collagen has been widely used as a very promising material to regenerate various tissues. It is a chief component of the extracellular matrix, and encourages various biological effects conducive to tissue regeneration. However, poor mechanical stability, low processability, and high level of water absorption can lead to impaired control of growth factor release and have impeded the use of collagen as a functional biomedical scaffold. Here, to overcome the shortcomings of collagen scaffolds, we have additively manufactured collagen/polycaprolactone (PCL) biocomposites supplemented with a bioceramic (hydroxyapatite (HA)/β-tricalcium-phosphate (TCP)) and two growth factors (recombinant human bone morphogenetic protein-2 [rhBMP-2] and platelet-rich plasma [PRP]). Various weight fractions of PCL in the collagen/PCL composites were manipulated to select optimal growth factor release and highly active cellular responses. After the optimal concentration of PCL in the collagen/PCL scaffold was determined, biocomposites supplemented with bioceramic/growth-factors were fabricated. Continuously released growth factors were assumed to increase the in vitro cellular activities of the osteoblast-like cells (MG63) cultured on the biocomposites. In vitro cellular responses, including osteogenic activities, were examined, and results showed that compared to the HA/TCP/rhBMP-2 supplemented scaffold the HA/TCP/PRP biocomposites provide significantly high cellular activities (cell proliferation: >1.3-fold) and mineralization (calcium deposition: >1.4-fold, osteocalcin: >2.6-fold) sufficient for regenerating bone tissue. Copyright © 2017. Published by Elsevier B.V.

Effects of ingestion of collagen peptide on collagen fibrils and glycosaminoglycans in the dermis.

PubMed

Matsuda, Naoya; Koyama, Yoh-ichi; Hosaka, Yoshinao; Ueda, Hiromi; Watanabe, Takafumi; Araya, Takayuki; Irie, Shinkichi; Takehana, Kazushige

2006-06-01

In order to investigate the effects of collagen peptide ingestion on fibroblasts and the extracellular matrix in the dermis, collagen peptide was administered orally to pigs at 0.2 g/kg body weight/d for 62 d, and its effects were compared with those of lactalbumin and water controls. Fibroblast density, and diameter and density of collagen fibrils were significantly larger in the collagen peptide group than in the lactalbumin and water control groups. The two major components of dermal glycosaminoglycans, hyaluronic acid and dermatan sulfate, which are present in the inter-fibrillar space, did not differ significantly among the three groups. However, the ratio of dermatan sulfate, which is derived from fibril-bound decorin, was largest in the collagen peptide group. These results suggest that ingestion of collagen peptide induces increased fibroblast density and enhances formation of collagen fibrils in the dermis in a protein-specific manner.

Effects of isopropanol on collagen fibrils in new parchment

PubMed Central

2012-01-01

Background Isopropanol is widely used by conservators to relax the creases and folds of parchment artefacts. At present, little is known of the possible side effects of the chemical on parchments main structural component- collagen. This study uses X-ray Diffraction to investigate the effects of a range of isopropanol concentrations on the dimensions of the nanostructure of the collagen component of new parchment. Results It is found in this study that the packing features of the collagen molecules within the collagen fibril are altered by exposure to isopropanol. The results suggest that this chemical treatment can induce a loss of structural water from the collagen within parchment and thus a rearrangement of intermolecular bonding. This study also finds that the effects of isopropanol treatment are permanent to parchment artefacts and cannot be reversed with rehydration using deionised water. Conclusions This study has shown that isopropanol induces permanent changes to the packing features of collagen within parchment artefacts and has provided scientific evidence that its use to remove creases and folds on parchment artefacts will cause structural change that may contribute to long-term deterioration of parchment artefacts. This work provides valuable information that informs conservation practitioners regarding the use of isopropanol on parchment artefacts. PMID:22462769

Genetics Home Reference: otospondylomegaepiphyseal dysplasia

MedlinePlus

... instructions for making one component of type XI collagen, which is a complex molecule that gives structure ... support the body's joints and organs. Type XI collagen is found in cartilage, a tough but flexible ...

Simultaneous characterization of pancreatic stellate cells and other pancreatic components within three-dimensional tissue environment during chronic pancreatitis

NASA Astrophysics Data System (ADS)

Hu, Wenyan; Fu, Ling

2013-05-01

Pancreatic stellate cells (PSCs) and other pancreatic components that play a critical role in exocrine pancreatic diseases are generally identified separately by conventional studies, which provide indirect links between these components. Here, nonlinear optical microscopy was evaluated for simultaneous characterization of these components within a three-dimensional (3-D) tissue environment, primarily based on multichannel detection of intrinsic optical emissions and cell morphology. Fresh rat pancreatic tissues harvested at 1 day, 7 days, and 28 days after induction of chronic pancreatitis were imaged, respectively. PSCs, inflammatory cells, blood vessels, and collagen fibers were identified simultaneously. The PSCs at day 1 of chronic pancreatitis showed significant enlargement compared with those in normal pancreas (p<0.001, analysis of variance linear contrast; n=8 for each group). Pathological events relating to these components were observed, including presence of inflammatory cells, deposited collagen, and phenotype conversion of PSCs. We demonstrate that label-free nonlinear optical microscopy is an efficient tool for dissecting PSCs and other pancreatic components coincidently within 3-D pancreatic tissues. It is a prospect for intravital observation of dynamic events under natural physiological conditions, and might help uncover the key mechanisms of exocrine pancreatic diseases, leading to more effective treatments.

Matrix composition and mechanics of decellularized lung scaffolds.

PubMed

Petersen, Thomas H; Calle, Elizabeth A; Colehour, Maegen B; Niklason, Laura E

2012-01-01

The utility of decellularized native tissues for tissue engineering has been widely demonstrated. Here, we examine the production of decellularized lung scaffolds from native rodent lung using two different techniques, principally defined by use of either the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or sodium dodecyl sulfate (SDS). All viable cellular material is removed, including at least 99% of DNA. Histochemical staining and mechanical testing indicate that collagen and elastin are retained in the decellularized matrices with CHAPS-based decellularization, while SDS-based decellularization leads to loss of collagen and decline in mechanical strength. Quantitative assays confirm that most collagen is retained with CHAPS treatment but that about 80% of collagen is lost with SDS treatment. In contrast, for both detergent methods, at least 60% of elastin content is lost along with about 95% of native proteoglycan content. Mechanical testing of the decellularized scaffolds indicates that they are mechanically similar to native lung using CHAPS decellularization, including retained tensile strength and elastic behavior, demonstrating the importance of collagen and elastin in lung mechanics. With SDS decellularization, the mechanical integrity of scaffolds is significantly diminished with some loss of elastic function as well. Finally, a simple theoretical model of peripheral lung matrix mechanics is consonant with our experimental findings. This work demonstrates the feasibility of producing a decellularized lung scaffold that can be used to study lung matrix biology and mechanics, independent of the effects of cellular components. Copyright © 2011 S. Karger AG, Basel.

Evaluation of inflammatory processes by FTIR spectroscopy.

PubMed

Rodrigues, Laís Morandini; Carvalho, Luís Felipe das Chagas E Silva; Bonnier, Franck; Anbinder, Ana Lia; Martinho, Herculano da Silva; Almeida, Janete Dias

2018-04-01

Fourier transform infrared (FTIR) spectroscopy is a powerful diagnosis technique and has been used to identify patterns of molecular changes based on vibration modes. The objective of this study was to evaluate inflammatory fibrous hyperplasia (IFH) lesions and oral normal mucosa (NM) initially with histopathological exam and then using micro-FTIR spectroscopy to analyse the samples. Eleven IFH and 11 NM samples were analysed at five different points to cover the largest area possible by the micro-FTIR technique. Bands were observed between 970 and 1743 cm -1 which corresponded to different structural components like collagen, lipids, fatty acids, proteins and amino acids. Spectral bands were more intense mostly for IFH lesions, including collagen bands, which are an important component of inflammatory fibrous hyperplasia. This study demonstrated that differentiation in the inflammatory tissue was observed in FTIR spectral differences, in terms of biochemical composition.

Collagen XVII: A Shared Antigen in Neurodermatological Interactions?

PubMed Central

2013-01-01

Collagen XVII is a nonfibril-forming transmembrane collagen, which functions as both a matrix protein and a cell-surface receptor. It is particularly copious in the skin, where it is known to be a structural component of hemidesmosomes. In addition, collagen XVII has been found to be present in the central nervous system, thus offering an explanation for the statistical association between bullous pemphigoid, in which autoimmunity is directed against dermal collagen XVII, and neurological diseases. In support of the hypothesis that collagen XVII serves as a shared antigen mediating an immune response between skin and brain, research on animal and human tissue, as well as numerous epidemiological and case studies, is presented. PMID:23878581

Determination of three-dimensional molecular orientation of type-I collagen by circularly-polarized second harmonic generation imaging

NASA Astrophysics Data System (ADS)

Zhuo, Guan-Yu; Hung, Wei-Han; Kao, Fu-Jen

2017-04-01

The content of collagen is up to 30% existing in mammals. It supports the main component of connective tissues such as skin, ligament, and cartilage. Among various types of collagen, type-I collagen is of the most abundance and has been broadly studied due to the importance in bioscience. Second harmonic generation (SHG) microscopy is an effective tool used to study the collagen organization without labeling. In this study, we used circular polarization instead of linear polarization to retrieve three-dimensional (3D) molecular orientation of type-I collagen with only two cross polarized SHG images without acquiring an image stack of varying polarization.

Improving nanoparticle diffusion through tumor collagen matrix by photo-thermal gold nanorods

NASA Astrophysics Data System (ADS)

Raeesi, Vahid; Chan, Warren C. W.

2016-06-01

Collagen (I) impairs the targeting of nanoparticles to tumor cells by obstructing their diffusion inside dense tumor interstitial matrix. This potentially makes large nanoparticles (>50 nm) reside near the tumor vessels and thereby compromises their functionality. Here we propose a strategy to locally improve nanoparticle transport inside collagen (I) component of the tumor tissue. We first used heat generating gold nanorods to alter collagen (I) matrix by local temperature elevation. We then explored this impact on the transport of 50 nm and 120 nm inorganic nanoparticles inside collagen (I). We demonstrated an increase in average diffusivity of 50 nm and 120 nm in the denatured collagen (I) by ~14 and ~21 fold, respectively, compared to intact untreated collagen (I) matrix. This study shows how nanoparticle-mediated hyperthermia inside tumor tissue can improve the transport of large nanoparticles through collagen (I) matrix. The ability to increase nanoparticles diffusion inside tumor stroma allows their targeting or other functionalities to take effect, thereby significantly improving cancer therapeutic or diagnostic outcome.Collagen (I) impairs the targeting of nanoparticles to tumor cells by obstructing their diffusion inside dense tumor interstitial matrix. This potentially makes large nanoparticles (>50 nm) reside near the tumor vessels and thereby compromises their functionality. Here we propose a strategy to locally improve nanoparticle transport inside collagen (I) component of the tumor tissue. We first used heat generating gold nanorods to alter collagen (I) matrix by local temperature elevation. We then explored this impact on the transport of 50 nm and 120 nm inorganic nanoparticles inside collagen (I). We demonstrated an increase in average diffusivity of 50 nm and 120 nm in the denatured collagen (I) by ~14 and ~21 fold, respectively, compared to intact untreated collagen (I) matrix. This study shows how nanoparticle-mediated hyperthermia inside tumor tissue can improve the transport of large nanoparticles through collagen (I) matrix. The ability to increase nanoparticles diffusion inside tumor stroma allows their targeting or other functionalities to take effect, thereby significantly improving cancer therapeutic or diagnostic outcome. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08463f

Glycosaminoglycans contribute to extracellular matrix fiber recruitment and arterial wall mechanics.

PubMed

Mattson, Jeffrey M; Turcotte, Raphaël; Zhang, Yanhang

2017-02-01

Elastic and collagen fibers are well known to be the major load-bearing extracellular matrix (ECM) components of the arterial wall. Studies of the structural components and mechanics of arterial ECM generally focus on elastin and collagen fibers, and glycosaminoglycans (GAGs) are often neglected. Although GAGs represent only a small component of the vessel wall ECM, they are considerably important because of their diverse functionality and their role in pathological processes. The goal of this study was to study the mechanical and structural contributions of GAGs to the arterial wall. Biaxial tensile testing was paired with multiphoton microscopic imaging of elastic and collagen fibers in order to establish the structure-function relationships of porcine thoracic aorta before and after enzymatic GAG removal. Removal of GAGs results in an earlier transition point of the nonlinear stress-strain curves [Formula: see text]. However, stiffness was not significantly different after GAG removal treatment, indicating earlier but not absolute stiffening. Multiphoton microscopy showed that when GAGs are removed, the adventitial collagen fibers are straighter, and both elastin and collagen fibers are recruited at lower levels of strain, in agreement with the mechanical change. The amount of stress relaxation also decreased in GAG-depleted arteries [Formula: see text]. These findings suggest that the interaction between GAGs and other ECM constituents plays an important role in the mechanics of the arterial wall, and GAGs should be considered in addition to elastic and collagen fibers when studying arterial function.

Evaluation of tissue components in the peripheral nervous system using Sirius red staining and immunohistochemistry: a comparative study (human, pig, rat).

PubMed

Kaemmer, D; Bozkurt, A; Otto, J; Junge, K; Klink, C; Weis, J; Sellhaus, B; O'Dey, D M; Pallua, N; Jansen, M; Schumpelick, V; Klinge, U

2010-06-30

Little is known about species differences in the peripheral nerve system and quantitative evaluation of main tissue components has rarely been done. Nevertheless, animal models are used for example in pain research without exact knowledge of degree of fibrosis in pathological states which would determine possible treatment options. It would therefore be of crucial interest to describe the degree of fibrosis and the remaining functional nerve tissue as exact as possible. In the present study we evaluated collagen (stroma) and nerve fiber (parenchyma) composition of peripheral nerves in three species (human, rat, pig) and used digital colour-separation and analysis for collagen type differentiation and quantification of immuno-positive-stained area. We found similar ratios of collagen types I and III in epineurium and similar immuno-positive area for staining of neurofilament and S-100beta. In contrast, we measured significantly different ratios of collagen type I to type III in the endoneurium. This combined analysis of the main tissue components of peripheral nerves could be an easy-to-use tool in evaluating changes during damage caused by scaring, systemic disease or compression syndromes. The calculated collagen type I/III ratio may serve as an objective diagnostic value for the description or as prognostic marker for therapeutic approaches in peripheral nerve pathology. However, in particular studies of collagen accumulation in nerves, species dependant differences have to be considered. Copyright 2010 Elsevier B.V. All rights reserved.

Radicals initiated by gamma-rays in collagen and its main components

NASA Astrophysics Data System (ADS)

Kornacka, Ewa Maria; Przybytniak, Grażyna; Zimek, Zbigniew

2018-01-01

Radical products generated by gamma-rays were identified in collagen and in microcrystalline powders of glycyne, L-proline and L-hydroksyproline. Reagents irradiated at 77 K were studied by EPR spectroscopy in the range of 100-350 K using temperature control system. Two radical centers found in collagen were localized in proline ring at α and γ positions to the carbonyl group. There were neither terminal radicals generated by chain scission nor transient products disrupting hydrogen bonding system of irradiated collagen.

Collagen Membrane and Immune Response in Guided Bone Regeneration: Recent Progress and Perspectives.

PubMed

Chu, Chenyu; Deng, Jia; Sun, Xianchang; Qu, Yili; Man, Yi

2017-10-01

Collagen is one of the important components of collagen membranes as well as the extracellular matrix (ECM). Most previous studies have focused on combining collagen membranes with various cross-linking agents, grafting materials, and cytokines to enhance their mechanical properties and bioactivities. Moreover, collagen membranes are often designed to minimize foreign body reactions involving macrophages. However, macrophages were recently found to play a pivotal role during bone regeneration based on their polarization into both proinflammatory and anti-inflammatory phenotypes. Because of the abilities to modulate macrophage polarization and mediate the balance of proinflammatory and anti-inflammatory microenvironments, immune-responsive collagen membranes may be an innovative strategy for promoting bone regeneration. Herein, following a brief review of collagen membranes and the background of macrophages, recent modulations and studies of immune-responsive collagen are described to express the potential of collagen interacting with macrophages and the necessity of further studies in the field of immune-responsive collagen membranes.

Multi-component nanofibrous scaffolds with tunable properties for bone tissue engineering

NASA Astrophysics Data System (ADS)

Jose, Moncy V.

Bone is a highly complex tissue which is an integral part of vertebrates and hence any damage has a major negative effect on the quality of life. Tissue engineering is regarded as an ideal route to resolve the issues related to the scarcity of tissue and organ for transplantation. Apart from cell line and growth factors, the choice of materials and fabrication technique for scaffold are equally important. The goal of this work was to develop a multi-component nanofibrous scaffold based on a synthetic polymer (poly(lactic-co-glycolide) (PLGA)), a biopolymer (collagen) and a biomineral (nano-hydroxyapatite (nano-HA)) by electrospinning technique, which mimics the nanoscopic, chemical, and anisotropic features of bone. Preliminary studies involved fabrication of nanocomposite scaffolds based on PLGA and nano-HA. Morphological and mechanical characterizations revealed that at low concentrations, nano-HA acted as reinforcements, whereas at higher concentrations the presence of aggregation was detrimental to the scaffold. Hydrolytic degradation studies revealed the scaffold had a little mass loss and the mechanical property was maintained for a period of 6 weeks. This study was followed by evaluation of a blend system based on PLGA and collagen. Collagen addition provides hydrophilicity and the necessary cell binding sites in PLGA. The structural characterization revealed that the blend had limited interactions between the two components. The mechanical characterization revealed that with increasing collagen concentration, there was a decline in mechanical properties. However, crosslinking of the blend system, with carbodiimide (EDC) resulted in improving the mechanical properties of the scaffolds. A multi-component system was developed by adding different concentrations of nano-HA to a fixed PLGA/collagen blend composition (80/20). Morphological and mechanical characterizations revealed properties similar to the PLGA/HA system. Cyto-compatibility studies revealed favorable cell adhesion and proliferation. Protein adsorption studies showed the higher surface area as well as the presence of collagen resulted in higher fibronectin and vitronectin adsorption. Crosslinking by EDC resulted in enhanced mechanical property in hydrated state and enhanced degradation stability. These results suggest that such a multi-component system can take advantage of the mechanical benefit available from the individual components and also provide specific biological cues necessary for a successful scaffold.

Reciprocal signalling by Notch-Collagen V-CALCR retains muscle stem cells in their niche.

PubMed

Baghdadi, Meryem B; Castel, David; Machado, Léo; Fukada, So-Ichiro; Birk, David E; Relaix, Frederic; Tajbakhsh, Shahragim; Mourikis, Philippos

2018-05-01

The cell microenvironment, which is critical for stem cell maintenance, contains both cellular and non-cellular components, including secreted growth factors and the extracellular matrix 1-3 . Although Notch and other signalling pathways have previously been reported to regulate quiescence of stem cells 4-9 , the composition and source of molecules that maintain the stem cell niche remain largely unknown. Here we show that adult muscle satellite (stem) cells in mice produce extracellular matrix collagens to maintain quiescence in a cell-autonomous manner. Using chromatin immunoprecipitation followed by sequencing, we identified NOTCH1/RBPJ-bound regulatory elements adjacent to specific collagen genes, the expression of which is deregulated in Notch-mutant mice. Moreover, we show that Collagen V (COLV) produced by satellite cells is a critical component of the quiescent niche, as depletion of COLV by conditional deletion of the Col5a1 gene leads to anomalous cell cycle entry and gradual diminution of the stem cell pool. Notably, the interaction of COLV with satellite cells is mediated by the Calcitonin receptor, for which COLV acts as a surrogate local ligand. Systemic administration of a calcitonin derivative is sufficient to rescue the quiescence and self-renewal defects found in COLV-null satellite cells. This study reveals a Notch-COLV-Calcitonin receptor signalling cascade that maintains satellite cells in a quiescent state in a cell-autonomous fashion, and raises the possibility that similar reciprocal mechanisms act in diverse stem cell populations.

Cell-ECM Interactions During Cancer Invasion

NASA Astrophysics Data System (ADS)

Jiang, Yi

The extracellular matrix (ECM), a fibrous material that forms a network in a tissue, significantly affects many aspects of cellular behavior, including cell movement and proliferation. Transgenic mouse tumor studies indicate that excess collagen, a major component of ECM, enhances tumor formation and invasiveness. Clinically, tumor associated collagen signatures are strong markers for breast cancer survival. However, the underlying mechanisms are unclear since the properties of ECM are complex, with diverse structural and mechanical properties depending on various biophysical parameters. We have developed a three-dimensional elastic fiber network model, and parameterized it with in vitro collagen mechanics. Using this model, we study ECM remodeling as a result of local deformation and cell migration through the ECM as a network percolation problem. We have also developed a three-dimensional, multiscale model of cell migration and interaction with ECM. Our model reproduces quantitative single cell migration experiments. This model is a first step toward a fully biomechanical cell-matrix interaction model and may shed light on tumor associated collagen signatures in breast cancer. This work was partially supported by NIH-U01CA143069.

Differential Actions of the Endocytic Collagen Receptor uPARAP/Endo180 and the Collagenase MMP-2 in Bone Homeostasis

PubMed Central

Madsen, Daniel H.; Jürgensen, Henrik J.; Ingvarsen, Signe; Melander, Maria C.; Albrechtsen, Reidar; Hald, Andreas; Holmbeck, Kenn; Bugge, Thomas H.; Behrendt, Niels; Engelholm, Lars H.

2013-01-01

A well-coordinated remodeling of uncalcified collagen matrices is a pre-requisite for bone development and homeostasis. Collagen turnover proceeds through different pathways, either involving extracellular reactions exclusively, or being dependent on endocytic processes. Extracellular collagen degradation requires the action of secreted or membrane attached collagenolytic proteases, whereas the alternative collagen degradation pathway proceeds intracellularly after receptor-mediated uptake and delivery to the lysosomes. In this study we have examined the functional interplay between the extracellular collagenase, MMP-2, and the endocytic collagen receptor, uPARAP, by generating mice with combined deficiency of both components. In both uPARAP-deficient and MMP-2-deficient adult mice the length of the tibia and femur was decreased, along with a reduced bone mineral density and trabecular bone quality. An additional decrease in bone length was observed when combining the two deficiencies, pointing to both components being important for the remodeling processes in long bone growth. In agreement with results found by others, a different effect of MMP-2 deficiency was observed in the distinct bone structures of the calvaria. These membranous bones were found to be thickened in MMP-2-deficient mice, an effect likely to be related to an accompanying defect in the canalicular system. Surprisingly, both of the latter defects in MMP-2-deficient mice were counteracted by concurrent uPARAP deficiency, demonstrating that the collagen receptor does not support the same matrix remodeling processes as the MMP in the growth of the skull. We conclude that both uPARAP and MMP-2 take part in matrix turnover processes important for bone growth. However, in some physiological situations, these two components do not support the same step in the growth process. PMID:23940733

Second Harmonic Generation Confocal Microscopy of Collagen Type I from Rat Tendon Cryosections

PubMed Central

Theodossiou, Theodossis A.; Thrasivoulou, Christopher; Ekwobi, Chidi; Becker, David L.

2006-01-01

We performed second harmonic generation (SHG) imaging of collagen in rat-tendon cryosections, using femtosecond laser scanning confocal microscopy, both in backscattering and transmission geometries. SHG transmission images of collagen fibers were spatially resolved due to a coherent, directional SHG component. This effect was enhanced with the use of an index-matching fluid (ni = 1.52). The average SHG intensity oscillated with wavelength in the backscattered geometry (isotropic SHG component), whereas the spectral profile was consistent with quasi-phase-matching conditions in transmission geometry (forward propagating, coherent SHG component) around 440 nm (λp = 880 nm). Collagen type I from bovine Achilles tendon was imaged for SHG in the backscattered geometry and its first-order effective nonlinear coefficient was determined (\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\vert}d_{{\\mathrm{eff}}}{\\vert}\\approx 0.085({\\pm}0.025){\\times}10^{-12}{\\mathrm{mV}}^{-1}\\end{equation*}\\end{document}) by comparison to samples of inorganic materials with known effective nonlinear coefficients (LiNbO3 and LiIO3). The SHG spectral response of collagen type I from bovine Achilles tendon matched that of the rat-tendon cryosections in backscattered geometry. Collagen types I, II, and VI powders (nonfibrous) did not show any detectable SHG, indicating a lack of noncentrosymmetric crystalline structure at the molecular level. The various stages of collagen thermal denaturation were investigated in rat-tendon cryosections using SHG and bright-field imaging. Thermal denaturation resulted in the gradual destruction of the SHG signal. PMID:17130233

Characterizing of tissue microstructure with single-detector polarization-sensitive optical coherence tomography

NASA Astrophysics Data System (ADS)

Liu, Bin; Harman, Michelle; Giattina, Susanne; Stamper, Debra L.; Demakis, Charles; Chilek, Mark; Raby, Stephanie; Brezinski, Mark E.

2006-06-01

Assessing tissue birefringence with imaging modality polarization-sensitive optical coherence tomography (PS-OCT) could improve the characterization of in vivo tissue pathology. Among the birefringent components, collagen may provide invaluable clinical information because of its alteration in disorders ranging from myocardial infarction to arthritis. But the features required of clinical imaging modality in these areas usually include the ability to assess the parameter of interest rapidly and without extensive data analysis, the characteristics that single-detector PS-OCT demonstrates. But beyond detecting organized collagen, which has been previously demonstrated and confirmed with the appropriate histological techniques, additional information can potentially be gained with PS-OCT, including collagen type, form versus intrinsic birefringence, the collagen angle, and the presence of multiple birefringence materials. In part I, we apply the simple but powerful fast-Fourier transform (FFT) to both PS-OCT mathematical modeling and in vitro bovine meniscus for improved PS-OCT data analysis. The FFT analysis yields, in a rapid, straightforward, and easily interpreted manner, information on the presence of multiple birefringent materials, distinguishing the true anatomical structure from patterns in image resulting from alterations in the polarization state and identifying the tissue/phantom optical axes. Therefore the use of the FFT analysis of PS-OCT data provides information on tissue composition beyond identifying the presence of organized collagen in real time and directly from the image without extensive mathematical manipulation or data analysis. In part II, Helistat phantoms (collagen type I) are analyzed with the ultimate goal of improved tissue characterization. This study, along with the data in part I, advance the insights gained from PS-OCT images beyond simply determining the presence or absence of birefringence.

Type IV collagen is a novel DEJ biomarker that is reduced by radiotherapy.

PubMed

McGuire, J D; Gorski, J P; Dusevich, V; Wang, Y; Walker, M P

2014-10-01

The dental basement membrane (BM) is composed of collagen types IV, VI, VII, and XVII, fibronectin, and laminin and plays an inductive role in epithelial-mesenchymal interactions during tooth development. The BM is degraded and removed during later-stage tooth morphogenesis; however, its original position defines the location of the dentin-enamel junction (DEJ) in mature teeth. We recently demonstrated that type VII collagen is a novel component of the inner enamel organic matrix layer contiguous with the DEJ. Since it is frequently co-expressed with and forms functional complexes with type VII collagen, we hypothesized that type IV collagen should also be localized to the DEJ in mature human teeth. To identify collagen IV, we first evaluated defect-free erupted teeth from various donors. To investigate a possible stabilizing role, we also evaluated extracted teeth exposed to high-dose radiotherapy--teeth that manifest post-radiotherapy DEJ instability. We now show that type IV collagen is a component within the morphological DEJ of posterior and anterior teeth from individuals aged 18 to 80 yr. Confocal microscopy revealed that immunostained type IV collagen was restricted to the 5- to 10-µm-wide optical DEJ, while collagenase treatment or previous in vivo tooth-level exposure to > 60 Gray irradiation severely reduced immunoreactivity. This assignment was confirmed by Western blotting with whole-tooth crown and enamel extracts. Without reduction, type IV collagen contained macromolecular α-chains of 225 and 250 kDa. Compositionally, our results identify type IV collagen as the first macromolecular biomarker of the morphological DEJ of mature teeth. Given its network structure and propensity to stabilize the dermal-epidermal junction, we propose that a collagen-IV-enriched DEJ may, in part, explain its well-known fracture toughness, crack propagation resistance, and stability. In contrast, loss of type IV collagen may represent a biochemical rationale for the DEJ instability observed following oral cancer radiotherapy. © International & American Associations for Dental Research.

Type IV Collagen is a Novel DEJ Biomarker that is Reduced by Radiotherapy

PubMed Central

McGuire, J.D.; Gorski, J.P.; Dusevich, V.; Wang, Y.; Walker, M.P.

2014-01-01

The dental basement membrane (BM) is composed of collagen types IV, VI, VII, and XVII, fibronectin, and laminin and plays an inductive role in epithelial-mesenchymal interactions during tooth development. The BM is degraded and removed during later-stage tooth morphogenesis; however, its original position defines the location of the dentin-enamel junction (DEJ) in mature teeth. We recently demonstrated that type VII collagen is a novel component of the inner enamel organic matrix layer contiguous with the DEJ. Since it is frequently co-expressed with and forms functional complexes with type VII collagen, we hypothesized that type IV collagen should also be localized to the DEJ in mature human teeth. To identify collagen IV, we first evaluated defect-free erupted teeth from various donors. To investigate a possible stabilizing role, we also evaluated extracted teeth exposed to high-dose radiotherapy – teeth that manifest post-radiotherapy DEJ instability. We now show that type IV collagen is a component within the morphological DEJ of posterior and anterior teeth from individuals aged 18 to 80 yr. Confocal microscopy revealed that immunostained type IV collagen was restricted to the 5- to 10-µm-wide optical DEJ, while collagenase treatment or previous in vivo tooth-level exposure to > 60 Gray irradiation severely reduced immunoreactivity. This assignment was confirmed by Western blotting with whole-tooth crown and enamel extracts. Without reduction, type IV collagen contained macromolecular α-chains of 225 and 250 kDa. Compositionally, our results identify type IV collagen as the first macromolecular biomarker of the morphological DEJ of mature teeth. Given its network structure and propensity to stabilize the dermal-epidermal junction, we propose that a collagen-IV-enriched DEJ may, in part, explain its well-known fracture toughness, crack propagation resistance, and stability. In contrast, loss of type IV collagen may represent a biochemical rationale for the DEJ instability observed following oral cancer radiotherapy. PMID:25146181

Regulation of Corneal Stroma Extracellular Matrix Assembly

PubMed Central

Chen, Shoujun; Mienaltowski, Michael J.; Birk, David E.

2014-01-01

The transparent cornea is the major refractive element of the eye. A finely controlled assembly of the stromal extracellular matrix is critical to corneal function, as well as in establishing the appropriate mechanical stability required to maintain corneal shape and curvature. In the stroma, homogeneous, small diameter collagen fibrils, regularly packed with a highly ordered hierarchical organization, are essential for function. This review focuses on corneal stroma assembly and the regulation of collagen fibrillogenesis. Corneal collagen fibrillogenesis involves multiple molecules interacting in sequential steps, as well as interactions between keratocytes and stroma matrix components. The stroma has the highest collagen V:I ratio in the body. Collagen V regulates the nucleation of protofibril assembly, thus controlling the number of fibrils and assembly of smaller diameter fibrils in the stroma. The corneal stroma is also enriched in small leucine-rich proteoglycans (SLRPs) that cooperate in a temporal and spatial manner to regulate linear and lateral collagen fibril growth. In addition, the fibril-associated collagens (FACITs) such as collagen XII and collagen XIV have roles in the regulation of fibril packing and inter-lamellar interactions. A communicating keratocyte network contributes to the overall and long-range regulation of stromal extracellular matrix assembly, by creating micro-domains where the sequential steps in stromal matrix assembly are controlled. Keratocytes control the synthesis of extracellular matrix components, which interact with the keratocytes dynamically to coordinate the regulatory steps into a cohesive process. Mutations or deficiencies in stromal regulatory molecules result in altered interactions and deficiencies in both transparency and refraction, leading to corneal stroma pathobiology such as stromal dystrophies, cornea plana and keratoconus. PMID:25819456

Genetics Home Reference: Weissenbacher-Zweymüller syndrome

MedlinePlus

... instructions for making one component of type XI collagen, which is a complex molecule that gives structure ... support the body's joints and organs. Type XI collagen is found in cartilage, a tough but flexible ...

From Nano to Macro: Studying the Hierarchical Structure of the Corneal Extracellular Matrix

PubMed Central

Quantock, Andrew J.; Winkler, Moritz; Parfitt, Geraint J.; Young, Robert D.; Brown, Donald J.; Boote, Craig; Jester, James V.

2014-01-01

In this review, we discuss current methods for studying ocular extracellular matrix (ECM) assembly from the ‘nano’ to the ‘macro’ levels of hierarchical organization. Since collagen is the major structural protein in the eye, providing mechanical strength and controlling ocular shape, the methods presented focus on understanding the molecular assembly of collagen at the nanometer level using x-ray scattering through to the millimeter to centimeter level using nonlinear optical (NLO) imaging of second harmonic generated (SHG) signals. Three-dimensional analysis of ECM structure is also discussed, including electron tomography, serial block face scanning electron microscopy (SBF-SEM) and digital image reconstruction. Techniques to detect non-collagenous structural components of the ECM are also presented, and these include immunoelectron microscopy and staining with cationic dyes. Together, these various approaches are providing new insights into the structural blueprint of the ocular ECM, and in particular that of the cornea, which impacts upon our current understanding of the control of corneal shape, pathogenic mechanisms underlying ectatic disorders of the cornea and the potential for corneal tissue engineering. PMID:25819457

Natural healing-inspired collagen-targeting surgical protein glue for accelerated scarless skin regeneration.

PubMed

Jeon, Eun Young; Choi, Bong-Hyuk; Jung, Dooyup; Hwang, Byeong Hee; Cha, Hyung Joon

2017-07-01

Skin scarring after deep dermal injuries is a major clinical problem due to the current therapies limited to established scars with poor understanding of healing mechanisms. From investigation of aberrations within the extracellular matrix involved in pathophysiologic scarring, it was revealed that one of the main factors responsible for impaired healing is abnormal collagen reorganization. Here, inspired by the fundamental roles of decorin, a collagen-targeting proteoglycan, in collagen remodeling, we created a scar-preventive collagen-targeting glue consisting of a newly designed collagen-binding mussel adhesive protein and a specific glycosaminoglycan. The collagen-targeting glue specifically bound to type I collagen in a dose-dependent manner and regulated the rate and the degree of fibrillogenesis. In a rat skin excisional model, the collagen-targeting glue successfully accelerated initial wound regeneration as defined by effective reepithelialization, neovascularization, and rapid collagen synthesis. Moreover, the improved dermal collagen architecture was demonstrated by uniform size of collagen fibrils, their regular packing, and a restoration of healthy tissue component. Collectively, our natural healing-inspired collagen-targeting glue may be a promising therapeutic option for improving the healing rate with high-quality and effective scar inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

In situ hybridization reveals that type I and III collagens are produced by pericytes in the anterior pituitary gland of rats.

PubMed

Fujiwara, Ken; Jindatip, Depicha; Kikuchi, Motoshi; Yashiro, Takashi

2010-12-01

Type I and III collagens widely occur in the rat anterior pituitary gland and are the main components of the extracellular matrix (ECM). Although ECM components possibly play an important role in the function of the anterior pituitary gland, little is known about collagen-producing cells. Type I collagen is a heterotrimer of two α1(I) chains (the product of the col1a1 gene) and one α2(I) chain (the product of the col1a2 gene). Type III collagen is a homotrimer of α1(III) chains (the product of the col3a1 gene). We used in situ hybridization with digoxigenin-labeled cRNA probes to examine the expression of col1a1, col1a2, and col3a1 mRNAs in the pituitary gland of adult rats. mRNA expression for these collagen genes was clearly observed, and cells expressing col1a1, col1a2, and col3a1 mRNA were located around capillaries in the gland. We also investigated the possible double-staining of collagen mRNA and pituitary hormones, S-100 protein (a marker of folliculo-stellate cells), or desmin (a marker of pericytes). Col1a1 and col3a1 mRNA were identified in desmin-immunopositive cells. Thus, only pericytes produce type I and III collagens in the rat anterior pituitary gland.

Keratocyte behavior in three-dimensional photopolymerizable poly(ethylene glycol) hydrogels

PubMed Central

Thibault, Richard; Ambrose, Winnette McIntosh; Schein, Oliver D.; Chakravarti, Shukti; Elisseeff, Jennifer

2015-01-01

The goal of this study was to evaluate three-dimensional (3-D) poly(ethylene glycol) (PEG) hydrogels as a culture system for studying corneal keratocytes. Bovine keratocytes were subcultured in DMEM/F-12 containing 10% fetal bovine serum (FBS) through passage 5. Primary keratocytes (P0) and corneal fibroblasts from passages 1 (P1) and 3 (P3) were photoencapsulated at various cell concentrations in PEG hydrogels via brief exposure to light. Additional hydrogels contained adhesive YRGDS and nonadhesive YRDGS peptides. Hydrogel constructs were cultured in DMEM/F-12 with 10% FBS for 2 and 4 weeks. Cell viability was assessed by DNA quantification and vital staining. Biglycan, type I collagen, type III collagen, keratocan and lumican expression were determined by reverse transcriptase–polymerase chain reaction. Deposition of type I collagen, type III collagen and keratan sulfate (KS)-containing matrix components was visualized using confocal microscopy. Keratocytes in a monolayer lost their stellate morphology and keratocan expression, displayed elongated cell bodies, and up-regulated biglycan, type I collagen and type III collagen characteristic of corneal fibroblasts. Encapsulated keratocytes remained viable for 4 weeks with spherical morphologies. Hydrogels supported production of KS, type I collagen and type III collagen matrix components. PEG-based hydrogels can support keratocyte viability and matrix production. 3-D hydrogel culture can stabilize but not restore the keratocyte phenotype. This novel application of PEG hydrogels has potential use in the study of corneal keratocytes in a 3-D environment. PMID:18567550

Development of a nanofiltration method for bone collagen 14C AMS dating

NASA Astrophysics Data System (ADS)

Boudin, Mathieu; Boeckx, Pascal; Buekenhoudt, Anita; Vandenabeele, Peter; Van Strydonck, Mark

2013-01-01

Radiocarbon dating of bones is usually performed on the collagen fraction. However, this collagen can contain exogenous molecules, including humic substances (HSs) and/or other soil components that may have a different age than the bone. Incomplete removal can result in biased 14C dates. Ultrafiltration of collagen, dissolved as gelatin (molecular weight (MW) ∼100,000 Dalton), has received considerable attention to obtain more reliable dates. Ultrafiltration is an effective method of removal of low-molecular weight contaminants from bone collagen but it does not remove high-molecular weight contaminants, such as cross-linked humic collagen complexes. However, comparative dating studies have raised the question whether this cleaning step itself may introduce contamination with carbon from the filters used. In this study, a nanofiltration method was developed using a ceramic filter to avoid a possible extraneous carbon contamination introduced by the filter. This method should be applicable to various protein materials e.g. collagen, silk, wool, leather and should be able to remove low-molecular and high molecular weight HSs. In this study bone collagen was hot acid hydrolyzed to amino acids and nanofiltrated. A filter with a molecular weight cutoff (MWCO) of 450 Dalton was chosen in order to collect the amino acids in the permeate and the HSs in the retentate. Two pilot studies were set up. Two nanofiltration types were tested in pilot study 1: dead end and cross flow filtration. Humic substance (HS)-solutions with fossil carbon and modern hydrolyzed collagen contaminated with HSs were filtrated and analyzed with spectrofluorescence to determine the HS removal. Cross flow nanofiltration showed the most efficient HS removal. A second pilot study based upon these results was set up wherein only cross flow filtration was performed. 14C measurements of the permeates of hydrolyzed modern collagen contaminated with fossil HSs demonstrate a significant but incomplete removal of HSs (between 63% and 85%).

Hyperlipidemia affects multiscale structure and strength of murine femur.

PubMed

Ascenzi, Maria-Grazia; Lutz, Andre; Du, Xia; Klimecky, Laureen; Kawas, Neal; Hourany, Talia; Jahng, Joelle; Chin, Jesse; Tintut, Yin; Nackenhors, Udo; Keyak, Joyce

2014-07-18

To improve bone strength prediction beyond limitations of assessment founded solely on the bone mineral component, we investigated the effect of hyperlipidemia, present in more than 40% of osteoporotic patients, on multiscale structure of murine bone. Our overarching purpose is to estimate bone strength accurately, to facilitate mitigating fracture morbidity and mortality in patients. Because (i) orientation of collagen type I affects, independently of degree of mineralization, cortical bone׳s micro-structural strength; and, (ii) hyperlipidemia affects collagen orientation and μCT volumetric tissue mineral density (vTMD) in murine cortical bone, we have constructed the first multiscale finite element (mFE), mouse-specific femoral model to study the effect of collagen orientation and vTMD on strength in Ldlr(-/-), a mouse model of hyperlipidemia, and its control wild type, on either high fat diet or normal diet. Each µCT scan-based mFE model included either element-specific elastic orthotropic properties calculated from collagen orientation and vTMD (collagen-density model) by experimentally validated formulation, or usual element-specific elastic isotropic material properties dependent on vTMD-only (density-only model). We found that collagen orientation, assessed by circularly polarized light and confocal microscopies, and vTMD, differed among groups and that microindentation results strongly correlate with elastic modulus of collagen-density models (r(2)=0.85, p=10(-5)). Collagen-density models yielded (1) larger strains, and therefore lower strength, in simulations of 3-point bending and physiological loading; and (2) higher correlation between mFE-predicted strength and 3-point bending experimental strength, than density-only models. This novel method supports ongoing translational research to achieve the as yet elusive goal of accurate bone strength prediction. Copyright © 2014 Elsevier Ltd. All rights reserved.

Enhanced migration of murine fibroblast-like 3T3-L1 preadipocytes on type I collagen-coated dish is reversed by silibinin treatment.

PubMed

Liu, Xiaoling; Xu, Qian; Liu, Weiwei; Yao, Guodong; Zhao, Yeli; Xu, Fanxing; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Yamato, Masayuki; Ikejima, Takashi

2018-04-01

Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, whereas excessive migration is often responsible for impaired adipose tissue related with obesity and fibrotic diseases. Type I collagen (collagen I) is the most abundant component of extracellular matrix and has been shown to regulate fibroblast migration in vitro, but its role in adipose tissue is not known. Silibinin is a bioactive natural flavonoid with antioxidant and antimetastasis activities. In this study, we found that type I collagen coating promoted the proliferation and migration of murine 3T3-L1 preadipocytes in a dose-dependent manner, implying that collagen I could be an extracellular signal. Regarding the mechanisms of collagen I-stimulated 3T3-L1 migration, we found that NF-κB p65 is activated, including the increased nuclear translocation of NF-κB p65 as well as the upregulation of NF-κB p65 phosphorylation and acetylation, accompanied by the increased expressions of proinflammatory factors and the generation of reactive oxygen species (ROS). Reduction of collagen I-enhanced migration of cells by treatment with silibinin was associated with suppression of NF-κB p65 activity and ROS generation, and negatively correlated with the increasing sirt1 expression. Taken together, the enhanced migration of 3T3-L1 cells induced on collagen I-coated dish is mediated by the activation of NF-κB p65 function and ROS generation that can be alleviated with silibinin by upregulation of sirt1, leading to the repression of NF-κB p65 function and ROS generation.

Collagen matrix as a tool in studying fibroblastic cell behavior.

PubMed

Kanta, Jiří

2015-01-01

Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes.

Collagen matrix as a tool in studying fibroblastic cell behavior

PubMed Central

Kanta, Jiří

2015-01-01

Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes. PMID:25734486

Developments in purification methods for obtaining and evaluation of collagen derived endogenous angioinhibitors

PubMed Central

Gunda, Venugopal; Verma, Raj K.; Pawar, Smita C.; Sudhakar, Yakkanti A.

2013-01-01

Collagen constitutes one of the vital components of the basement membrane scaffolds. Non-collagenous domains (NC1) derived from collagens exhibit potent anti-angiogenic properties, thus attaining significance in regulation of angiogenesis promoted diseases. Individual NC1 domains essential for anti-angiogenic evaluations are generally obtained through purification of individual non-collagenous domains, which have undergone steady developments for enhancing the yields, purpose of biological evaluations and solubility based on the nature of different NC1 domains. This review focuses on the method developments in obtaining biologically active NC1 domains and for specific evaluations in different scenarios. PMID:24215863

Targeting Discoidin Domain Receptors in Prostate Cancer

DTIC Science & Technology

2016-08-01

DDRs), a set of kinase receptors that signal in response to collagen . The project’s goal is to define the expression and therapeutic potential of...antibody, which blocks receptor activation by collagen I. Mice were inoculated with PC3 cells and anti-DDR1 or control antibody treatment. The study... collagen , the major organic component of the bone extracellular matrix. Purpose: To investigate the expression, therapeutic potential, and

Collagen structure of tendon relates to function.

PubMed

Franchi, Marco; Trirè, Alessandra; Quaranta, Marilisa; Orsini, Ester; Ottani, Victoria

2007-03-30

A tendon is a tough band of fibrous connective tissue that connects muscle to bone, designed to transmit forces and withstand tension during muscle contraction. Tendon may be surrounded by different structures: 1) fibrous sheaths or retinaculae; 2) reflection pulleys; 3) synovial sheaths; 4) peritendon sheaths; 5) tendon bursae. Tendons contain a) few cells, mostly represented by tenoblasts along with endothelial cells and some chondrocytes; b) proteoglycans (PGs), mainly decorin and hyaluronan, and c) collagen, mostly type I. Tendon is a good example of a high ordered extracellular matrix in which collagen molecules assemble into filamentous collagen fibrils (formed by microfibrils) which aggregate to form collagen fibers, the main structural components. It represents a multihierarchical structure as it contains collagen molecules arranged in fibrils then grouped in fibril bundles, fascicles and fiber bundles that are almost parallel to the long axis of the tendon, named as primary, secondary and tertiary bundles. Collagen fibrils in tendons show prevalently large diameter, a D-period of about 67 nm and appear built of collagen molecules lying at a slight angle (< 5 degrees). Under polarized light microscopy the collagen fiber bundles appear crimped with alternative dark and light transverse bands. In recent studies tendon crimps observed via SEM and TEM show that the single collagen fibrils suddenly changing their direction contain knots. These knots of collagen fibrils inside each tendon crimp have been termed "fibrillar crimps", and even if they show different aspects they all may fulfil the same functional role. As integral component of musculoskeletal system, the tendon acts to transmit muscle forces to the skeletal system. There is no complete understanding of the mechanisms in transmitting/absorbing tensional forces within the tendon; however it seems likely that a flattening of tendon crimps may occur at a first stage of tendon stretching. Increasing stretching, other transmission mechanisms such as an interfibrillar coupling via PGs linkages and a molecular gliding within the fibrils structure may be involved.

Study in Mice Links Key Signaling Molecule to Underlying Cause of Osteogenesis Imperfecta

MedlinePlus

... by mutations in a gene that codes for collagen, an abundant structural component of bone. This type ... linked to defects in enzymes that help process collagen to its mature form. These types of OI ...

Molecular organization of various collagen fragments as revealed by atomic force microscopy and diffusion-ordered NMR spectroscopy.

PubMed

Stötzel, Sabine; Schurink, Marloes; Wienk, Hans; Siebler, Uschi; Burg-Roderfeld, Monika; Eckert, Thomas; Kulik, Bianca; Wechselberger, Rainer; Sewing, Judith; Steinmeyer, Jürgen; Oesser, Steffen; Boelens, Rolf; Siebert, Hans-Christian

2012-09-17

Heterogeneous mixtures of collagen fragments can be used as nutrition supplement or as key ingredients for ointments with therapeutic relevance in wound healing. Some mixtures of collagen fragments are referred to as collagen hydrolysates owing to the production process with hydrolytic enzymes. Since the precise composition of collagen hydrolysates is generally unknown, it is of interest to analyze samples containing various collagen fragments with appropriate biophysical methods. Any product optimization without a profound knowledge concerning the size and the molecular weight distribution of its components is nearly impossible. It turned out that a combination of AFM methods with NMR techniques is exceptionally suited to examine the size range and the aggregation behavior of the collagen fragments in the hydrolysates of fish, jellyfish, chicken, porcine and bovine collagen. Supported by molecular modeling calculations, the AFM and NMR experiments provide a detailed knowledge about the composition of collagen hydrolysates and collagen ointments. Furthermore, the data allow a correlation between the size of the fragments and their potential bioactivity. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Body wall structure in the starfish Asterias rubens.

PubMed

Blowes, Liisa M; Egertová, Michaela; Liu, Yankai; Davis, Graham R; Terrill, Nick J; Gupta, Himadri S; Elphick, Maurice R

2017-09-01

The body wall of starfish is composed of magnesium calcite ossicles connected by collagenous tissue and muscles and it exhibits remarkable variability in stiffness, which is attributed to the mechanical mutability of the collagenous component. Using the common European starfish Asterias rubens as an experimental animal, here we have employed a variety of techniques to gain new insights into the structure of the starfish body wall. The structure and organisation of muscular and collagenous components of the body wall were analysed using trichrome staining. The muscle system comprises interossicular muscles as well as muscle strands that connect ossicles with the circular muscle layer of the coelomic lining. The collagenous tissue surrounding the ossicle network contains collagen fibres that form loop-shaped straps that wrap around calcite struts near to the surface of ossicles. The 3D architecture of the calcareous endoskeleton was visualised for the first time using X-ray microtomography, revealing the shapes and interactions of different ossicle types. Furthermore, analysis of the anatomical organisation of the ossicles indicates how changes in body shape may be achieved by local contraction/relaxation of interossicular muscles. Scanning synchrotron small-angle X-ray diffraction (SAXD) scans of the starfish aboral body wall and ambulacrum were used to study the collagenous tissue component at the fibrillar level. Collagen fibrils in aboral body wall were found to exhibit variable degrees of alignment, with high levels of alignment probably corresponding to regions where collagenous tissue is under tension. Collagen fibrils in the ambulacrum had a uniformly low degree of orientation, attributed to macrocrimp of the fibrils and the presence of slanted as well as horizontal fibrils connecting antimeric ambulacral ossicles. Body wall collagen fibril D-period lengths were similar to previously reported mammalian D-periods, but were significantly different between the aboral and ambulacral samples. The overlap/D-period length ratio within fibrils was higher than reported for mammalian tissues. Collectively, the data reported here provide new insights into the anatomy of the body wall in A. rubens and a foundation for further studies investigating the structural basis of the mechanical properties of echinoderm body wall tissue composites. © 2017 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.

Regulation of corneal stroma extracellular matrix assembly.

PubMed

Chen, Shoujun; Mienaltowski, Michael J; Birk, David E

2015-04-01

The transparent cornea is the major refractive element of the eye. A finely controlled assembly of the stromal extracellular matrix is critical to corneal function, as well as in establishing the appropriate mechanical stability required to maintain corneal shape and curvature. In the stroma, homogeneous, small diameter collagen fibrils, regularly packed with a highly ordered hierarchical organization, are essential for function. This review focuses on corneal stroma assembly and the regulation of collagen fibrillogenesis. Corneal collagen fibrillogenesis involves multiple molecules interacting in sequential steps, as well as interactions between keratocytes and stroma matrix components. The stroma has the highest collagen V:I ratio in the body. Collagen V regulates the nucleation of protofibril assembly, thus controlling the number of fibrils and assembly of smaller diameter fibrils in the stroma. The corneal stroma is also enriched in small leucine-rich proteoglycans (SLRPs) that cooperate in a temporal and spatial manner to regulate linear and lateral collagen fibril growth. In addition, the fibril-associated collagens (FACITs) such as collagen XII and collagen XIV have roles in the regulation of fibril packing and inter-lamellar interactions. A communicating keratocyte network contributes to the overall and long-range regulation of stromal extracellular matrix assembly, by creating micro-domains where the sequential steps in stromal matrix assembly are controlled. Keratocytes control the synthesis of extracellular matrix components, which interact with the keratocytes dynamically to coordinate the regulatory steps into a cohesive process. Mutations or deficiencies in stromal regulatory molecules result in altered interactions and deficiencies in both transparency and refraction, leading to corneal stroma pathobiology such as stromal dystrophies, cornea plana and keratoconus. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy.

PubMed

Fields, Mark; Spencer, Nicholas; Dudhia, Jayesh; McMillan, Paul F

2017-06-01

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple-helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ∼60-80°C for hydrated samples. We report high temperature Raman spectra for freeze-dried cartilage samples that show an increase in laser-excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H 2 O is retained within the freeze-dried tissue. This is confirmed by thermogravimetric analysis that show 5-7 wt% H 2 O remaining within freeze-dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re-hydration following recovery indicate that the capacity of the denatured collagen to re-absorb water is reduced. Our results are important for revealing the presence of bound H 2 O within the collagen component of connective tissue even after freeze-drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure. © 2017 Wiley Periodicals, Inc.

In vitro synthesis of tensioned synoviocyte bioscaffolds for meniscal fibrocartilage tissue engineering.

PubMed

Warnock, Jennifer J; Baker, Lindsay; Ballard, George A; Ott, Jesse

2013-12-03

Meniscal injury is a common cause of lameness in the dog. Tissue engineered bioscaffolds may be a treatment option for meniscal incompetency, and ideally would possess meniscus- like extracellular matrix (ECM) and withstand meniscal tensile hoop strains. Synovium may be a useful cell source for meniscal tissue engineering because of its natural role in meniscal deficiency and its in vitro chondrogenic potential. The objective of this study is to compare meniscal -like extracellular matrix content of hyperconfluent synoviocyte cell sheets ("HCS") and hyperconfluent synoviocyte sheets which have been tensioned over wire hoops (tensioned synoviocyte bioscaffolds, "TSB") and cultured for 1 month. Long term culture with tension resulted in higher GAG concentration, higher chondrogenic index, higher collagen concentration, and type II collagen immunoreactivity in TSB versus HCS. Both HCS and TSB were immunoreactive for type I collagen, however, HCS had mild, patchy intracellular immunoreactivity while TSB had diffuse moderate immunoreactivity over the entire bisocaffold. The tissue architecture was markedly different between TSB and HCS, with TSB containing collagen organized in bands and sheets. Both HCS and TSB expressed alpha smooth muscle actin and displayed active contractile behavior. Double stranded DNA content was not different between TSB and HCS, while cell viability decreased in TSB. Long term culture of synoviocytes with tension improved meniscal- like extra cellular matrix components, specifically, the total collagen content, including type I and II collagen, and increased GAG content relative to HCS. Future research is warranted to investigate the potential of TSB for meniscal tissue engineering.

Fibroblast populated collagen lattices exhibit opposite biophysical conditions by fibrin or hyaluronic acid supplementation.

PubMed

Chopin-Doroteo, Mario; Salgado-Curiel, Rosa M; Pérez-González, José; Marín-Santibáñez, Benjamín M; Krötzsch, Edgar

2018-06-01

Fibrin and hyaluronic acid are important components of the provisional wound matrix. Through interactions with fibroblasts, they provide biophysical cues that regulate the viscoelastic properties of the extracellular matrix. To understand the roles of fibrin and hyaluronic acid in a collagenous environment, we used fibroblast populated collagen lattices (collagen, collagen-fibrin, and collagen-hyaluronic acid). Compared with collagen and collagen-hyaluronic acid cultures, collagen-fibrin cultures showed less contraction, which is correlated with increased elastic (G') and complex (|G*|) moduli, and reduced proportions of dendritic fibroblasts, despite increased αv integrin expression. Stiffness decreased during culture in collagen-fibrin environment, meanwhile phase shift (δ) values increased, clearly associated with the rise in fibrinolytic and gelatinolytic activities. These processes changed the viscoelastic properties of the system toward G' and |G*| values observed on day 5 in collagen cultures. Although less collagen turnover was observed in collagen-fibrin cultures than in collagen and collagen-hyaluronic acid cultures, collagen neosynthesis was apparently insufficient to contribute to the overall viscoelastic properties of the system. Collagen-hyaluronic acid cultures showed very limited changes during time. Firstly, they exhibited the highest δ values, suggesting an increase in the viscous behavior due to the hygroscopic properties of hyaluronic acid. These results showed that fibrin and hyaluronic acid not only affect differently the viscoelastic properties of the culture, they can tune fibroblastic activity by regulating cell attachment and extracellular matrix remodeling. Copyright © 2018 Elsevier Ltd. All rights reserved.

Molecular dynamics simulations on networks of heparin and collagen.

PubMed

Kulke, Martin; Geist, Norman; Friedrichs, Wenke; Langel, Walter

2017-06-01

Synthetic scaffolds containing collagen (Type I) are of increasing interest for bone tissue engineering, especially for highly porous biomaterials in combination with glycosaminoglycans. In experiments the integration of heparin during the fibrillogenesis resulted in different types of collagen fibrils, but models for this aggregation on a molecular scale were only tentative. We conducted molecular dynamic simulations investigating the binding of heparin to collagen and the influence of the telopeptides during collagen aggregation. This aims at explaining experimental findings on a molecular level. Novel structures for N- and C-telopeptides were developed with the TIGER2 replica exchange algorithm and dihedral principle component analysis. We present an extended statistical analysis of the mainly electrostatic interaction between heparin and collagen and identify several binding sites. Finally, we propose a molecular mechanism for the influence of glycosaminoglycans on the morphology of collagen fibrils. Proteins 2017; 85:1119-1130. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Polarization-resolved second-harmonic generation imaging for liver fibrosis assessment without labeling

NASA Astrophysics Data System (ADS)

Lin, Jian; Pan, Shiying; Zheng, Wei; Huang, Zhiwei

2013-10-01

We apply the polarization-resolved second-harmonic generation (PR-SHG) microscopy to investigate the changes of collagen typings (type I vs type III) and collagen fibril orientations of liver tissue in bile-duct-ligation (BDL) rat models. The PR-SHG results show that the second-order susceptibility tensor ratios (χ31/χ15 and χ33/χ15) of collagen fibers increase with liver fibrotic progression after BDL surgery, reflecting an increase of the type III collagen component with the severity of liver fibrosis; and the square root of the collagen type III to type I ratio linearly correlates (R2 = 0.98) with histopathological scores. Furthermore, the collagen fibril orientations become more random with liver fibrosis transformation as compared to normal liver tissue. This work demonstrates that PR-SHG microscopy has the potential for label-free diagnosis and characterization of liver fibrosis based on quantitative analysis of collagen typings and fibril orientations.

Distribution of collagens type V and VI in the normal human alveolar mucosa: an immunoelectronmicroscopic study using ultrathin frozen sections.

PubMed

Rabanus, J P; Gelderblom, H R; Schuppan, D; Becker, J

1991-05-01

The ultrastructural localization of collagens type V and VI in normal human gingival mucosa was investigated by immunoelectron microscopy. Twenty biopsies were fixed in dimethylsuberimidate and shock-frozen in slush nitrogen. Collagen type V was mainly located to meshworks of uniform nonstriated microfibrils of 12 to 20 nm width, which preferentially appeared in larger spaces between cross-striated major collagen fibrils. Occasionally single microfibrils of collagen type V fanned out from the ends of major collagen fibrils, which may indicate a role as a core fibril. Collagen type V was not found in the subepithelial basement membrane and the immediately adjacent stroma. Collagen type VI was detected in a loose reticular network of unbanded microfilaments that were morphologically distinguishable by knoblike protrusions every 100-110 nm. These microfilaments were found in the vicinity, but not as an intrinsic component, of the subepithelial basement membrane. Single filaments of collagen type VI filaments appeared to form bridges between neighboring cross-striated major collagen fibrils, suggesting an interconnecting role for this collagen type. The method presented appears to be excellently suited to study the normal and pathological supramolecular organization of the oral extracellular matrix.

Manipulation of in vitro collagen matrix architecture for scaffolds of improved physiological relevance

NASA Astrophysics Data System (ADS)

Hapach, Lauren A.; VanderBurgh, Jacob A.; Miller, Joseph P.; Reinhart-King, Cynthia A.

2015-12-01

Type I collagen is a versatile biomaterial that is widely used in medical applications due to its weak antigenicity, robust biocompatibility, and its ability to be modified for a wide array of applications. As such, collagen has become a major component of many tissue engineering scaffolds, drug delivery platforms, and substrates for in vitro cell culture. In these applications, collagen constructs are fabricated to recapitulate a diverse set of conditions. Collagen fibrils can be aligned during or post-fabrication, cross-linked via numerous techniques, polymerized to create various fibril sizes and densities, and copolymerized into a wide array of composite scaffolds. Here, we review approaches that have been used to tune collagen to better recapitulate physiological environments for use in tissue engineering applications and studies of basic cell behavior. We discuss techniques to control fibril alignment, methods for cross-linking collagen constructs to modulate stiffness, and composite collagen constructs to better mimic physiological extracellular matrix.

Noninvasive Quantitative Imaging of Collagen Microstructure in Three-Dimensional Hydrogels Using High-Frequency Ultrasound

PubMed Central

Mercado, Karla P.; Helguera, María; Hocking, Denise C.

2015-01-01

Collagen I is widely used as a natural component of biomaterials for both tissue engineering and regenerative medicine applications. The physical and biological properties of fibrillar collagens are strongly tied to variations in collagen fiber microstructure. The goal of this study was to develop the use of high-frequency quantitative ultrasound to assess collagen microstructure within three-dimensional (3D) hydrogels noninvasively and nondestructively. The integrated backscatter coefficient (IBC) was employed as a quantitative ultrasound parameter to detect, image, and quantify spatial variations in collagen fiber density and diameter. Collagen fiber microstructure was varied by fabricating hydrogels with different collagen concentrations or polymerization temperatures. IBC values were computed from measurements of the backscattered radio-frequency ultrasound signals collected using a single-element transducer (38-MHz center frequency, 13–47 MHz bandwidth). The IBC increased linearly with increasing collagen concentration and decreasing polymerization temperature. Parametric 3D images of the IBC were generated to visualize and quantify regional variations in collagen microstructure throughout the volume of hydrogels fabricated in standard tissue culture plates. IBC parametric images of corresponding cell-embedded collagen gels showed cell accumulation within regions having elevated collagen IBC values. The capability of this ultrasound technique to noninvasively detect and quantify spatial differences in collagen microstructure offers a valuable tool to monitor the structural properties of collagen scaffolds during fabrication, to detect functional differences in collagen microstructure, and to guide fundamental research on the interactions of cells and collagen matrices. PMID:25517512

Noninvasive Quantitative Imaging of Collagen Microstructure in Three-Dimensional Hydrogels Using High-Frequency Ultrasound.

PubMed

Mercado, Karla P; Helguera, María; Hocking, Denise C; Dalecki, Diane

2015-07-01

Collagen I is widely used as a natural component of biomaterials for both tissue engineering and regenerative medicine applications. The physical and biological properties of fibrillar collagens are strongly tied to variations in collagen fiber microstructure. The goal of this study was to develop the use of high-frequency quantitative ultrasound to assess collagen microstructure within three-dimensional (3D) hydrogels noninvasively and nondestructively. The integrated backscatter coefficient (IBC) was employed as a quantitative ultrasound parameter to detect, image, and quantify spatial variations in collagen fiber density and diameter. Collagen fiber microstructure was varied by fabricating hydrogels with different collagen concentrations or polymerization temperatures. IBC values were computed from measurements of the backscattered radio-frequency ultrasound signals collected using a single-element transducer (38-MHz center frequency, 13-47 MHz bandwidth). The IBC increased linearly with increasing collagen concentration and decreasing polymerization temperature. Parametric 3D images of the IBC were generated to visualize and quantify regional variations in collagen microstructure throughout the volume of hydrogels fabricated in standard tissue culture plates. IBC parametric images of corresponding cell-embedded collagen gels showed cell accumulation within regions having elevated collagen IBC values. The capability of this ultrasound technique to noninvasively detect and quantify spatial differences in collagen microstructure offers a valuable tool to monitor the structural properties of collagen scaffolds during fabrication, to detect functional differences in collagen microstructure, and to guide fundamental research on the interactions of cells and collagen matrices.

The pearl oyster Pinctada fucata martensii genome and multi-omic analyses provide insights into biomineralization

PubMed Central

Fan, Guangyi; Jiao, Yu; Zhang, He; Huang, Ronglian; Zheng, Zhe; Bian, Chao; Deng, Yuewen; Wang, Qingheng; Wang, Zhongduo; Liang, Xinming; Liang, Haiying; Shi, Chengcheng; Zhao, Xiaoxia; Sun, Fengming; Hao, Ruijuan; Bai, Jie; Liu, Jialiang; Chen, Wenbin; Liang, Jinlian; Liu, Weiqing; Xu, Zhe; Shi, Qiong; Xu, Xun

2017-01-01

Abstract Nacre, the iridescent material found in pearls and shells of molluscs, is formed through an extraordinary process of matrix-assisted biomineralization. Despite recent advances, many aspects of the biomineralization process and its evolutionary origin remain unknown. The pearl oyster Pinctada fucata martensii is a well-known master of biomineralization, but the molecular mechanisms that underlie its production of shells and pearls are not fully understood. We sequenced the highly polymorphic genome of the pearl oyster and conducted multi-omic and biochemical studies to probe nacre formation. We identified a large set of novel proteins participating in matrix-framework formation, many in expanded families, including components similar to that found in vertebrate bones such as collagen-related VWA-containing proteins, chondroitin sulfotransferases, and regulatory elements. Considering that there are only collagen-based matrices in vertebrate bones and chitin-based matrices in most invertebrate skeletons, the presence of both chitin and elements of collagen-based matrices in nacre suggests that elements of chitin- and collagen-based matrices have deep roots and might be part of an ancient biomineralizing matrix. Our results expand the current shell matrix-framework model and provide new insights into the evolution of diverse biomineralization systems. PMID:28873964

M2-like macrophages are responsible for collagen degradation through a mannose receptor–mediated pathway

PubMed Central

Madsen, Daniel H.; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E.; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S.; Brenner, David A.; Burgdorf, Sven; Engelholm, Lars H.; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto

2013-01-01

Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase–dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor–associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process. PMID:24019537

The solubilization of bone and dentin collagens by pepsin. Effect of cross-linkages and non-collagen components.

PubMed

Carmichael, D J; Dodd, C M; Veis, A

1977-03-28

Bone and dentin collagen are less susceptible to solubilization by pepsin digestion then is skin collagen. Digestion at 4 degrees C for 72 h solubilized only 35.3% of bovine cortical bone and 5.6% of bovine dentin compared with nearly 100% dissolution of bovine skin. Sodium dodecyl sulfate-acrylamide gel electrophoresis and molecular sieve chromatography showed that, for bone and dentin, intact alpha chains and cross-linked aggregates of beta, gamma and higher weight remained intact after pepsin solubilization but lower molecular weight fragments also were prevalent indicating chain scission in helical regions. Electron microscopic examination of segment long spacing precipitates of the soluble collagens confirmed the presence of solubilized polymerized collagen. The principal reducible cross-link in both bone and dentin was the precursor of dihydroxylsinonorleucine and this cross-link was also present in the solubilized collagens. Small amounts of non-collagenous proteins and glycosaminoglycans of different compositions in dentin and bone resisted extraction before pepsin digestion. However, the differences in solubilization of the collagens have been related to differences in cross-linkage placement.

Persistence of bone collagen cross-links in skeletons of the Nuraghi population living in Sardinia 1500-1200 B.C

NASA Technical Reports Server (NTRS)

Wojtowicz, A.; Yamauchi, M.; Montella, A.; Bandiera, P.; Sotowski, R.; Ostrowski, K.

1999-01-01

Bone collagen has a specific molecular ultrastructure which can be proved by birefringence. This protein, forming the main organic component of bone tissue, is known to survive millennia in paleontological bones and teeth. Birefringence of bone collagen obtained from the skeletons of the Nuraghi population living in Sardinia c-ca 1500 years B.C. was found previously by the use of polarizing microscopy [1]. In this paper, using high pressure liquid chromatography (HPLC) techniques, we show the existence of bone collagen cross-links preserved in Nuraghi skeletons after more than 3000 years.

Direct detection of crosslinks of collagen and elastin in the hydrolysates of human yellow ligament using single-column high performance liquid chromatography.

PubMed

Chen, J R; Takahashi, M; Kushida, K; Suzuki, M; Suzuki, K; Horiuchi, K; Nagano, A

2000-02-15

Collagen and elastin are recognized as two major connective tissue proteins of human yellow ligament. In both collagen and elastin there are many kinds of intra- or intermolecular crosslinks. Pyridinoline (Pyr) and deoxypyridinoline (Dpyr) are mature crosslinks which maintain the structure of the collagen fibril. Desmosine (Des) and isodesmosine (Isodes) represent the major crosslinking components of elastin. Pentosidine (Pen), which is a senescent crosslink and one of the advanced glycation end products, accumulates with age in tissue proteins including collagen. We developed a direct and one-injection HPLC method to measure Pyr, Dpyr, Des, Isodes, and Pen in the hydrolysate of human yellow ligament. This method used one column and two detectors. Recovery rates of Pyr, Dpyr, Pen, Des, and Isodes were 86.4-98.3, 83.6-96.8, 78.7-95.6, 83.6-97.9, and 85.6-99.3%, respectively (n = 8). The intraassay coefficients of variation for Pyr, Dpyr, Pen, Des, and Isodes were 3.7, 4.1, 5.4, 4.5, and 4.7%, respectively (n = 8), and the interassay coefficients of variation for Pyr, Dpyr, Pen, Des, and Isodes were 4.4, 5.1, 4.9, 4.6 and 4.1%, respectively. Linear regression analysis showed the linearity (r = 0.99, P = 0.0001) of calibration line for each Pyr, Dpyr, Pen, Des, and Isodes. Using this method, we investigated age-related changes in the crosslinks of collagen and elastin in human yellow ligament. There was a significant correlation between Pen and age, but no correlations with Pyr, Dpyr, Des, and Isodes. We believe that this method is useful for investigating the content of these crosslinks in both collagen and elastin under various conditions. Copyright 2000 Academic Press.

The bioactive acidic serine- and aspartate-rich motif peptide.

PubMed

Minamizaki, Tomoko; Yoshiko, Yuji

2015-01-01

The organic component of the bone matrix comprises 40% dry weight of bone. The organic component is mostly composed of type I collagen and small amounts of non-collagenous proteins (NCPs) (10-15% of the total bone protein content). The small integrin-binding ligand N-linked glycoprotein (SIBLING) family, a NCP, is considered to play a key role in bone mineralization. SIBLING family of proteins share common structural features and includes the arginine-glycine-aspartic acid (RGD) motif and acidic serine- and aspartic acid-rich motif (ASARM). Clinical manifestations of gene mutations and/or genetically modified mice indicate that SIBLINGs play diverse roles in bone and extraskeletal tissues. ASARM peptides might not be primary responsible for the functional diversity of SIBLINGs, but this motif is suggested to be a key domain of SIBLINGs. However, the exact function of ASARM peptides is poorly understood. In this article, we discuss the considerable progress made in understanding the role of ASARM as a bioactive peptide.

Morphometric changes in the aortic arch with advancing age in fetal to mature thoroughbred horses.

PubMed

Endoh, Chihiro; Matsuda, Kazuya; Okamoto, Minoru; Tsunoda, Nobuo; Taniyama, Hiroyuki

2017-03-28

Aortic rupture is a well recognized cause of sudden death in thoroughbred horses. Some microscopic lesions, such as those caused by cystic medial necrosis and medionecrosis, can lead to aortic rupture. However, these microscopic lesions are also observed in normal horses. On the other hand, a previous study of aortic rupture suggested that underlying elastin and collagen deposition disorders might be associated with aortic rupture. Therefore, the purpose of this study was to compare the structural components of the tunica media of the aortic arch, which is composed of elastin, collagen, smooth muscle cells and mucopolysaccharides (MPS), in fetal to mature thoroughbred horses. The percentage area of elastin was greatest in the young horses and subsequently decreased with aging. The percentage area of collagen increased with aging, and the elderly horses (aged ≥20) exhibited significantly higher percentage areas of collagen than the young horses. The percentage area of smooth muscle cells did not change with age. The percentage area of MPS was inversely proportional to the percentage area of elastin. The fetuses exhibited a markedly larger percentage area of MPS than the mature horses. We concluded that the medial changes seen in the aortic arch, which included a reduction in the amount of elastin and increases in the amounts of collagen and MPS, were age-related variations.

Imaging denatured collagen strands in vivo and ex vivo via photo-triggered hybridization of caged collagen mimetic peptides.

PubMed

Li, Yang; Foss, Catherine A; Pomper, Martin G; Yu, S Michael

2014-01-31

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i) to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Collagen organization in canine myxomatous mitral valve disease: an x-ray diffraction study.

PubMed

Hadian, Mojtaba; Corcoran, Brendan M; Han, Richard I; Grossmann, J Günter; Bradshaw, Jeremy P

2007-10-01

Collagen fibrils, a major component of mitral valve leaflets, play an important role in defining shape and providing mechanical strength and flexibility. Histopathological studies show that collagen fibrils undergo dramatic changes in the course of myxomatous mitral valve disease in both dogs and humans. However, little is known about the detailed organization of collagen in this disease. This study was designed to analyze and compare collagen fibril organization in healthy and lesional areas of myxomatous mitral valves of dogs, using synchrotron small-angle x-ray diffraction. The orientation, density, and alignment of collagen fibrils were mapped across six different valves. The findings reveal a preferred collagen alignment in the main body of the leaflets between two commissures. Qualitative and quantitative analysis of the data showed significant differences between affected and lesion-free areas in terms of collagen content, fibril alignment, and total tissue volume. Regression analysis of the amount of collagen compared to the total tissue content at each point revealed a significant relationship between these two parameters in lesion-free but not in affected areas. This is the first time this technique has been used to map collagen fibrils in cardiac tissue; the findings have important applications to human cardiology.

NMR Studies of Cartilage Dynamics, Diffusion, Degradation

NASA Astrophysics Data System (ADS)

Huster, Daniel; Schiller, Jurgen; Naji, Lama; Kaufmann Jorn; Arnold, Klaus

An increasing number of people is suffering from rheumatic diseases, and, therefore, methods of early diagnosis of joint degeneration are urgently required. For their establishment, however, an improved knowledge about the molecular organisation of cartilage would be helpful. Cartilage consists of three main components: Water, collagen and chondroitin sulfate (CS) that is (together with further polysaccharides and proteins) a major constituent of the proteoglycans of cartilage. 1H and 13C MAS (magic-angle spinning) NMR (nuclear magnetic resonance) opened new perspectives for the study of the macromolecular components in cartilage. We have primarily studied the mobilities of CS and collagen in bovine nasal and pig articular cartilage (that differ significantly in their collagen/polysaccharide content) by measuring 13C NMR relaxation times as well as the corresponding 13C CP (cross polarisation) MAS NMR spectra. These data clearly indicate that the mobility of cartilage macromolecules is broadly distributed from almost completely rigid (collagen) to highly mobile (polysaccharides), which lends cartilage its mechanical strength and shock-absorbing properties.

Digesting a Path Forward: The Utility of Collagenase Tumor Treatment for Improved Drug Delivery.

PubMed

Dolor, Aaron; Szoka, Francis C

2018-06-04

Collagen and hyaluronan are the most abundant components of the extracellular matrix (ECM) and their overexpression in tumors is linked to increased tumor growth and metastasis. These ECM components contribute to a protective tumor microenvironment by supporting a high interstitial fluid pressure and creating a tortuous setting for the convection and diffusion of chemotherapeutic small molecules, antibodies, and nanoparticles in the tumor interstitial space. This review focuses on the research efforts to deplete extracellular collagen with collagenases to normalize the tumor microenvironment. Although collagen synthesis inhibitors are in clinical development, the use of collagenases is contentious and clinically untested in cancer patients. Pretreatment of murine tumors with collagenases increased drug uptake and diffusion 2-10-fold. This modest improvement resulted in decreased tumor growth, but the benefits of collagenase treatment are confounded by risks of toxicity from collagen breakdown in healthy tissues. In this review, we evaluate the published in vitro and in vivo benefits and limitations of collagenase treatment to improve drug delivery.

Suppression of hedgehog signaling regulates hepatic stellate cell activation and collagen secretion.

PubMed

Li, Tao; Leng, Xi-Sheng; Zhu, Ji-Ye; Wang, Gang

2015-01-01

Hepatic stellate cells (HSCs) play an important role in liver fibrosis. This study investigates the expression of hedgehog in HSC and the role of hedgehog signaling on activation and collagen secretion of HSC. Liver ex vivo perfusion with collagenase IV and density gradient centrifugation were used to isolate HSC. Expression of hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 in HSC were detected by RT-PCR. Hedgehog siRNA vectors targeting Ihh, Smo and Gli2 were constructed and transfected into HSC respectively. Suppression of hedgehog signaling were detected by SYBR Green fluorescence quantitative RT-PCR. Effects of hedgehog signaling inhibition on HSC activation and collagen I secretion were analyzed. Hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 were expressed in HSC. siRNA vectors targeting Ihh, Smo and Gli2 were successfully constructed and decreased target gene expression. Suppression of hedgehog signaling significantly decreased the expression of α-SMA in HSC (P<0.01). Collagen type I secretion of HSC were also significantly decreased (P<0.01). In summary, HSC activation and collagen secretion can be regulated by hedgehog signaling. Hedgehog may play a role in the pathogenesis of liver fibrosis.

Quantitative characterization of collagen in the fibrotic capsule surrounding implanted polymeric microparticles through second harmonic generation imaging

DOE PAGES

Akilbekova, Dana; Bratlie, Kaitlin M.; Abraham, Thomas

2015-06-30

The collagenous capsule formed around an implant will ultimately determine the nature of its in vivo fate. To provide a better understanding of how surface modifications can alter the collagen orientation and composition in the fibrotic capsule, we used second harmonic generation (SHG) microscopy to evaluate collagen organization and structure generated in mice subcutaneously injected with chemically functionalized polystyrene particles. SHG is sensitive to the orientation of a molecule, making it a powerful tool for measuring the alignment of collagen fibers. Additionally, SHG arises from the second order susceptibility of the interrogated molecule in response to the electric field. Variationmore » in these tensor components distinguishes different molecular sources of SHG, providing collagen type specificity. Here, we demonstrated the ability of SHG to differentiate collagen type I and type III quantitatively and used this method to examine fibrous capsules of implanted polystyrene particles. Data presented in this work shows a wide range of collagen fiber orientations and collagen compositions in response to surface functionalized polystyrene particles. Dimethylamino functionalized particles were able to form a thin collagenous matrix resembling healthy skin. These findings have the potential to improve the fundamental understanding of how material properties influence collagen organization and composition quantitatively.« less

Quantitative Characterization of Collagen in the Fibrotic Capsule Surrounding Implanted Polymeric Microparticles through Second Harmonic Generation Imaging.

PubMed

Akilbekova, Dana; Bratlie, Kaitlin M

2015-01-01

The collagenous capsule formed around an implant will ultimately determine the nature of its in vivo fate. To provide a better understanding of how surface modifications can alter the collagen orientation and composition in the fibrotic capsule, we used second harmonic generation (SHG) microscopy to evaluate collagen organization and structure generated in mice subcutaneously injected with chemically functionalized polystyrene particles. SHG is sensitive to the orientation of a molecule, making it a powerful tool for measuring the alignment of collagen fibers. Additionally, SHG arises from the second order susceptibility of the interrogated molecule in response to the electric field. Variation in these tensor components distinguishes different molecular sources of SHG, providing collagen type specificity. Here, we demonstrated the ability of SHG to differentiate collagen type I and type III quantitatively and used this method to examine fibrous capsules of implanted polystyrene particles. Data presented in this work shows a wide range of collagen fiber orientations and collagen compositions in response to surface functionalized polystyrene particles. Dimethylamino functionalized particles were able to form a thin collagenous matrix resembling healthy skin. These findings have the potential to improve the fundamental understanding of how material properties influence collagen organization and composition quantitatively.

Quantitative Characterization of Collagen in the Fibrotic Capsule Surrounding Implanted Polymeric Microparticles through Second Harmonic Generation Imaging

PubMed Central

Akilbekova, Dana; Bratlie, Kaitlin M.

2015-01-01

The collagenous capsule formed around an implant will ultimately determine the nature of its in vivo fate. To provide a better understanding of how surface modifications can alter the collagen orientation and composition in the fibrotic capsule, we used second harmonic generation (SHG) microscopy to evaluate collagen organization and structure generated in mice subcutaneously injected with chemically functionalized polystyrene particles. SHG is sensitive to the orientation of a molecule, making it a powerful tool for measuring the alignment of collagen fibers. Additionally, SHG arises from the second order susceptibility of the interrogated molecule in response to the electric field. Variation in these tensor components distinguishes different molecular sources of SHG, providing collagen type specificity. Here, we demonstrated the ability of SHG to differentiate collagen type I and type III quantitatively and used this method to examine fibrous capsules of implanted polystyrene particles. Data presented in this work shows a wide range of collagen fiber orientations and collagen compositions in response to surface functionalized polystyrene particles. Dimethylamino functionalized particles were able to form a thin collagenous matrix resembling healthy skin. These findings have the potential to improve the fundamental understanding of how material properties influence collagen organization and composition quantitatively. PMID:26125551

Quantitative characterization of collagen in the fibrotic capsule surrounding implanted polymeric microparticles through second harmonic generation imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akilbekova, Dana; Bratlie, Kaitlin M.; Abraham, Thomas

The collagenous capsule formed around an implant will ultimately determine the nature of its in vivo fate. To provide a better understanding of how surface modifications can alter the collagen orientation and composition in the fibrotic capsule, we used second harmonic generation (SHG) microscopy to evaluate collagen organization and structure generated in mice subcutaneously injected with chemically functionalized polystyrene particles. SHG is sensitive to the orientation of a molecule, making it a powerful tool for measuring the alignment of collagen fibers. Additionally, SHG arises from the second order susceptibility of the interrogated molecule in response to the electric field. Variationmore » in these tensor components distinguishes different molecular sources of SHG, providing collagen type specificity. Here, we demonstrated the ability of SHG to differentiate collagen type I and type III quantitatively and used this method to examine fibrous capsules of implanted polystyrene particles. Data presented in this work shows a wide range of collagen fiber orientations and collagen compositions in response to surface functionalized polystyrene particles. Dimethylamino functionalized particles were able to form a thin collagenous matrix resembling healthy skin. These findings have the potential to improve the fundamental understanding of how material properties influence collagen organization and composition quantitatively.« less

In vitro characterization of a collagen scaffold enzymatically cross-linked with a tailored elastin-like polymer.

PubMed

Garcia, Yolanda; Hemantkumar, Naik; Collighan, Russell; Griffin, Martin; Rodriguez-Cabello, Jose Carlos; Pandit, Abhay

2009-04-01

Collagen, the main structural component of the extracellular matrix (ECM), provides tensile stiffness to different structures and organs against rupture. However, collagen tissue-engineered implants are hereto still lacking in mechanical strength. Attempts to create stiffer scaffolds have resulted in increased brittleness of the material, reducing the versatility of the original component. The hypothesis behind this research is that the introduction of an elastic element in the scaffold will enhance the mechanical properties of the collagen-based scaffolds, as elastin does in the ECM to prevent irreversible deformation. In this study, an elastin-like polymer (ELP) designed and synthesized using recombinant DNA methodology is used with the view to providing increased proteolytic resistance and increased functionality to the scaffolds by carrying specific sequences for microbial transglutaminase cross-linking, endothelial cell adhesion, and drug delivery. Evaluation of the effects that cross-linking ELP-collagen has on the physicochemical properties of the scaffold such as porosity, presence of cross-linking, thermal behavior, and mechanical strength demonstrated that the introduction of enzymatically resistant covalent bonds between collagen and ELP increases the mechanical strength of the scaffolds in a dose-dependent manner without significantly affecting the porosity or thermal properties of the original scaffold. Importantly, the scaffolds also showed selective behavior, in a dose (ELP)-dependent manner toward human umbilical vein endothelial cells and smooth muscle cells when compared to fibroblasts.

Substitution of aspartate for glycine 103 of the type II collagen triple helical domain: Identification of the minimal mutation which can produce Kniest dysplasia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilkin, D.J.; Rimoin, D.L.; Cohn, D.H.

1994-09-01

Kniest dysplasia is an autosomal dominant chondrodysplasia which results from mutations in the gene for type II collagen, COL2A1. Characteristics of the disorder include a short trunk and extremities, mid-face hypoplasia, cleft palate, myopia, retinal detachment, and hearing loss. Recently, deletions of all or part of exon 12 have been identified in individuals with Kniest dysplasia, suggesting that mutations within this region of the protein may primarily result in the Kniest dysplasia phenotype. We used SSCP to analyze an amplified genomic DNA fragment containing exon 12 from 7 individuals with Kniest dysplasia. An abnormality was identified in one patient. DNAmore » sequence analysis demonstrated that the patient was heterozygous for a G to A transition that implied substitution of glycine{sup 103} of the triple helix by aspartate. The mutation was not observed in DNA from either of the proband`s parents. Protein microsequencing demonstrated expression of the abnormal allele in the proband`s cartilage, indicating that the Kniest phenotype results from the presence of abnormal type II collagen molecules in the extracellular matrix. These data demonstrate the minimal mutation which can produce Kniest dysplasia and further support the hypothesis that alteration of a domain which includes the region encoded by exon 12 in the type II collagen protein leads to this disorder. Experiments designed to identify specific effects that mutations in this region have on intermolecular interactions among abnormal type II collagen molecules and other components of the cartilage extracellular matrix may clarify the underlying pathophysiology of Kniest dysplasia.« less

Signal transduction by beta1 integrin receptors in human chondrocytes in vitro: collaboration with the insulin-like growth factor-I receptor.

PubMed

Shakibaei, M; John, T; De Souza, P; Rahmanzadeh, R; Merker, H J

1999-09-15

We have examined the mechanism by which collagen-binding integrins co-operate with insulin-like growth factor-I (IGF-I) receptors (IGF-IR) to regulate chondrocyte phenotype and differentiation. Adhesion of chondrocytes to anti-beta1 integrin antibodies or collagen type II leads to phosphorylation of cytoskeletal and signalling proteins localized at focal adhesions, including alpha-actinin, vinculin, paxillin and focal adhesion kinase (FAK). These stimulate docking proteins such as Shc (Src-homology collagen). Moreover, exposure of collagen type II-cultured chondrocytes to IGF-I leads to co-immunoprecipitation of Shc protein with the IGF-IR and with beta1, alpha1 and alpha5 integrins, but not with alpha3 integrin. Shc then associates with growth factor receptor-bound protein 2 (Grb2), an adaptor protein and extracellular signal-regulated kinase. The expression of the docking protein Shc occurs only when chondrocytes are bound to collagen type II or integrin antibodies and increases when IGF-I is added, suggesting a collaboration between integrins and growth factors in a common/shared biochemical signalling pathway. Furthermore, these results indicate that focal adhesion assembly may facilitate signalling via Shc, a potential common target for signal integration between integrin and growth-factor signalling regulatory pathways. Thus, the collagen-binding integrins and IGF-IR co-operate to regulate focal adhesion components and these signalling pathways have common targets (Shc-Grb2 complex) in subcellular compartments, thereby linking to the Ras-mitogen-activated protein kinase signalling pathway. These events may play a role during chondrocyte differentiation.

Chum salmon egg extracts induce upregulation of collagen type I and exert antioxidative effects on human dermal fibroblast cultures.

PubMed

Yoshino, Atsushi; Polouliakh, Natalia; Meguro, Akira; Takeuchi, Masaki; Kawagoe, Tatsukata; Mizuki, Nobuhisa

2016-01-01

Components of fish roe possess antioxidant and antiaging activities, making them potentially very beneficial natural resources. Here, we investigated chum salmon eggs (CSEs) as a source of active ingredients, including vitamins, unsaturated fatty acids, and proteins. We incubated human dermal fibroblast cultures for 48 hours with high and low concentrations of CSE extracts and analyzed changes in gene expression. Cells treated with CSE extract showed concentration-dependent upregulation of collagen type I genes and of multiple antioxidative genes, including OXR1, TXNRD1, and PRDX family genes. We further conducted in silico phylogenetic footprinting analysis of promoter regions. These results suggested that transcription factors such as acute myeloid leukemia-1a and cyclic adenosine monophosphate response element-binding protein may be involved in the observed upregulation of antioxidative genes. Our results support the idea that CSEs are strong candidate sources of antioxidant materials and cosmeceutically effective ingredients.

Mechanical forces regulate the interactions of fibronectin and collagen I in extracellular matrix.

PubMed

Kubow, Kristopher E; Vukmirovic, Radmila; Zhe, Lin; Klotzsch, Enrico; Smith, Michael L; Gourdon, Delphine; Luna, Sheila; Vogel, Viola

2015-08-14

Despite the crucial role of extracellular matrix (ECM) in directing cell fate in healthy and diseased tissues--particularly in development, wound healing, tissue regeneration and cancer--the mechanisms that direct the assembly and regulate hierarchical architectures of ECM are poorly understood. Collagen I matrix assembly in vivo requires active fibronectin (Fn) fibrillogenesis by cells. Here we exploit Fn-FRET probes as mechanical strain sensors and demonstrate that collagen I fibres preferentially co-localize with more-relaxed Fn fibrils in the ECM of fibroblasts in cell culture. Fibre stretch-assay studies reveal that collagen I's Fn-binding domain is responsible for the mechano-regulated interaction. Furthermore, we show that Fn-collagen interactions are reciprocal: relaxed Fn fibrils act as multivalent templates for collagen assembly, but once assembled, collagen fibres shield Fn fibres from being stretched by cellular traction forces. Thus, in addition to the well-recognized, force-regulated, cell-matrix interactions, forces also tune the interactions between different structural ECM components.

The collagen receptor uPARAP/Endo180 in tissue degradation and cancer (Review)

PubMed Central

MELANDER, MARIA C.; JÜRGENSEN, HENRIK J.; MADSEN, DANIEL H.; ENGELHOLM, LARS H.; BEHRENDT, NIELS

2015-01-01

The collagen receptor uPARAP/Endo180, the product of the MRC2 gene, is a central component in the collagen turnover process governed by various mesenchymal cells. Through the endocytosis of collagen or large collagen fragments, this recycling receptor serves to direct basement membrane collagen as well as interstitial collagen to lysosomal degradation. This capacity, shared only with the mannose receptor from the same protein family, endows uPARAP/Endo180 with a critical role in development and homeostasis, as well as in pathological disruptions of the extracellular matrix structure. Important pathological functions of uPARAP/Endo180 have been identified in various cancers and in several fibrotic conditions. With a particular focus on matrix turnover in cancer, this review presents the necessary background for understanding the function of uPARAP/Endo180 at the molecular and cellular level, followed by an in-depth survey of the available knowledge of the expression and role of this receptor in various types of cancer and other degenerative diseases. PMID:26316068

Jellyfish collagen scaffolds for cartilage tissue engineering.

PubMed

Hoyer, Birgit; Bernhardt, Anne; Lode, Anja; Heinemann, Sascha; Sewing, Judith; Klinger, Matthias; Notbohm, Holger; Gelinsky, Michael

2014-02-01

Porous scaffolds were engineered from refibrillized collagen of the jellyfish Rhopilema esculentum for potential application in cartilage regeneration. The influence of collagen concentration, salinity and temperature on fibril formation was evaluated by turbidity measurements and quantification of fibrillized collagen. The formation of collagen fibrils with a typical banding pattern was confirmed by atomic force microscopy and transmission electron microscopy analysis. Porous scaffolds from jellyfish collagen, refibrillized under optimized conditions, were fabricated by freeze-drying and subsequent chemical cross-linking. Scaffolds possessed an open porosity of 98.2%. The samples were stable under cyclic compression and displayed an elastic behavior. Cytotoxicity tests with human mesenchymal stem cells (hMSCs) did not reveal any cytotoxic effects of the material. Chondrogenic markers SOX9, collagen II and aggrecan were upregulated in direct cultures of hMSCs upon chondrogenic stimulation. The formation of typical extracellular matrix components was further confirmed by quantification of sulfated glycosaminoglycans. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Self-healing Characteristics of Collagen Coatings with Respect to Surface Abrasion

PubMed Central

Kim, Chang-Lae; Kim, Dae-Eun

2016-01-01

A coating based on collagen with self-healing properties was developed for applications in mechanical components that are prone to abrasion due to contact with a counter surface. The inherent swelling behavior of collagen in water was exploited as the fundamental mechanism behind self-healing of a wear scar formed on the surface. The effects of freeze-drying process and water treatment of the collagen coatings on their mechanical and self-healing properties were analyzed. Water was also used as the medium to trigger the self-healing effect of the collagen coatings after the wear test. It was found that collagen coatings without freeze-drying did not demonstrate any self-healing effect whereas the coatings treated by freeze-drying process showed remarkable self-healing effect. Overall, collagen coatings that were freeze-dried and water treated showed the best friction and self-healing properties. Repeated self-healing ability of these coatings with respect to wear scar was also demonstrated. It was also confirmed that the self-healing property of the collagen coating was effective over a relatively wide range of temperature. PMID:27010967

Self-healing Characteristics of Collagen Coatings with Respect to Surface Abrasion

NASA Astrophysics Data System (ADS)

Kim, Chang-Lae; Kim, Dae-Eun

2016-03-01

A coating based on collagen with self-healing properties was developed for applications in mechanical components that are prone to abrasion due to contact with a counter surface. The inherent swelling behavior of collagen in water was exploited as the fundamental mechanism behind self-healing of a wear scar formed on the surface. The effects of freeze-drying process and water treatment of the collagen coatings on their mechanical and self-healing properties were analyzed. Water was also used as the medium to trigger the self-healing effect of the collagen coatings after the wear test. It was found that collagen coatings without freeze-drying did not demonstrate any self-healing effect whereas the coatings treated by freeze-drying process showed remarkable self-healing effect. Overall, collagen coatings that were freeze-dried and water treated showed the best friction and self-healing properties. Repeated self-healing ability of these coatings with respect to wear scar was also demonstrated. It was also confirmed that the self-healing property of the collagen coating was effective over a relatively wide range of temperature.

Viscoelastic properties of pressure overload hypertrophied myocardium: effect of serine protease treatment

NASA Technical Reports Server (NTRS)

Stroud, Jason D.; Baicu, Catalin F.; Barnes, Mary A.; Spinale, Francis G.; Zile, Michael R.

2002-01-01

To determine whether and to what extent one component of the extracellular matrix, fibrillar collagen, contributes causally to abnormalities in viscoelasticity, collagen was acutely degraded by activation of endogenous matrix metalloproteinases (MMPs) with the serine protease plasmin. Papillary muscles were isolated from normal cats and cats with right ventricular pressure overload hypertrophy (POH) induced by pulmonary artery banding. Plasmin treatment caused MMP activation, collagen degradation, decreased the elastic stiffness constant, and decreased the viscosity constant in both normal and POH muscles. Thus, whereas many mechanisms may contribute to the abnormalities in myocardial viscoelasticity in the POH myocardium, changes in fibrillar collagen appear to play a predominant role.

Collagen in the spicule organic matrix of the gorgonian Leptogorgia virgulata

NASA Technical Reports Server (NTRS)

Kingsley, R. J.; Tsuzaki, M.; Watabe, N.; Mechanic, G. L.

1990-01-01

Decalcification of the calcareous spicules from the gorgonian Leptogorgia virgulata reveals an organic matrix that may be divided into water insoluble and soluble fractions. The insoluble fraction displays characteristics typical of collagen, which is an unusual component of an invertebrate calcium carbonate structure. This matrix fraction exhibits a collagenous amino acid profile and behavior upon SDS-PAGE. Furthermore, the reducible crosslink, dihydroxylysinonorleucine (DHLNL), is detected in this fraction. The composition of the matrix varies seasonally; i.e., the collagenous composition is most prevalent in the summer. These results indicate that the insoluble matrix is a dynamic structure. Potential roles of this matrix in spicule calcification are discussed.

Photothermal-modulated drug delivery and magnetic relaxation based on collagen/poly(γ-glutamic acid) hydrogel.

PubMed

Cho, Sun-Hee; Kim, Ahreum; Shin, Woojung; Heo, Min Beom; Noh, Hyun Jong; Hong, Kwan Soo; Cho, Jee-Hyun; Lim, Yong Taik

2017-01-01

Injectable and stimuli-responsive hydrogels have attracted attention in molecular imaging and drug delivery because encapsulated diagnostic or therapeutic components in the hydrogel can be used to image or change the microenvironment of the injection site by controlling various stimuli such as enzymes, temperature, pH, and photonic energy. In this study, we developed a novel injectable and photoresponsive composite hydrogel composed of anticancer drugs, imaging contrast agents, bio-derived collagen, and multifaceted anionic polypeptide, poly (γ-glutamic acid) (γ-PGA). By the introduction of γ-PGA, the intrinsic temperature-dependent phase transition behavior of collagen was modified to a low viscous sol state at room temperature and nonflowing gel state around body temperature. The modified temperature-dependent phase transition behavior of collagen/γ-PGA hydrogels was also evaluated after loading of near-infrared (NIR) fluorophore, indocyanine green (ICG), which could transform absorbed NIR photonic energy into thermal energy. By taking advantage of the abundant carboxylate groups in γ-PGA, cationic-charged doxorubicin (Dox) and hydrophobic MnFe 2 O 4 magnetic nanoparticles were also incorporated successfully into the collagen/γ-PGA hydrogels. By illumination of NIR light on the collagen/γ-PGA/Dox/ICG/MnFe 2 O 4 hydrogels, the release kinetics of Dox and magnetic relaxation of MnFe 2 O 4 nanoparticles could be modulated. The experimental results suggest that the novel injectable and NIR-responsive collagen/γ-PGA hydrogels developed in this study can be used as a theranostic platform after loading of various molecular imaging probes and therapeutic components.

Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis.

PubMed

Chapman, Mark A; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David; Lieber, Richard L

2017-02-01

Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173-183, 2009; Kjaer M. Physiol Rev 84: 649-98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins-fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell. Copyright © 2017 the American Physiological Society.

Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis

PubMed Central

Chapman, Mark A.; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David

2017-01-01

Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173–183, 2009; Kjaer M. Physiol Rev 84: 649–98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins—fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell. PMID:27881411

Collagen esterification enhances the function and survival of pancreatic β cells in 2D and 3D culture systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ko, Jae Hyung; Kim, Yang Hee; Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul

Collagen, one of the most important components of the extracellular matrix (ECM), may play a role in the survival of pancreatic islet cells. In addition, chemical modifications that change the collagen charge profile to a net positive charge by esterification have been shown to increase the adhesion and proliferation of various cell types. The purpose of this study was to characterize and compare the effects of native collagen (NC) and esterified collagen (EC) on β cell function and survival. After isolation by the collagenase digestion technique, rat islets were cultured with NC and EC in 2 dimensional (2D) and 3more » dimensional (3D) environments for a long-term duration in vitro. The cells were assessed for islet adhesion, morphology, viability, glucose-induced insulin secretion, and mRNA expression of glucose metabolism-related genes, and visualized by scanning electron microscopy (SEM). Islet cells attached tightly in the NC group, but islet cell viability was similar in both the NC and EC groups. Glucose-stimulated insulin secretion was higher in the EC group than in the NC group in both 2D and 3D culture. Furthermore, the mRNA expression levels of glucokinase in the EC group were higher than those in the NC group and were associated with glucose metabolism and insulin secretion. Finally, SEM observation confirmed that islets had more intact component cells on EC sponges than on NC sponges. These results indicate that modification of collagen may offer opportunities to improve function and viability of islet cells. - Highlights: • We changed the collagen charge profile to a net positive charge by esterification. • Islets cultured on esterified collagen improved survival in both 2D and 3D culture. • Islets cultured on esterified collagen enhanced glucose-stimulated insulin release. • High levels of glucokinase mRNA may be associated with increased insulin release.« less

High-fat diet amplifies renal renin angiotensin system expression, blood pressure elevation, and renal dysfunction caused by Ceacam1 null deletion.

PubMed

Li, Caixia; Culver, Silas A; Quadri, Syed; Ledford, Kelly L; Al-Share, Qusai Y; Ghadieh, Hilda E; Najjar, Sonia M; Siragy, Helmy M

2015-11-01

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAMl), a substrate of the insulin receptor tyrosine kinase, regulates insulin action by promoting insulin clearance. Global null mutation of Ceacam1 gene (Cc1(-/-)) results in features of the metabolic syndrome, including insulin resistance, hyperinsulinemia, visceral adiposity, elevated blood pressure, and albuminuria. It also causes activation of the renal renin-angiotensin system (RAS). In the current study, we tested the hypothesis that high-fat diet enhances the expression of RAS components. Three-month-old wild-type (Cc1(+/+)) and Cc1(-/-) mice were fed either a regular or a high-fat diet for 8 wk. At baseline under regular feeding conditions, Cc1(-/-) mice exhibited higher blood pressure, urine albumin-to-creatinine ratio (UACR), and renal expression of angiotensinogen, renin/prorenin, angiotensin-converting enzyme, (pro)renin receptor, angiotensin subtype AT1 receptor, angiotensin II, and elevated PI3K phosphorylation, as detected by p85α (Tyr(508)) immunostaining, inflammatory response, and the expression of collagen I and collagen III. In Cc1(+/+) mice, high-fat diet increased blood pressure, UACR, the expression of angiotensin-converting enzyme and angiotensin II, PI3K phosphorylation, inflammatory response, and the expression of collagen I and collagen III. In Cc1(-/-) mice, high-fat intake further amplified these parameters. Immunohistochemical staining showed increased p-PI3K p85α (Tyr(508)) expression in renal glomeruli, proximal, distal, and collecting tubules of Cc1(-/-) mice fed a high-fat diet. Together, this demonstrates that high-fat diet amplifies the permissive effect of Ceacam1 deletion on renal expression of all RAS components, PI3K phosphorylation, inflammation, and fibrosis. Copyright © 2015 the American Physiological Society.

Propolis Modifies Collagen Types I and III Accumulation in the Matrix of Burnt Tissue.

PubMed

Olczyk, Pawel; Wisowski, Grzegorz; Komosinska-Vassev, Katarzyna; Stojko, Jerzy; Klimek, Katarzyna; Olczyk, Monika; Kozma, Ewa M

2013-01-01

Wound healing represents an interactive process which requires highly organized activity of various cells, synthesizing cytokines, growth factors, and collagen. Collagen types I and III, serving as structural and regulatory molecules, play pivotal roles during wound healing. The aim of this study was to compare the propolis and silver sulfadiazine therapeutic efficacy throughout the quantitative and qualitative assessment of collagen types I and III accumulation in the matrix of burnt tissues. Burn wounds were inflicted on pigs, chosen for the evaluation of wound repair because of many similarities between pig and human skin. Isolated collagen types I and III were estimated by the surface plasmon resonance method with a subsequent collagenous quantification using electrophoretic and densitometric analyses. Propolis burn treatment led to enhanced collagens and its components expression, especially during the initial stage of the study. Less expressed changes were observed after silver sulfadiazine (AgSD) application. AgSD and, with a smaller intensity, propolis stimulated accumulation of collagenous degradation products. The assessed propolis therapeutic efficacy, throughout quantitatively and qualitatively analyses of collagen types I and III expression and degradation in wounds matrix, may indicate that apitherapeutic agent can generate favorable biochemical environment supporting reepithelization.

Role of Mesenchymal-Derived Stem Cells in Stimulating Dormant Tumor Cells to Proliferate and Form Clinical Metastases

DTIC Science & Technology

2016-07-01

tumor dormancy we have determined the break in dormancy is dependent on collagen and other fibrotic extracellular matrix components for the induction... collagen to induce a break from dormancy compared to dormant D2.0R cells revealed a set of genes that overlap with published dormancy gene sets. We...dormant D2.0R”) and proliferate when cultured in matrigel supplemented with collagen type-1 (“proliferative D2.0R”) (Barkan, Cancer Research, 2008, 68

In Situ Imaging of Tissue Remodeling with Collagen Hybridizing Peptides

PubMed Central

2017-01-01

Collagen, the major structural component of nearly all mammalian tissues, undergoes extensive proteolytic remodeling during developmental states and a variety of life-threatening diseases such as cancer, myocardial infarction, and fibrosis. While degraded collagen could be an important marker of tissue damage, it is difficult to detect and target using conventional tools. Here, we show that a designed peptide (collagen hybridizing peptide: CHP), which specifically hybridizes to the degraded, unfolded collagen chains, can be used to image degraded collagen and inform tissue remodeling activity in various tissues: labeled with 5-carboxyfluorescein and biotin, CHPs enabled direct localization and quantification of collagen degradation in isolated tissues within pathologic states ranging from osteoarthritis and myocardial infarction to glomerulonephritis and pulmonary fibrosis, as well as in normal tissues during developmental programs associated with embryonic bone formation and skin aging. The results indicate the general correlation between the level of collagen remodeling and the amount of denatured collagen in tissue and show that the CHP probes can be used across species and collagen types, providing a versatile tool for not only pathology and developmental biology research but also histology-based disease diagnosis, staging, and therapeutic screening. This study lays the foundation for further testing CHP as a targeting moiety for theranostic delivery in various animal models. PMID:28877431

Type VII Collagen Expression in the Human Vitreoretinal Interface, Corpora Amylacea and Inner Retinal Layers

PubMed Central

Wullink, Bart; Pas, Hendri H.; Van der Worp, Roelofje J.; Kuijer, Roel; Los, Leonoor I.

2015-01-01

Type VII collagen, as a major component of anchoring fibrils found at basement membrane zones, is crucial in anchoring epithelial tissue layers to their underlying stroma. Recently, type VII collagen was discovered in the inner human retina by means of immunohistochemistry, while proteomic investigations demonstrated type VII collagen at the vitreoretinal interface of chicken. Because of its potential anchoring function at the vitreoretinal interface, we further assessed the presence of type VII collagen at this site. We evaluated the vitreoretinal interface of human donor eyes by means of immunohistochemistry, confocal microscopy, immunoelectron microscopy, and Western blotting. Firstly, type VII collagen was detected alongside vitreous fibers6 at the vitreoretinal interface. Because of its known anchoring function, it is likely that type VII collagen is involved in vitreoretinal attachment. Secondly, type VII collagen was found within cytoplasmic vesicles of inner retinal cells. These cells resided most frequently in the ganglion cell layer and inner plexiform layer. Thirdly, type VII collagen was found in astrocytic cytoplasmic inclusions, known as corpora amylacea. The intraretinal presence of type VII collagen was confirmed by Western blotting of homogenized retinal preparations. These data add to the understanding of vitreoretinal attachment, which is important for a better comprehension of common vitreoretinal attachment pathologies. PMID:26709927

Increased Lymphatic Vessel Length Is Associated With the Fibroblast Reticulum and Disease Severity in Usual Interstitial Pneumonia and Nonspecific Interstitial Pneumonia

PubMed Central

Cosgrove, Gregory P.; Janssen, William J.; Huie, Tristan J.; Burnham, Ellen L.; Heinz, David E.; Curran-Everett, Douglas; Sahin, Hakan; Schwarz, Marvin I.; Cool, Carlyne D.; Groshong, Steve D.; Geraci, Mark W.; Tuder, Rubin M.; Hyde, Dallas M.; Henson, Peter M.

2012-01-01

Background: Lymphangiogenesis responds to tissue injury as a key component of normal wound healing. The development of fibrosis in the idiopathic interstitial pneumonias may result from abnormal wound healing in response to injury. We hypothesize that increased lymphatic vessel (LV) length, a marker of lymphangiogenesis, is associated with parenchymal components of the fibroblast reticulum (organizing collagen, fibrotic collagen, and fibroblast foci), and its extent correlates with disease severity. Methods: We assessed stereologically the parenchymal structure of fibrotic lungs and its associated lymphatic network, which was highlighted immunohistochemically in age-matched samples of usual interstitial pneumonia (UIP), nonspecific interstitial pneumonia (NSIP) with FVC < 80%, COPD with a Global Initiative for Obstructive Lung Disease stage 0, and normal control lungs. Results: LV length density, as opposed to vessel volume density, was found to be associated with organizing and fibrotic collagen density (P < .0001). Length density of LVs and the volume density of organizing and fibrotic collagen were significantly associated with severity of both % FVC (P < .001) and diffusing capacity of the lung for carbon monoxide (P < .001). Conclusions: Severity of disease in UIP and NSIP is associated with increased LV length and is strongly associated with components of the fibroblast reticulum, namely organizing and fibrotic collagen, which supports a pathogenic role of LVs in these two diseases. Furthermore, the absence of definable differences between UIP and NSIP suggests that LVs are a unifying mechanism for the development of fibrosis in these fibrotic lung diseases. PMID:22797508

Second harmonic generation microscopy analysis of extracellular matrix changes in human idiopathic pulmonary fibrosis

PubMed Central

Tilbury, Karissa; Hocker, James; Wen, Bruce L.; Sandbo, Nathan; Singh, Vikas; Campagnola, Paul J.

2014-01-01

Abstract. Patients with idiopathic fibrosis (IPF) have poor long-term survival as there are limited diagnostic/prognostic tools or successful therapies. Remodeling of the extracellular matrix (ECM) has been implicated in IPF progression; however, the structural consequences on the collagen architecture have not received considerable attention. Here, we demonstrate that second harmonic generation (SHG) and multiphoton fluorescence microscopy can quantitatively differentiate normal and IPF human tissues. For SHG analysis, we developed a classifier based on wavelet transforms, principle component analysis, and a K-nearest-neighbor algorithm to classify the specific alterations of the collagen structure observed in IPF tissues. The resulting ROC curves obtained by varying the numbers of principal components and nearest neighbors yielded accuracies of >95%. In contrast, simpler metrics based on SHG intensity and collagen coverage in the image provided little or no discrimination. We also characterized the change in the elastin/collagen balance by simultaneously measuring the elastin autofluorescence and SHG intensities and found that the IPF tissues were less elastic relative to collagen. This is consistent with known mechanical consequences of the disease. Understanding ECM remodeling in IPF via nonlinear optical microscopy may enhance our ability to differentiate patients with rapid and slow progression and, thus, provide better prognostic information. PMID:25134793

Myofibroblast secretome and its auto-/paracrine signaling

PubMed Central

Bomb, Ritin; Heckle, Mark R.; Sun, Yao; Mancarella, Salvatore; Guntaka, Ramareddy V.; Gerling, Ivan C.; Weber, Karl T.

2016-01-01

Summary Myofibroblasts (myoFb) are phenotypically transformed, contractile fibroblast-like cells expressing α-smooth muscle actin microfilaments. They are integral to collagen fibrillogenesis with scar tissue formation at sites of repair irrespective of the etiologic origins of injury or tissue involved. MyoFb can persist long after healing is complete, where their ongoing turnover of collagen accounts for a progressive structural remodeling of an organ (a.k.a. fibrosis, sclerosis or cirrhosis). Such persistent metabolic activity is derived from a secretome consisting of requisite components in the de novo generation of angiotensin (Ang) II. Autocrine and paracrine signaling induced by tissue AngII is expressed via AT1 receptor ligand binding to respectively promote: i) regulation of myoFb collagen synthesis via the fibrogenic cytokine TGF-β1-Smad pathway; and ii) dedifferentiation and protein degradation of atrophic myocytes immobilized and ensnared by fibrillar collagen at sites of scarring. Several cardioprotective strategies in the prevention of fibrosis and involving myofibroblasts are considered. They include: inducing myoFb apoptosis through inactivation of antiapoptotic proteins; AT1 receptor antagonist to interfere with auto-/paracrine myoFb signaling or to induce counterregulatory expression of ACE2; and attacking the AngII-AT1R-TGF-β1-Smad pathway by antibody or the use of triplex-forming oligonucleotides. PMID:26818589

Immunoadsorption for collagen and rheumatic diseases.

PubMed

Yamaji, Ken

2017-10-01

The field of therapeutics has seen remarkable progress in the recent years, which has made mainstream drug treatment possible for collagen and rheumatic diseases. However, treatment of intractable cases where drug effectiveness is poor is a challenge. Furthermore, organ damage, concurrent illnesses or allergic reactions make adequate drug therapy impossible. For such cases, therapeutic apheresis is very significant, and it is important how this should be valued related to drug therapies. Therapeutic apheresis for collagen and rheumatic diseases involves the removal of factors that cause and exacerbate the disease; the aim of immunoadsorption, in particular, is to improve the clinical condition of patients with autoimmune disease by selectively removing pathogenic immune complexes and autoantibodies from their plasma. Immunoadsorption, in particular, unlike plasma exchange and DFPP, utilizes a high-affinity column that selectively removes autoantibodies and immune complexes, leaving other plasma components intact. There is no need to replenish fresh frozen plasma or blood products such as albumin and gamma globulin preparations. Immunoadsorption is thus superior in terms of safety, as the risk of infection or allergic reaction relating to these preparations can be avoided. We anticipate future investigations of application of synchronized therapy using drugs and therapeutic apheresis, most notably immunoadsorption, in combination to treat intractable clinical conditions such as collagen and rheumatic diseases. In this paper, our discussion includes the indications for immunoadsorption such as collagen and rheumatic diseases, the relevant conditions and types, as well as the latest understanding related to methods and clinical efficacy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Visualization of predentine matrix components and endocytic structures in rat incisor odontoblasts with tannic acid.

PubMed

Goldberg, M; Septier, D

1989-12-01

Rat incisor odontoblasts and predentine fixed with tannic acid-glutaraldehyde-osmium tetroxide (Tago) were compared with those obtained by prior incubation in tannic acid-Ringer before conventional fixation with glutaraldehyde-osmium-tetroxide (Tari) The Tago method allowed visualization of complex glycoconjugates along the plasma membrane, in the pericellular spaces and in the intercellular predentine matrix. The non-collagenous proteins, proteoglycans and lipids were seen as granules and thin filaments located between the collagen fibers and at their surface. The collagen fibers themselves were also stained. The Tari method which was used to visualize exocytosis, mainly revealed endocytosis in the form of large intracellular vacuoles containing tannic acid and stained proteoglycans. It is suggested that tannic acid-Ringer incubation prior to fixation increases the endocytosis of the matrix components, which acculumates in these large vesicles.

Hyperunstable matrix proteins in the byssus of Mytilus galloprovincialis.

PubMed

Sagert, Jason; Waite, J Herbert

2009-07-01

The marine mussel Mytilus galloprovincialis is tethered to rocks in the intertidal zone by a holdfast known as the byssus. Functioning as a shock absorber, the byssus is composed of threads, the primary molecular components of which are collagen-containing proteins (preCOLs) that largely dictate the higher order self-assembly and mechanical properties of byssal threads. The threads contain additional matrix components that separate and perhaps lubricate the collagenous microfibrils during deformation in tension. In this study, the thread matrix proteins (TMPs), a glycine-, tyrosine- and asparagine-rich protein family, were shown to possess unique repeated sequence motifs, significant transcriptional heterogeneity and were distributed throughout the byssal thread. Deamidation was shown to occur at a significant rate in a recombinant TMP and in the byssal thread as a function of time. Furthermore, charge heterogeneity presumably due to deamidation was observed in TMPs extracted from threads. The TMPs were localized to the preCOL-containing secretory granules in the collagen gland of the foot and are assumed to provide a viscoelastic matrix around the collagenous fibers in byssal threads.

Live imaging of collagen deposition during skin development and repair in a collagen I - GFP fusion transgenic zebrafish line.

PubMed

Morris, Josephine L; Cross, Stephen J; Lu, Yinhui; Kadler, Karl E; Lu, Yongbo; Dallas, Sarah L; Martin, Paul

2018-06-06

Fibrillar collagen is a major component of many tissues but has been difficult to image in vivo using transgenic approaches because of problems associated with establishing cells and organisms that generate GFP-fusion collagens that can polymerise into functional fibrils. Here we have developed and characterised GFP and mCherry collagen-I fusion zebrafish lines with basal epidermal-specific expression. We use these lines to reveal the dynamic nature of collagen-I fibril deposition beneath the developing embryonic epidermis, as well as the repair of this collagen meshwork following wounding. Transmission electron microscope studies show that these transgenic lines faithfully reproduce the collagen ultrastructure present in wild type larval skin. During skin development we show that collagen I is deposited by basal epidermal cells initially in fine filaments that are largely randomly orientated but are subsequently aligned into a cross-hatch, orthogonal sub-epithelial network by embryonic day 4. Following skin wounding, we see that sub-epidermal collagen is re-established in the denuded domain, initially as randomly orientated wisps that subsequently become bonded to the undamaged collagen and aligned in a way that recapitulates developmental deposition of sub-epidermal collagen. Crossing our GFP-collagen line against one with tdTomato marking basal epidermal cell membranes reveals how much more rapidly wound re-epithelialisation occurs compared to the re-deposition of collagen beneath the healed epidermis. By use of other tissue specific drivers it will be possible to establish zebrafish lines to enable live imaging of collagen deposition and its remodelling in various other organs in health and disease. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The nature of the mineral component of bone and the mechanism of calcification.

PubMed

Glimcher, M J

1987-01-01

From the physical chemical standpoint, the formation of a solid phase of Ca-P in bone represents a phase transformation, a process exemplified by the formation of ice from water. Considering the structural complexity and abundance of highly organized macromolecules in the cells and extracellular tissue spaces of mineralized tissues generally and in bone particularly, it is inconceivable that this phase transformation occurs by homogeneous nucleation, i.e., without the active participation of an organic component acting as a nucleator. This is almost surely true in biologic mineralization in general. Electron micrographs and low-angle neutron and X-ray diffraction studies clearly show that calcification of collagen fibrils occurs in an extremely intimate and highly organized fashion: initiation of crystal formation within the collagen fibrils in the hole zone region, with the long axes (c-axis) of the crystals aligned roughly parallel to the long axis of the fibril within which they are located. Crystals are initially formed in hole zone regions within individual fibrils separated by unmineralized regions. Calcification is initiated in spatially distinct nucleation sites. This indicates that such regions within a single, undirectional fibril represents independent sites for heterogeneous nucleation. Clearly, sites where mineralization is initiated in adjacent collagen fibrils are even further separated, emphasizing even more clearly that the process of progressive calcification of the collagen fibrils and therefore of the tissue is characterized principally by the presence of increasing numbers of independent nucleation sites within additional hole zone regions of the collagen fibrils. The increase in the mass of Ca-P apatite accrues principally by multiplication of more crystals, mostly by secondary nucleation from the crystals initially deposited in the hole zone region. Very little additional growth of the crystals occurs with time, the additional increase in mineral mass being principally the result of increase in the number of crystals (multiplication), not size of the crystals (crystal growth). The crystals within the collagen fibers grow in number and possibly in size to extend into the overlap zone of the collagen fibrils ("pores") so that all of the available space within the fibrils, which has possibly expanded in volume from its uncalcified level, is eventually occupied by the mineral crystals. It must be recognized that the calcification of separate tissue components and compartments (collagen, mitochondria, matrix vesicles) must be an independent physical chemical event.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of Calendula officinalis on human gingival fibroblasts.

PubMed

Saini, Pragtipal; Al-Shibani, Nouf; Sun, Jun; Zhang, Weiping; Song, Fengyu; Gregson, Karen S; Windsor, L Jack

2012-04-01

Calendula officinalis is commonly called the marigold. It is a staple topical remedy in homeopathic medicine. It is rich in quercetin, carotenoids, lutein, lycopene, rutin, ubiquinone, xanthophylls, and other anti-oxidants. It has anti-inflammatory properties. Quercetin, one of the active components in Calendula, has been shown to inhibit recombinant human matrix metalloproteinase (MMP) activity and decrease the expression of tumor necrosis factor-α, interleukin-1β (IL), IL-6 and IL-8 in phorbol 12-myristate 13-acetate and calcium ionophore-stimulated human mast cells. To examine the effects of Calendula on human gingival fibroblast (HGF) mediated collagen degradation and MMP activity. Lactate dehydrogenate assays were performed to determine the non-toxic concentrations of Calendula, doxycycline and quercetin. Cell-mediated collagen degradation assays were performed to examine the inhibitory effect on cell-mediated collagen degradation. Gelatin zymography was performed to examine their effects on MMP-2 activity. The experiments were repeated three times and ANOVA used for statistical analyses. Calendula at 2-3% completely inhibited the MMP-2 activity in the zymograms. Doxycycline inhibited HGF-mediated collagen degradation at 0.005, 0.01, 0.02 and 0.05%, and MMP-2 activity completely at 0.05%. Quercetin inhibited HGF-mediated collagen degradation at 0.005, 0.01 and 0.02%, and MMP-2 activity in a dose-dependent manner. Calendula inhibited HGF-mediated collagen degradation and MMP-2 activity more than the same correlated concentration of pure quercetin. Calendula inhibits HGF-mediated collagen degradation and MMP-2 activity more than the corresponding concentration of quercetin. This may be attributed to additional components in Calendula other than quercetin. Published by Elsevier Ltd.

Ingestion of onion soup high in quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in man: a pilot study.

PubMed

Hubbard, Gary P; Wolffram, Siegfried; de Vos, Ric; Bovy, Arnaud; Gibbins, Jonathan M; Lovegrove, Julie A

2006-09-01

Epidemiological data suggest that those who consume a diet rich in quercetin-containing foods may have a reduced risk of CVD. Furthermore, in vitro and ex vivo studies have observed the inhibition of collagen-induced platelet activation by quercetin. The aim of the present study was to investigate the possible inhibitory effects of quercetin ingestion from a dietary source on collagen-stimulated platelet aggregation and signalling. A double-blind randomised cross-over pilot study was undertaken. Subjects ingested a soup containing either a high or a low amount of quercetin. Plasma quercetin concentrations and platelet aggregation and signalling were assessed after soup ingestion. The high-quercetin soup contained 69 mg total quercetin compared with the low-quercetin soup containing 5 mg total quercetin. Plasma quercetin concentrations were significantly higher after high-quercetin soup ingestion than after low-quercetin soup ingestion and peaked at 2.59 (sem 0.42) mumol/l. Collagen-stimulated (0.5 mug/ml) platelet aggregation was inhibited after ingestion of the high-quercetin soup in a time-dependent manner. Collagen-stimulated tyrosine phosphorylation of a key component of the collagen-signalling pathway via glycoprotein VI, Syk, was significantly inhibited by ingestion of the high-quercetin soup. The inhibition of Syk tyrosine phosphorylation was correlated with the area under the curve for the high-quercetin plasma profile. In conclusion, the ingestion of quercetin from a dietary source of onion soup could inhibit some aspects of collagen-stimulated platelet aggregation and signalling ex vivo. This further substantiates the epidemiological data suggesting that those who preferentially consume high amounts of quercetin-containing foods have a reduced risk of thrombosis and potential CVD risk.

Collagen Fingerprinting: A New Screening Technique for Radiocarbon Dating Ancient Bone.

PubMed

Harvey, Virginia L; Egerton, Victoria M; Chamberlain, Andrew T; Manning, Phillip L; Buckley, Michael

2016-01-01

Collagen is the dominant organic component of bone and is intimately locked within the hydroxyapatite structure of this ubiquitous biomaterial that dominates archaeological and palaeontological assemblages. Radiocarbon analysis of extracted collagen is one of the most common approaches to dating bone from late Pleistocene or Holocene deposits, but dating is relatively expensive compared to other biochemical techniques. Numerous analytical methods have previously been investigated for the purpose of screening out samples that are unlikely to yield reliable dates including histological analysis, UV-stimulated fluorescence and, most commonly, the measurement of percentage nitrogen (%N) and ratio of carbon to nitrogen (C:N). Here we propose the use of collagen fingerprinting (also known as Zooarchaeology by Mass Spectrometry, or ZooMS, when applied to species identification) as an alternative screening method for radiocarbon dating, due to its ability to provide information on collagen presence and quality, alongside species identification. The method was tested on a series of sub-fossil bone specimens from cave systems on Cayman Brac (Cayman Islands), chosen due to the observable range in diagenetic alteration, and in particular, the extent of mineralisation. Six (14)C dates, of 18 initial attempts, were obtained from remains of extinct hutia, Capromys sp. (Rodentia; Capromyidae), recovered from five distinct caves on Cayman Brac, and ranging from 393 ± 25 to 1588 ± 26 radiocarbon years before present (yr BP). All of the bone samples that yielded radiocarbon dates generated excellent collagen fingerprints, and conversely those that gave poor fingerprints also failed dating. Additionally, two successfully fingerprinted bone samples were screened out from a set of 81. Both subsequently generated (14)C dates, demonstrating successful utilisation of ZooMS as an alternative screening mechanism to identify bone samples that are suitable for 1(4)C analysis.

Collagen Fingerprinting: A New Screening Technique for Radiocarbon Dating Ancient Bone

PubMed Central

Harvey, Virginia L.; Egerton, Victoria M.; Chamberlain, Andrew T.; Manning, Phillip L.; Buckley, Michael

2016-01-01

Collagen is the dominant organic component of bone and is intimately locked within the hydroxyapatite structure of this ubiquitous biomaterial that dominates archaeological and palaeontological assemblages. Radiocarbon analysis of extracted collagen is one of the most common approaches to dating bone from late Pleistocene or Holocene deposits, but dating is relatively expensive compared to other biochemical techniques. Numerous analytical methods have previously been investigated for the purpose of screening out samples that are unlikely to yield reliable dates including histological analysis, UV-stimulated fluorescence and, most commonly, the measurement of percentage nitrogen (%N) and ratio of carbon to nitrogen (C:N). Here we propose the use of collagen fingerprinting (also known as Zooarchaeology by Mass Spectrometry, or ZooMS, when applied to species identification) as an alternative screening method for radiocarbon dating, due to its ability to provide information on collagen presence and quality, alongside species identification. The method was tested on a series of sub-fossil bone specimens from cave systems on Cayman Brac (Cayman Islands), chosen due to the observable range in diagenetic alteration, and in particular, the extent of mineralisation. Six 14C dates, of 18 initial attempts, were obtained from remains of extinct hutia, Capromys sp. (Rodentia; Capromyidae), recovered from five distinct caves on Cayman Brac, and ranging from 393 ± 25 to 1588 ± 26 radiocarbon years before present (yr BP). All of the bone samples that yielded radiocarbon dates generated excellent collagen fingerprints, and conversely those that gave poor fingerprints also failed dating. Additionally, two successfully fingerprinted bone samples were screened out from a set of 81. Both subsequently generated 14C dates, demonstrating successful utilisation of ZooMS as an alternative screening mechanism to identify bone samples that are suitable for 14C analysis. PMID:26938469

Skin aging and menopause : implications for treatment.

PubMed

Raine-Fenning, Nicholas J; Brincat, Mark P; Muscat-Baron, Yves

2003-01-01

The skin is one of the largest organs of the body, which is significantly affected by the aging process and menopause. The significant changes sustained by the skin during the menopause are due to the effect sustained on the skin's individual components. The estrogen receptor has been detected on the cellular components of the skin. Accordingly, dermal cellular metabolism is influenced by the hypoestrogenoemic state of menopause leading to changes in the collagen content, alterations in the concentration of glycoaminoglycans and most importantly the water content. Consequently changes in these basic components leads to an alteration in function compatible with skin aging. Changes in the skin collagen leads to diminished elasticity and skin strength. Collagen content may be measured by various methods such as direct skin biopsy, skin blister assessment for collagen markers and skin thickness measurement. All these variables indicate a reduction in collagen content following menopause. This may be reversed with the administration of estrogen given both topically and systemically.A reduction in hydrophilic glycoaminglycans leads to a direct reduction in water content, which influences the skin turgor. These effects on glycoaminoglycans, due to the hypoestrogenia, have been clearly shown in animal studies and appeared to be rapidly reversed with the application of estrogens. The sum total of these basic effects on the skin leads to wrinkles, the skin condition typifying skin aging.Structures resident in the skin are likewise influenced by menopause. Changes to the cutaneous vascular reactivity are noted following menopause. Capillary blood flow velocity decreases significantly in postmenopausal women. Postmenopausal flushing is due to profound vasodilatation in the dermal papillae. Hair growth is also influenced by the hormonal milieu and consequently hair loss has been associated with the beginning of menopause. Treatments administered for menopause, in particular hormone replacement therapy, appear to alter its effects on the basic components of the skin as well as the more complex structures residing in the skin, consequently retarding the skin aging process.

Molecular and ultrastructural studies of a fibrillar collagen from octocoral (Cnidaria).

PubMed

Orgel, Joseph P R O; Sella, Ido; Madhurapantula, Rama S; Antipova, Olga; Mandelberg, Yael; Kashman, Yoel; Benayahu, Dafna; Benayahu, Yehuda

2017-09-15

We report here the biochemical, molecular and ultrastructural features of a unique organization of fibrillar collagen extracted from the octocoral Sarcophyton ehrenbergi Collagen, the most abundant protein in the animal kingdom, is often defined as a structural component of extracellular matrices in metazoans. In the present study, collagen fibers were extracted from the mesenteries of S. ehrenbergi polyps. These fibers are organized as filaments and further compacted as coiled fibers. The fibers are uniquely long, reaching an unprecedented length of tens of centimeters. The diameter of these fibers is 9±0.37 μm. The amino acid content of these fibers was identified using chromatography and revealed close similarity in content to mammalian type I and II collagens. The ultrastructural organization of the fibers was characterized by means of high-resolution microscopy and X-ray diffraction. The fibers are composed of fibrils and fibril bundles in the range of 15 to 35 nm. These data indicate a fibrillar collagen possessing structural aspects of both types I and II collagen, a highly interesting and newly described form of fibrillar collagen organization. © 2017. Published by The Company of Biologists Ltd.

The role of the non-collagenous matrix in tendon function.

PubMed

Thorpe, Chavaunne T; Birch, Helen L; Clegg, Peter D; Screen, Hazel R C

2013-08-01

Tendon consists of highly ordered type I collagen molecules that are grouped together to form subunits of increasing diameter. At each hierarchical level, the type I collagen is interspersed with a predominantly non-collagenous matrix (NCM) (Connect. Tissue Res., 6, 1978, 11). Whilst many studies have investigated the structure, organization and function of the collagenous matrix within tendon, relatively few have studied the non-collagenous components. However, there is a growing body of research suggesting the NCM plays an important role within tendon; adaptations to this matrix may confer the specific properties required by tendons with different functions. Furthermore, age-related alterations to non-collagenous proteins have been identified, which may affect tendon resistance to injury. This review focuses on the NCM within the tensional region of developing and mature tendon, discussing the current knowledge and identifying areas that require further study to fully understand structure-function relationships within tendon. This information will aid in the development of appropriate techniques for tendon injury prevention and treatment. © 2013 The Authors. International Journal of Experimental Pathology © 2013 International Journal of Experimental Pathology.

A spectral approach for the quantitative description of cardiac collagen network from nonlinear optical imaging.

PubMed

Masè, Michela; Cristoforetti, Alessandro; Avogaro, Laura; Tessarolo, Francesco; Piccoli, Federico; Caola, Iole; Pederzolli, Carlo; Graffigna, Angelo; Ravelli, Flavia

2015-01-01

The assessment of collagen structure in cardiac pathology, such as atrial fibrillation (AF), is essential for a complete understanding of the disease. This paper introduces a novel methodology for the quantitative description of collagen network properties, based on the combination of nonlinear optical microscopy with a spectral approach of image processing and analysis. Second-harmonic generation (SHG) microscopy was applied to atrial tissue samples from cardiac surgery patients, providing label-free, selective visualization of the collagen structure. The spectral analysis framework, based on 2D-FFT, was applied to the SHG images, yielding a multiparametric description of collagen fiber orientation (angle and anisotropy indexes) and texture scale (dominant wavelength and peak dispersion indexes). The proof-of-concept application of the methodology showed the capability of our approach to detect and quantify differences in the structural properties of the collagen network in AF versus sinus rhythm patients. These results suggest the potential of our approach in the assessment of collagen properties in cardiac pathologies related to a fibrotic structural component.

Mechanical properties of complex biological systems using AFM-based force spectroscopy

NASA Astrophysics Data System (ADS)

Graham, John Stephen

An atomic force microscope (AFM) was designed and built to study the mechanical properties of small collagen fibrils and the plasma membrane of living cells. Collagen is a major component of bone, skin and connective tissues, and is abundant in the extracellular matrix (ECM). Because of its abundance, an understanding of how disease affects collagen mechanics is crucial in disease prevention efforts. Two levels of type I collagen structure were investigated, subfibrils (on the order of 1 mum in length) and longer fibrils. Comparisons were made between measurements of wild-type (wt) collagen and collagen from the mouse model of osteogenesis imperfecta (OI). Significant differences between OI and wt collagen were observed, primarily that intermolecular bonds in OI collagen fibrils are weaker than in wt, or not ruptured, as in the case of OI subfibrils. As cells interact with collagen in the ECM, the mechanical properties of the plasma membrane are also of great interest. Membrane tethers were extracted from living cells under varied conditions in order to assess the contributions of membrane-associated macromolecules such as the actin cytoskeleton and the glycocalyx, and intracellular signaling. Tether extraction force was found to be sensitive to all of these altered conditions, suggesting that tether extraction may be used to monitor various cellular processes.

Limitations in Bonding to Dentin and Experimental Strategies to Prevent Bond Degradation

PubMed Central

Liu, Y.; Tjäderhane, L.; Breschi, L.; Mazzoni, A.; Li, N.; Mao, J.; Pashley, D.H.; Tay, F.R.

2011-01-01

The limited durability of resin-dentin bonds severely compromises the lifetime of tooth-colored restorations. Bond degradation occurs via hydrolysis of suboptimally polymerized hydrophilic resin components and degradation of water-rich, resin-sparse collagen matrices by matrix metalloproteinases (MMPs) and cysteine cathepsins. This review examined data generated over the past three years on five experimental strategies developed by different research groups for extending the longevity of resin-dentin bonds. They include: (1) increasing the degree of conversion and esterase resistance of hydrophilic adhesives; (2) the use of broad-spectrum inhibitors of collagenolytic enzymes, including novel inhibitor functional groups grafted to methacrylate resins monomers to produce anti-MMP adhesives; (3) the use of cross-linking agents for silencing the activities of MMP and cathepsins that irreversibly alter the 3-D structures of their catalytic/allosteric domains; (4) ethanol wet-bonding with hydrophobic resins to completely replace water from the extrafibrillar and intrafibrillar collagen compartments and immobilize the collagenolytic enzymes; and (5) biomimetic remineralization of the water-filled collagen matrix using analogs of matrix proteins to progressively replace water with intrafibrillar and extrafibrillar apatites to exclude exogenous collagenolytic enzymes and fossilize endogenous collagenolytic enzymes. A combination of several of these strategies should result in overcoming the critical barriers to progress currently encountered in dentin bonding. PMID:21220360

Changes in Structure, Interstitial Cajal-like Cells and Apoptosis of Smooth Muscle Cells in Congenital Ureteropelvic Junction Obstruction

PubMed Central

Mehrazma, Mitra; Tanzifi, Parin; Rakhshani, Naser

2014-01-01

Objective: The goal of this study is to evaluate some structural changes in muscular, collagenous and neural components as well as expression of Cajal-like cells and apoptosis of smooth muscle cells in congenital ureteropelvic junction obstruction (UPJO). Methods: Tissue specimens were obtained from 25 patients with UPJO and compared with normal ureteropelvic junction regions of 19 autopsies. In paraffin embedded sections the amount of Cajal-like cells, density of nerve fibers and smooth muscle cell apoptosis (using immunohistochemical staining) were determined. Collagen deposition and muscular components were stained by Trichrome-Masson staining and evaluated by image analysis techniques. Arrangement of muscular bundles was also evaluated qualitatively. Findings : The number of Cajal-like cells was significantly lower in patients than in controls. The apoptotic score and mean number of nerve fibers were not statistically different for the two groups. Arrangement of muscular fibers was more irregular in patients than in controls (P<0.001). Collagen deposition was significantly higher in patients than in controls (P<0.001). The mean amount of muscular component was lower in patients than in normal ones. (P= 0.09) Conclusion: We found significant pathologic changes in congenital ureteropelvic junction obstruction such as decrease in Cajal-like cells, increase in collagen deposition and irregular arrangement of muscle fibers. PMID:25793054

Role of Mesenchymal-Derived Stem Cells in Stimulating Dormant Tumor Cells to Proliferate and Form Clinical Metastases

DTIC Science & Technology

2016-07-01

have determined the break in dormancy is dependent on collagen and other fibrotic extracellular matrix components for the induction of a proliferative...state in these dormant D2.0R breast cancer cell lines. Performing gene expression array on these dormant D2.0R cells exposed to collagen to induce a...D2.0R”) and proliferate when cultured in matrigel supplemented with collagen type-1 (“proliferative D2.0R”) (Barkan, Cancer Research, 2008, 68(15

Molecular Mechanisms of Stress-Responsive Changes in Collagen and Elastin Networks in Skin.

PubMed

Aziz, Jazli; Shezali, Hafiz; Radzi, Zamri; Yahya, Noor Azlin; Abu Kassim, Noor Hayaty; Czernuszka, Jan; Rahman, Mohammad Tariqur

2016-01-01

Collagen and elastin networks make up the majority of the extracellular matrix in many organs, such as the skin. The mechanisms which are involved in the maintenance of homeostatic equilibrium of these networks are numerous, involving the regulation of genetic expression, growth factor secretion, signalling pathways, secondary messaging systems, and ion channel activity. However, many factors are capable of disrupting these pathways, which leads to an imbalance of homeostatic equilibrium. Ultimately, this leads to changes in the physical nature of skin, both functionally and cosmetically. Although various factors have been identified, including carcinogenesis, ultraviolet exposure, and mechanical stretching of skin, it was discovered that many of them affect similar components of regulatory pathways, such as fibroblasts, lysyl oxidase, and fibronectin. Additionally, it was discovered that the various regulatory pathways intersect with each other at various stages instead of working independently of each other. This review paper proposes a model which elucidates how these molecular pathways intersect with one another, and how various internal and external factors can disrupt these pathways, ultimately leading to a disruption in collagen and elastin networks. © 2016 S. Karger AG, Basel.

Contributions of Raman spectroscopy to the understanding of bone strength.

PubMed

Mandair, Gurjit S; Morris, Michael D

2015-01-01

Raman spectroscopy is increasingly commonly used to understand how changes in bone composition and structure influence tissue-level bone mechanical properties. The spectroscopic technique provides information on bone mineral and matrix collagen components and on the effects of various matrix proteins on bone material properties as well. The Raman spectrum of bone not only contains information on bone mineral crystallinity that is related to bone hardness but also provides information on the orientation of mineral crystallites with respect to the collagen fibril axis. Indirect information on collagen cross-links is also available and will be discussed. After a short introduction to bone Raman spectroscopic parameters and collection methodologies, advances in in vivo Raman spectroscopic measurements for animal and human subject studies will be reviewed. A discussion on the effects of aging, osteogenesis imperfecta, osteoporosis and therapeutic agents on bone composition and mechanical properties will be highlighted, including genetic mouse models in which structure-function and exercise effects are explored. Similarly, extracellular matrix proteins, proteases and transcriptional proteins implicated in the regulation of bone material properties will be reviewed.

Comparison of thermal properties of fish collagen and bovine collagen in the temperature range 298-670K.

PubMed

Gauza-Włodarczyk, Marlena; Kubisz, Leszek; Mielcarek, Sławomir; Włodarczyk, Dariusz

2017-11-01

The increased interest in fish collagen is a consequence of the risk of exposure to Creutzfeld-Jacob disease (CJD) and the bovine spongiform encephalopathy (BSE), whose occurrence is associated with prions carried by bovine collagen. Collagen is the main biopolymer in living organisms and the main component of the skin and bones. Until the discovery of the BSE, bovine collagen had been widely used. The BSE epidemic increased the interest in new sources of collagen such as fish skin collagen (FSC) and its properties. Although the thermal properties of collagen originating from mammals have been well described, less attention has been paid to the thermal properties of FSC. Denaturation temperature is a particularly important parameter, depending on the collagen origin and hydration level. In the reported experiment, the free water and bound water release processes along with thermal denaturation process were studied by means of the differential scanning calorimetry (DSC). Measurements were carried out using a DSC 7 instrument (Elmer-Perkin), in the temperature range 298-670K. The study material was FSC derived by acidic hydration method. The bovine Achilles tendon (BAT) collagen type I was used as the control material. The thermograms recorded revealed both, exothermic and endothermic peaks. For both materials, the peaks in the temperature range of 330-360K were assigned to the release of free water and bound water. The denaturation temperatures of FSC and BAT collagen were determined as 420K and 493K, respectively. Thermal decomposition process was observed at about 500K for FSC and at about 510K for BAT collagen. These results show that FSC is less resistant to high temperature than BAT collagen. Copyright © 2017 Elsevier B.V. All rights reserved.

Physiological regulation of extracellular matrix collagen and elastin in the arterial wall of rats by noradrenergic tone and angiotensin II.

PubMed

Dab, Houcine; Kacem, Kamel; Hachani, Rafik; Dhaouadi, Nadra; Hodroj, Wassim; Sakly, Mohsen; Randon, Jacques; Bricca, Giampiero

2012-03-01

The interactions between the effects of the sympathetic nervous system (SNS) and angiotensin II (ANG II) on vascular extracellular matrix (ECM) synthesis were determined in rats. The mRNA and protein content of collagen I, collagen III and elastin in the abdominal aorta (AA) and femoral artery (FA) was investigated in Wistar-Kyoto rats treated for 5 weeks with guanethidine, a sympathoplegic, losartan, an ANG II AT1 receptor (AT1R) blocker, or both. The effects of noradrenaline (NE) and ANG II on collagen III and elastin mRNA, and the receptor involved, were tested in cultured vascular smooth muscle cells (VSMCs) in vitro. Guanethidine increased collagen types I and III and decreased elastin, while losartan had an opposite effect, although without effect on collagen III. The combination of treatments abrogated changes induced by simple treatment with collagen I and elastin, but increased collagen III mRNA in AA and not in FA. NE stimulated collagen III mRNA via β receptors and elastin via α1 and α2 receptors. ANG II stimulated collagen III but inhibited elastin mRNA via AT1R. Overall, SNS and ANG II exert opposite and antagonistic effects on major components of ECM in the vascular wall. This may be of relevance for the choice of a therapeutic strategy in vascular diseases.

Quantitative analysis of the synthesis and secretion of type VII collagen in cultured human dermal fibroblasts with a sensitive sandwich enzyme-linked immunoassay.

PubMed

Amano, Satoshi; Ogura, Yuki; Akutsu, Nobuko; Nishiyama, Toshio

2007-02-01

Type VII collagen is the major component of anchoring fibrils in the epidermal basement membrane. Its expression has been analyzed by immunostaining or Northern blotting, but rarely at the protein level. In this study, we have quantitatively examined the effects of ascorbic acid and various cytokines/growth factors on the protein synthesis and secretion of type VII collagen by human dermal fibroblasts in culture, using a developed, highly sensitive sandwich enzyme-linked immunoassay with two kinds of specific monoclonal antibodies against the non-collagenous domain-1. Ascorbic acid and its derivative induced a twofold increase in type VII collagen synthesis, and markedly increased the secretion of type VII collagen into the medium when compared with the control culture. This effect was not influenced by the presence of transforming growth factor-beta1 (TGF-beta1). The synthesis of type VII collagen was elevated by TGF-beta1, platelet-derived growth factor, tumor necrosis factor-alpha, and interleukin-1beta, but not by TGF-alpha. Thus, our data indicate that the synthesis and secretion of type VII collagen in human dermal fibroblasts are regulated by ascorbate and the enhancement of type VII collagen gene expression by cytokines/growth factors is accompanied with elevated production of type VII collagen at the protein level.

Deamidation of collagen.

PubMed

Hurtado, Pilar Perez; O'Connor, Peter B

2012-03-20

Collagen is the major component of skin, tendons, ligaments, teeth, and bones, it provides the framework that holds most multicellular animals together, and collagen type I constitutes the major fibrillar collagen of bone. Because of the complexity of collagen's structure, the study of post-translational modifications such as deamidation for this protein is challenging. Although there is no evidence of this protein being used for age assessment, it has been shown that deamidation of collagen is remarkably increased in old bones from mammals. Nonspectrometric methodologies have been used for the determination of the extent of deamidation as a measure of the amount of amide nitrogen released in ammonia as well as constant rates for deamidation of asparagine in collagen. In general, these methodologies required more sample and separation processes. To understand if collagen plays a significant role in the aging process of fossil materials, a simpler and more accurate method is needed to determine the extent of deamidation at the whole protein level. The present work shows a method to determine the extent of deamidation in collagen using Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) along with collisionally activated dissociation (CAD) and electron capture dissociation (ECD). The measured deamidation half-life for three different tryptic peptides from collagen (I) ranged from 2000 to 6000 s under high temperature conditions (∼62 °C) and pH 7.5.

Peptide tessellation yields micrometre-scale collagen triple helices

NASA Astrophysics Data System (ADS)

Tanrikulu, I. Caglar; Forticaux, Audrey; Jin, Song; Raines, Ronald T.

2016-11-01

Sticky-ended DNA duplexes can associate spontaneously into long double helices; however, such self-assembly is much less developed with proteins. Collagen is the most prevalent component of the extracellular matrix and a common clinical biomaterial. As for natural DNA, the ~103-residue triple helices (~300 nm) of natural collagen are recalcitrant to chemical synthesis. Here we show how the self-assembly of short collagen-mimetic peptides (CMPs) can enable the fabrication of synthetic collagen triple helices that are nearly a micrometre in length. Inspired by the mathematics of tessellations, we derive rules for the design of single CMPs that self-assemble into long triple helices with perfect symmetry. Sticky ends thus created are uniform across the assembly and drive its growth. Enacting this design yields individual triple helices that, in length, match or exceed those in natural collagen and are remarkably thermostable, despite the absence of higher-order association. The symmetric assembly of CMPs provides an enabling platform for the development of advanced materials for medicine and nanotechnology.

Collagen Self-Assembly on Orthopedic Magnesium Biomaterials Surface and Subsequent Bone Cell Attachment

PubMed Central

Zhao, Nan; Zhu, Donghui

2014-01-01

Magnesium (Mg) biomaterials are a new generation of biodegradable materials and have promising potential for orthopedic applications. After implantation in bone tissues, these materials will directly interact with extracellular matrix (ECM) biomolecules and bone cells. Type I collagen, the major component of bone ECM, forms the architecture scaffold that provides physical support for bone cell attachment. However, it is still unknown how Mg substrate affects collagen assembly on top of it as well as subsequent cell attachment and growth. Here, we studied the effects of collagen monomer concentration, pH, assembly time, and surface roughness of two Mg materials (pure Mg and AZ31) on collagen fibril formation. Results showed that formation of fibrils would not initiate until the monomer concentration reached a certain level depending on the type of Mg material. The thickness of collagen fibril increased with the increase of assembly time. The structures of collagen fibrils formed on semi-rough surfaces of Mg materials have a high similarity to that of native bone collagen. Next, cell attachment and growth after collagen assembly were examined. Materials with rough surface showed higher collagen adsorption but compromised bone cell attachment. Interestingly, surface roughness and collagen structure did not affect cell growth on AZ31 for up to a week. Findings from this work provide some insightful information on Mg-tissue interaction at the interface and guidance for future surface modifications of Mg biomaterials. PMID:25303459

Development of a pericardial acellular matrix biomaterial: biochemical and mechanical effects of cell extraction.

PubMed

Courtman, D W; Pereira, C A; Kashef, V; McComb, D; Lee, J M; Wilson, G J

1994-06-01

There is evidence to suggest that the cellular components of homografts and bioprosthetic xenografts may contribute to calcification or immunogenic reactions. A four-step detergent and enzymatic extraction process has been developed to remove cellular components from bovine pericardial tissue. The process results in an acellular matrix material consisting primarily of elastin, insoluble collagen, and tightly bound glycosaminoglycans. Light and electron microscopy confirmed that nearly all cellular constituents are removed without ultrastructural evidence of damage to fibrous components. Collagen denaturation temperatures remained unaltered. Biochemical analysis confirmed the retention of collagen and elastin and some differential extraction of glycosaminoglycans. Low strain rate fracture testing and high strain rate viscoelastic characterization showed that, with the exception of slightly increased stress relaxation, the mechanical properties of the fresh tissue were preserved in the pericardial acellular matrix. Crosslinking of the material in glutaraldehyde or poly(glycidyl ether) produced mechanical changes consistent with the same treatments of fresh tissue. The pericardial acellular matrix is a promising approach to the production of biomaterials for heart valve or cardiovascular patching applications.

Progesterone and gravidity differentially regulate expression of extracellular matrix components in the pregnant rat myometrium.

PubMed

Shynlova, Oksana; Mitchell, Jennifer A; Tsampalieros, Anne; Langille, B Lowell; Lye, Stephen J

2004-04-01

Myometrial growth and remodeling during pregnancy depends on increased synthesis of interstitial matrix proteins. We hypothesize that the presence of mechanical tension in a specific hormonal environment regulates the expression of extracellular matrix (ECM) components in the uterus. Myometrial tissue was collected from pregnant rats on Gestational Days 0, 12, 15, 17, 19, 21, 22, 23 (labor), and 1 day postpartum and ECM expression was analyzed by Northern blotting. Expression of fibronectin, laminin beta2, and collagen IV mRNA was low during early gestation but increased dramatically on Day 23 during labor. Expression of fibrillar collagens (type I and III) peaked Day 19 and decreased near term. In contrast, elastin mRNA remained elevated from midgestation onward. Injection of progesterone (P4) on Days 20-23 (to maintain elevated plasma P4 levels) delayed the onset of labor, caused dramatic reductions in the levels of fibronectin and laminin mRNA, and prevented the fall of collagen III mRNA levels on Day 23. Treatment of pregnant rats with the progesterone receptor antagonist RU486 on Day 19 induced preterm labor on Day 20 and a premature increase in mRNA levels of collagen IV, fibronectin, and laminin. Analysis of the uterine tissue from unilaterally pregnant rats revealed that most of the changes in ECM gene expression occurred specifically in the gravid horn. Our results show a decrease in expression of fibrillar collagens and a coordinated temporal increase in expression of components of the basement membrane near term associated with decreased P4 and increased mechanical tension. These ECM changes contribute to myometrial growth and remodeling during late pregnancy and the preparation for the synchronized contractions of labor.

Preparation and characterization of collagen/PLA, chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering.

PubMed

Haaparanta, Anne-Marie; Järvinen, Elina; Cengiz, Ibrahim Fatih; Ellä, Ville; Kokkonen, Harri T; Kiviranta, Ilkka; Kellomäki, Minna

2014-04-01

In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.

Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium

PubMed Central

Sillanpää, Jouko; Nallapareddy, Sreedhar R.; Prakash, Vittal P.; Qin, Xiang; Hook, Magnus; Weinstock, George M.; Murray, Barbara E.

2009-01-01

SUMMARY Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about pathogenic determinants of this organism. We have previously identified a cell wall anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad spectrum binding to extracellular matrix proteins. Here, we analyzed the draft genome of strain TX0016 for potential MSCRAMMs (microbial surface component recognizing adhesive matrix molecules). Genome-based bioinformatics identified 22 predicted cell wall anchored E. faeciumsurface proteins (Fms) of which 15 (including Acm) have typical characteristics of MSCRAMMs including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one (Fms10, redesignated Scm for second collagen adhesin of E. faeciu m) revealed that recombinant Scm65 (A- and B-domains) and Scm36 (A-domain) bound efficiently to collagen type V in a concentration dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism of recombinant Scm36 and of Acm37 indicated that these proteins are rich in β-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; 9 of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated as EbpCfm), detected a “ladder” pattern of high-molecular weight protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpCfm is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpAfm), fms5 (ebpBfm) and ebpCfm are co-transcribed, consistent with pilus-encoding gene clusters of other gram-positive bacteria. All 15 genes occurred frequently in 30 clinically-derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen. PMID:18832325

Library Screen Identifies Enterococcus faecalis CcpA, the Catabolite Control Protein A, as an Effector of Ace, a Collagen Adhesion Protein Linked to Virulence

PubMed Central

Gao, Peng; Pinkston, Kenneth L.; Bourgogne, Agathe; Cruz, Melissa R.; Garsin, Danielle A.; Murray, Barbara E.

2013-01-01

The Enterococcus faecalis cell wall-anchored protein Ace is an important virulence factor involved in cell adhesion and infection. Expression of Ace on the cell surface is affected by many factors, including stage of growth, culture temperature, and environmental components, such as serum, urine, and collagen. However, the mechanisms that regulate or modulate Ace display are not well understood. With interest in identifying genes associated with Ace expression, we utilized a whole-cell enzyme-linked immunosorbent assay (ELISA)-based screening method to identify mutants from a transposon insertion mutant library which exhibited distinct Ace surface expression profiles. We identified a ccpA insertion mutant which showed significantly decreased levels of Ace surface expression at early growth phase versus those of wild-type OG1RF. Confirmation of the observation was achieved through flow cytometry and complementation analysis. Compared to the wild type, the E. faecalis ccpA mutant had an impaired ability to adhere to collagen when grown to early exponential phase, consistent with the lack of Ace expression in the early growth phase. As a key component of carbon catabolite regulation, CcpA has been previously reported to play a critical role in regulating expression of proteins involved in E. faecalis carbohydrate uptake and utilization. Our discovery is the first to associate CcpA with the production of a major E. faecalis virulence factor, providing new insights into the regulation of E. faecalis pathogenesis. PMID:23974022

Analysis of molecular pathways in pancreatic ductal adenocarcinomas with a bioinformatics approach.

PubMed

Wang, Yan; Li, Yan

2015-01-01

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death worldwide. Our study aimed to reveal molecular mechanisms. Microarray data of GSE15471 (including 39 matching pairs of pancreatic tumor tissues and patient-matched normal tissues) was downloaded from Gene Expression Omnibus (GEO) database. We identified differentially expressed genes (DEGs) in PDAC tissues compared with normal tissues by limma package in R language. Then GO and KEGG pathway enrichment analyses were conducted with online DAVID. In addition, principal component analysis was performed and a protein-protein interaction network was constructed to study relationships between the DEGs through database STRING. A total of 532 DEGs were identified in the 38 PDAC tissues compared with 33 normal tissues. The results of principal component analysis of the top 20 DEGs could differentiate the PDAC tissues from normal tissues directly. In the PPI network, 8 of the 20 DEGs were all key genes of the collagen family. Additionally, FN1 (fibronectin 1) was also a hub node in the network. The genes of the collagen family as well as FN1 were significantly enriched in complement and coagulation cascades, ECM-receptor interaction and focal adhesion pathways. Our results suggest that genes of collagen family and FN1 may play an important role in PDAC progression. Meanwhile, these DEGs and enriched pathways, such as complement and coagulation cascades, ECM-receptor interaction and focal adhesion may be important molecular mechanisms involved in the development and progression of PDAC.

Drosophila type IV collagen mutation associates with immune system activation and intestinal dysfunction.

PubMed

Kiss, Márton; Kiss, András A; Radics, Monika; Popovics, Nikoletta; Hermesz, Edit; Csiszár, Katalin; Mink, Mátyás

2016-01-01

The basal lamina (BM) contains numerous components with a predominance of type IV collagens. Clinical manifestations associated with mutations of the human COL4A1 gene include perinatal cerebral hemorrhage and porencephaly, hereditary angiopathy, nephropathy, aneurysms and muscle cramps (HANAC), ocular dysgenesis, myopathy, Walker–Warburg syndrome and systemic tissue degeneration. In Drosophila, the phenotype associated with dominant temperature sensitive mutations of col4a1 include severe myopathy resulting from massive degradation of striated muscle fibers, and in the gut, degeneration of circular visceral muscle cells and epithelial cells following detachment from the BM. In order to determine the consequences of altered BMfunctions due to aberrant COL4A1 protein, we have carried out a series of tests using Drosophila DTS-L3 mutants from our allelic series of col4a1 mutations with confirmed degeneration of various cell types and lowest survival rate among the col4a1 mutant lines at restrictive temperature. Results demonstrated epithelial cell degeneration in the gut, shortened gut, enlarged midgut with multiple diverticulae, intestinal dysfunction and shortened life span. Midgut immunohistochemistry analyses confirmed altered expression and distribution of BM components integrin PSI and PSII alpha subunits, laminin gamma 1, and COL4A1 both in larvae and adults. Global gene expression analysis revealed activation of the effector AMP genes of the primary innate immune system including Metchnikowin, Diptericin, Diptericin B, and edin that preceded morphological changes. Attacin::GFP midgut expression pattern further supported these changes. An increase in ROS production and changes in gut bacterial flora were also noted and may have further enhanced an immune response. The phenotypic features of Drosophila col4a1 mutants confirmed an essential role for type IV collagen in maintaining epithelial integrity, gut morphology and intestinal function and suggest that aberrant structure and function of the COL4A1 protein may also be a significant factor in modulating immunity.

Noninvasive evaluation of collagen and hemoglobin contents and scattering property of in vivo keloid scars and normal skin using diffuse reflectance spectroscopy: pilot study

NASA Astrophysics Data System (ADS)

Tseng, Sheng-Hao; Hsu, Chao-Kai; Yu-Yun Lee, Julia; Tzeng, Shih-Yu; Chen, Wan-Rung; Liaw, Yu-Kai

2012-07-01

Collagen is a rich component in skin that provides skin structure integrity; however, its contribution to the absorption and scattering properties of various types of skin has not been extensively studied. We considered the contribution of the collagen to the absorption spectrum of in vivo normal skin and keloids of 12 subjects derived from our diffuse reflectance spectroscopy (DRS) system in the wavelength range from 550 to 860 nm. It was found that the collagen concentration, the hemoglobin oxygen saturation, and the reduced scattering coefficient of keloids were remarkably different from that of normal skin. Our results suggest that our DRS system could assist clinicians in understanding the functional and structural condition of keloid scars. In the future, we will evaluate the accuracy of our system in the keloid diagnosis and investigate the applicability of our system for other skin-collagen-related studies.

Osmotic pressure induced tensile forces in tendon collagen

NASA Astrophysics Data System (ADS)

Masic, Admir; Bertinetti, Luca; Schuetz, Roman; Chang, Shu-Wei; Metzger, Till Hartmut; Buehler, Markus J.; Fratzl, Peter

2015-01-01

Water is an important component of collagen in tendons, but its role for the function of this load-carrying protein structure is poorly understood. Here we use a combination of multi-scale experimentation and computation to show that water is an integral part of the collagen molecule, which changes conformation upon water removal. The consequence is a shortening of the molecule that translates into tensile stresses in the range of several to almost 100 MPa, largely surpassing those of about 0.3 MPa generated by contractile muscles. Although a complete drying of collagen would be relevant for technical applications, such as the fabrication of leather or parchment, stresses comparable to muscle contraction already occur at small osmotic pressures common in biological environments. We suggest, therefore, that water-generated tensile stresses may play a role in living collagen-based materials such as tendon or bone.

Decellularized heart ECM hydrogel using supercritical carbon dioxide for improved angiogenesis.

PubMed

Seo, Yoojin; Jung, Youngmee; Kim, Soo Hyun

2018-02-01

Initial angiogenesis within the first 3 days is critical for healing ischemic diseases such as myocardial infarction. Recently, decellularized extracellular matrix (dECM) has been reported to provide tissue-derived ECM components and can be used as a scaffold for cell delivery for angiogenesis in tissue engineering. Decellularization by various detergents such as sodium dodecyl sulfate (SDS) and triton X-100 can remove the cell nuclei in tissue organs. However, this leads to ECM structure denaturation, decreased presence of various ECM proteins and cytokines, and loss of mechanical properties. To overcome these limitations, in this study, we developed a supercritical carbon dioxide and ethanol co-solvent (scCO 2 -EtOH) decellularization method, which is a detergent-free system that prevents ECM structure disruption and retains various angiogenic proteins in the heart dECM, and tested on rat heart tissues. The heart tissue was placed into the scCO 2 reactor and decellularized at 37 °C and 350 bar. After scCO 2 -EtOH treatment, the effects were evaluated by DNA, collagen, and glycosaminoglycan (GAG) quantification and hematoxylin and eosin and immunofluorescence staining to determine the absence of nucleic acids and preservation of heart ECM components. Similar to the native group, the scCO 2 -EtOH group contained more ECM components such as collagen, GAGs, collagen I, laminin, and fibronectin and angiogenic factors including vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor and others in comparison to the detergent group. In addition, to estimate angiogenesis of the dECM hydrogels, the neutralized dECM solution was injected in a rat subcutaneous layer (n = 6 in each group: collagen, scCO 2 -EOH, and detergent group), after which the solution naturally formed gelation in the subcutaneous layer. After 3 days, the gels were harvested and estimated by immunofluorescence staining and the ImageJ program for angiogenesis analysis. Consequently, blood vessel formation and density of vWF and α-SMA in the scCO 2 -EtOH group were significantly greater than that in the collagen group. Here we suggest that heart-derived decellularized extracellular matrix (dECM) with scCO 2 -EtOH treatment is a highly promising angiogenic material for healing in ischemic disease. Supercritical carbon dioxide (scCO 2 ) in a supercritical phase has low viscosity and high diffusivity between gas and liquid properties and is known to be affordable, non-toxic, and eco-friendly. Therefore, scCO 2 extraction technology has been extensively used in commercial and industrial fields. Recently, decellularized extracellular matrix (dECM) was applied to tissue engineering and regenerative medicine as a scaffold, therapeutic material, and bio-ink for 3D printing. Moreover, the general decellularization method using detergents has limitations including eliminating tissue-derived ECM components and disrupting their structures after decellularization. To overcome these limitations, heart tissues were treated with scCO 2 -EtOH for decellularization, resulting in preserving of tissue due to the various ECM and angiogenic factors derived. In addition, initiation of angiogenesis was highly induced even after 3 days of injection. Copyright © 2017. Published by Elsevier Ltd.

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yano, Hiroyuki; Division of Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593; Hamanaka, Ryoji

Highlights: Black-Right-Pointing-Pointer We examine how radiation affects the expression level and signal pathway of collagen. Black-Right-Pointing-Pointer TGF-{beta}1 mRNA is elevated earlier than those of collagen genes after irradiation. Black-Right-Pointing-Pointer Smad pathway mediates the expression of collagen in radiation induced fibrosis. Black-Right-Pointing-Pointer MAPK pathways are not affected in the expression of collagen after irradiation. -- Abstract: Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Realmore » time RT-RCR showed that both {alpha}1and {alpha}2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-{beta}1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-{beta} receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of {alpha}2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.« less

Laminin and collagen modulate expression of the small leucine-rich proteoglycan fibromodulin in rat anterior pituitary gland.

PubMed

Syaidah, Rahimi; Horiguchi, Kotaro; Fujiwara, Ken; Tsukada, Takehiro; Kikuchi, Motoshi; Yashiro, Takashi

2013-11-01

The anterior pituitary is a complex organ consisting of five types of hormone-producing cells, non–hormone-producing cells such as folliculostellate (FS) cells and vascular cells (endothelial cells and pericytes). We have previously shown that FS cells and pericytes produce fibromodulin, a small leucine-rich proteoglycan (SLRP). SLRPs are major proteoglycans of the extracellular matrix (ECM) and are important in regulating cell signaling pathways and ECM assembly. However, the mechanism regulating fibromodulin expression in the anterior pituitary has not been elucidated. Here, we investigate whether fibromodulin expression is modulated by major anterior pituitary ECM components such as laminin and type I collagen. Using transgenic rats expressing green fluorescent protein (GFP) specifically in FS cells, we examine fibromodulin expression in GFP-positive (FS cells) and GFP-negative cells (e.g., pericytes, endocrine cells and endothelial cells). Immunostaining and Western blot analysis were used to assess protein expression in the presence and absence of laminin or type I collagen. We confirmed fibromodulin expression in the pituitary and observed the up-regulation of fibromodulin in FS cells in the presence of ECM components. However, neither laminin nor type I collagen affected expression in GFP-negative cells. This suggests that laminin and type I collagen support the function of FS cells by increasing fibromodulin protein expression in the anterior pituitary.

Bi-layered collagen nano-structured membrane prototype (collagen matrix 10826(®)) for oral soft tissue regeneration: an "in vitro" study.

PubMed

Nocini, Pier Francesco; Zanotti, Guglielmo; Castellani, Roberto; Grasso, Silvia; Cristofaro, Maria Giulia; De Santis, Daniele

2013-06-01

To evaluate fundamental cell functions, such as adhesion, IL-6 production and proliferation of human gingival keratinocytes cultured on a newly engineered collagen matrix (CM-10826) and to assess the degree of specific biocompatibility of this new device. Primary cultures of human keratinocytes were derived "in vitro" from biopsies of independent donors. Their true epithelial origin was ensured by the expression of cytokeratin 14. Adhesion, proliferation and production of IL-6 cytokine was then measured in the presence or absence of CM-10826 activity or of its relevant components. Functional tests revealed that keratinocytes adhered to CM-10826 and up-regulated their basal IL-6 production. The type of keratinocytes used expressed cytokeratin 14. Proliferation experiments demonstrated that the best cellular response was observed in the presence of Collagen I, the main component of CM-10826. No undesired effects were observed as for keratinocyte viability, morphology or differentiation. Our results demonstrate that CM-10826 has a favourable biological effect on the "in vitro" response of gingival keratinocytes in terms of IL-6 production, cell growth and adhesion. These findings may encourage a possible use of this collagen membrane as a tissue which, alone, may substitute for autologous gingival grafts thereby overcoming the limitations of autologous tissue. © 2012 John Wiley & Sons A/S.

Biomimetic component coating on 3D scaffolds using high bioactivity of mesoporous bioactive ceramics

PubMed Central

Yun, Hui-suk; Kim, Sang-Hyun; Khang, Dongwoo; Choi, Jungil; Kim, Hui-hoon; Kang, Minji

2011-01-01

Background Mesoporous bioactive glasses (MBGs) are very attractive materials for use in bone tissue regeneration because of their extraordinarily high bone-forming bioactivity in vitro. That is, MBGs may induce the rapid formation of hydroxy apatite (HA) in simulated body fluid (SBF), which is a major inorganic component of bone extracellular matrix (ECM) and comes with both good osteoconductivity and high affinity to adsorb proteins. Meanwhile, the high bioactivity of MBGs may lead to an abrupt initial local pH variation during the initial Ca ion-leaching from MBGs at the initial transplant stage, which may induce unexpected negative effects on using them in in vivo application. In this study we suggest a new way of using MBGs in bone tissue regeneration that can improve the strength and make up for the weakness of MBGs. We applied the outstanding bone-forming bioactivity of MBG to coat the main ECM components HA and collagen on the MBG-polycarplolactone (PCL) composite scaffolds for improving their function as bone scaffolds in tissue regeneration. This precoating process can also expect to reduce initial local pH variation of MBGs. Methods and materials The MBG-PCL scaffolds were immersed in the mixed solution of the collagen and SBF at 37°C for 24 hours. The coating of ECM components on the MBG-PCL scaffolds and the effect of ECM coating on in vitro cell behaviors were confirmed. Results The ECM components were fully coated on MBG-PCL scaffolds after immersing in SBF containing dilute collagen-I solution only for 24 hours due to the high bone-forming bioactivity of MBG. Both cell affinity and osteoconductivity of MBG-PCL scaffolds were dramatically enhanced by this precoating process. Conclusion The precoating process of ECM components on MBG-PCL scaffold using a high bioactivity of MBG was not only effective in enhancing the functionality of scaffolds but also effective in eliminating the unexpected side effect. The MBG-PCL scaffold-coated ECM components ideally satisfied the required conditions of scaffold in tissue engineering, including 3D well-interconnected pore structures with high porosity, good bioactivity, enhanced cell affinity, biocompatibility, osteoconductivity, and sufficient mechanical properties, and promise excellent potential application in the field of biomaterials. PMID:22072886

Chum salmon egg extracts induce upregulation of collagen type I and exert antioxidative effects on human dermal fibroblast cultures

PubMed Central

Yoshino, Atsushi; Polouliakh, Natalia; Meguro, Akira; Takeuchi, Masaki; Kawagoe, Tatsukata; Mizuki, Nobuhisa

2016-01-01

Components of fish roe possess antioxidant and antiaging activities, making them potentially very beneficial natural resources. Here, we investigated chum salmon eggs (CSEs) as a source of active ingredients, including vitamins, unsaturated fatty acids, and proteins. We incubated human dermal fibroblast cultures for 48 hours with high and low concentrations of CSE extracts and analyzed changes in gene expression. Cells treated with CSE extract showed concentration-dependent upregulation of collagen type I genes and of multiple antioxidative genes, including OXR1, TXNRD1, and PRDX family genes. We further conducted in silico phylogenetic footprinting analysis of promoter regions. These results suggested that transcription factors such as acute myeloid leukemia-1a and cyclic adenosine monophosphate response element-binding protein may be involved in the observed upregulation of antioxidative genes. Our results support the idea that CSEs are strong candidate sources of antioxidant materials and cosmeceutically effective ingredients. PMID:27621603

Assessment of hyaline cartilage matrix composition using near infrared spectroscopy.

PubMed

Palukuru, Uday P; McGoverin, Cushla M; Pleshko, Nancy

2014-09-01

Changes in the composition of the extracellular matrix (ECM) are characteristic of injury or disease in cartilage tissue. Various imaging modalities and biochemical techniques have been used to assess the changes in cartilage tissue but lack adequate sensitivity, or in the case of biochemical techniques, result in destruction of the sample. Fourier transform near infrared (FT-NIR) spectroscopy has shown promise for the study of cartilage composition. In the current study NIR spectroscopy was used to identify the contributions of individual components of cartilage in the NIR spectra by assessment of the major cartilage components, collagen and chondroitin sulfate, in pure component mixtures. The NIR spectra were obtained using homogenous pellets made by dilution with potassium bromide. A partial least squares (PLS) model was calculated to predict composition in bovine cartilage samples. Characteristic absorbance peaks between 4000 and 5000 cm(-1) could be attributed to components of cartilage, i.e. collagen and chondroitin sulfate. Prediction of the amount of collagen and chondroitin sulfate in tissues was possible within 8% (w/dw) of values obtained by gold standard biochemical assessment. These results support the use of NIR spectroscopy for in vitro and in vivo applications to assess matrix composition of cartilage tissues, especially when tissue destruction should be avoided. Copyright © 2014. Published by Elsevier B.V.

A Structural Finite Element Model for Lamellar Unit of Aortic Media Indicates Heterogeneous Stress Field After Collagen Recruitment

PubMed Central

Thunes, James R.; Pal, Siladitya; Fortunato, Ronald N.; Phillippi, Julie A.; Gleason, Thomas G.; Vorp, David A.; Maiti, Spandan

2016-01-01

Incorporation of collagen structural information into the study of biomechanical behavior of ascending thoracic aortic (ATA) wall tissue should provide better insight into the pathophysiology of ATA. Structurally motivated constitutive models that include fiber dispersion and recruitment can successfully capture overall mechanical response of the arterial wall tissue. However, these models cannot examine local microarchitectural features of the collagen network, such as the effect of fiber disruptions and interaction between fibrous and non-fibrous components, which may influence emergent biomechanical properties of the tissue. Motivated by this need, we developed a finite element based three-dimensional structural model of the lamellar units of the ATA media that directly incorporates the collagen fiber microarchitecture. The fiber architecture was computer generated utilizing network features, namely fiber orientation distribution, intersection density and areal concentration, obtained from image analysis of multiphoton microscopy images taken from human aneurysmal ascending thoracic aortic media specimens with bicuspid aortic valve (BAV) phenotype. Our model reproduces the typical J-shaped constitutive response of the aortic wall tissue. We found that the stress state in the non-fibrous matrix was homogeneous until the collagen fibers were recruited, but became highly heterogeneous after that event. The degree of heterogeneity was dependent upon local network architecture with high stresses observed near disrupted fibers. The magnitude of non-fibrous matrix stress at higher stretch levels was negatively correlated with local fiber density. The localized stress concentrations, elucidated by this model, may be a factor in the degenerative changes in aneurysmal ATA tissue. PMID:27113538

Poisson's ratio of collagen fibrils measured by small angle X-ray scattering of strained bovine pericardium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wells, Hannah C.; Sizeland, Katie H.; Kayed, Hanan R.

Type I collagen is the main structural component of skin, tendons, and skin products, such as leather. Understanding the mechanical performance of collagen fibrils is important for understanding the mechanical performance of the tissues that they make up, while the mechanical properties of bulk tissue are well characterized, less is known about the mechanical behavior of individual collagen fibrils. In this study, bovine pericardium is subjected to strain while small angle X-ray scattering (SAXS) patterns are recorded using synchrotron radiation. The change in d-spacing, which is a measure of fibril extension, and the change in fibril diameter are determined frommore » SAXS. The tissue is strained 0.25 (25%) with a corresponding strain in the collagen fibrils of 0.045 observed. The ratio of collagen fibril width contraction to length extension, or the Poisson's ratio, is 2.1 ± 0.7 for a tissue strain from 0 to 0.25. This Poisson's ratio indicates that the volume of individual collagen fibrils decreases with increasing strain, which is quite unlike most engineering materials. This high Poisson's ratio of individual fibrils may contribute to high Poisson's ratio observed for tissues, contributing to some of the remarkable properties of collagen-based materials.« less

Tomographic imaging of bone composition using coherently scattered x rays

NASA Astrophysics Data System (ADS)

Batchelar, Deidre L.; Dabrowski, W.; Cunningham, Ian A.

2000-04-01

Bone tissue consists primarily of calcium hydroxyapatite crystals (bone mineral) and collagen fibrils. Bone mineral density (BMD) is commonly used as an indicator of bone health. Techniques available at present for assessing bone health provide a measure of BMD, but do not provide information about the degree of mineralization of the bone tissue. This may be adequate for assessing diseases in which the collagen-mineral ratio remains constant, as assumed in osteoporosis, but is insufficient when the mineralization state is known to change, as in osteomalacia. No tool exists for the in situ examination of collagen and hydroxyapatite density distributions independently. Coherent-scatter computed tomography (CSCT) is a technique we are developing that produces images of the low- angle scatter properties of tissue. These depend on the molecular structure of the scatterer making it possible to produce material-specific maps of each component in a conglomerate. After corrections to compensate for exposure fluctuations, self-attenuation of scatter and the temporal response of the image intensifier, material-specific images of mineral, collagen, fat and water distributions are obtained. The gray-level in these images provides the volumetric density of each component independently.

Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perumal, Shiamalee; Antipova, Olga; Orgel, Joseph P.R.O.

We describe the molecular structure of the collagen fibril and how it affects collagen proteolysis or 'collagenolysis.' The fibril-forming collagens are major components of all mammalian connective tissues, providing the structural and organizational framework for skin, blood vessels, bone, tendon, and other tissues. The triple helix of the collagen molecule is resistant to most proteinases, and the matrix metalloproteinases that do proteolyze collagen are affected by the architecture of collagen fibrils, which are notably more resistant to collagenolysis than lone collagen monomers. Until now, there has been no molecular explanation for this. Full or limited proteolysis of the collagen fibrilmore » is known to be a key process in normal growth, development, repair, and cell differentiation, and in cancerous tumor progression and heart disease. Peptide fragments generated by collagenolysis, and the conformation of exposed sites on the fibril as a result of limited proteolysis, regulate these processes and that of cellular attachment, but it is not known how or why. Using computational and molecular visualization methods, we found that the arrangement of collagen monomers in the fibril (its architecture) protects areas vulnerable to collagenolysis and strictly governs the process. This in turn affects the accessibility of a cell interaction site located near the cleavage region. Our observations suggest that the C-terminal telopeptide must be proteolyzed before collagenase can gain access to the cleavage site. Collagenase then binds to the substrate's 'interaction domain,' which facilitates the triple-helix unwinding/dissociation function of the enzyme before collagenolysis.« less

Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis.

PubMed

Perumal, Shiamalee; Antipova, Olga; Orgel, Joseph P R O

2008-02-26

We describe the molecular structure of the collagen fibril and how it affects collagen proteolysis or "collagenolysis." The fibril-forming collagens are major components of all mammalian connective tissues, providing the structural and organizational framework for skin, blood vessels, bone, tendon, and other tissues. The triple helix of the collagen molecule is resistant to most proteinases, and the matrix metalloproteinases that do proteolyze collagen are affected by the architecture of collagen fibrils, which are notably more resistant to collagenolysis than lone collagen monomers. Until now, there has been no molecular explanation for this. Full or limited proteolysis of the collagen fibril is known to be a key process in normal growth, development, repair, and cell differentiation, and in cancerous tumor progression and heart disease. Peptide fragments generated by collagenolysis, and the conformation of exposed sites on the fibril as a result of limited proteolysis, regulate these processes and that of cellular attachment, but it is not known how or why. Using computational and molecular visualization methods, we found that the arrangement of collagen monomers in the fibril (its architecture) protects areas vulnerable to collagenolysis and strictly governs the process. This in turn affects the accessibility of a cell interaction site located near the cleavage region. Our observations suggest that the C-terminal telopeptide must be proteolyzed before collagenase can gain access to the cleavage site. Collagenase then binds to the substrate's "interaction domain," which facilitates the triple-helix unwinding/dissociation function of the enzyme before collagenolysis.

Novel bio-synthetic hybrid materials and coculture systems for musculoskeletal tissue engineering

NASA Astrophysics Data System (ADS)

Lee, Hyeseung Janice

Tissue Engineering is a truly exciting field of this age, trying to regenerate and repair impaired tissues. Unlike the old artificial implants, tissue engineering aims at making a long-term functional biological replacement. One strategy for such tissue engineering requires the following three components: cells, scaffolds, and soluble factors. Cells are cultured in a three-dimensional (3D) scaffold with medium containing various soluble factors. Once a tissue is developed in vitro, then it is implanted in vivo. The overall goal of this thesis was to develop novel bio-synthetic hybrid scaffolds and coculture system for musculoskeletal tissue engineering. The most abundant cartilage extracellular matrix (ECM) components are collagen and glycosaminoglycan (GAG), which are the natural scaffold for chondrocytes. As two different peptides, collagen mimetic peptide (CMP) and hyaluronic acid binding peptide (HABPep) were previously shown to bind to collagen and hyaluronic acid (HA) of GAG, respectively, it was hypothesized that immobilizing CMP and HABP on 3D scaffold would results in an interaction between ECM components and synthetic scaffolds via peptide-ECM bindings. CMP or HABPep-conjugated photopolymerizable poly(ethylene oxide) diacrylate (PEODA) hydrogels were synthesized and shown to retain encapsulated collagen or HA, respectively. This result supported that conjugated CMP and HABPep can interact with collagen and HA, respectively, and can serve as biological linkers in 3D synthetic hydrogels. When chondrocytes or mesenchymal stem cells (MSCs) were seeded, cells in CMP-conjugated scaffolds produced significantly more amount of type II collagen and GAG, compared to those in control scaffolds. Moreover, MSCs cultured in CMP-conjugated scaffolds exhibited lower level of hypertrophic markers, cbfa-1 and type X collagen. These results demonstrated that enhanced interaction between collagen and scaffold via CMP improves chondrogenesis of chondrocytes and MSCs and further reduces hypertrophy of differentiating MSCs. On the other hand, although cells in HABPep-conjugated scaffolds produced less ECM components, they survived and proliferated significantly more than those in control, resulting in overall increase in ECM contents per scaffold. Once implanted in vivo, HABPep-conjugated constructs increased GAG and type II collagen contents further, compared to those of the control hydrogel. These results showed that enhanced interaction between HA and scaffold via HABPep improved the in vitro culture expansion of MSCs and further ECM production in vivo. Effects of cell-secreted bioactive factors via cell-cell communication on stem cell differentiation were also investigated in 3D bilayer system. First, when mesenchymal progenitor cells (MPCs) were cocultured with ES-derived cells (ESDC), morphogenetic factors secreted by ESDCs showed a potential to improve MPC chondrogenesis in both control and chondrogenic medium by increasing not only MPC's chondrogenic gene expression, but also ECM production. Moreover, the effect of ESDC cell-mediated chondrogenesis of MSC could not be mimicked by chondrogenic medium supplemented with TGF-beta1 and dexamethasone. Secondly, coculturing hepatic cells enhanced specific chondrogenic differentiation of ES cells in the 3D bilayer system. These studies demonstrated that cell-secreted soluble factors can be used to guide stem cell differentiation.

Nutritional composition and total collagen content of three commercially important edible jellyfish.

PubMed

Khong, Nicholas M H; Yusoff, Fatimah Md; Jamilah, B; Basri, Mahiran; Maznah, I; Chan, Kim Wei; Nishikawa, Jun

2016-04-01

The study aimed to evaluate nutraceutical potential of three commercially significant edible jellyfish species (Acromitus hardenbergi, Rhopilema hispidum and Rhopilema esculentum). The bell and oral arms of these jellyfishes were analyzed for their proximate composition, calorific value, collagen content, amino acid profile, chemical score and elemental constituent. In general, all jellyfish possessed low calorific values (1.0-4.9 kcal/g D.W.) and negligible fat contents (0.4-1.8 g/100 g D.W.), while protein (20.0-53.9 g/100 g D.W.) and minerals (15.9-57.2g/100g D.W.) were found to be the richest components. Total collagen content of edible jellyfish varied from 122.64 to 693.92 mg/g D.W., accounting for approximately half its total protein content. The dominant amino acids in both bell and oral arms of all jellyfish studied includes glycine, glutamate, threonine, proline, aspartate and arginine, while the major elements were sodium, potassium, chlorine, magnesium, sulfur, zinc and silicon. Among the jellyfish, A. hardenbergi exhibited significantly higher total amino acids, chemical scores and collagen content (p<0.05) compared to R. hispidum and R. esculentum. Having good protein quality and low calories, edible jellyfish is an appealing source of nutritive ingredients for the development of oral formulations, nutricosmetics and functional food. Copyright © 2015 Elsevier Ltd. All rights reserved.

Type VII Collagen is Enriched in the Enamel Organic Matrix Associated with the Dentin-Enamel Junction of Mature Human Teeth

PubMed Central

McGuire, Jacob D.; Walker, Mary P.; Mousa, Ahmad; Wang, Yong; Gorski, Jeff P.

2014-01-01

The inner enamel region of erupted teeth is known to exhibit higher fracture toughness and crack growth resistance than bulk phase enamel. However, an explanation for this behavior has been hampered by the lack of compositional information for the residual enamel organic matrix. Since enamel-forming ameloblasts are known to express type VII collagen and type VII collagen null mice display abnormal amelogenesis, the aim of this study was to determine whether type VII collagen is a component of the enamel organic matrix at the dentin-enamel junction (DEJ) of mature human teeth. Immunofluorescent confocal microscopy of demineralized tooth sections localized type VII collagen to the organic matrix surrounding individual enamel rods near the DEJ. Morphologically, immunoreactive type VII collagen helical-bundles resembled the gnarled-pattern of enamel rods detected by Coomassie Blue staining. Western blotting of whole crown or enamel matrix extracts also identified characteristic Mr=280 and 230 kDa type VII dimeric forms, which resolved into 75 and 25 kDa bands upon reduction. As expected, the collagenous domain of type VII collagen was resistant to pepsin digestion, but was susceptible to purified bacterial collagenase. These results demonstrate the inner enamel organic matrix in mature teeth contains macromolecular type VII collagen. Based on its physical association with the DEJ and its well-appreciated capacity to complex with other collagens, we hypothesize that enamel embedded type VII collagen fibrils may contribute not only to the structural resilience of enamel, but may also play a role in bonding enamel to dentin. PMID:24594343

Collagen in organ development

NASA Technical Reports Server (NTRS)

Hardman, P.; Spooner, B. S.

1992-01-01

It is important to know whether microgravity will adversely affect developmental processes. Collagens are macromolecular structural components of the extracellular matrix (ECM) which may be altered by perturbations in gravity. Interstitial collagens have been shown to be necessary for normal growth and morphogenesis in some embryonic organs, and in the mouse salivary gland, the biosynthetic pattern of these molecules changes during development. Determination of the effects of microgravity on epithelial organ development must be preceded by crucial ground-based studies. These will define control of normal synthesis, secretion, and deposition of ECM macromolecules and the relationship of these processes to morphogenesis.

[Selective removal of tannins from Polygonum cuspidatum extracts using collagen fiber adsorbent].

PubMed

Li, Juan; Liao, Xuepin; Shu, Xingxu; Shi, Bi

2010-03-01

To investigate the selective removal of tannins from Polygonum cuspidatum extracts by using collagen fiber adsorbent, and to evaluate the adsorption and desorption performances of collagen fiber adsorbent to tannins. The adsorbent was prepared from bovine skin collagen fiber through crosslinking reaction of glutaraldehyde, and then used for the selective removal of tannins from P. cuspidatum extracts. Gelatin-turbidity method, gelatin-ultraviolet spectrometry method and HPLC were used for detection of tannins in the solutions. Ethanol-water solutions with varying concentration were used to test their desorption ability of tannins in order to choose proper desorption solution. On the basis of batch experimental results, the column adsorption and desorption tests were carried out, by using gelatin-turbidity method for detection of tannins. The collagen fiber adsorbent exhibited excellent adsorption selectivity to tannins. It was found that tannins of P. cuspidatum were completely removed, while nearly no adsorption of active components (resveratrol as representative) was found. Moreover, the collagen fiber adsorbent could be regenerated by using 30% ethanol-water solution and then reused. The collagen fiber adsorbent can be considered as a promising material for selective removal of tannins from P. cuspidatum extracts.

Aspartic Acid Racemization and Collagen Degradation Markers Reveal an Accumulation of Damage in Tendon Collagen That Is Enhanced with Aging*

PubMed Central

Thorpe, Chavaunne T.; Streeter, Ian; Pinchbeck, Gina L.; Goodship, Allen E.; Clegg, Peter D.; Birch, Helen L.

2010-01-01

Little is known about the rate at which protein turnover occurs in living tendon and whether the rate differs between tendons with different physiological roles. In this study, we have quantified the racemization of aspartic acid to calculate the age of the collagenous and non-collagenous components of the high strain injury-prone superficial digital flexor tendon (SDFT) and low strain rarely injured common digital extensor tendon (CDET) in a group of horses with a wide age range. In addition, the turnover of collagen was assessed indirectly by measuring the levels of collagen degradation markers (collagenase-generated neoepitope and cross-linked telopeptide of type I collagen). The fractional increase in d-Asp was similar (p = 0.7) in the SDFT (5.87 × 10−4/year) and CDET (5.82 × 10−4/year) tissue, and d/l-Asp ratios showed a good correlation with pentosidine levels. We calculated a mean (±S.E.) collagen half-life of 197.53 (±18.23) years for the SDFT, which increased significantly with horse age (p = 0.03) and was significantly (p < 0.001) higher than that for the CDET (34.03 (±3.39) years). Using similar calculations, the half-life of non-collagenous protein was 2.18 (±0.41) years in the SDFT and was significantly (p = 0.04) lower than the value of 3.51 (±0.51) years for the CDET. Collagen degradation markers were higher in the CDET and suggested an accumulation of partially degraded collagen within the matrix with aging in the SDFT. We propose that increased susceptibility to injury in older individuals results from an inability to remove partially degraded collagen from the matrix leading to reduced mechanical competence. PMID:20308077

Preferential Enhancement of Sensory and Motor Axon Regeneration by Combining Extracellular Matrix Components with Neurotrophic Factors

PubMed Central

Santos, Daniel; González-Pérez, Francisco; Giudetti, Guido; Micera, Silvestro; Udina, Esther; Del Valle, Jaume; Navarro, Xavier

2016-01-01

After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM) components and neurotrophic factors (NTFs) exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF)/neurotrophin-3 (NT3) or collagen, fibronectin and brain-derived neurotrophic factor (BDNF) selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (MP) (LM + MP.NGF/NT3), or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF). Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervation. PMID:28036084

[Water-exchange processes in hyaline cartilage and its basic components in a normal state and in osteoarthritis].

PubMed

Nikolaeva, S S; Chkhol, K Z; Bykov, V A; Roshchina, A A; Iakovleva, L V; Koroleva, O A; Omel'ianenko, N P; Rebrov, L B

2000-01-01

The content of different forms of tissue water was studied in the normal articular cartilage and osteoarthrosis cartilage and its structural components: collagen, potassium hyaluronate, sodium chondroitinsulphate and its complexes. In the components of cartilage matrix a few of fractions of bound water different in the strength of binding are present. At the maximal humidity, all water in collagen binds with the active groups of biopolymers and in the glycosaminoglycans, in addition to bound water, are present, two crystal forms of freezing water (free water) at least. The quantity of free water in the collagen-chondroitin sulphat membrane, is increased with the increase of chondroitin sulphate. In the collagen-hyaluronate complex, fraction of free water is found only at the low concentration of hyaluronate kalium. It was shown that in the hyalin cartilage, in different from the other connective tissue (skin, achilles tendon), the most part of water is free water and its quantity is increased in the osteoarthrosis. It is supposed that the rearrangement of binding and free-water fractions in the osteoarthrosis is the result of deficiency of hyaluronic acid and therefore this may be regarded in the improvement of methods of treatment. This scientific and methodical approach allow to receive information on the forms and binding energy of water in the biological tissues, which is absorbed from fluids and steam phase and determine characters of the pathological changes.

Distinct functions of the laminin β LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila

PubMed Central

2014-01-01

Background The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles. Results Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos we obtained multiple mutants for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin β and collagen IV α1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV α2 chain is detected during late embryogenesis. Conclusions Assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. Our data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the β6/β3/β8 interface of the laminin β LN domain is highly critical for formation of contiguous cardiac ECM layers. Certain mutations in the collagen IV triple helix-forming domain may exert a semi-dominant effect leading to an overall weakening of ECM structures as well as intracellular accumulation of collagen and other molecules, thus paralleling observations made in other organisms and in connection with collagen-related diseases. PMID:24935095

Ultrastructure of the bovine nuchal ligament.

PubMed Central

Morocutti, M; Raspanti, M; Ottani, V; Govoni, P; Ruggeri, A

1991-01-01

Nuchal ligament is composed almost exclusively of elastic fibres and collagen fibrils, interwoven very closely and lying parallel to the main ligament axis. Elastic fibres are very large, straight and roughly cylindrical; the collagenous matrix consists of septa of diminishing size forming a 3-dimensional matrix that envelops fibre bundles as well as individual elastic fibres. In all areas examined, collagen fibrils are of very uniform size and, on replicas, they reveal a spiral subfibrillar arrangement with an inclination angle of 17 degrees. Collagen fibrils appear to adhere to the elastic fibres very closely, conforming to their irregular shape. Sometimes they impinge directly upon the elastic fibres, while in other cases a space is visible between collagen fibrils and elastic fibres that contains a rich fabric of intermediate filaments. The collagen-elastin complex of the ligamentum nuchae may be considered a fibre-reinforced composite material comprising tough fibres immersed in an amorphous elastic matrix. Its mechanical behaviour is the result of the combined properties of its components and their interactions. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:1810923

Treponema denticola Outer Membrane Enhances the Phagocytosis of Collagen-Coated Beads by Gingival Fibroblasts

PubMed Central

Battikhi, Tulin; Lee, Wilson; McCulloch, Christopher A. G.; Ellen, Richard P.

1999-01-01

Human gingival fibroblasts (HGFs) degrade collagen fibrils in physiological processes by phagocytosis. Since Treponema denticola outer membrane (OM) extract perturbs actin filaments, important structures in phagocytosis, we determined whether the OM affects collagen phagocytosis in vitro by HGFs. Phagocytosis was measured by flow cytometric assessment of internalized collagen-coated fluorescent latex beads. Confluent HGFs pretreated with T. denticola ATCC 35405 OM exhibited an increase in the percentage of collagen phagocytic cells (phagocytosis index [PI]) and in the number of beads per phagocytosing cell (phagocytic capacity [PC]) compared with untreated controls. The enhancement was swift (within 15 min) and was still evident after 1 day. PI and PC of HGFs for bovine serum albumin (BSA)-coated beads were also increased, indicating a global increase in phagocytic processes. These results contrasted those for control OM from Veillonella atypica ATCC 17744, which decreased phagocytosis. The T. denticola OM-induced increase in bead uptake was eliminated by heating the OM and by depolymerization of actin filaments by cytochalasin D treatment of HGFs. Fluid-phase accumulation of lucifer yellow was enhanced in a saturable, concentration-dependent, transient manner by the T. denticola OM. Our findings were not due to HGF detachment or cytotoxicity in response to the T. denticola OM treatment since the HGFs exhibited minimal detachment from the substratum; they did not take up propidium iodide; and there was no change in their size, granularity, or content of sub-G1 DNA. We conclude that a heat-sensitive component(s) in T. denticola OM extract stimulates collagen phagocytosis and other endocytic processes such as nonspecific phagocytosis and pinocytosis by HGFs. PMID:10024564

A clinicopathological study of the expression of extracellular matrix components in urothelial carcinoma.

PubMed

Ioachim, Elli; Michael, Michalis; Stavropoulos, Nicolaos E; Kitsiou, Evangelia; Salmas, Marios; Malamou-Mitsi, Vasiliki

2005-03-01

To measure the immunohistochemical expression of the extracellular matrix (ECM) components tenascin, fibronectin, collagen type IV and laminin in urothelial carcinomas, and to correlate their expression with clinicopathological features to clarify the prognostic value of these molecules and their role in tumour progression. Tumour specimens obtained during transurethral resection of bladder tumour (TURBT) from 103 patients (82 men and 2 1 women, mean age 66.7 years, range 27-89) were studied retrospectively. The expression of tenascin, fibronectin, collagen type IV and laminin was correlated with clinicopathological features (tumour grade and stage, multiplicity, simultaneous in situ component, the proliferative activity as estimated by the two proliferation associated indices, Ki-67 and proliferating cell nuclear antigen, the recurrence rate, and the progression of invading tumour). Specimens investigated for tenascin expression from patients with superficial bladder cancers were categorized into 28 treated by TURBT only and 53 who had TURBT followed by intravesical instillations of interferon. Cytoplasmic tenascin expression was detected in tumour cells in 20% of specimens. Tenascin was expressed in the tumour stroma in 76% of specimens, and was positively correlated with tumour grade and stage. Stromal tenascin expression was positively correlated with proliferative activity, and with the expression of fibronectin and collagen type IV. Fibronectin was expressed in the tumour stroma in 89% of specimens and was positively correlated with tumour stage, proliferative activity, and expression of collagen type IV and laminin. Collagen type IV was expressed in 93% of specimens, and was positively correlated with tumour grade and stage. Laminin was expressed in 78% of specimens and had no significant correlation with the clinicopathological features. Patients treated with TURBT alone and who had low levels of tenascin had a longer tumour-free interval than those with high levels of tenascin. Levels of tenascin might be valuable for predicting the risk of early recurrence. The expression of tenascin, fibronectin and collagen type IV seems to be correlated with more aggressive tumour behaviour. Furthermore, their interrelationships could indicate that they are involved in the remodelling of bladder cancer tissue, probably influencing tumour progression.

Diffusion of MMPs on the Surface of Collagen Fibrils: The Mobile Cell Surface – Collagen Substratum Interface

PubMed Central

Collier, Ivan E.; Legant, Wesley; Marmer, Barry; Lubman, Olga; Saffarian, Saveez; Wakatsuki, Tetsuro; Elson, Elliot; Goldberg, Gregory I.

2011-01-01

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions. PMID:21912660

Different fibroblast subpopulations of the eye: a therapeutic target to prevent postoperative fibrosis in glaucoma therapy.

PubMed

Stahnke, Thomas; Löbler, Marian; Kastner, Christian; Stachs, Oliver; Wree, Andreas; Sternberg, Katrin; Schmitz, Klaus-Peter; Guthoff, Rudolf

2012-07-01

The aim of this study is the characterization of fibroblasts mainly responsible for fibrosis processes associated with trabeculectomy or microstent implantation for glaucoma therapy. Therefore we isolated human primary fibroblasts from choroidea, sclera, Tenon capsule, and orbital fat tissues. These fibroblast subpopulations were analysed in vitro for expression of the extracellular matrix components which are responsible for postoperative scarring in glaucoma therapy. For scarring the proteins of the collagen family are predominant and so we focused on the expression of collagen I, collagen III and collagen VI in every fibroblast subpopulation. Also, the extracellular matrix protein fibronectin which crosslinks collagen fibres or other extracellular matrix components and cell surfaces, was analyzed. Collagen I, III and VI were prominent in every fibroblast subpopulation. The highest amounts of collagen III were found in hCF and hOF, whereas the signal in hSF and hTF was negligible. Additionally, there is a link between scarring processes and proliferating potential of fibroblasts, in case of microstent implantation triggered through the infiltration of inflammatory cells. Thus we analyzed fibroblast subpopulations for the presence of TGF-β1 which is one of the most important cytokines involved in proliferation processes. TGF-β1 was prominent in all fibroblast subpopulations with lowest expression in hCF cultures. To prevent postoperative fibroblast proliferation we analyzed in vitro the proliferation-inhibitors paclitaxel and mitomycin C which are potential candidates in drug eluting drainage systems on ocular fibroblast subpopulations. These inhibitors arrest fibroblast proliferation and viability, being, however, not very specific and have a cytotoxic potential also on healthy tissues surrounding the microstent outflow area. Significant differences in protein synthesis of fibroblasts subpopulations which could be specific targets for inhibition may help to find out fibroblast specific inhibitors to prevent postoperative scarring and could prevent patients from secondary surgery after microstent implantation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Collagen: Biochemistry, biomechanics, biotechnology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nimni, M.E.

1988-01-01

This book is an up-to-date reference for new ideas, information, and concepts in collagen research. The first volume emphasizes the relationship between the molecular structure and function of collagen, including descriptions of collagen types which exist in tissues as well as how these molecules organize into fibrils and the nature of the chemical crosslinks which stabilize them. In Volume II the biomechanical behavior of various specialized tissues, abnormal accumulation of collagen in the form of scars of fibrous infiltration are examined/and wound healing, tissue regulation and repair are covered in detail. Volume III explores the increasing application of collagen technologymore » to the field of bioprosthesis, including the production of heart valve bioprosthesis, blood vessels, ligament substitutes, and bone substitutes.« less

Molecular Pathogenesis of Chlamydia Disease Complications: Epithelial-Mesenchymal Transition and Fibrosis.

PubMed

Igietseme, Joseph U; Omosun, Yusuf; Nagy, Tamas; Stuchlik, Olga; Reed, Matthew S; He, Qing; Partin, James; Joseph, Kahaliah; Ellerson, Debra; George, Zenas; Goldstein, Jason; Eko, Francis O; Bandea, Claudiu; Pohl, Jan; Black, Carolyn M

2018-01-01

The reproductive system complications of genital chlamydial infection include fallopian tube fibrosis and tubal factor infertility. However, the molecular pathogenesis of these complications remains poorly understood. The induction of pathogenic epithelial-mesenchymal transition (EMT) through microRNA (miRNA) dysregulation was recently proposed as the pathogenic basis of chlamydial complications. Focusing on fibrogenesis, we investigated the hypothesis that chlamydia-induced fibrosis is caused by EMT-driven generation of myofibroblasts, the effector cells of fibrosis that produce excessive extracellular matrix (ECM) proteins. The results revealed that the targets of a major category of altered miRNAs during chlamydial infection are key components of the pathophysiological process of fibrogenesis; these target molecules include collagen types I, III, and IV, transforming growth factor β (TGF-β), TGF-β receptor 1 (TGF-βR1), connective tissue growth factor (CTGF), E-cadherin, SRY-box 7 (SOX7), and NFAT (nuclear factor of activated T cells) kinase dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1a (Dyrk1a). Chlamydial induction of EMT resulted in the generation of α-smooth muscle actin (α-SMA)-positive myofibroblasts that produced ECM proteins, including collagen types I and III and fibronectin. Furthermore, the inhibition of EMT prevented the generation of myofibroblasts and production of ECM proteins during chlamydial infection. These findings may provide useful avenues for targeting EMT or specific components of the EMT pathways as a therapeutic intervention strategy to prevent chlamydia-related complications. Copyright © 2017 American Society for Microbiology.

Extracellular matrix components expression in human pluripotent stem cell-derived retinal organoids recapitulates retinogenesis in vivo and reveals an important role for IMPG1 and CD44 in the development of photoreceptors and interphotoreceptor matrix.

PubMed

Felemban, Majed; Dorgau, Birthe; Hunt, Nicola Claire; Hallam, Dean; Zerti, Darin; Bauer, Roman; Ding, Yuchun; Collin, Joseph; Steel, David; Krasnogor, Natalio; Al-Aama, Jumana; Lindsay, Susan; Mellough, Carla; Lako, Majlinda

2018-05-17

The extracellular matrix (ECM) plays an important role in numerous processes including cellular proliferation, differentiation, migration, maturation, adhesion guidance and axonal growth. To date, there has been no detailed analysis of the ECM distribution during retinal ontogenesis in humans and the functional importance of many ECM components is poorly understood. In this study, the expression of key ECM components in adult mouse and monkey retina, developing and adult human retina and retinal organoids derived from human pluripotent stem cells was studied. Our data indicate that basement membrane ECMs (Fibronectin and Collagen IV) were expressed in Bruch's membrane and the inner limiting membrane of the developing human retina, whilst the hyalectins (Versican and Brevican), cluster of differentiation 44 (CD44), photoreceptor-specific ECMs Interphotoreceptor Matrix Proteoglycan 1 (IMPG1) and Interphotoreceptor Matrix Proteoglycan 2 (IMPG2) were detected in the developing interphotoreceptor matrix (IPM). The expression of IMPG1, Versican and Brevican in the developing IPM was conserved between human developing retina and human pluripotent stem cell-derived retinal organoids. Blocking the action of CD44 and IMPG1 in pluripotent stem cell derived retinal organoids affected the development of photoreceptors, their inner/outer segments and connecting cilia and disrupted IPM formation, with IMPG1 having an earlier and more significant impact. Together, our data suggest an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation during human retinogenesis. The expression and the role of many extracellular matrix (ECM) components during human retinal development is not fully understood. In this study, expression of key ECM components (Collagen IV, Fibronectin, Brevican, Versican, IMPG1 and IMPG2) was investigated during human retinal ontogenesis. Collagen IV and Fibronectin were expressed in Bruch's membrane; whereas Brevican, Versican, IMPG1 & IMPG2 in the developing interphotoreceptor matrix (IPM). Retinal organoids were successfully generated from pluripotent stem cells. The expression of ECM components was examined in the retinal organoids and found to recapitulate human retinal development in vivo. Using functional blocking experiments, we were able to highlight an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation. Copyright © 2018 Acta Materialia Inc. All rights reserved.

Localization of extracellular matrix components in developing mouse salivary glands by confocal microscopy

NASA Technical Reports Server (NTRS)

Hardman, P.; Spooner, B. S.

1992-01-01

The importance of the extracellular matrix (ECM) in epithelial-mesenchymal interactions in developing organisms is well established. Proteoglycans and interstitial collagens are required for the growth, morphogenesis, and differentiation of epithelial organs and the distribution of these molecules has been described. However, much less is known about other ECM macromolecules in developing epithelial organs. We used confocal microscopy to examine the distribution of laminin, heparan sulfate (BM-1) proteoglycan, fibronectin, and collagen types I, IV, and V, in mouse embryonic salivary glands. Organ rudiments were isolated from gestational day 13 mouse embryos and cultured for 24, 48, or 72 hours. Whole mounts were stained by indirect immunofluorescence and then examined using a Zeiss Laser Scan Microscope. We found that each ECM component examined had a distinct distribution and that the distribution of some molecules varied with culture time. Laminin was mainly restricted to the basement membrane. BM-1 proteoglycan was concentrated in the basement membrane and also formed a fine network throughout the mesenchyme. Type IV collagen was mainly located in the basement membrane of the epithelium, but it was also present throughout the mesenchyme. Type V collagen was distributed throughout the mesenchyme at 24 hours, but at 48 hours was principally located in the basement membrane. Type I collagen was distributed throughout the mesenchyme at all culture times, and accumulated in the clefts and particularly at the epithelial-mesenchymal interface as time in culture increased. Fibronectin was observed throughout the mesenchyme at all times.

Manipulation of valve composition to elucidate the role of collagen in aortic valve calcification

PubMed Central

2014-01-01

Background Extracellular matrix (ECM) disarray is found in calcific aortic valvular disease (CAVD), yet much remains to be learned about the role of individual ECM components in valvular interstitial cell (VIC) function and dysfunction. Previous clinical analyses have shown that calcification is associated with decreased collagen content, while previous in vitro work has suggested that the presence of collagen attenuates the responsiveness of VICs to pro-calcific stimuli. The current study uses whole leaflet cultures to examine the contributions of endogenous collagen in regulating the phenotype and calcification of VICs. Methods A “top-down” approach was used to characterize changes in VIC phenotype in response to collagen alterations in the native 3D environment. Collagen-deficient leaflets were created via enzymatic treatment and cultured statically for six days in vitro. After culture, leaflets were harvested for analysis of DNA, proliferation, apoptosis, ECM composition, calcification, and gene/protein expression. Results In general, disruption of collagen was associated with increased expression of disease markers by VICs in whole organ leaflet culture. Compared to intact control leaflets, collagen-deficient leaflets demonstrated increased VIC proliferation and apoptosis, increased expression of disease-related markers such as alpha-smooth muscle actin, alkaline phosphatase, and osteocalcin, and an increase in calcification as evidenced by positive von Kossa staining. Conclusions These results indicate that disruption of the endogenous collagen structure in aortic valves is sufficient to stimulate pathological consequences in valve leaflet cultures, thereby highlighting the importance of collagen and the valve extracellular matrix in general in maintaining homeostasis of the valve phenotype. PMID:24581344

Collagen gel protects L929 cells from TNFα-induced death by activating NF-κB.

PubMed

Wang, Hong-Ju; Li, Meng-Qi; Liu, Wei-Wei; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2017-09-01

Type I collagen is one of the most abundant components of extracellular matrix. We previously illustrated that murine fibrosarcoma L929 cells grew well on type I collagen gel and escaped from TNFα-induced cell death. In this study, we investigated the mechanism underlying the protective effect of collagen gel. We used western blot, confocal microscopy, MTT assay and flow cytometry by introducing fluorescence staining to determine the expression levels of nuclear factor kappa B (NF-κB), inhibitory ratio and autophagy. L929 cells on collagen gel showed higher expression of NF-κB in the nucleus. Inhibition of NF-κB with pyrrolidine dithiocarbamate hydrochloride (PDTC) or knockdown by NF-κB-siRNA canceled the protective effect of collagen gel on L929 cells from TNFα-induced death, suggesting for the role of NF-κB in the protection from cell death. We found a new aspect of the effect of PDTC on L929 cells cultured on collagen gel. PDTC alone without TNFα induced apoptosis in the L929 cells cultured on collagen gel but not the cells on plastic dish. The apoptosis induction of the L929 cells cultured on collagen gel with PDTC was repressed by inhibiting autophagy with chloroquine, an autophagy inhibitor, suggesting that autophagy contributes to the death induced by the treatment with PDTC. Possible underlying mechanism of this finding is discussed. NF-κB played an important role in protecting the L929 cells cultured on collagen gel from TNFα-induced death.

Type VII collagen is enriched in the enamel organic matrix associated with the dentin-enamel junction of mature human teeth.

PubMed

McGuire, Jacob D; Walker, Mary P; Mousa, Ahmad; Wang, Yong; Gorski, Jeff P

2014-06-01

The inner enamel region of erupted teeth is known to exhibit higher fracture toughness and crack growth resistance than bulk phase enamel. However, an explanation for this behavior has been hampered by the lack of compositional information for the residual enamel organic matrix. Since enamel-forming ameloblasts are known to express type VII collagen and type VII collagen null mice display abnormal amelogenesis, the aim of this study was to determine whether type VII collagen is a component of the enamel organic matrix at the dentin-enamel junction (DEJ) of mature human teeth. Immunofluorescent confocal microscopy of demineralized tooth sections localized type VII collagen to the organic matrix surrounding individual enamel rods near the DEJ. Morphologically, immunoreactive type VII collagen helical-bundles resembled the gnarled-pattern of enamel rods detected by Coomassie Blue staining. Western blotting of whole crown or enamel matrix extracts also identified characteristic Mr=280 and 230 kDa type VII dimeric forms, which resolved into 75 and 25 kDa bands upon reduction. As expected, the collagenous domain of type VII collagen was resistant to pepsin digestion, but was susceptible to purified bacterial collagenase. These results demonstrate the inner enamel organic matrix in mature teeth contains macromolecular type VII collagen. Based on its physical association with the DEJ and its well-appreciated capacity to complex with other collagens, we hypothesize that enamel embedded type VII collagen fibrils may contribute not only to the structural resilience of enamel, but may also play a role in bonding enamel to dentin. Copyright © 2014 Elsevier Inc. All rights reserved.

Isolation and characterization of acid-soluble collagen from the scales of marine fishes from Japan and Vietnam.

PubMed

Minh Thuy, Le Thi; Okazaki, Emiko; Osako, Kazufumi

2014-04-15

Acid-soluble collagen (ASC) was successfully extracted from the scales of lizard fish (Saurida spp.) and horse mackerel (Trachurus japonicus) from Japan and Vietnam and grey mullet (Mugil cephalis), flying fish (Cypselurus melanurus) and yellowback seabream (Dentex tumifrons) from Japan. ASC yields were about 0.43-1.5% (on a dry weight basis), depending on the species. The SDS-PAGE profile showed that the ASCs were type I collagens, and consisted of two different α chains, α1 and α2, as well as a β component. ASC of horse mackerel from Vietnam contained a higher imino acid level than that from Japan. ASC denaturation temperature (Td) ranged from 26 to 29 °C, depending on fish species and imino acid content (p<0.01). Maximal solubility of individual collagens was observed at pHs 1-3. Collagen solubility decreased sharply at NaCl concentrations >0.4M, regardless of fish type. Copyright © 2013 Elsevier Ltd. All rights reserved.

Inhibition of collagen synthesis by select calcium and sodium channel blockers can be mitigated by ascorbic acid and ascorbyl palmitate

PubMed Central

Ivanov, Vadim; Ivanova, Svetlana; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

2016-01-01

Calcium, sodium and potassium channel blockers are widely prescribed medications for a variety of health problems, most frequently for cardiac arrhythmias, hypertension, angina pectoris and other disorders. However, chronic application of channel blockers is associated with numerous side effects, including worsening cardiac pathology. For example, nifedipine, a calcium-channel blocker was found to be associated with increased mortality and increased risk for myocardial infarction. In addition to the side effects mentioned above by different channel blockers, these drugs can cause arterial wall damage, thereby contributing to vascular wall structure destabilization and promoting events facilitating rupture of plaques. Collagen synthesis is regulated by ascorbic acid, which is also essential for its optimum structure as a cofactor in lysine and proline hydroxylation, a precondition for optimum crosslinking of collagen and elastin. Therefore, the main objective in this study was to evaluate effects of various types of channel blockers on intracellular accumulation and cellular functions of ascorbate, specifically in relation to formation and extracellular deposition of major collagen types relevant for vascular function. Effects of select Na- and Ca- channel blockers on collagen synthesis and deposition were evaluated in cultured human dermal fibroblasts and aortic smooth muscle cells by immunoassay. All channel blockers tested demonstrated inhibitory effects on collagen type I deposition to the ECM by fibroblasts, each to a different degree. Ascorbic acid significantly increased collagen I ECM deposition. Nifedipine (50 µM), a representative of channel blockers tested, significantly reduced ascorbic acid and ascorbyl palmitate-dependent ECM deposition of collagen type l and collagen type lV by cultured aortic smooth muscle cells. In addition, nifedipine (50 µM) significantly reduced ascorbate-dependent collagen type l and type lV synthesis by cultured aortic smooth muscle cells, assayed by measuring intracellular collagen content. We observed increased intracellular levels of ascorbate under supplementation with elevated doses of ascorbic acid, as well as its lipid soluble derivative ascorbyl palmitate. Nifedipine reduced ascorbic acid intracellular influx in cultured aortic smooth muscle cells with nifedipine (50 µM) compared to control. Adverse effects of nifedipine were neutralized either by an increased level of cell supplementation with ascorbic acid or by substituting it with ascorbyl palmitate. These studies suggest that adverse effects of channel blockers could be caused by their weakening the arterial wall integrity by interfering with proper extracellular matrix formation. In conclusion, these studies confirm the adverse effects of channel blockers on collagen type l and lV deposition, the key ECM components essential for maintaining optimal structural integrity of the arterial walls. Ascorbate supplementation reversed channel blocker inhibition of these collagen types synthesis and deposition. The results of this study imply the benefits of ascorbate and ascorbate palmitate supplementation in medical management of cardiovascular disease in order to compensate for adverse effects of channel blockers. PMID:27335688

Inhibition of collagen synthesis by select calcium and sodium channel blockers can be mitigated by ascorbic acid and ascorbyl palmitate.

PubMed

Ivanov, Vadim; Ivanova, Svetlana; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

2016-01-01

Calcium, sodium and potassium channel blockers are widely prescribed medications for a variety of health problems, most frequently for cardiac arrhythmias, hypertension, angina pectoris and other disorders. However, chronic application of channel blockers is associated with numerous side effects, including worsening cardiac pathology. For example, nifedipine, a calcium-channel blocker was found to be associated with increased mortality and increased risk for myocardial infarction. In addition to the side effects mentioned above by different channel blockers, these drugs can cause arterial wall damage, thereby contributing to vascular wall structure destabilization and promoting events facilitating rupture of plaques. Collagen synthesis is regulated by ascorbic acid, which is also essential for its optimum structure as a cofactor in lysine and proline hydroxylation, a precondition for optimum crosslinking of collagen and elastin. Therefore, the main objective in this study was to evaluate effects of various types of channel blockers on intracellular accumulation and cellular functions of ascorbate, specifically in relation to formation and extracellular deposition of major collagen types relevant for vascular function. Effects of select Na- and Ca- channel blockers on collagen synthesis and deposition were evaluated in cultured human dermal fibroblasts and aortic smooth muscle cells by immunoassay. All channel blockers tested demonstrated inhibitory effects on collagen type I deposition to the ECM by fibroblasts, each to a different degree. Ascorbic acid significantly increased collagen I ECM deposition. Nifedipine (50 µM), a representative of channel blockers tested, significantly reduced ascorbic acid and ascorbyl palmitate-dependent ECM deposition of collagen type l and collagen type lV by cultured aortic smooth muscle cells. In addition, nifedipine (50 µM) significantly reduced ascorbate-dependent collagen type l and type lV synthesis by cultured aortic smooth muscle cells, assayed by measuring intracellular collagen content. We observed increased intracellular levels of ascorbate under supplementation with elevated doses of ascorbic acid, as well as its lipid soluble derivative ascorbyl palmitate. Nifedipine reduced ascorbic acid intracellular influx in cultured aortic smooth muscle cells with nifedipine (50 µM) compared to control. Adverse effects of nifedipine were neutralized either by an increased level of cell supplementation with ascorbic acid or by substituting it with ascorbyl palmitate. These studies suggest that adverse effects of channel blockers could be caused by their weakening the arterial wall integrity by interfering with proper extracellular matrix formation. In conclusion, these studies confirm the adverse effects of channel blockers on collagen type l and lV deposition, the key ECM components essential for maintaining optimal structural integrity of the arterial walls. Ascorbate supplementation reversed channel blocker inhibition of these collagen types synthesis and deposition. The results of this study imply the benefits of ascorbate and ascorbate palmitate supplementation in medical management of cardiovascular disease in order to compensate for adverse effects of channel blockers.

Biomimetic Proteoglycan Interactions with Type I Collagen Investigated via 2D and 3D TEM

NASA Astrophysics Data System (ADS)

Moorehead, Carli

Collagen is one of the leading components in extracellular matrix (ECM), providing durability, structural integrity, and functionality for many tissues. Regulation of collagen fibrillogenesis and degradation is important in the treatment of a number of diseases from orthopedic injuries to genetic deficiencies. Recently, novel, biocompatible, semi-synthetic biomimetic proteoglycans (BPGs) were developed, which consist of an enzymatically resistant synthetic polymer core and natural chondroitin sulfate bristles. It was demonstrated that BPGs affect type I collagen fibrillogenesis in vitro, as reflected by their impact delaying the kinetic formation of gels similar to native PGs. This indicates that the morphology of collagen scaffolds as well as endogenous ECM could also be modulated by these proteoglycan mimics. However, the imaging modality used previously, reflectance confocal microscopy, did not yield the resolution necessary to spatially localize BPGs within the collagen network or investigate the effect of BPGs on the quality of collagen fibrils produced in an in vitro fibrillogenesis model which is important for understanding the method of interaction. Consequently, a histological technique, electron tomography, was adapted and utilized to 3D image the nano-scale structures within this simplified tissue model. BPGs were found to aid in lateral growth and enhance fibril banding periodicity resulting in structures more closely resembling those in tissue, in addition to attaching to the collagen surface despite the lack of a protein core.

Human elastin polypeptides improve the biomechanical properties of three-dimensional matrices through the regulation of elastogenesis.

PubMed

Boccafoschi, Francesca; Ramella, Martina; Sibillano, Teresa; De Caro, Liberato; Giannini, Cinzia; Comparelli, Roberto; Bandiera, Antonella; Cannas, Mario

2015-03-01

The replacement of diseased tissues with biological substitutes with suitable biomechanical properties is one of the most important goal in tissue engineering. Collagen represents a satisfactory choice for scaffolds. Unfortunately, the lack of elasticity represents a restriction to a wide use of collagen for several applications. In this work, we studied the effect of human elastin-like polypeptide (HELP) as hybrid collagen-elastin matrices. In particular, we studied the biomechanical properties of collagen/HELP scaffolds considering several components involved in ECM remodeling (elastin, collagen, fibrillin, lectin-like receptor, metalloproteinases) and cell phenotype (myogenin, myosin heavy chain) with particular awareness for vascular tissue engineering applications. Elastin and collagen content resulted upregulated in collagen-HELP matrices, even showing an improved structural remodeling through the involvement of proteins to a ECM remodeling activity. Moreover, the hybrid matrices enhanced the contractile activity of C2C12 cells concurring to improve the mechanical properties of the scaffold. Finally, small-angle X-ray scattering analyses were performed to enable a very detailed analysis of the matrices at the nanoscale, comparing the scaffolds with native blood vessels. In conclusion, our work shows the use of recombinant HELP, as a very promising complement able to significantly improve the biomechanical properties of three-dimensional collagen matrices in terms of tensile stress and elastic modulus. © 2014 Wiley Periodicals, Inc.

Biphasic Scaffolds from Marine Collagens for Regeneration of Osteochondral Defects.

PubMed

Bernhardt, Anne; Paul, Birgit; Gelinsky, Michael

2018-03-13

Collagens of marine origin are applied increasingly as alternatives to mammalian collagens in tissue engineering. The aim of the present study was to develop a biphasic scaffold from exclusively marine collagens supporting both osteogenic and chondrogenic differentiation and to find a suitable setup for in vitro chondrogenic and osteogenic differentiation of human mesenchymal stroma cells (hMSC). Biphasic scaffolds from biomimetically mineralized salmon collagen and fibrillized jellyfish collagen were fabricated by joint freeze-drying and crosslinking. Different experiments were performed to analyze the influence of cell density and TGF-β on osteogenic differentiation of the cells in the scaffolds. Gene expression analysis and analysis of cartilage extracellular matrix components were performed and activity of alkaline phosphatase was determined. Furthermore, histological sections of differentiated cells in the biphasic scaffolds were analyzed. Stable biphasic scaffolds from two different marine collagens were prepared. An in vitro setup for osteochondral differentiation was developed involving (1) different seeding densities in the phases; (2) additional application of alginate hydrogel in the chondral part; (3) pre-differentiation and sequential seeding of the scaffolds and (4) osteochondral medium. Spatially separated osteogenic and chondrogenic differentiation of hMSC was achieved in this setup, while osteochondral medium in combination with the biphasic scaffolds alone was not sufficient to reach this ambition. Biphasic, but monolithic scaffolds from exclusively marine collagens are suitable for the development of osteochondral constructs.

Formation of Highly Aligned Collagen Nanofibers by Continuous Cyclic Stretch of a Collagen Hydrogel Sheet.

PubMed

Nam, Eunryel; Lee, Won Chul; Takeuchi, Shoji

2016-07-01

A collagen sheet with highly aligned collagen fibers is fabricated by continuous cyclic stretch. The rearrangement of the collagen fibers depends on the different process parameters of the cyclic stretch, including magnitude, frequency, and period of stretch. The collagen fibers are aligned perpendicularly to the direction of the stretch. Corneal stromal cells and smooth muscle cells cultivated on the highly aligned collagen sheet show alignment along the collagen fibers without the stretch during culture. Thus, the sheet can be a suitable scaffold for use in regenerative medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The role of lipopolysaccharide injected systemically in the reactivation of collagen-induced arthritis in mice

PubMed Central

Yoshino, Shin; Ohsawa, Motoyasu

2000-01-01

We investigated the role of bacterial lipopolysaccharide (LPS) in the reactivation of autoimmune disease by using collagen-induced arthritis (CIA) in mice in which autoimmunity to the joint cartilage component type II collagen (CII) was involved.CIA was induced by immunization with CII emulsified with complete Freund's adjuvant at the base of the tail (day 0) followed by a booster injection on day 21. Varying doses of LPS from E. coli were i.p. injected on day 50.Arthritis began to develop on day 25 after immunization with CII and reached a peak on day 35. Thereafter, arthritis subsided gradually but moderate joint inflammation was still observed on day 50. An i.p. injection of LPS on day 50 markedly reactivated arthritis on a dose-related fashion. Histologically, on day 55, there were marked oedema of synovium which had proliferated by the day of LPS injection, new formation of fibrin, and intense infiltration of neutrophils accompanied with a large number of mononuclear cells. The reactivation of CIA by LPS was associated with increases in anti-CII IgG and IgG2a antibodies as well as various cytokines including IL-12, IFN-γ, IL-1β, and TNF-α. LPS from S. enteritidis, S. typhimurium, and K. neumoniae and its component, lipid A from E. coli also reactivated the disease. Polymyxin B sulphate suppressed LPS- or lipid A-induced reactivation of CIA.These results suggest that LPS may play an important role in the reactivation of autoimmune joint inflammatory diseases such as rheumatoid arthritis in humans. PMID:10742285

Effect of laser soldering irradiation on covalent bonds of pure collagen.

PubMed

Constantinescu, Mihai A; Alfieri, Alex; Mihalache, George; Stuker, Florian; Ducray, Angélique; Seiler, Rolf W; Frenz, Martin; Reinert, Michael

2007-03-01

Laser tissue welding and soldering is being increasingly used in the clinical setting for defined surgical procedures. The exact induced changes responsible for tensile strength are not yet fully investigated. To further improve the strength of the bonding, a better understanding of the laser impact at the subcellular level is necessary. The goal of this study was to analyze whether the effect of laser irradiation on covalent bonding in pure collagen using irradiances typically applied for tissue soldering. Pure rabbit and equine type I collagen were subjected to laser irradiation. In the first part of the study, rabbit and equine collagen were compared using identical laser and irradiation settings. In the second part of the study, equine collagen was irradiated at increasing laser powers. Changes in covalent bonding were studied indirectly using the sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique. Tensile strengths of soldered membranes were measured with a calibrated tensile force gauge. In the first experiment, no differences between the species-specific collagen bands were noted, and no changes in banding were found on SDS-PAGE after laser irradiation. In the second experiment, increasing laser irradiation power showed no effect on collagen banding in SDS-PAGE. Finally, the laser tissue soldering of pure collagen membranes showed virtually no determinable tensile strength. Laser irradiation of pure collagen at typical power settings and exposure times generally used in laser tissue soldering does not induce covalent bonding between collagen molecules. This is true for both rabbit and equine collagen proveniences. Furthermore, soldering of pure collagen membranes without additional cellular components does not achieve the typical tensile strength reported in native, cell-rich tissues. This study is a first step in a better understanding of laser impact at the molecular level and might prove useful in engineering of combined collagen-soldering matrix membranes for special laser soldering applications.

Distribution of collagenous colitis: utility of flexible sigmoidoscopy.

PubMed Central

Tanaka, M; Mazzoleni, G; Riddell, R H

1992-01-01

We investigated the distribution of the collagen band in 33 patients with collagenous colitis to estimate the likelihood of the disease being diagnosed in biopsy specimens from the left side of the colon, such as those obtained using flexible sigmoidoscopy. To be included in this study patients had a subepithelial collagen band greater than or equal to 10 microns, an increase in chronic inflammatory cells in the same specimen, and diarrhoea for which there was no other apparent cause. In 17 patients undergoing full colonoscopy with a thickened collagen band, collagenous colitis was frequently patchy, even though overall the thickened collagen band was almost equally distributed throughout the colon. Rectal biopsy specimens showed a normal collagen band in 73% of patients, while a thickened collagen band was found in 82% of patients in at least one specimen from the left side of the colon. Three patients had a thickened collagen band only in the caecum. In three of eight rectal biopsy specimens with a normal collagen band there was no mucosal inflammation to raise the possibility of proximal disease, although all but one specimen with a normal collagen band from the sigmoid and descending colon were inflamed. Rectal biopsy alone is therefore a relatively poor method of making the diagnosis. Flexible sigmoidoscopy with multiple biopsy specimens from several sites is a reasonable initial investigation but not sufficient to exclude collagenous colitis when based on the presence of a thickened collagen band alone. Should left sided biopsy specimens show a normal collagen band but an inflamed mucosa, total colonoscopy with multiple specimens including the caecum may be required to establish the diagnosis. Images Figure 1 Figure 2 PMID:1740280

Guided tissue regeneration with heterologous materials in primary subtalar arthrodesis: a case report.

PubMed

Frangez, Igor; Kasnik, Tea; Cimerman, Matej; Smrke, Dragica Maja

2016-05-03

Calcaneal fractures are relatively rare and difficult to treat. Treatment options vary based on the type of fracture and the surgeon's experiences. In recent years, surgical procedures have increasingly been used due to the better long-term results. We present a case where guided tissue regeneration was performed in a calcaneal fracture that needed primary subtalar arthrodesis. We used the principles of guided tissue regeneration from oral surgery to perform primary subtalar arthrodesis and minimize the risk of non-union. We used a heterologous collagen membrane, which acts as a mechanical barrier and protects the bone graft from the invasion of unwanted cells that could lead to non-union. The collagenous membrane also has osteoconductive properties and is therefore able to increase the osteoblast proliferation rate. A 62-year-old Caucasian woman sustained multiple fractures of her lower limbs and spine after a fall from a ladder. Her left calcaneus had a comminuted multifragmental fracture (Sanders type IV) with severe destruction of the cartilage of her subtalar joint and depression of the Böhler's angle. Therefore, we performed primary arthrodesis of her subtalar joint with elevation of the Böhler's angle using a 7.3 mm titanium screw, a heterologous cortico-cancellous collagenated pre-hydrated bone mix, a heterologous cancellous collagenated bone wedge, and a heterologous collagen membrane (Tecnoss®, Italy). The graft was fully incorporated 12 weeks after the procedure and a year and a half later our patient walks without limping. We present a new use of guided tissue regeneration with heterologous materials that can be used to treat extensive bone defects after bone injuries. We believe that guided tissue regeneration using heterologous materials, including a heterologous collagen membrane that presents a mechanical barrier between soft tissues and bone as well as a stimulative component that enhances bone formation, could be more often used in bone surgery.

Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities.

PubMed

Igoucheva, Olga; Alexeev, Vitali; Halabi, Carmen M; Adams, Sheila M; Stoilov, Ivan; Sasaki, Takako; Arita, Machiko; Donahue, Adele; Mecham, Robert P; Birk, David E; Chu, Mon-Li

2015-08-28

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Histochemical and Immunohistochemical Study of α-SMA, Collagen, and PCNA in Epithelial Ovarian Neoplasm

PubMed

Anggorowati, Nungki; Ratna Kurniasari, Chatarina; Damayanti, Karina; Cahyanti, Titik; Widodo, Irianiwati; Ghozali, Ahmad; Romi, Muhammad Mansyur; Sari, Dwi Cahyani Ratna; Arfian, Nur

2017-03-01

Background: Alpha-smooth muscle actin (α-SMA) is an isoform of actin, positive in myofibroblasts and is an epithelial to mesenchymal transition (EMT) marker. EMT is a process by which tumor cells develop to be more hostile and able to metastasize. Progression of tumor cells is always followed by cell composition and extracellular matrix component alteration. Increased α-SMA expression and collagen alteration may predict the progressivity of ovarian neoplasms. Objective: The aim of this research was to analyse the characteristic of α-SMA and collagen in tumor cells and stroma of ovarian neoplasms. In this study, PCNA (proliferating cell nuclear antigen) expression was also investigated. Methods: Thirty samples were collected including serous, mucinous, endometrioid, and clear cell subtypes. The expression of α-SMA and PCNA were calculated in cells and stroma of ovarian tumors. Collagen was detected using Sirius Red staining and presented as area fraction. Results: The overexpressions of α-SMA in tumor cells were only detected in serous and clear cell ovarian carcinoma. The histoscore of α-SMA was higher in malignant than in benign or borderline ovarian epithelial neoplasms (105.3±129.9 vs. 17.3±17.1, P=0.011; mean±SD). Oppositely, stromal α-SMA and collagen area fractions were higher in benign than in malignant tumors (27.2±6.6 vs 20.5±8.4, P=0.028; 31.0±5.6 vs. 23.7±6.4, P=0.04). The percentages of epithelial and stromal PCNA expressions were not significantly different between benign and malignant tumors. Conclusion: Tumor cells of serous and clear cell ovarian carcinoma exhibit mesenchymal characteristic as shown by α-SMA positive expression. This expression might indicate that these subtypes were more aggressive. This research showed that collagen and α-SMA area fractions in stroma were higher in benign than in malignant neoplasms. 10.22034/APJCP.2017.18.3.667

Differences in extracellular matrix proteins between Friesian horses with aortic rupture, unaffected Friesians and Warmblood horses.

PubMed

Ploeg, M; Gröne, A; van de Lest, C H A; Saey, V; Duchateau, L; Wolsein, P; Chiers, K; Ducatelle, R; van Weeren, P R; de Bruijn, M; Delesalle, C

2017-09-01

Unlike in Warmblood horses, aortic rupture is quite common in Friesian horses, in which a hereditary trait is suspected. The aortic connective tissue in affected Friesians shows histological changes such as medial necrosis, elastic fibre fragmentation, mucoid material accumulation and fibrosis with aberrant collagen morphology. However, ultrastructural examination of the collagen fibres of the mid-thoracic aorta has been inconclusive in further elucidating the pathogenesis of the disease. To assess several extracellular matrix (ECM) components biochemically in order to explore a possible underlying breed-related systemic ECM defect in Friesians with aortic rupture. Cadaver study. Tissues from affected Friesians (n = 18), unaffected Friesians (n = 10) and Warmblood horses (n = 30) were compared. Samples were taken from the thoracic aorta at the level of the rupture site, from two locations caudal to the rupture and from the deep digital flexor tendon. Total collagen content, post-translational modifications of collagen formation including lysine hydroxylation, and hydroxylysylpyridinoline (HP), lysylpyridinoline (LP) and pyrrole cross-links were analysed. Additionally, elastin cross-links, glycosaminoglycan content and matrix metalloproteinase (MMP) activity were assessed. Significantly increased MMP activity and increased LP and HP cross-linking, lysine hydroxylation and elastin cross-linking were found at the site of rupture in affected Friesians. These changes may reflect processes involved in healing and aneurysm formation. Unaffected Friesians had less lysine hydroxylation and pyrrole cross-linking within the tendons compared with Warmblood horses. No differences in the matrix of the aorta were found between normal Warmbloods and Friesian horses. Small sample size. The differences in collagen parameters in tendon tissue may reflect differences in connective tissue metabolism between Friesians and Warmblood horses. © 2017 EVJ Ltd.

Type XVII collagen (BP180) can function as a cell-matrix adhesion molecule via binding to laminin 332

PubMed Central

Van den Bergh, F.; Eliason, S.L.; Giudice, G.J.

2010-01-01

Collagen XVII (COL17) is a transmembrane glycoprotein that is expressed on the basal surface of basal epidermal keratinocytes. Previous observations have led to the hypothesis that an interaction between COL17 and laminin 332, an extracellular matrix protein, contributes to the attachment of the basal keratinocyte to the basement membrane. In order to isolate and manipulate COL17 interactions with ECM components, we induced COL17 expression in two cells lines, SK-MEL1 and K562, that exhibit little or no capacity to attach to our test substrates, including laminin 332, types I and IV collagen, and fibronectin. Cells expressing high levels of COL17 preferentially adhered to a laminin 332 matrix, and, to a lesser extent, type IV collagen, while showing little or no binding to type I collagen or fibronectin. A quantitative analysis of cell adhesive forces revealed that, compared with COL17-negative cells, COL17-positive cells required over 7-fold greater force to achieve 50% detachment from a laminin 332 substrate. When a cell preparation (either K562 or SK-MEL1) with heterogeneous COL17 expression levels was allowed to attach to a laminin 332 matrix, the COL17-positive and COL17-negative cells differentially sorted to the bound and unbound cell fractions, respectively. COL17-dependent attachment to laminin 332 could be reduced or abolished by siRNA-mediated knockdown of COL17 expression or by adding to the assay wells specific antibodies against COL17 or laminin 332. These findings provide strong support for the hypothesis that cell surface COL17 can interact with laminin 332 and, together, participate in the adherence of a cell to the extracellular matrix. PMID:21034821

Inhibitory effects of Atractylodis lanceae rhizoma and Poria on collagen- or thromboxane A2-induced aggregation in rabbit platelets.

PubMed

Nasu, Yuiko; Iwashita, Masaya; Saito, Masaki; Fushiya, Shinji; Nakahata, Norimichi

2009-05-01

Kami-shoyo-san (Jia-Wei-Xiao-Yao-San), Toki-shakuyaku-san (Dang-Gui-Shao-Yao-San) and Toki-shigyaku-ka-goshuyu-shokyo-to (Dang-Gui-Si-Ni-Jia-Wu-Zhu-Yu-Sheng-Jiang-Tang) are Kampo (traditional Chinese) medicines which are traditionally and effectively used for the treatment of chilly sensation (Hiesho) in Japan, but the active components and their detailed mechanisms have not yet been clarified. Etiologies of Hiesho include poor peripheral blood circulation and platelet aggregability contributes to peripheral blood circulation; therefore, we investigated the effect of Kampo medicines on platelet aggregation using rabbit platelets in vitro. Collagen and U46619, a thromboxane A(2) receptor agonist, caused rabbit platelet aggregation, which was potently inhibited by pretreatment of platelets with Kami-shoyo-san and Toki-shakuyaku-san in vitro. Toki-shigyaku-ka-goshuyu-shokyo-to, however, did not significantly inhibit collagen- or U46619-induced platelet aggregation. Therefore, we examined the effect on platelet aggregation of two herbal medicines, Atractylodis Lanceae Rhizoma and Poria, both of which are contained in Kami-shoyo-san and Toki-shakuyaku-san but not in Toki-shigyaku-ka-goshuyu-shokyo-to. As the results indicate, Atractylodis Lanceae Rhizoma inhibited platelet aggregation induced by collagen but not by U46619. Poria effectively inhibited U46619-induced platelet aggregation and it partially inhibited collagen-induced platelet aggregation. On the other hand, Atractylodis Lanceae Rhizoma and Poria did not inhibit adrenaline/adenosine diphosphate- or adrenaline/serotonin-induced platelet aggregation. These results suggest the possibility that the inhibition of platelet aggregation by two Kampo medicines, Kami-shoyo-san and Toki-shakuyaku-san, is one of the mechanisms underlying the improvement of Hiesho. Furthermore, Atractylodis Lanceae Rhizoma and Poria are possible herbal medicines for the inhibition of platelet aggregation.

Immunohistochemical study of extracellular matrices and elastic fibers in a human sternoclavicular joint.

PubMed

Shimada, K; Takeshige, N; Moriyama, H; Miyauchi, Y; Shimada, S; Fujimaki, E

1997-12-01

In this study, we clarified the distribution of elastic and oxytalan fibers in a human sternoclavicular joint (SCJ) using a color image system and in extracellular matrices using immunoperoxidase staining. Fine elastic fibers (EFs) were scattered in the fibrous layer of the sternoclavicular disk. This articular disk was composed of a collagenous bundle on the sternum side of the articular disk in the SCJ and cellular components including connective tissue on the clavicular side of the articular disk. The thickness of the disk gradually increased from the inferior to superior portion. Collagen fibers type I, III and V and other extracellular matrices (ECMs) were detected in the hypertrophic zone in the clavicular and sternum side of the SCJ and in the connective tissue of the articulatio condylar. On the cervical surface of the articular disk, cellular activity was higher than on the sternum surface.

Characterization of the viscoelastic behavior of a simplified collagen micro-fibril based on molecular dynamics simulations.

PubMed

Ghodsi, Hossein; Darvish, Kurosh

2016-10-01

Collagen fibril is a major component of connective tissues such as bone, tendon, blood vessels, and skin. The mechanical properties of this highly hierarchical structure are greatly influenced by the presence of covalent cross-links between individual collagen molecules. This study investigates the viscoelastic behavior of a collagen lysine-lysine cross-link based on creep simulations with applied forces in the range or 10 to 2000pN using steered molecular dynamics (SMD). The viscoelastic model of the cross-link was combined with a system composed by two segments of adjacent collagen molecules hence representing a reduced viscoelastic model for a simplified micro-fibril. It was found that the collagen micro-fibril assembly had a steady-state Young׳s modulus ranging from 2.24 to 3.27GPa, which is in agreement with reported experimental measurements. The propagation of longitudinal force wave along the molecule was implemented by adding a delay element to the model. The force wave speed was found to be correlated with the speed of one-dimensional elastic waves in rods. The presented reduced model with three degrees of freedom can serve as a building block for developing models of the next level of hierarchy, i.e., a collagen fibril. Copyright © 2016 Elsevier Ltd. All rights reserved.

Collagen-Proteoglycan Relationships in Epiphyseal Cartilage

PubMed Central

Eisenstein, Reuben; Larsson, Sven-Erik; Sorgente, Nino; Kuettner, Klaus E.

1973-01-01

Columnar and hypertrophic zones of calf scapular cartilage were studied before and after extraction with 3 M guanidinium chloride (GuCl) and digestion with enzymes which degrade various components of the extracellular matrix. Morphologic and chemical analysis suggests that there are at least two anatomic pools of proteoglycan in this tissue. One, which resides between collagen fibrils, is extractable with GuCl. Another appears attached to collagen by strong bonds and is apparently not extractable with GuCl. This type of collagen-proteoglycan relationship is possibly restricted to epiphyseal cartilage. The morphology of the lacuna is different in the columnar and hypertrophic zones. Proteoglycans in the distal hypertrophic zone are less resistant to GuCl extraction. ImagesFig 9Fig 10Fig 11Fig 1Fig 2Fig 3Fig 4Fig 5Fig 6Fig 7Fig 8 PMID:4357177

Cell-populated collagen lattice contraction model for the investigation of fibroblast collagen interactions.

PubMed

Ehrlich, H Paul; Moyer, Kurtis E

2013-01-01

The fibroblast-populated collagen lattice (FPCL) was intended to act as the dermal component for "skin-equivalent" or artificial skin developed for skin grafting burn patients. The "skin-equivalent" was clinically unsuccessful as a skin graft, but today it is successfully used as a dressing for the management of chronic wounds. The FPCL has, however, become an instrument for investigating cell-connective tissue interactions within a three-dimensional matrix. Through the capacity of cell compaction of collagen fibrils, the FPCL undergoes a reduction in volume referred to as lattice contraction. Lattice contraction proceeds by cell-generated forces that reduce the water mass between collagen fibers, generating a closer relationship between collagen fibers. The compaction of collagen fibers is responsible for the reduction in the FPCL volume. Cell-generated forces through the linkage of collagen fibers with fibroblast's cytoskeletal actin-rich microfilament structures are responsible for the completion of the collagen matrix compaction. The type of culture dish used to cast FPCL as well as the cell number will dictate the mechanism for compacting collagen matrices. Fibroblasts, at moderate density, cast as an FPCL within a petri dish and released from the surface of the dish soon after casting compact collagen fibers through cell tractional forces. Fibroblasts at moderate density cast as an FPCL within a tissue culture dish and not released for 4 days upon release show rapid lattice contraction through a mechanism of cell contraction forces. Fibroblasts at high density cast in an FPCL within a petri dish, released from the surface of the dish soon after casting, compact a collagen lattice very rapidly through forces related to cell elongation. The advantage of the FPCL contraction model is the study of cells in the three-dimensional environment, which is similar to the environment from which these cells were isolated. In this chapter methods are described for manufacturing collagen lattices, which assess the three forces involved in compacting and/or organizing collagen fibrils into thicker collagen fibers. The clinical relevance of the FPCL contraction model is related to advancing our understanding of wound contraction and scar contracture.

Characterization of the column and autocellular junctions that define the vasculature of gill lamellae.

PubMed

Kato, Akira; Nakamura, Korefumi; Kudo, Hisayuki; Tran, Yen Ha; Yamamoto, Yoko; Doi, Hiroyuki; Hirose, Shigehisa

2007-09-01

Novel adhesion junctions have been characterized that are formed at the interface between pillar cells and collagen columns, both of which are essential constituents of the gill lamellae in fish. We termed these junctions the "column junction" and "autocellular junction" and determined their molecular compositions by immunofluorescence microscopy using pufferfish. We visualized collagen columns by concanavalin A staining and found that the components of integrin-mediated cell-matrix adhesion, such as talin, vinculin, paxillin, and fibronectin, were concentrated on plasma membranes surrounding collagen columns (column membranes). This connection is analogous to the focal adhesion of cultured mammalian cells, dense plaque of smooth muscle cells, and myotendinous junction of skeletal muscle cells. We named this connection the "column junction." In the cytoplasm near the column, actin fibers, actinin, and a phosphorylated myosin light chain of 20 kDa are densely located, suggesting the contractile nature of pillar cells. The membrane infoldings surrounding the collagen columns were found to be connected by the autocellular junction, whose components are highly tyrosine-phosphorylated and contain the tight junction protein ZO-1. This study represents the first molecular characterization and fluorescence visualization of the column and autocellular junctions involved in both maintaining structural integrity and the hemodynamics of the branchial lamellae.

Alteration of the bone tissue material properties in type 1 diabetes mellitus: A Fourier transform infrared microspectroscopy study.

PubMed

Mieczkowska, Aleksandra; Mansur, Sity Aishah; Irwin, Nigel; Flatt, Peter R; Chappard, Daniel; Mabilleau, Guillaume

2015-07-01

Type 1 diabetes mellitus (T1DM) is a severe disorder characterized by hyperglycemia and hypoinsulinemia. A higher occurrence of bone fractures has been reported in T1DM, and although bone mineral density is reduced in this disorder, it is also thought that bone quality may be altered in this chronic pathology. Vibrational microscopies such as Fourier transform infrared microspectroscopy (FTIRM) represent an interesting approach to study bone quality as they allow investigation of the collagen and mineral compartment of the extracellular matrix in a specific bone location. However, as spectral feature arising from the mineral may overlap with those of the organic component, the demineralization of bone sections should be performed for a full investigation of the organic matrix. The aims of the present study were to (i) develop a new approach, based on the demineralization of thin bone tissue section to allow a better characterization of the bone organic component by FTIRM, (ii) to validate collagen glycation and collagen integrity in bone tissue and (iii) to better understand what alterations of tissue material properties in newly forming bone occur in T1DM. The streptozotocin-injected mouse (150 mg/kg body weight, injected at 8 weeks old) was used as T1DM model. Animals were randomly allocated to control (n = 8) or diabetic (n = 10) groups and were sacrificed 4 weeks post-STZ injection. Bones were collected at necropsy, embedded in polymethylmethacrylate and sectioned prior to examination by FTIRM. FTIRM collagen parameters were collagen maturity (area ratio between 1660 and 1690 cm(-1) subbands), collagen glycation (area ratio between the 1032 cm(-1) subband and amide I) and collagen integrity (area ratio between the 1338 cm(-1) subband and amide II). No significant differences in the mineral compartment of the bone matrix could be observed between controls and STZ-injected animals. On the other hand, as compared with controls, STZ-injected animals presented with significant higher value for collagen maturity (17%, p = 0.0048) and collagen glycation (99%, p = 0.0121), while collagen integrity was significantly lower by 170% (p = 0.0121). This study demonstrated the profound effect of early T1DM on the organic compartment of the bone matrix in newly forming bone. Further studies in humans are required to ascertain whether T1DM also lead to similar effect on the quality of the bone matrix. Copyright © 2015 Elsevier Inc. All rights reserved.

Icaritin Inhibits Collagen Degradation-Related Factors and Facilitates Collagen Accumulation in Atherosclerotic Lesions: A Potential Action for Plaque Stabilization

PubMed Central

Zhang, Zong-Kang; Li, Jie; Yan, De-Xin; Leung, Wing-Nang; Zhang, Bao-Ting

2016-01-01

Most acute coronary syndromes result from rupture of vulnerable atherosclerotic plaques. The collagen content of plaques may critically affect plaque stability. This study tested whether Icaritin (ICT), an intestinal metabolite of Epimedium-derived flavonoids, could alter the collagen synthesis/degradation balance in atherosclerotic lesions. Rabbits were fed with an atherogenic diet for four months. Oral administration of ICT (10 mg·kg−1·day−1) was started after two months of an atherogenic diet and lasted for two months. The collagen degradation-related parameters, including macrophages accumulation, content and activity of interstitial collagenase-1 (MMP-1), and the collagen synthesis-related parameters, including amount and distribution of smooth muscle cells (SMC) and collagen mRNA/protein levels, were evaluated in the aorta. ICT reduced plasma lipid levels, inhibited macrophage accumulation, lowered MMP-1 mRNA and protein expression, and suppressed proteolytic activity of pro-MMP-1 and MMP-1 in the aorta. ICT changed the distribution of the SMCs towards the fibrous cap of lesions without increasing the amount of SMCs. Higher collagen protein content in lesions and aorta homogenates was observed with ICT treatment compared with the atherogenic diet only, without altered collagen mRNA level. These results suggest that ICT could inhibit the collagen degradation-related factors and facilitate collagen accumulation in atherosclerotic lesions, indicating a new potential of ICT in atherosclerotic plaques. PMID:26828485

Assessment of prokaryotic collagen-like sequences derived from streptococcal Scl1 and Scl2 proteins as a source of recombinant GXY polymers.

PubMed

Han, Runlin; Zwiefka, Antoni; Caswell, Clayton C; Xu, Yi; Keene, Douglas R; Lukomska, Ewa; Zhao, Zhihong; Höök, Magnus; Lukomski, Slawomir

2006-08-01

Collagen triple helix, composed of the repeating Gly-Xaa-Yaa (GXY) sequence, is a structural element found in all multicellular animals and also in some prokaryotes. Long GXY polymers are highly regarded components used in food, cosmetic, biomedical, and pharmaceutical industries. In this study, we explore a new concept for the production of recombinant GXY polymers which are based on the sequence of "prokaryotic collagens", the streptococcal collagen-like proteins Scl1 and Scl2. Analysis of 50 Scl variants identified the amino acid distribution and GXY-repeat usage that are involved in the stabilization of the triple helix in Scls. Using circular dichroism spectroscopy and electron microscopy, we show that significantly different recombinant rScl polypeptides form stable, unhydroxylated homotrimeric triple helices that can be produced both intra- and extracellularly in the Escherichia coli. These rScl constructs containing 20 to 129 GXY repeats had mid-point melting temperatures between 32 and 39 degrees C. Altogether, Scl-derived collagens, which are different from the mammalian collagens, can form stable triple helices under physiological conditions and can be used for the production of recombinant GXY polymers with a wide variety of potential applications.

Strain-Specific Induction of Endometrial Periglandular Fibrosis in Mice Exposed During Adulthood to the Endocrine Disrupting Chemical Bisphenol A

PubMed Central

Kendziorski, Jessica A.; Belcher, Scott M.

2015-01-01

The aim of this study was to compare effects of bisphenol A (BPA) on collagen accumulation in uteri of two mouse strains. Adult C57Bl/6N and CD-1 mice were exposed to dietary BPA (0.004–40 mg/kg/day) or 17α-ethinyl estradiol (0.00002–0.001 mg/kg/day) as effect control. An equine endometrosis-like phenotype with increased gland nesting and periglandular collagen accumulation was characteristic of unexposed C57Bl/6N, but not CD-1, endometrium. BPA non-monotonically increased gland nest density and periglandular collagen accumulation in both strains. Increased collagen I and III expression, decreased matrix metalloproteinase 2 (MMP2) and MMP14 expression, and increased immune response were associated with the endometrosis phenotype in the C57Bl/6N strain and the 30 ppm BPA CD-1 group. The association between the pro-collagen shift in increased collagen expression and decreased MMP2 expression and activity implies that strain differences and BPA exposure salter regulation of endometrial remodeling and contributes to increased fibrosis, a component of several human uterine diseases. PMID:26307436

Identification of Pyridinoline Trivalent Collagen Cross-Links by Raman Microspectroscopy.

PubMed

Gamsjaeger, Sonja; Robins, Simon P; Tatakis, Dimitris N; Klaushofer, Klaus; Paschalis, Eleftherios P

2017-06-01

Intermolecular cross-linking of bone collagen is intimately related to the way collagen molecules are arranged in a fibril, imparts certain mechanical properties to the fibril, and may be involved in the initiation of mineralization. Raman microspectroscopy allows the analysis of minimally processed bone blocks and provides simultaneous information on both the mineral and organic matrix (mainly type I collagen) components, with a spatial resolution of ~1 μm. The aim of the present study was to validate Raman spectroscopic parameters describing one of the major mineralizing type I trivalent cross-links, namely pyridinoline (PYD). To achieve this, a series of collagen cross-linked peptides with known PYD content (as determined by HPLC analysis), human bone, porcine skin, predentin and dentin animal model tissues were analyzed by Raman microspectroscopy. The results of the present study confirm that it is feasible to monitor PYD trivalent collagen cross-links by Raman spectroscopic analysis in mineralized tissues, exclusively through a Raman band ~1660 wavenumbers. This allows determination of the relative PYD content in undecalcified bone tissues with a spatial resolution of ~1 μm, thus enabling correlations with histologic and histomorphometric parameters.

Conducting polymers with immobilised fibrillar collagen for enhanced neural interfacing.

PubMed

Liu, Xiao; Yue, Zhilian; Higgins, Michael J; Wallace, Gordon G

2011-10-01

Conducting polymers with pendant functionality are advantageous in various bionic and organic bioelectronic applications, as they allow facile incorporation of bio-regulative cues to provide bio-mimicry and conductive environments for cell growth, differentiation and function. In this work, polypyrrole substrates doped with chondroitin sulfate (CS), an extracellular matrix molecule bearing carboxylic acid moieties, were electrochemically synthesized and conjugated with type I collagen. During the coupling process, the conjugated collagen formed a 3-dimensional fibrillar matrix in situ at the conducting polymer interface, as evidenced by atomic force microscopy (AFM) and fluorescence microscopy under aqueous physiological conditions. Cyclic voltammetry (CV) and impedance measurement confirmed no significant reduction in the electroactivity of the fibrillar collagen-modified conducting polymer substrates. Rat pheochromocytoma (nerve) cells showed increased differentiation and neurite outgrowth on the fibrillar collagen, which was further enhanced through electrical stimulation of the underlying conducting polymer substrate. Our study demonstrates that the direct coupling of ECM components such as collagen, followed by their further self-assembly into 3-dimensional matrices, has the potential to improve the neural-electrode interface of implant electrodes by encouraging nerve cell attachment and differentiation. Copyright © 2011 Elsevier Ltd. All rights reserved.

New recommendations for measuring collagen solubility.

PubMed

Latorre, María E; Lifschitz, Adrian L; Purslow, Peter P

2016-08-01

The heat-solubility of intramuscular collagen is usually conducted in 1/4 Ringer's solution at pH7.4, despite this ionic strength and pH being inappropriate for post-rigor meat. The current work studied the percentage of soluble collagen and hydrothermal isometric tension characteristics of perimysial strips on bovine semitendinosus muscles in either 1/4 Ringer's solution, distilled water, PBS, or a solution of the same salt concentration as 1/4 Ringer's but at pH5.6. Values of % soluble collagen were lower at pH7.4 than 5.6. Increasing ionic strength reduced % soluble collagen. The maximum perimysial isometric tension was independent of the bathing medium, but the percent relaxation was higher at pH7.4 than at pH5.6, and increased with ionic strength of the media. It is recommended that future measurements of collagen solubility and tests on connective tissue components of post-rigor meat should be carried out in a solution of concentrations NaCl and KCl equivalent to those in 1/4 Ringer's, but at pH5.6, a pH relevant to post-rigor meat. Copyright © 2016 Elsevier Ltd. All rights reserved.

Age-related collagen turnover of the interstitial matrix and basement membrane: Implications of age- and sex-dependent remodeling of the extracellular matrix.

PubMed

Kehlet, Stephanie N; Willumsen, Nicholas; Armbrecht, Gabriele; Dietzel, Roswitha; Brix, Susanne; Henriksen, Kim; Karsdal, Morten A

2018-01-01

The extracellular matrix (ECM) plays a vital role in maintaining normal tissue function. Collagens are major components of the ECM and there is a tight equilibrium between degradation and formation of these proteins ensuring tissue health and homeostasis. As a consequence of tissue turnover, small collagen fragments are released into the circulation, which act as important biomarkers in the study of certain tissue-related remodeling factors in health and disease. The aim of this study was to establish an age-related collagen turnover profile of the main collagens of the interstitial matrix (type I and III collagen) and basement membrane (type IV collagen) in healthy men and women. By using well-characterized competitive ELISA-assays, we assessed specific fragments of degraded (C1M, C3M, C4M) and formed (PINP, Pro-C3, P4NP7S) type I, III and IV collagen in serum from 617 healthy men and women ranging in ages from 22 to 86. Subjects were divided into 5-year age groups according to their sex and age. Groups were compared using Kruskal-Wallis adjusted for Dunn's multiple comparisons test and Mann-Whitney t-test. Age-specific changes in collagen turnover was most profound for type I collagen. PINP levels decreased in men with advancing age, whereas in women, the level decreased in early adulthood followed by an increase around the age of menopause (age 40-60). Sex-specific changes in type I, III and IV collagen turnover was present at the age around menopause (age 40-60) with women having an increased turnover. In summary, collagen turnover is affected by age and sex with the interstitial matrix and the basement membrane being differently regulated. The observed changes needs to be accounted for when measuring ECM related biomarkers in clinical studies.

Gentamicin induces functional type VII collagen in recessive dystrophic epidermolysis bullosa patients

PubMed Central

Woodley, David T.; Cogan, Jon; Hou, Yingping; Lyu, Chao; Marinkovich, M. Peter; Keene, Douglas

2017-01-01

BACKGROUND. Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable disease caused by mutations in the gene encoding type VII collagen, the major component of anchoring fibrils (AF). We previously demonstrated that gentamicin produced functional type VII collagen in RDEB cells harboring nonsense mutations. Herein, we determined whether topical or intradermal gentamicin administration induces type VII collagen and AFs in RDEB patients. METHODS. A double-blind, placebo-controlled pilot trial assessed safety and efficacy of topical and intradermal gentamicin in 5 RDEB patients with nonsense mutations. The topical arm tested 0.1% gentamicin ointment or placebo application 3 times daily at 2 open erosion sites for 2 weeks. The intradermal arm tested daily intradermal injection of gentamicin solution (8 mg) or placebo into 2 intact skin sites for 2 days in 4 of 5 patients. Primary outcomes were induction of type VII collagen and AFs at the test sites and safety assessment. A secondary outcome assessed wound closure of topically treated erosions. RESULTS. Both topical and intradermal gentamicin administration induced type VII collagen and AFs at the dermal-epidermal junction of treatment sites. Newly created type VII collagen varied from 20% to 165% of that expressed in normal human skin and persisted for 3 months. Topical gentamicin corrected dermal-epidermal separation, improved wound closure, and reduced blister formation. There were no untoward side effects from gentamicin treatments. Type VII collagen induction did not generate anti–type VII collagen autoantibodies in patients’ blood or skin. CONCLUSION. Topical and intradermal gentamicin suppresses nonsense mutations and induces type VII collagen and AFs in RDEB patients. Gentamicin therapy may provide a readily available treatment for RDEB patients with nonsense mutations. TRIAL REGISTRATION. ClinicalTrials.gov NCT02698735. FUNDING. Epidermolysis Bullosa Research Partnership, Epidermolysis Bullosa Medical Research Foundation, NIH, and VA Merit Award. PMID:28691931

[The genetics of collagen diseases].

PubMed

Kaplan, J; Maroteaux, P; Frezal, J

1986-01-01

Heritable disorders of collagen include Ehler-Danlos syndromes (11 types are actually known), Larsen syndrome and osteogenesis imperfecta. Their clinical, genetic and biochemical features are reviewed. Marfan syndrome is closely related to heritable disorders of collagen.

Shortening of the Lactobacillus paracasei subsp. paracasei BGNJ1-64 AggLb Protein Switches Its Activity from Auto-aggregation to Biofilm Formation.

PubMed

Miljkovic, Marija; Bertani, Iris; Fira, Djordje; Jovcic, Branko; Novovic, Katarina; Venturi, Vittorio; Kojic, Milan

2016-01-01

AggLb is the largest (318.6 kDa) aggregation-promoting protein of Lactobacillus paracasei subsp. paracasei BGNJ1-64 responsible for forming large cell aggregates, which causes auto-aggregation, collagen binding and pathogen exclusion in vitro. It contains an N-terminus leader peptide, followed by six successive collagen binding domains, 20 successive repeats (CnaB-like domains) and an LPXTG sorting signal at the C-terminus for cell wall anchoring. Experimental information about the roles of the domains of AggLb is currently unknown. To define the domain that confers cell aggregation and the key domains for interactions of specific affinity between AggLb and components of the extracellular matrix, we constructed a series of variants of the aggLb gene and expressed them in Lactococcus lactis subsp. lactis BGKP1-20 using a lactococcal promoter. All of the variants contained a leader peptide, an inter collagen binding-CnaB domain region (used to raise an anti-AggLb antibody), an anchor domain and a different number of collagen binding and CnaB-like domains. The role of the collagen binding repeats of the N-terminus in auto-aggregation and binding to collagen and fibronectin was confirmed. Deletion of the collagen binding repeats II, III, and IV resulted in a loss of the strong auto-aggregation, collagen and fibronectin binding abilities whereas the biofilm formation capability was increased. The strong auto-aggregation, collagen and fibronectin binding abilities of AggLb were negatively correlated to biofilm formation.

Cigarette smoke extract induces select matrix metalloproteinases and integrin expression in periodontal ligament fibroblasts.

PubMed

Bulmanski, Zachary; Brady, Matthew; Stoute, Diana; Lallier, Thomas E

2012-06-01

The periodontal ligament (PDL) is the connective tissue that anchors the cementum of the teeth to the alveolar bone. PDL fibroblasts are responsible for the production of collagen and remodeling of the PDL. Periodontal disease is increased among smokers in both incidence and severity. This study examines the direct effect of smoking on PDL fibroblasts and their production of various matrix components and remodeling enzymes. PDL cells were plated for 1 day and then treated with various concentrations of cigarette smoke extract (CSE). Survival of PDL cells was quantified after exposure to CSE, and their ability to contract three-dimensional collagen gels was examined. Changes in transcript expression after CSE treatment was compared using reverse transcription-polymerase chain reaction analysis for matrix metalloproteinases (MMPs), collagens, and integrins. Treatment with CSE-induced cell death at concentrations of ≥5%. PDL-cell-induced collagen gel contraction was reduced at concentrations of 1.5% CSE. Treatment with CSE selectively increased the expression of collagen Vα3 and decreased collagen XIα1. CSE increased the expression of MMP1 and MMP3 and, to a lesser extent, MMP2 and MMP8. CSE also increased the expression of integrins α1, α2, and α10 (collagen receptors) and α9 (a tenascin receptor). This study shows that cigarette smoking has local effects on the cells of the PDL. CSE reduced survival of PDL cells and their ability to contract collagen matrices. CSE also altered the expression of molecules known to provide the structural integrity of the ligament by altering collagen synthesis and remodeling as well as cell adhesion.

Atomic force imaging microscopy investigation of the interaction of ultraviolet radiation with collagen thin films

NASA Astrophysics Data System (ADS)

Stylianou, A.; Yova, D.; Alexandratou, E.; Petri, A.

2013-02-01

Collagen is the major fibrous protein in the extracellular matrix and consists a significant component of skin, bone, cartilage and tendon. Due to its unique properties, it has been widely used as scaffold or culture substrate for tissue regeneration or/and cell-substrate interaction studies. The ultraviolet light-collagen interaction investigations are crucial for the improvement of many applications such as that of the UV irradiation in the field of biomaterials, as sterilizing and photo-cross-linking method. The aim of this paper was to investigate the mechanisms of UV-collagen interactions by developing a collagen-based, well characterized, surface with controlled topography of collagen thin films in the nanoscale range. The methodology was to quantify the collagen surface modification induced on ultraviolet radiation and correlate it with changes induced in cells. Surface nanoscale characterization was performed by Atomic Force Microscopy (AFM) which is a powerful tool and offers quantitative and qualitative information with a non-destructive manner. In order to investigate cells behavior, the irradiated films were used for in vitro cultivation of human skin fibroblasts and the cells morphology, migration and alignment were assessed with fluorescence microscopy imaging and image processing methods. The clarification of the effects of UV light on collagen thin films and the way of cells behavior to the different modifications that UV induced to the collagen-based surfaces will contribute to the better understanding of cell-matrix interactions in the nanoscale and will assist the appropriate use of UV light for developing biomaterials.

Assessment of coronary plaque collagen with polarization sensitive optical coherence tomography (PS-OCT)

NASA Astrophysics Data System (ADS)

Giattina, Susanne D.; Courtney, Brian K.; Herz, Paul R.; Harman, Michelle; Shortkroff, Sonya; Stamper, Debra L.; Liu, Bin; Fujimoto, James G.; Brezinski, Mark E.

2006-02-01

Current evidence indicates that most plaques classified as vulnerable or ruptured plaques do not lead to unstable angina or myocardial infarction. Improved methods are needed to risk stratify plaques to identify those which lead to most acute coronary syndromes. Collagen depletion in the intima overlying lipid collections appears to be a critical component of unstable plaques. In this study, we use polarization sensitive optical coherence tomography (PS-OCT) for the assessment of coronary plaque collagen. Collagen is birefringent, meaning that different polarization states travel through it at different velocities. Changes in PS-OCT images are a measure of tissue birefringence. Twenty-two coronary artery segments were imaged with PS-OCT and analyzed by picrosirius staining (a measure of collagen intensity and fiber size) and trichrome blue. The regression plot between PS-OCT changes and measured collagen yielded a correlation coefficient value of 0.475 (p<0.002). Good correlation was noted between two blinded investigators both with respect to PS-OCT measurements as well as luminosity as assessed by picrosirius. The predictive value of a PS-OCT measurement of negligible birefringence (less than 33% change) for minimal collagen was 93% while the predictive value of high birefringence (greater than 66% change) for high collagen concentrations was 89%. The effect of fiber type (chemical composition) was minimal relative to the effect due to fiber concentration. The capability of PS-OCT to assess plaque collagen content, in addition to its ability to generate high resolution structural assessments, make it a potentially powerful technology for identifying high risk plaques.

Electrochemically Preadsorbed Collagen Promotes Adult Human Mesenchymal Stem Cell Adhesion

PubMed Central

Benavidez, Tomás E.; Wechsler, Marissa E.; Farrer, Madeleine M.; Bizios, Rena

2016-01-01

The present article reports on the effect of electric potential on the adsorption of collagen type I (the most abundant component of the organic phase of bone) onto optically transparent carbon electrodes (OTCE) and its mediation on subsequent adhesion of adult, human, mesenchymal stem cells (hMSCs). For this purpose, adsorption of collagen type I was investigated as a function of the protein concentration (0.01, 0.1, and 0.25 mg/mL) and applied potential (open circuit potential [OCP; control], +400, +800, and +1500 mV). The resulting substrate surfaces were characterized using spectroscopic ellipsometry, atomic force microscopy, and cyclic voltammetry. Adsorption of collagen type I onto OTCE was affected by the potential applied to the sorbent surface and the concentration of protein. The higher the applied potential and protein concentration, the higher the adsorbed amount (Γcollagen). It was also observed that the application of potential values higher than +800 mV resulted in the oxidation of the adsorbed protein. Subsequent adhesion of hMSCs on the OTCEs (precoated with the collagen type I films) under standard cell culture conditions for 2 h was affected by the extent of collagen preadsorbed onto the OTCE substrates. Specifically, enhanced hMSCs adhesion was observed when the Γcollagen was the highest. When the collagen type I was oxidized (under applied potential equal to +1500 mV), however, hMSCs adhesion was decreased. These results provide the first correlation between the effects of electric potential on protein adsorption and subsequent modulation of anchorage-dependent cell adhesion. PMID:26549607

Abnormal Type I Collagen Post-translational Modification and Crosslinking in a Cyclophilin B KO Mouse Model of Recessive Osteogenesis Imperfecta

PubMed Central

Cabral, Wayne A.; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R.; Chang, Weizhong; Perosky, Joseph E.; Makareeva, Elena N.; Mertz, Edward L.; Leikin, Sergey; Tomer, Kenneth B.; Kozloff, Kenneth M.; Eyre, David R.; Yamauchi, Mitsuo; Marini, Joan C.

2014-01-01

Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib−/− mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2–11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib−/− fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink pattern was associated with decreased collagen deposition into matrix in culture, altered fibril structure in tissue, and reduced bone strength. These studies demonstrate novel consequences of the indirect regulatory effect of CyPB on collagen hydroxylation, impacting collagen glycosylation, crosslinking and fibrillogenesis, which contribute to maintaining bone mechanical properties. PMID:24968150

Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.

PubMed

Cabral, Wayne A; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R; Chang, Weizhong; Perosky, Joseph E; Makareeva, Elena N; Mertz, Edward L; Leikin, Sergey; Tomer, Kenneth B; Kozloff, Kenneth M; Eyre, David R; Yamauchi, Mitsuo; Marini, Joan C

2014-06-01

Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink pattern was associated with decreased collagen deposition into matrix in culture, altered fibril structure in tissue, and reduced bone strength. These studies demonstrate novel consequences of the indirect regulatory effect of CyPB on collagen hydroxylation, impacting collagen glycosylation, crosslinking and fibrillogenesis, which contribute to maintaining bone mechanical properties.

Loss of SPARC dysregulates basal lamina assembly to disrupt larval fat body homeostasis in Drosophila melanogaster.

PubMed

Shahab, Jaffer; Baratta, Cristina; Scuric, Bianca; Godt, Dorothea; Venken, Koen J T; Ringuette, Maurice J

2015-04-01

SPARC is a collagen-binding glycoprotein whose functions during early development are unknown. We previously reported that SPARC is expressed in Drosophila by hemocytes and the fat body (FB) and enriched in basal laminae (BL) surrounding tissues, including adipocytes. We sought to explore if SPARC is required for proper BL assembly in the FB. SPARC deficiency leads to larval lethality, associated with remodeling of the FB. In the absence of SPARC, FB polygonal adipocytes assume a spherical morphology. Loss-of-function clonal analyses revealed a cell-autonomous accumulation of BL components around mutant cells that include collagen IV (Col lV), Laminin, and Perlecan. Ultrastructural analyses indicate SPARC-deficient adipocytes are surrounded by an aberrant accumulation of a fibrous extracellular matrix. Our data indicate a critical requirement for SPARC for the proper BL assembly in Drosophila FB. Since Col IV within the BL is a prime determinant of cell shape, the rounded appearance of SPARC-deficient adipocytes is due to aberrant assembly of Col IV. © 2014 Wiley Periodicals, Inc.

Methods for the visualization and analysis of extracellular matrix protein structure and degradation.

PubMed

Leonard, Annemarie K; Loughran, Elizabeth A; Klymenko, Yuliya; Liu, Yueying; Kim, Oleg; Asem, Marwa; McAbee, Kevin; Ravosa, Matthew J; Stack, M Sharon

2018-01-01

This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders. © 2018 Elsevier Inc. All rights reserved.

Tendon and ligament injuries: the genetic component

PubMed Central

September, Alison V; Schwellnus, Martin P; Collins, Malcolm

2007-01-01

Tendons and ligaments within the upper and lower limbs are some of the more common sites of musculoskeletal injuries during physical activity. Several extrinsic and intrinsic factors have been shown to be associated with these injuries. More recently, studies have suggested that there is also, at least in part, a genetic component to the Achilles tendon, rotator cuff and anterior cruciate ligament injuries. However, specific genes have not been suggested to be associated with rotator cuff or anterior cruciate ligament injuries. Sequence variants of the tenascin C (TNC) gene, on the other hand, have been shown to be associated with Achilles tendinopathies and Achilles tendon ruptures, whereas a variant of the collagen V α 1 (COL5A1) gene has also been shown to be associated with Achilles tendinopathies. Both genes encode for important structural components of tendons and ligaments. The COL5A1 gene encodes for a component of type V collagen, which has an important role in regulating collagen fibre assembly and fibre diameters. The TNC gene, on the other hand, encodes for TNC, which regulates the tissue's response to mechanical load. To date, only variants in two genes have been shown to be associated with Achilles tendon injuries. In addition, although specific genes have not been identified, investigators have suggested that there is also a genetic component to both rotator cuff and anterior cruciate ligament injuries. In future, specific genotypes associated with increased risk of injury to specific tendons and ligaments can prevent these injuries by identifying individuals at higher risk. PMID:17261551

Deficiency of CRTAP in non-lethal recessive osteogenesis imperfecta reduces collagen deposition into matrix.

PubMed

Valli, M; Barnes, A M; Gallanti, A; Cabral, W A; Viglio, S; Weis, M A; Makareeva, E; Eyre, D; Leikin, S; Antoniazzi, F; Marini, J C; Mottes, M

2012-11-01

Deficiency of any component of the ER-resident collagen prolyl 3-hydroxylation complex causes recessive osteogenesis imperfecta (OI). The complex modifies the α1(I)Pro986 residue and contains cartilage-associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and cyclophilin B (CyPB). Fibroblasts normally secrete about 10% of CRTAP. Most CRTAP mutations cause a null allele and lethal type VII OI. We identified a 7-year-old Egyptian boy with non-lethal type VII OI and investigated the effects of his null CRTAP mutation on collagen biochemistry, the prolyl 3-hydroxylation complex, and collagen in extracellular matrix. The proband is homozygous for an insertion/deletion in CRTAP (c.118_133del16insTACCC). His dermal fibroblasts synthesize fully overmodified type I collagen, and 3-hydroxylate only 5% of α1(I)Pro986. CRTAP transcripts are 10% of control. CRTAP protein is absent from proband cells, with residual P3H1 and normal CyPB levels. Dermal collagen fibril diameters are significantly increased. By immunofluorescence of long-term cultures, we identified a severe deficiency (10-15% of control) of collagen deposited in extracellular matrix, with disorganization of the minimal fibrillar network. Quantitative pulse-chase experiments corroborate deficiency of matrix deposition, rather than increased matrix turnover. We conclude that defects of extracellular matrix, as well as intracellular defects in collagen modification, contribute to the pathology of type VII OI. © 2011 John Wiley & Sons A/S.

Enhancement of neurite outgrowth in neuron cancer stem cells by growth on 3-D collagen scaffolds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Chih-Hao; Neurosurgery, Department of Surgery, Kaohsiung Veterans General Hospital, Taiwan, ROC; Department of Biomedical Engineering, I-Shou University, Taiwan, ROC

Highlights: Black-Right-Pointing-Pointer Neuron cancer stem cells (NCSCs) behave high multiply of growth on collagen scaffold. Black-Right-Pointing-Pointer Enhancement of NCSCs neurite outgrowth on porous collagen scaffold. Black-Right-Pointing-Pointer 3-D collagen culture of NCSCs shows an advance differentiation than 2-D culture. -- Abstract: Collagen is one component of the extracellular matrix that has been widely used for constructive remodeling to facilitate cell growth and differentiation. The 3-D distribution and growth of cells within the porous scaffold suggest a clinical significance for nerve tissue engineering. In the current study, we investigated proliferation and differentiation of neuron cancer stem cells (NCSCs) on a 3-D porousmore » collagen scaffold that mimics the natural extracellular matrix. We first generated green fluorescence protein (GFP) expressing NCSCs using a lentiviral system to instantly monitor the transitions of morphological changes during growth on the 3-D scaffold. We found that proliferation of GFP-NCSCs increased, and a single cell mass rapidly grew with unrestricted expansion between days 3 and 9 in culture. Moreover, immunostaining with neuronal nuclei (NeuN) revealed that NCSCs grown on the 3-D collagen scaffold significantly enhanced neurite outgrowth. Our findings confirmed that the 80 {mu}m porous collagen scaffold could enhance attachment, viability and differentiation of the cancer neural stem cells. This result could provide a new application for nerve tissue engineering and nerve regeneration.« less

Internal strain drives spontaneous periodic buckling in collagen and regulates remodeling.

PubMed

Dittmore, Andrew; Silver, Jonathan; Sarkar, Susanta K; Marmer, Barry; Goldberg, Gregory I; Neuman, Keir C

2016-07-26

Fibrillar collagen, an essential structural component of the extracellular matrix, is remarkably resistant to proteolysis, requiring specialized matrix metalloproteinases (MMPs) to initiate its remodeling. In the context of native fibrils, remodeling is poorly understood; MMPs have limited access to cleavage sites and are inhibited by tension on the fibril. Here, single-molecule recordings of fluorescently labeled MMPs reveal cleavage-vulnerable binding regions arrayed periodically at ∼1-µm intervals along collagen fibrils. Binding regions remain periodic even as they migrate on the fibril, indicating a collective process of thermally activated and self-healing defect formation. An internal strain relief model involving reversible structural rearrangements quantitatively reproduces the observed spatial patterning and fluctuations of defects and provides a mechanism for tension-dependent stabilization of fibrillar collagen. This work identifies internal-strain-driven defects that may have general and widespread regulatory functions in self-assembled biological filaments.

Combining nano-physical and computational investigations to understand the nature of "aging" in dermal collagen.

PubMed

Ahmed, Tarek; Nash, Anthony; Clark, Kristina En; Ghibaudo, Marion; de Leeuw, Nora H; Potter, Anne; Stratton, Richard; Birch, Helen L; Enea Casse, Ramona; Bozec, Laurent

2017-01-01

The extracellular matrix of the dermis is a complex, dynamic system with the various dermal components undergoing individual physiologic changes as we age. Age-related changes in the physical properties of collagen were investigated in particular by measuring the effect of aging, most likely due to the accumulation of advanced glycation end product (AGE) cross-links, on the nanomechanical properties of the collagen fibril using atomic force microscope nano-indentation. An age-related decrease in the Young's modulus of the transverse fibril was observed (from 8.11 to 4.19 GPa in young to old volunteers, respectively, P <0.001). It is proposed that this is due to a change in the fibril density caused by age-related differences in water retention within the fibrils. The new collagen-water interaction mechanism was verified by electronic structure calculations, showing it to be energetically feasible.

Epidermolysis Bullosa Acquitsita

PubMed Central

Gupta, Rishu; Woodley, David T.; Chen, Mei

2011-01-01

EBA is a rare, acquired, chronic subepidermal bullous disease of the skin and mucosa characterized by autoantibodies to type VII collagen structures, a major component of anchoring fibrils, that attach the epidermis onto the dermis. EBA patients have tissue-bound as well as circulating anti-type VII collagen autoantibodies that attack type VII collagen and result in a reduction or perturbation of normally functioning anchoring fibrils. Patients with EBA have skin fragility, blisters, erosions, scars, milia, and nail loss: all features reminiscent of genetic dystrophic epidermolysis bullosa. These anti-type VII collagen antibodies are “pathogenic” because when injected into mice, the mice develop an EBA-like blistering disease. In addition to the classical mechanobullous presentation, EBA also has several other distinct clinical syndromes similar to bullous pemphigoid, Brunsting-Perry pemphigoid, or cicatricial pemphigoid. Although treatment for EBA is often unsatisfactory, some therapeutic success has been achieved with colchicine, dapsone, plasmaphoresis, photopheresis, infliximab, and intravenous immunoglobulin. PMID:22137228

Physics of soft hyaluronic acid-collagen type II double network gels

NASA Astrophysics Data System (ADS)

Morozova, Svetlana; Muthukumar, Murugappan

2015-03-01

Many biological hydrogels are made up of multiple interpenetrating, charged components. We study the swelling, elastic diffusion, mechanical, and optical behaviors of 100 mol% ionizable hyaluronic acid (HA) and collagen type II fiber networks. Dilute, 0.05-0.5 wt% hyaluronic acid networks are extremely sensitive to solution salt concentration, but are stable at pH above 2. When swelled in 0.1M NaCl, single-network hyaluronic acid gels follow scaling laws relevant to high salt semidilute solutions; the elastic shear modulus G' and diffusion constant D scale with the volume fraction ϕ as G' ~ϕ 9 / 4 and D ~ϕ 3 / 4 , respectively. With the addition of a collagen fiber network, we find that the hyaluronic acid network swells to suspend the rigid collagen fibers, providing extra strength to the hydrogel. Results on swelling equilibria, elasticity, and collective diffusion on these double network hydrogels will be presented.

Internal strain drives spontaneous periodic buckling in collagen and regulates remodeling

PubMed Central

Dittmore, Andrew; Silver, Jonathan; Sarkar, Susanta K.; Marmer, Barry; Goldberg, Gregory I.; Neuman, Keir C.

2016-01-01

Fibrillar collagen, an essential structural component of the extracellular matrix, is remarkably resistant to proteolysis, requiring specialized matrix metalloproteinases (MMPs) to initiate its remodeling. In the context of native fibrils, remodeling is poorly understood; MMPs have limited access to cleavage sites and are inhibited by tension on the fibril. Here, single-molecule recordings of fluorescently labeled MMPs reveal cleavage-vulnerable binding regions arrayed periodically at ∼1-µm intervals along collagen fibrils. Binding regions remain periodic even as they migrate on the fibril, indicating a collective process of thermally activated and self-healing defect formation. An internal strain relief model involving reversible structural rearrangements quantitatively reproduces the observed spatial patterning and fluctuations of defects and provides a mechanism for tension-dependent stabilization of fibrillar collagen. This work identifies internal–strain-driven defects that may have general and widespread regulatory functions in self-assembled biological filaments. PMID:27402741

Long-term natural history and complications of collagenous colitis.

PubMed

Freeman, Hugh J

2012-09-01

Microscopic forms of colitis have been described, including collagenous colitis, a possibly heterogeneous disorder. Collagenous colitis most often appears to have an entirely benign clinical course that usually responds to limited treatment. Sometimes significant extracolonic disorders, especially arthritis, spondylitis, thyroiditis and skin disorders, such as pyoderma gangrenosum, dominate the clinical course and influence the treatment strategy. However, rare fatalities have been reported and several complications, some severe, have been attributed directly to the colitis. Toxic colitis and toxic megacolon may develop. Concomitant gastric and small intestinal inflammatory disorders have been described including celiac disease and more extensive collagenous inflammatory disease. Colonic ulceration has been associated with the use of nonsteroidal anti-inflammatory drugs, while other forms of inflammatory bowel disease, including ulcerative colitis and Crohn disease, may evolve directly from collagenous colitis. Submucosal 'dissection', colonic fractures, or mucosal tears and perforation, possibly from air insufflation during colonoscopy, have been reported. Similar changes may result from increased intraluminal pressures that may occur during radiological imaging of the colon. Neoplastic disorders of the colon may also occur during the course of collagenous colitis, including colon carcinoma and neuroendocrine tumours (ie, carcinoids). Finally, lymphoproliferative disease has been reported.

Collagenous gastritis: a morphologic and immunohistochemical study of 40 patients.

PubMed

Arnason, Thomas; Brown, Ian S; Goldsmith, Jeffrey D; Anderson, William; O'Brien, Blake H; Wilson, Claire; Winter, Harland; Lauwers, Gregory Y

2015-04-01

Collagenous gastritis is a rare condition defined histologically by a superficial subepithelial collagen layer. This study further characterizes the morphologic spectrum of collagenous gastritis by evaluating a multi-institutional series of 40 patients (26 female and 14 male). The median age at onset was 16 years (range 3-89 years), including 24 patients (60%) under age 18. Twelve patients (30%) had associated celiac disease, collagenous sprue, or collagenous colitis. Hematoxylin and eosin slides were reviewed in biopsies from all patients and tenascin, gastrin, eotaxin, and IgG4/IgG immunohistochemical stains were applied to a subset. The distribution of subepithelial collagen favored the body/fundus in pediatric patients and the antrum in adults. There were increased surface intraepithelial lymphocytes (>25 lymphocytes/100 epithelial cells) in five patients. Three of these patients had associated celiac and/or collagenous sprue/colitis, while the remaining two had increased duodenal lymphocytosis without specific etiology. An eosinophil-rich pattern (>30 eosinophils/high power field) was seen in 21/40 (52%) patients. Seven patients' biopsies demonstrated atrophy of the gastric corpus mucosa. Tenascin immunohistochemistry highlighted the subepithelial collagen in all 21 specimens evaluated and was a more sensitive method of collagen detection in biopsies from two patients with subtle subepithelial collagen. No increased eotaxin expression was identified in 16 specimens evaluated. One of the twenty-three biopsies tested had increased IgG4-positive cells (100/high power field) with an IgG4/IgG ratio of 55%. In summary, collagenous gastritis presents three distinct histologic patterns including a lymphocytic gastritis-like pattern, an eosinophil-rich pattern, and an atrophic pattern. Eotaxin and IgG4 were not elevated enough to implicate these pathways in the pathogenesis. Tenascin immunohistochemistry can be used as a sensitive method of collagen detection.

Distribution of Basement Membrane Molecules, Laminin and Collagen Type IV, in Normal and Degenerated Cartilage Tissues.

PubMed

Foldager, Casper Bindzus; Toh, Wei Seong; Gomoll, Andreas H; Olsen, Bjørn Reino; Spector, Myron

2014-04-01

The objective of the present study was to investigate the presence and distribution of 2 basement membrane (BM) molecules, laminin and collagen type IV, in healthy and degenerative cartilage tissues. Normal and degenerated tissues were obtained from goats and humans, including articular knee cartilage, the intervertebral disc, and meniscus. Normal tissue was also obtained from patella-tibial enthesis in goats. Immunohistochemical analysis was performed using anti-laminin and anti-collagen type IV antibodies. Human and goat skin were used as positive controls. The percentage of cells displaying the pericellular presence of the protein was graded semiquantitatively. When present, laminin and collagen type IV were exclusively found in the pericellular matrix, and in a discrete layer on the articulating surface of normal articular cartilage. In normal articular (hyaline) cartilage in the human and goat, the proteins were found co-localized pericellularly. In contrast, in human osteoarthritic articular cartilage, collagen type IV but not laminin was found in the pericellular region. Nonpathological fibrocartilaginous tissues from the goat, including the menisci and the enthesis, were also positive for both laminin and collagen type IV pericellularly. In degenerated fibrocartilage, including intervertebral disc, as in degenerated hyaline cartilage only collagen type IV was found pericellularly around chondrocytes but with less intense staining than in non-degenerated tissue. In calcified cartilage, some cells were positive for laminin but not type IV collagen. We report differences in expression of the BM molecules, laminin and collagen type IV, in normal and degenerative cartilaginous tissues from adult humans and goats. In degenerative tissues laminin is depleted from the pericellular matrix before collagen type IV. The findings may inform future studies of the processes underlying cartilage degeneration and the functional roles of these 2 extracellular matrix proteins, normally associated with BM.

Ex vivo multiscale quantitation of skin biomechanics in wild-type and genetically-modified mice using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Bancelin, Stéphane; Lynch, Barbara; Bonod-Bidaud, Christelle; Ducourthial, Guillaume; Psilodimitrakopoulos, Sotiris; Dokládal, Petr; Allain, Jean-Marc; Schanne-Klein, Marie-Claire; Ruggiero, Florence

2015-12-01

Soft connective tissues such as skin, tendon or cornea are made of about 90% of extracellular matrix proteins, fibrillar collagens being the major components. Decreased or aberrant collagen synthesis generally results in defective tissue mechanical properties as the classic form of Elhers-Danlos syndrome (cEDS). This connective tissue disorder is caused by mutations in collagen V genes and is mainly characterized by skin hyperextensibility. To investigate the relationship between the microstructure of normal and diseased skins and their macroscopic mechanical properties, we imaged and quantified the microstructure of dermis of ex vivo murine skin biopsies during uniaxial mechanical assay using multiphoton microscopy. We used two genetically-modified mouse lines for collagen V: a mouse model for cEDS harboring a Col5a2 deletion (a.k.a. pN allele) and the transgenic K14-COL5A1 mice which overexpress the human COL5A1 gene in skin. We showed that in normal skin, the collagen fibers continuously align with stretch, generating the observed increase in mechanical stress. Moreover, dermis from both transgenic lines exhibited altered collagen reorganization upon traction, which could be linked to microstructural modifications. These findings show that our multiscale approach provides new crucial information on the biomechanics of dermis that can be extended to all collagen-rich soft tissues.

Hydrogels for lung tissue engineering: Biomechanical properties of thin collagen-elastin constructs.

PubMed

Dunphy, Siobhán E; Bratt, Jessica A J; Akram, Khondoker M; Forsyth, Nicholas R; El Haj, Alicia J

2014-10-01

In this study, collagen-elastin constructs were prepared with the aim of producing a material capable of mimicking the mechanical properties of a single alveolar wall. Collagen has been used in a wide range of tissue engineering applications; however, due to its low mechanical properties its use is limited to non load-bearing applications without further manipulation using methods such as cross-linking or mechanical compression. Here, it was hypothesised that the addition of soluble elastin to a collagen hydrogel could improve its mechanical properties. Hydrogels made from collagen only and collagen plus varying amounts elastin were prepared. Young׳s modulus of each membrane was measured using the combination of a non-destructive indentation and a theoretical model previously described. An increase in Young׳s modulus was observed with increasing concentration of elastin. The use of non-destructive indentation allowed for online monitoring of the elastic moduli of cell-seeded constructs over 8 days. The addition of lung fibroblasts into the membrane increased the stiffness of the hydrogels further and cell-seeded collagen hydrogels were found to have a stiffness equal to the theoretical value for a single alveolar wall (≈5kPa). Through provision of some of the native extracellular matrix components of the lung parenchyma these scaffolds may be able to provide an initial building block toward the regeneration of new functional lung tissue. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of Silicone on the Collagen Fibrillogenesis and Stability

PubMed Central

Kadziński, Leszek; Prokopowicz, Magdalena; Jakóbkiewicz-Banecka, Joanna; Gabig-Cimińska, Magdalena; Łukasiak, Jerzy; Banecki, Bogdan

2015-01-01

Collagen, the most abundant protein in mammals, is able to form fibrils, which have central role in tissue repair, fibrosis, and tumor invasion. As a component of skin, tendons, and cartilages, this protein contacts with any implanted materials. An inherent problem associated with implanted prostheses is their propensity to be coated with host proteins shortly after implantation. Also, silicone implants undergoing relatively long periods of contact with blood can lead to formation of thrombi and emboli. In this paper, we demonstrate the existence of interactions between siloxanes and collagen. Low-molecular-weight cyclic siloxane (hexamethylcyclotrisiloxane—D3) and polydimethylsiloxanes (PDMS) forming linear chains, ranging in viscosity from 20 to 12,000 cSt, were analyzed. We show that D3 as well as short-chain PDMS interact with collagen, resulting in a decrease in fibrillogenesis. However, loss of collagen native structure does not occur because of these interactions. Rather, collagen seems to be sequestered in its native form in an interlayer formed by collagen–siloxane complexes. On the other hand, silicone molecules with longer chains (i.e., PDMS with viscosity of 1000 and 12,000 cSt, the highest viscosity analyzed here) demonstrate little interaction with this protein and do not seem to affect collagen activity. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1275–1281, 2015 PMID:25589402

Immunofluorescence localization of dissociation supernatant and extracellular matrix components in Lytechinus pictus sectioned embryos. M.S. Thesis

NASA Technical Reports Server (NTRS)

Garciaflack, Ana Leticia

1988-01-01

Indirect immunofluorescence was used to localize specific extracellular components in embryos of the sea urchin Lytechinus pictus. Hyalin and S2 (a group of components found in the disaggregation supernatant from Strongylocentrotus purpuratus blastulae) were uniformly present at all stages (unfertilized up to 32 hr) except hyalin could not be detected at the 12 hour early blastula stage. Laminin was found in 16 cell, 32 cell, 6 hour, 18 hour, 24 hour, and 32 hour stages, with especially bright fluorescence at 18 hours. Collagen I was present at all stages (freshly fertilized up to 32 hour) except little was detected at 12 hours. Fibronectin was uniformly present in blastocoelar fibers stained with anto-collagen I and anti-fibronectin. These results were compared with those for S. purpuratus to produce an overview of the localization of specific extracellular matrix components during development of two species of sea urchins. The results set the stage for future studies that will examine the function of these components at the various developmental stages.

Colorectal laterally spreading tumors show characteristic expression of cell polarity factors, including atypical protein kinase C λ/ι, E-cadherin, β-catenin and basement membrane component.

PubMed

Ichikawa, Yasushi; Nagashima, Yoji; Morioka, Kaori; Akimoto, Kazunori; Kojima, Yasuyuki; Ishikawa, Takashi; Goto, Ayumu; Kobayashi, Noritoshi; Watanabe, Kazuteru; Ota, Mitsuyoshi; Fujii, Shoichi; Kawamata, Mayumi; Takagawa, Ryo; Kunizaki, Chikara; Takahashi, Hirokazu; Nakajima, Atsushi; Maeda, Shin; Shimada, Hiroshi; Inayama, Yoshiaki; Ohno, Shigeo; Endo, Itaru

2014-09-01

Colorectal flat-type tumors include laterally spreading tumors (LSTs) and flat depressed-type tumors. The former of which shows a predominant lateral spreading growth rather than an invasive growth. The present study examined the morphological characteristics of LSTs, in comparison with polypoid- or flat depressed-type tumors, along with the expression of atypical protein kinase C (aPKC) λ/ι, a pivotal cell polarity regulator, and the hallmarks of cell polarity, as well as with type IV collagen, β-catenin and E-cadherin. In total, 37 flat-type (24 LSTs and 13 flat depressed-type tumors) and 20 polypoid-type colorectal tumors were examined. The LSTs were classified as 15 LST adenoma (LST-A) and nine LST cancer in adenoma (LST-CA). An immunohistochemical examination was performed on aPKC λ/ι, type IV collagen, β-catenin and E-cadherin. The LST-A and -CA showed a superficial replacing growth pattern, with expression of β-catenin and E-cadherin in the basolateral membrane and type IV collagen along the basement membrane. In addition, 86.6% of LST-A and 55.6% of LST-CA showed aPKC λ/ι expression of 1+ (weak to normal intensity staining in the cytoplasm compared with the normal epithelium). Furthermore, ~45% of the polypoid-type adenomas showed 2+ (moderate intensity staining in the cytoplasm and/or nucleus) and 66.7% of the polypoid-type cancer in adenoma were 3+ (strong intensity staining in the cytoplasm and nucleus). A statistically significant positive correlation was observed between the expression of aPKC λ/ι and β-catenin (r=0.842; P<0.001), or type IV collagen (r=0.823; P<0.001). The LSTs showed a unique growth pattern, different from the expanding growth pattern presented by a polypoid tumor and invasive cancer. The growth characteristics of LST appear to be caused by adequate coexpression of β-catenin, type IV collagen and aPKC λ/ι.

Frequency-dependent micromechanics of cellularized biopolymer networks

NASA Astrophysics Data System (ADS)

Jones, Chris; Kim, Jihan; McIntyre, David; Sun, Bo

Mechanical interactions between cells and the extracellular matrix (ECM) influence many cellular behaviors such as growth, differentiation, and migration. These are dynamic processes in which the cells actively remodel the ECM. Reconstituted collagen gel is a common model ECM for studying cell-ECM interactions in vitro because collagen is the most abundant component of mammalian ECM and gives the ECM its material stiffness. We embed micron-sized particles in collagen and use holographic optical tweezers to apply forces to the particles in multiple directions and over a range of frequencies up to 10 Hz. We calculate the local compliance and show that it is dependent on both the direction and frequency of the applied force. Performing the same measurement on many particles allows us to characterize the spatial inhomogeneity of the mechanical properties and shows that the compliance decreases at higher frequencies. Performing these measurements on cell-populated collagen gels shows that cellular remodeling of the ECM changes the mechanical properties of the collagen and we investigate whether this change is dependent on the local strain and distance from nearby cells.

The triple helix of collagens - an ancient protein structure that enabled animal multicellularity and tissue evolution.

PubMed

Fidler, Aaron L; Boudko, Sergei P; Rokas, Antonis; Hudson, Billy G

2018-04-09

The cellular microenvironment, characterized by an extracellular matrix (ECM), played an essential role in the transition from unicellularity to multicellularity in animals (metazoans), and in the subsequent evolution of diverse animal tissues and organs. A major ECM component are members of the collagen superfamily -comprising 28 types in vertebrates - that exist in diverse supramolecular assemblies ranging from networks to fibrils. Each assembly is characterized by a hallmark feature, a protein structure called a triple helix. A current gap in knowledge is understanding the mechanisms of how the triple helix encodes and utilizes information in building scaffolds on the outside of cells. Type IV collagen, recently revealed as the evolutionarily most ancient member of the collagen superfamily, serves as an archetype for a fresh view of fundamental structural features of a triple helix that underlie the diversity of biological activities of collagens. In this Opinion, we argue that the triple helix is a protein structure of fundamental importance in building the extracellular matrix, which enabled animal multicellularity and tissue evolution. © 2018. Published by The Company of Biologists Ltd.

TANGO1 builds a machine for collagen export by recruiting and spatially organizing COPII, tethers and membranes

PubMed Central

Santos, António JM; Foresti, Ombretta; Zhang, Chong; Garcia-Parajo, Maria F; Campelo, Felix

2018-01-01

Collagen export from the endoplasmic reticulum (ER) requires TANGO1, COPII coats, and retrograde fusion of ERGIC membranes. How do these components come together to produce a transport carrier commensurate with the bulky cargo collagen? TANGO1 is known to form a ring that corrals COPII coats, and we show here how this ring or fence is assembled. Our data reveal that a TANGO1 ring is organized by its radial interaction with COPII, and lateral interactions with cTAGE5, TANGO1-short or itself. Of particular interest is the finding that TANGO1 recruits ERGIC membranes for collagen export via the NRZ (NBAS/RINT1/ZW10) tether complex. Therefore, TANGO1 couples retrograde membrane flow to anterograde cargo transport. Without the NRZ complex, the TANGO1 ring does not assemble, suggesting its role in nucleating or stabilising this process. Thus, coordinated capture of COPII coats, cTAGE5, TANGO1-short, and tethers by TANGO1 assembles a collagen export machine at the ER. PMID:29513218

Metal Ion-Assembled Micro-Collagen Heterotrimers

PubMed Central

LeBruin, Lyndelle Toni; Banerjee, Sunandan; O'Rourke, Bruce Delany; Case, Martin Ashley

2011-01-01

Collagen mimetic peptides (CMPs) provide critical insight into the assembly, stability and structure of the triple helical collagen protein. The majority of natural fibrous collagens are aab or abc heterotrimers, yet few examples of heterotrimeric CMPs have been reported. Previously CMP heterotrimers have only been accessible by total syntheses or by introducing complementary interstrand electrostatic or steric interactions. Here we describe an abc CMP heterotrimer in which each contributing CMP consists of only three amino acids: glycine, proline and 4-hydroxyproline. Assembly of the heterotrimeric triple helix is directed by a combination of metal-ion coordination to set the relative register of the CMPs, and minimization of valence frustration to direct heterotrimerization. Assembly of the four-component mixture is facile and extremely rapid, and equilibration to the abc heterotrimer occurs within a few hours at modestly elevated temperatures. The melting temperatures of the metal-assembled collagen trimers are higher by some 30 °C than the apopeptide assemblies. Two iterations of the design are described, and the outcomes suggest possibilities for designing self-assembling abc and abb heterotrimers. PMID:21590759

Three-dimensional structure of human lamellar bone: the presence of two different materials and new insights into the hierarchical organization.

PubMed

Reznikov, Natalie; Shahar, Ron; Weiner, Steve

2014-02-01

Lamellar bone is the most common bone type in humans. The predominant components of individual lamellae are plywood-like arrays of mineralized collagen fibrils aligned in different directions. Using a dual-beam electron microscope and the Serial Surface View (SSV) method we previously identified a small, but significantly different layer in rat lamellar bone, namely a disordered layer with collagen fibrils showing little or no preferred orientation. Here we present a 3D structural analysis of 12 SSV volumes (25 complete lamellae) from femora of 3 differently aged human individuals. We identify the ordered and disordered motifs in human bone as in the rat, with several significant differences. The ordered motif shows two major preferred orientations, perpendicular to the long axis of the bone, and aligned within 10-20° of the long axis, as well as fanning arrays. At a higher organizational level, arrays of ordered collagen fibrils are organized into 'rods' around 2 to 3μm in diameter, and the long axes of these 'rods' are parallel to the lamellar boundaries. Human bone also contains a disordered component that envelopes the rods and fills in the spaces between them. The disordered motif is especially well-defined between adjacent layers of rods. The disordered motif and its interfibrillar substance stain heavily with osmium tetroxide and Alcian blue indicating the presence of another organic component in addition to collagen. The canalicular network is confined to the disordered material, along with voids and individual collagen fibrils, some of which are also aligned more or less perpendicular to the lamellar boundaries. The organization of the ordered fibril arrays into rods enveloped in the continuous disordered structure was not observed in rat lamellar bone. We thus conclude that human lamellar bone is comprised of two distinct materials, an ordered material and a disordered material, and contains an additional hierarchical level of organization composed of arrays of ordered collagen fibrils, referred to as rods. This new structural information on human lamellar bone will improve our understanding of structure-mechanical function relations, mechanisms of mechano-sensing and the characterizations of bone pathologies. Copyright © 2013 Elsevier Inc. All rights reserved.

Making more matrix: enhancing the deposition of dermal-epidermal junction components in vitro and accelerating organotypic skin culture development, using macromolecular crowding.

PubMed

Benny, Paula; Badowski, Cedric; Lane, E Birgitte; Raghunath, Michael

2015-01-01

Skin is one of the most accessible tissues for experimental biomedical sciences, and cultured skin cells represent one of the longest-running clinical applications of stem cell therapy. However, culture-generated skin mimetic multicellular structures are still limited in their application by the time taken to develop these constructs in vitro and by their incomplete differentiation. The development of a functional dermal-epidermal junction (DEJ) is one of the most sought after aspects of cultured skin, and one of the hardest to recreate in vitro. At the DEJ, dermal fibroblasts and epidermal keratinocytes interact to form an interlinked basement membrane of extracellular matrix (ECM), which forms as a concerted action of both keratinocytes and fibroblasts. Successful formation of this basement membrane is essential for take and stability of cultured skin autografts. We studied interactive matrix production by monocultures and cocultures of primary human keratinocytes and fibroblasts in an attempt to improve the efficiency of basement membrane production in culture using mixed macromolecular crowding (mMMC); resulting ECM were enriched with the deposition of collagens I, IV, fibronectin, and laminin 332 (laminin 5) and also in collagen VII, the anchoring fibril component. Our in vitro data point to fibroblasts, rather than keratinocytes, as the major cellular contributors of the DEJ. Not only did we find more collagen VII production and deposition by fibroblasts in comparison to keratinocytes, but also observed that decellularized fibroblast ECM stimulated the production and deposition of collagen VII by keratinocytes, over and above that of keratinocyte monocultures. In confrontation cultures, keratinocytes and fibroblasts showed spontaneous segregation and demarcation of cell boundaries by DEJ protein deposition. Finally, mMMC was used in a classical organotypic coculture protocol with keratinocytes seeded over fibroblast-containing collagen gels. Applied during the submerged phase, mMMC was sufficient to accelerate the emergence of collagen VII along the de novo DEJ, together with stronger transglutaminase activity in the neoepidermis. Our findings corroborate the role of fibroblasts as important players in producing collagen VII and inducing collagen VII deposition in the DEJ, and that macromolecular crowding leads to organotypic epidermal differentiation in tissue culture in a significantly condensed time frame.

Amino acid composition in determination of collagen origin and assessment of physical factors effects.

PubMed

Gauza-Włodarczyk, Marlena; Kubisz, Leszek; Włodarczyk, Dariusz

2017-11-01

The amino acid composition of collagen is a characteristic feature of this protein. Collagen, irrespective of its origin, contains 19 amino acids, including hydroxyproline which does not occur in other proteins. Its atypical amino acid composition is characterized by high content of proline and glycine, as well as the absence of cysteine. This paper shows the comparison of qualitative composition of amino acids of fish skin (FS) collagen, bovine Achilles tendon (BAT) collagen, and bone collagen. Results demonstrate that FS collagen as well as BAT collagen contains no cysteine and significantly different amount of hydroxyproline. In BAT collagen hydroxyproline content is 30% higher than hydroxyproline content of FS collagen. In bone collagen the amount of hydroxyproline is two times more than in FS collagen. Furthermore, it is shown that sensitivity to radiation of individual amino acids varies and depends on the absorbed dose of ionizing radiation. The changes observed in the amino acid composition become very intense for the doses of 500kGy and 1000kGy. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of topical application of virgin coconut oil on skin components and antioxidant status during dermal wound healing in young rats.

PubMed

Nevin, K G; Rajamohan, T

2010-01-01

The present study was undertaken to evaluate the effect of a topical application of virgin coconut oil (VCO) on excision wounds in young rats. Three sets of experiments with 3 groups of female Sprague-Dawley rats each consisting of 6 animals were used for studying wound closure time, antioxidant status and biochemical parameters. Group 1 was the control; groups 2 and 3 were treated with 0.5 and 1.0 ml VCO, respectively, 24 h after wound creation for 10 days. After the experimental period, the healing property of VCO was evaluated by monitoring the time taken for complete epithelization as well as levels of various parameters of the wound's granulation tissue. The collagen solubility pattern, glycohydrolase activity, and histopathology of the granulation tissue were also analyzed. The antioxidant status during wound healing was monitored continuously for 14 days. VCO-treated wounds healed much faster, as indicated by a decreased time of complete epithelization and higher levels of various skin components. Pepsin-soluble collagen showed a significant increase in VCO- treated wounds, indicating a higher collagen cross-linking. Glycohydrolase activities were also found to be increased due to a higher turnover of collagen. Antioxidant enzyme activities, and reduced glutathione and malondialdehyde levels were found to be increased on the 10th day after wounding, which were found to have returned to normal levels on day 14 in the treated wounds. The lipid peroxide levels were found to be lower in the treated wounds. A histopathological study showed an increase in fibroblast proliferation and neovascularization in VCO-treated wounds compared to controls. The beneficial effect of VCO can be attributed to the cumulative effect of various biologically active minor components present in it. Copyright 2010 S. Karger AG, Basel.

Modifications of Erectile Tissue Components in the Penis during the Fetal Period

PubMed Central

Gallo, Carla B. M.; Costa, Waldemar S.; Furriel, Angelica; Bastos, Ana L.; Sampaio, Francisco J. B.

2014-01-01

Background The penile erectile tissue has a complex microscopic anatomy with important functions in the mechanism of penile erection. The knowledge of such structures is necessary for understanding the normal physiology of the adult penis. Therefore, it is important to know the changes of these penile structures during fetal development. This study aims to analyze the development of the main components of the erectile tissue, such as collagen, smooth muscle fibers and elastic system fibers, in human fetuses. Methodology/Principal Findings We studied the penises of 56 human fetuses aged 13 to 36 weeks post-conception (WPC). We used histochemical and immunohistochemical staining, as well as morphometric techniques to analyze the collagen, smooth muscle fibers and elastic system fibers in the corpus cavernosum and in the corpus spongiosum. These elements were identified and quantified as percentage by using the Image J software (NIH, Bethesda, USA). From 13 to 36 WPC, in the corpus cavernosum, the amount of collagen, smooth muscle fibers and elastic system fibers varied from 19.88% to 36.60%, from 4.39% to 29.76% and from 1.91% to 8.92%, respectively. In the corpus spongiosum, the amount of collagen, smooth muscle fibers and elastic system fibers varied from 34.65% to 45.89%, from 0.60% to 11.90% and from 3.22% to 11.93%, respectively. Conclusions We found strong correlation between the elements analyzed with fetal age, both in corpus cavernosum and corpus spongiosum. The growth rate of these elements was more intense during the second trimester (13 to 24 WPC) of gestation, both in corpus cavernosum and in corpus spongiosum. There is greater proportional amount of collagen in the corpus spongiosum than in corpus cavernosum during all fetal period. In the corpus spongiosum, there is about four times more collagen than smooth muscle fibers and elastic system fibers, during all fetal period studied. PMID:25170760

Constituents of neutrophil extracellular traps induce in vitro collagen formation in mare endometrium.

PubMed

Rebordão, Maria Rosa; Amaral, Ana; Lukasik, Karolina; Szóstek-Mioduchowska, Anna; Pinto-Bravo, Pedro; Galvão, António; Skarzynski, Dariusz J; Ferreira-Dias, Graça

2018-06-01

Neutrophil extracellular traps (NETs) are DNA complexes carrying nuclear and cytoplasmic proteins, such as elastase (ELA), cathepsin-G (CAT) and myeloperoxidase (MPO). Mare endometrosis is a chronic degenerative process characterized by excessive collagen in endometrium. While NETs fight bacteria that cause endometritis, they may trigger endometrial fibrogenesis. The aim was to evaluate the in vitro effect of some NETs components on mare endometrial fibrogenesis and determine its relationship with histopathology or estrous cycle. Endometrial explants were incubated with NETs components (ELA, CAT, MPO or oxytocin). Collagen type I (COL1) protein and type I and III (COL3) gene transcription were evaluated in follicular and mid-luteal phases endometria (Kenney and Doig type I/IIA and IIB/III). Increased COL1 occurred with all NETs proteins, although endometrial response to each NETs protease depended on estrous cycle and/or endometrial category. Since ELA enhanced COL1 production, NETs persistence might be linked to endometrosis. Estrous cycle influenced COL1 protein concentration and COL3 transcripts, suggesting that follicular phase may favor endometrial collagen production. However, luteal phase endometria with moderate or severe lesions may be also susceptible to fibrotic effects of NETs constituents. These data propose that NETs involvement in chronic endometritis in mares may act as putative endometrial fibrogenic mediators. Copyright © 2018 Elsevier Inc. All rights reserved.

The two-component system GrvRS (EtaRS) regulates ace expression in Enterococcus faecalis OG1RF.

PubMed

Roh, Jung Hyeob; Singh, Kavindra V; La Rosa, Sabina Leanti; Cohen, Ana Luisa V; Murray, Barbara E

2015-01-01

Expression of ace (adhesin to collagen of Enterococcus faecalis), encoding a virulence factor in endocarditis and urinary tract infection models, has been shown to increase under certain conditions, such as in the presence of serum, bile salts, urine, and collagen and at 46 °C. However, the mechanism of ace/Ace regulation under different conditions is still unknown. In this study, we identified a two-component regulatory system GrvRS as the main regulator of ace expression under these stress conditions. Using Northern hybridization and β-galactosidase assays of an ace promoter-lacZ fusion, we found transcription of ace to be virtually absent in a grvR deletion mutant under the conditions that increase ace expression in wild-type OG1RF and in the complemented strain. Moreover, a grvR mutant revealed decreased collagen binding and biofilm formation as well as attenuation in a murine urinary tract infection model. Here we show that GrvR plays a major role in control of ace expression and E. faecalis virulence. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Design and characterization of a biodegradable composite scaffold for ligament tissue engineering.

PubMed

Hayami, James W S; Surrao, Denver C; Waldman, Stephen D; Amsden, Brian G

2010-03-15

Herein we report on the development and characterization of a biodegradable composite scaffold for ligament tissue engineering based on the fundamental morphological features of the native ligament. An aligned fibrous component was used to mimic the fibrous collagen network and a hydrogel component to mimic the proteoglycan-water matrix of the ligament. The composite scaffold was constructed from cell-adherent, base-etched, electrospun poly(epsilon-caprolactone-co-D,L-lactide) (PCLDLLA) fibers embedded in a noncell-adherent photocrosslinked N-methacrylated glycol chitosan (MGC) hydrogel seeded with primary ligament fibroblasts. Base etching improved cellular adhesion to the PCLDLLA material. Cells within the MGC hydrogel remained viable (72 +/- 4%) during the 4-week culture period. Immunohistochemistry staining revealed ligament ECM markers collagen type I, collagen type III, and decorin organizing and accumulating along the PCLDLLA fibers within the composite scaffolds. On the basis of these results, it was determined that the composite scaffold design was a viable alternative to the current approaches used for ligament tissue engineering and merits further study. (c) 2009 Wiley Periodicals, Inc.

Lamina propria of the human vocal fold: histomorphometric study of collagen fibers.

PubMed

Prades, Jean-Michel; Dumollard, Jean Marc; Duband, Sébastien; Timoshenko, Andrei; Richard, Céline; Dubois, Marie Dominique; Martin, Christian; Peoc'h, Michel

2010-04-01

Since the seminal work of M. Hirano, which defined the three-layered lamina propria of the human vocal fold, there has been confusion in the labeling of each layer. Recent studies described the composition of fibers and interstitial molecules within the lamina propria leading to various biomechanical properties. However, collagen fibers appear as the most important structure component. We used an optical analysis and the picrosirius-polarization method to describe collagen fibers from six adult and two fetal human larynges fixed in formalin and frontally sectioned in the middle part of the vocal fold. The deep layer of the lamina propria is the most densely organized band of collagen fibers penetrating the superficial muscle bundles of the vocal muscle. The mean thickness of this layer is about 36% of the lamina propria and shows a network of strongly birefringent fibers (collagen type I and III). The superficial layer of the lamina propria is a narrow band of collagen fibers immediately below the basement membrane of the epithelium. The mean thickness of this layer is about 13% of the lamina propria and shows strong birefringent fibers. The intermediate layer is the less densely organized band between the deep and superficial layers. The mean thickness of this layer is about 51% of the lamina propria and shows clear, green weakly birefringent fibers characterized as collagen type III. The fetal lamina propria contains only a monolayer distribution of loose collagen fibers between the epithelium and the vocal muscle. These results help describe the distribution of collagen fibers within the lamina propria of the human vocal fold and have implications to understand the cover-body theory of voice production both in the adult and newborn.

Lysyl oxidase interacts with AGE signalling to modulate collagen synthesis in polycystic ovarian tissue

PubMed Central

Papachroni, Katerina K; Piperi, Christina; Levidou, Georgia; Korkolopoulou, Penelope; Pawelczyk, Leszek; Diamanti-Kandarakis, Evanthia; Papavassiliou, Athanasios G

2010-01-01

Abstract Connective tissue components – collagen types I, III and IV – surrounding the ovarian follicles undergo drastic changes during ovulation. Abnormal collagen synthesis and increased volume and density of ovarian stroma characterize the polycystic ovary syndrome (PCOS). During the ovulatory process, collagen synthesis is regulated by prolyl hydroxylase and lysyl oxidase (LOX) activity in ovarian follicles. LOX catalyzes collagen and elastin cross-linking and plays essential role in coordinating the control of ovarian extracellular matrix (ECM) during follicular development. We have recently shown accumulation of advanced glycation end products (AGEs), molecules that stimulate ECM production and abnormal collagen cross-linking, in ovarian tissue. However, the possible link between LOX and AGEs-induced signalling in collagen production and stroma formation in ovarian tissue from PCOS remains elusive. The present study investigates the hypothesis of AGE signalling pathway interaction with LOX gene activity in polycystic ovarian (PCO) tissue. We show an increased distribution and co-localization of LOX, collagen type IV and AGE molecules in the PCO tissue compared to control, as well as augmented expression of AGE signalling mediators/effectors, phospho(p)-ERK, phospho(p)-c-Jun and nuclear factor κB (NF-κB) in pathological tissue. Moreover, we demonstrate binding of AGE-induced transcription factors, NF-κB and activator protein-1 (AP-1) on LOX promoter, indicating a possible involvement of AGEs in LOX gene regulation, which may account for the documented increase in LOX mRNA and protein levels compared to control. These findings suggest that deposition of excess collagen in PCO tissue that induces cystogenesis may, in part, be due to AGE-mediated stimulation of LOX activity. PMID:19583806

Pancreatic Cancer Cells Enhance the Ability of Collagen Internalization during Epithelial–Mesenchymal Transition

PubMed Central

Ikenaga, Naoki; Ohuchida, Kenoki; Mizumoto, Kazuhiro; Akagawa, Shin; Fujiwara, Kenji; Eguchi, Daiki; Kozono, Shingo; Ohtsuka, Takao; Takahata, Shunichi; Tanaka, Masao

2012-01-01

Background Extracellular matrix (ECM) remodeling is predominantly mediated by fibroblasts using intracellular and extracellular pathways. Although it is well known that extracellular degradation of the ECM by proteases derived from cancer cells facilitates cellular invasion, the intracellular degradation of ECM components by cancer cells has not been clarified. The aim of this study was to characterize collagen internalization, which is the initial step of the intracellular degradation pathway in pancreatic cancer cells, in light of epithelial–mesenchymal transition (EMT). Methodology/Principal Findings We analyzed the function of collagen internalization in two pancreatic cancer cell lines, SUIT-2 and KP-2, and pancreatic stellate cells (PSCs) using Oregon Green 488-gelatin. PSCs had a strong ability for collagen uptake, and the pancreatic cancer cells also internalized collagen although less efficiently. The collagen internalization abilities of SUIT-2 and KP-2 cells were promoted by EMT induced by human recombinant transforming growth factor β1 (P<0.05). Expression of Endo180, a collagen uptake receptor, was high in mesenchymal pancreatic cancer cell lines, as determined by EMT marker expression (P<0.01). Quantitative RT-PCR and western blot analyses showed that Endo180 expression was also increased by EMT induction in SUIT-2 and KP-2 cells. Endo180 knockdown by RNA interference attenuated the collagen uptake (P<0.01) and invasive abilities (P<0.05) of SUIT-2 and KP-2 cells. Conclusions/Significance Pancreatic cancer cells are capable of collagen internalization, which is enhanced by EMT. This ECM clearance system may be a novel mechanism for cellular invasion and a potential therapeutic target in pancreatic cancer. PMID:22792318

When 1+1>2: Nanostructured composites for hard tissue engineering applications.

PubMed

Uskoković, Vuk

2015-12-01

Multicomponent, synergistic and multifunctional nanostructures have taken over the spotlight in the realm of biomedical nanotechnologies. The most prospective materials for bone regeneration today are almost exclusively composites comprising two or more components that compensate for the shortcomings of each one of them alone. This is quite natural in view of the fact that all hard tissues in the human body, except perhaps the tooth enamel, are composite nanostructures. This review article highlights some of the most prospective breakthroughs made in this research direction, with the hard tissues in main focus being those comprising bone, tooth cementum, dentin and enamel. The major obstacles to creating collagen/apatite composites modeled after the structure of bone are mentioned, including the immunogenicity of xenogeneic collagen and continuously failing attempts to replicate the biomineralization process in vitro. Composites comprising a polymeric component and calcium phosphate are discussed in light of their ability to emulate the soft/hard composite structure of bone. Hard tissue engineering composites created using hard material components other than calcium phosphates, including silica, metals and several types of nanotubes, are also discoursed on, alongside additional components deliverable using these materials, such as cells, growth factors, peptides, antibiotics, antiresorptive and anabolic agents, pharmacokinetic conjugates and various cell-specific targeting moieties. It is concluded that a variety of hard tissue structures in the body necessitates a similar variety of biomaterials for their regeneration. The ongoing development of nanocomposites for bone restoration will result in smart, theranostic materials, capable of acting therapeutically in direct feedback with the outcome of in situ disease monitoring at the cellular and subcellular scales. Progress in this research direction is expected to take us to the next generation of biomaterials, designed with the purpose of fulfilling Daedalus' dream - not restoring the tissues, but rather augmenting them. Copyright © 2015 Elsevier B.V. All rights reserved.

Bone Collagen: New Clues to its Mineralization Mechanism From Recessive Osteogenesis Imperfecta

PubMed Central

Eyre, David R.; Ann Weis, Mary

2013-01-01

Until 2006 the only mutations known to cause osteogenesis imperfecta (OI) were in the two genes coding for type I collagen chains. These dominant mutations affecting the expression or primary sequence of collagen α1(I) and α2(I) chains account for over 90% of OI cases. Since then a growing list of mutant genes causing the 5–10% of recessive cases has rapidly emerged. They include CRTAP, LEPRE1 and PPIB, which encode three proteins forming the prolyl 3-hydroxylase complex; PLOD2 and FKBP10, which encode respectively lysyl hydroxylase 2 and a foldase required for its activity in forming mature cross-links in bone collagen; SERPIN H1, which encodes the collagen chaperone HSP47; SERPIN F1, which encodes pigment epithelium-derived factor required for osteoid mineralization; and BMP1, which encodes the type I procollagen C-propeptidase. All cause fragile bone in infancy, which can include over-mineralization or under-mineralization defects as well as abnormal collagen post-translational modifications. Consistently both dominant and recessive variants lead to abnormal cross-linking chemistry in bone collagen. These recent discoveries strengthen the potential for a common pathogenic mechanism of misassembled collagen fibrils. Of the new genes identified, eight encode proteins required for collagen post-translational modification, chaperoning of newly synthesized collagen chains into native molecules or transport through the endoplasmic reticulum and Golgi for polymerization, cross-linking and mineralization. In reviewing these findings, we conclude that a common theme is emerging in the pathogenesis of brittle bone disease of mishandled collagen assembly with important insights on post-translational features of bone collagen that have evolved to optimize it as a biomineral template. PMID:23508630

Candidate Cell and Matrix Interaction Domains on the Collagen Fibril, the Predominant Protein of Vertebrates*S⃞

PubMed Central

Sweeney, Shawn M.; Orgel, Joseph P.; Fertala, Andrzej; McAuliffe, Jon D.; Turner, Kevin R.; Di Lullo, Gloria A.; Chen, Steven; Antipova, Olga; Perumal, Shiamalee; Ala-Kokko, Leena; Forlino, Antonella; Cabral, Wayne A.; Barnes, Aileen M.; Marini, Joan C.; Antonio, James D. San

2008-01-01

Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The “cell interaction domain” is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The “matrix interaction domain” may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging. PMID:18487200

A tissue adaptation model based on strain-dependent collagen degradation and contact-guided cell traction.

PubMed

Heck, T A M; Wilson, W; Foolen, J; Cilingir, A C; Ito, K; van Donkelaar, C C

2015-03-18

Soft biological tissues adapt their collagen network to the mechanical environment. Collagen remodeling and cell traction are both involved in this process. The present study presents a collagen adaptation model which includes strain-dependent collagen degradation and contact-guided cell traction. Cell traction is determined by the prevailing collagen structure and is assumed to strive for tensional homeostasis. In addition, collagen is assumed to mechanically fail if it is over-strained. Care is taken to use principally measurable and physiologically meaningful relationships. This model is implemented in a fibril-reinforced biphasic finite element model for soft hydrated tissues. The versatility and limitations of the model are demonstrated by corroborating the predicted transient and equilibrium collagen adaptation under distinct mechanical constraints against experimental observations from the literature. These experiments include overloading of pericardium explants until failure, static uniaxial and biaxial loading of cell-seeded gels in vitro and shortening of periosteum explants. In addition, remodeling under hypothetical conditions is explored to demonstrate how collagen might adapt to small differences in constraints. Typical aspects of all essentially different experimental conditions are captured quantitatively or qualitatively. Differences between predictions and experiments as well as new insights that emerge from the present simulations are discussed. This model is anticipated to evolve into a mechanistic description of collagen adaptation, which may assist in developing load-regimes for functional tissue engineered constructs, or may be employed to improve our understanding of the mechanisms behind physiological and pathological collagen remodeling. Copyright © 2014 Elsevier Ltd. All rights reserved.

Candidate Cell and Matrix Interaction Domains on the Collagen Fibril, the Predominant Protein of Vertebrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sweeney, Shawn M.; Orgel, Joseph P.; Fertala, Andrzej

Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The 'cell interaction domain' is proposed to regulatemore » dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The 'matrix interaction domain' may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.« less

Correlation of collagen synthesis with polarization-sensitive optical coherence tomography imaging of in vitro human atherosclerosis

NASA Astrophysics Data System (ADS)

Kuo, Wen-Chuan; Shyu, Jeou-Jong; Chou, Nai-Kuan; Lai, Chih-Ming; Tien, En-Kuang; Huang, Huan-Jang; Chou, Chien; Jan, Gwo-Jen

2005-04-01

Atherosclerosis is unquestionably the leading cause of morbidity and mortality in developed countries. In the mean time, the worldwide importance of acute vascular syndromes is increasing. Because collagen fiber is a critical component of atherosclerotic lesions; it constitutes up to 60% of the total atherosclerotic plaque protein. The uncontrolled collagen accumulation leads to arterial stenosis, whereas excessive collagen breakdown weakens plaques thereby making them prone to rupture finally. Thus, in this study, we present the first application, to our knowledge, of using polarization-sensitive optical coherence tomography (PS-OCT) in human atherosclerosis. We demonstrate this technique for imaging of intensity, birefringence, and fast-axis orientation simultaneously in atherosclerotic plaques. This in vitro study suggests that the birefringence change in plaque is due to the prominent deposition of collagen according to the correlation of PS-OCT images with histological counterpart. Moreover, we can acquire quantitative criteria based on the change of polarization of incident beam to estimate whether the collagen synthesized is "too much" or "not enough". Thus by combining of high resolution intensity imaging and birefringence detection makes PS-OCT could be a potentially powerful tool for early assessment of atherosclerosis appearance and the prediction of plaque rupture in clinic.

The Interface between Catalytic and Hemopexin Domains in Matrix Metalloproteinase-1 Conceals a Collagen Binding Exosite*

PubMed Central

Arnold, Laurence H.; Butt, Louise E.; Prior, Stephen H.; Read, Christopher M.; Fields, Gregg B.; Pickford, Andrew R.

2011-01-01

Matrix metalloproteinase-1 (MMP-1) is an instigator of collagenolysis, the catabolism of triple helical collagen. Previous studies have implicated its hemopexin (HPX) domain in binding and possibly destabilizing the collagen substrate in preparation for hydrolysis of the polypeptide backbone by the catalytic (CAT) domain. Here, we use biophysical methods to study the complex formed between the MMP-1 HPX domain and a synthetic triple helical peptide (THP) that encompasses the MMP-1 cleavage site of the collagen α1(I) chain. The two components interact with 1:1 stoichiometry and micromolar affinity via a binding site within blades 1 and 2 of the four-bladed HPX domain propeller. Subsequent site-directed mutagenesis and assay implicates blade 1 residues Phe301, Val319, and Asp338 in collagen binding. Intriguingly, Phe301 is partially masked by the CAT domain in the crystal structure of full-length MMP-1 implying that transient separation of the domains is important in collagen recognition. However, mutation of this residue in the intact enzyme disrupts the CAT-HPX interface resulting in a drastic decrease in binding activity. Thus, a balanced equilibrium between these compact and dislocated states may be an essential feature of MMP-1 collagenase activity. PMID:22030392

Collagen degradation and preservation of MMP-8 activity in human dentine matrix after demineralization.

PubMed

Hedenbjörk-Lager, Anders; Hamberg, Kristina; Pääkkönen, Virve; Tjäderhane, Leo; Ericson, Dan

2016-08-01

Dental caries is a process driven by acids produced by oral microorganisms followed by degradation of the dentine collagen matrix by proteolytic enzymes. Matrix metalloproteinases (MMPs) have been suggested to contribute to caries by degrading collagen. The aim of this study was to develop a method for generating demineralized dentine matrix substrate (DDM) maintaining MMP-8 bioactivity and no interference with later assays. Such a substrate would allow study of the effects of various treatments on MMP-8 activity and collagen degradation in demineralized dentine. Human dentine was powderized in a tissue grinder and frozen (-80°C). The powder was demineralized in dialysis tubes, using EDTA or acetic acid. The demineralized dentine matrix (DDM) was harvested and analyzed for collagen content using SDS-PAGE. The DDM was subsequently suspended in PBS or TESCA buffer. Protein, MMP-8 (ELISA) and collagen (HYP) was analyzed directly or after 1 wk. EDTA or acid demineralization of dentine using dialysis yielded a substrate rich in collagen coupled with preserved MMP-8 activity. Collagen degraded in room temperature, assessed by higher HYP amounts in the soluble fraction of DDM after one wk, indicating that the methods used preserved active DDM-components after the demineralization process. The presented demineralization methods both provided insoluble DDM substrates suitable for further intervention studies. However, it was found that the substrates differed depending on the demineralization method and buffers used. This needs further study to find an optimal technique for generating DDM with retained proteins as well as enzymatic bioactivity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fibrin gels exhibit improved biological, structural, and mechanical properties compared with collagen gels in cell-based tendon tissue-engineered constructs.

PubMed

Breidenbach, Andrew P; Dyment, Nathaniel A; Lu, Yinhui; Rao, Marepalli; Shearn, Jason T; Rowe, David W; Kadler, Karl E; Butler, David L

2015-02-01

The prevalence of tendon and ligament injuries and inadequacies of current treatments is driving the need for alternative strategies such as tissue engineering. Fibrin and collagen biopolymers have been popular materials for creating tissue-engineered constructs (TECs), as they exhibit advantages of biocompatibility and flexibility in construct design. Unfortunately, a few studies have directly compared these materials for tendon and ligament applications. Therefore, this study aims at determining how collagen versus fibrin hydrogels affect the biological, structural, and mechanical properties of TECs during formation in vitro. Our findings show that tendon and ligament progenitor cells seeded in fibrin constructs exhibit improved tenogenic gene expression patterns compared with their collagen-based counterparts for approximately 14 days in culture. Fibrin-based constructs also exhibit improved cell-derived collagen alignment, increased linear modulus (2.2-fold greater) compared with collagen-based constructs. Cyclic tensile loading, which promotes the maturation of tendon constructs in a previous work, exhibits a material-dependent effect in this study. Fibrin constructs show trending reductions in mechanical, biological, and structural properties, whereas collagen constructs only show improved tenogenic expression in the presence of mechanical stimulation. These findings highlight that components of the mechanical stimulus (e.g., strain amplitude or time of initiation) need to be tailored to the material and cell type. Given the improvements in tenogenic expression, extracellular matrix organization, and material properties during static culture, in vitro findings presented here suggest that fibrin-based constructs may be a more suitable alternative to collagen-based constructs for tissue-engineered tendon/ligament repair.

Fibrin Gels Exhibit Improved Biological, Structural, and Mechanical Properties Compared with Collagen Gels in Cell-Based Tendon Tissue-Engineered Constructs

PubMed Central

Dyment, Nathaniel A.; Lu, Yinhui; Rao, Marepalli; Shearn, Jason T.; Rowe, David W.; Kadler, Karl E.; Butler, David L.

2015-01-01

The prevalence of tendon and ligament injuries and inadequacies of current treatments is driving the need for alternative strategies such as tissue engineering. Fibrin and collagen biopolymers have been popular materials for creating tissue-engineered constructs (TECs), as they exhibit advantages of biocompatibility and flexibility in construct design. Unfortunately, a few studies have directly compared these materials for tendon and ligament applications. Therefore, this study aims at determining how collagen versus fibrin hydrogels affect the biological, structural, and mechanical properties of TECs during formation in vitro. Our findings show that tendon and ligament progenitor cells seeded in fibrin constructs exhibit improved tenogenic gene expression patterns compared with their collagen-based counterparts for approximately 14 days in culture. Fibrin-based constructs also exhibit improved cell-derived collagen alignment, increased linear modulus (2.2-fold greater) compared with collagen-based constructs. Cyclic tensile loading, which promotes the maturation of tendon constructs in a previous work, exhibits a material-dependent effect in this study. Fibrin constructs show trending reductions in mechanical, biological, and structural properties, whereas collagen constructs only show improved tenogenic expression in the presence of mechanical stimulation. These findings highlight that components of the mechanical stimulus (e.g., strain amplitude or time of initiation) need to be tailored to the material and cell type. Given the improvements in tenogenic expression, extracellular matrix organization, and material properties during static culture, in vitro findings presented here suggest that fibrin-based constructs may be a more suitable alternative to collagen-based constructs for tissue-engineered tendon/ligament repair. PMID:25266738

Effects of in vivo static compressive loading on aggrecan and type II and X collagens in the rat growth plate extracellular matrix.

PubMed

Cancel, Mathilde; Grimard, Guy; Thuillard-Crisinel, Delphine; Moldovan, Florina; Villemure, Isabelle

2009-02-01

Mechanical loads are essential to normal bone growth, but excessive loads can lead to progressive deformities. In addition, growth plate extracellular matrix remodelling is essential to regulate the normal longitudinal bone growth process and to ensure physiological bone mineralization. In order to investigate the effects of static compression on growth plate extracellular matrix using an in vivo animal model, a loading device was used to precisely apply a compressive stress of 0.2 MPa for two weeks on the seventh caudal vertebra (Cd7) of rats during the pubertal growth spurt. Control, sham and loaded groups were studied. Growth modulation was quantified based on calcein labelling, and three matrix components (type II and X collagens, and aggrecan) were assessed using immunohistochemistry/safranin-O staining. As well, extracellular matrix components and enzymes (MMP-3 and -13, ADAMTS-4 and -5) were studied by qRT-PCR. Loading reduced Cd7 growth by 29% (p<0.05) and 15% (p=0.07) when compared to controls and shams respectively. No significant change could be observed in the mRNA expression of collagens and the proteolytic enzyme MMP-13. However, MMP-3 was significantly increased in the loaded group as compared to the control group (p<0.05). No change was observed in aggrecan and ADAMTS-4 and -5 expression. Low immunostaining for type II and X collagens was observed in 83% of the loaded rats as compared to the control rats. This in vivo study shows that, during pubertal growth spurt, two-week static compression reduced caudal vertebrae growth rates; this mechanical growth modulation occurred with decreased type II and X collagen proteins in the growth plate.

Collagen-gelatin-genipin-hydroxyapatite composite scaffolds colonized by human primary osteoblasts are suitable for bone tissue engineering applications: in vitro evidences.

PubMed

Vozzi, G; Corallo, C; Carta, S; Fortina, M; Gattazzo, F; Galletti, M; Giordano, N

2014-05-01

The application of porous hydroxyapatite (HAp)-collagen as a bone tissue engineering scaffold represents a new trend of mimicking the specific bone extracellular matrix (ECM). The use of HAp in reconstructive surgery has shown that it is slowly invaded by host tissue. Therefore, implant compatibility may be augmented by seeding cells before implantation. Human primary osteoblasts were seeded onto innovative collagen-gelatin-genipin (GP)-HAp scaffolds containing respectively 10%, 20%, and 30% HAp. Cellular adhesion, proliferation, alkaline phosphatase (ALP) activity, osteopontin (OPN), and osteocalcin (OC) expressions were evaluated after 3, 7, 15, and 21 days. The three types of scaffolds showed increased cellular proliferation over time in culture (maximum at 21 days) but the highest was recorded in 10% HAp scaffolds. ALP activity was the highest in 10% HAp scaffolds in all the times of evaluation. OC and OPN resulted in higher concentration in 10% HAp scaffolds compared to 20% and 30% HAp (maximum at 21 days). Finally, scanning electron microscopy analysis showed progressive scaffolds adhesion and colonization from the surface to the inside from day 3 to day 21. In vitro attachment, proliferation, and colonization of human primary osteoblasts on collagen-GP-HAp scaffolds with different percentages of HAp (10%, 20%, and 30%) all increased over time in culture, but comparing different percentages of HAp, they seem to increase with decreasing of HAp component. Therefore, the mechanical properties (such as the stiffness due to the HAp%) coupled with a good biomimetic component (collagen) are the parameters to set up in composite scaffolds design for bone tissue engineering. Copyright © 2013 Wiley Periodicals, Inc.

Effect of Low-Level Laser Therapy in an Experimental Model of Osteoarthritis in Rats Evaluated Through Raman Spectroscopy

PubMed Central

Mangueira, Nilton Maciel; Xavier, Murilo; de Souza, Renato Aparecido; Salgado, Miguel Angel Castillo; Silveira, Landulfo

2015-01-01

Abstract Objective: This work aimed to investigate the biochemical changes associated with low-level laser therapy (LLLT) using 660 and 780 nm, on a well-established experimental model of osteoarthritis (OA) in the knees of rats with induced collagenase, using histomorphometry and Raman spectroscopy. Materials and methods: Thirty-six Wistar rats were divided into four groups: control (GCON, n=9), collagenase without treatment (GCOL, n=9), collagenase with LLLT 660 nm treatment (G660, n=8), and collagenase with LLLT 780 nm treatment (G780, n=10). LLLT protocol was: 30 mW power output, 10 sec irradiation time, 0.04 cm2 spot size, 0.3 J energy, 0.75 W/cm2 irradiance, and 7.5 J/cm2 fluence per session per day, during 14 days. Then, knees were withdrawn and submitted to histomorphometry and Raman spectroscopy analysis. Principal components analysis (PCA) and Mahalanobis distance were employed to characterize the spectral findings. Results: Histomorphometry revealed a significant increase in the amount of collagen III for the group irradiated with 660 nm. The Raman bands at 1247, 1273, and 1453 cm−1 (from principal component score PC2), attributed to collagen type II, and 1460 cm−1 (from PC3), attributed to collagen type III, suggested that the LLLT causes acceleration in cellular activity, especially on the cells that repair cartilage, accelerating the breakdown of cartilage destroyed by collagenase and stimulating the fibroblast to synthesize repairing collagen III. Conclusions: LLLT accelerated the initial breakdown of cartilage destroyed by collagenase and stimulated the fibroblast to synthesize the repairing collagen III, suggesting a beneficial effect of LLLT on OA. PMID:25714387

Extracorporeal human bone-like tissue generation

PubMed Central

Rosenberg, N.; Rosenberg, O.

2012-01-01

Objectives The need for bone tissue supplementation exists in a wide range of clinical conditions involving surgical reconstruction in limbs, the spine and skull. The bone supplementation materials currently used include autografts, allografts and inorganic matrix components; but these pose potentially serious side-effects. In particular the availability of the autografts is usually limited and their harvesting causes surgical morbidity. Therefore for the purpose of supplementation of autologous bone graft, we have developed a method for autologous extracorporeal bone generation. Methods Human osteoblast-like cells were seeded on porous granules of tricalcium phosphate and incubated in osteogenic media while exposed to mechanical stimulation by vibration in the infrasonic range of frequencies. The generated tissue was examined microscopically following haematoxylin eosin, trichrome and immunohistochemical staining. Results Following 14 days of incubation the generated tissue showed histological characteristics of bone-like material due to the characteristic eosinophilic staining, a positive staining for collagen trichrome and a positive specific staining for osteocalcin and collagen 1. Macroscopically, this tissue appeared in aggregates of between 0.5 cm and 2 cm. Conclusions We present evidence that the interaction of the cellular, inorganic and mechanical components in vitro can rapidly generate three-dimensional bone-like tissue that might be used as an autologous bone graft. PMID:23610651

Downregulation of connective tissue growth factor by three-dimensional matrix enhances ovarian carcinoma cell invasion.

PubMed

Barbolina, Maria V; Adley, Brian P; Kelly, David L; Shepard, Jaclyn; Fought, Angela J; Scholtens, Denise; Penzes, Peter; Shea, Lonnie D; Stack, M Sharon

2009-08-15

Epithelial ovarian carcinoma (EOC) is a leading cause of death from gynecologic malignancies, due mainly to the prevalence of undetected metastatic disease. The process of cell invasion during intraperitoneal anchoring of metastatic lesions requires concerted regulation of many processes, including modulation of adhesion to the extracellular matrix and localized invasion. Exploratory cDNA microarray analysis of early response genes (altered after 4 hr of 3D collagen culture) coupled with confirmatory real-time reverse-transcriptase polymerase chain reaction, multiple 3D cell culture matrices, Western blot, immunostaining, adhesion, migration and invasion assays were used to identify modulators of adhesion pertinent to EOC progression and metastasis. cDNA microarray analysis indicated a dramatic downregulation of connective tissue growth factor (CTGF) in EOC cells placed in invasion- mimicking conditions (3D Type I collagen). Examination of human EOC specimens revealed that CTGF expression was absent in 46% of the tested samples (n = 41), but was present in 100% of normal ovarian epithelium samples (n = 7). Reduced CTGF expression occurs in many types of cells and may be a general phenomenon displayed by cells encountering a 3D environment. CTGF levels were inversely correlated with invasion such that downregulation of CTGF increased, while its upregulation reduced collagen invasion. Cells adhered preferentially to a surface comprised of both collagen I and CTGF relative to either component alone using alpha6beta1 and alpha3beta1 integrins. Together these data suggest that downregulation of CTGF in EOC cells may be important for cell invasion through modulation of cell-matrix adhesion.

Downregulation of Connective Tissue Growth Factor by Three-Dimensional Matrix Enhances Ovarian Carcinoma Cell Invasion

PubMed Central

Barbolina, Maria V.; Adley, Brian P.; Kelly, David L.; Shepard, Jaclyn; Fought, Angela J.; Scholtens, Denise; Penzes, Peter; Shea, Lonnie D.; Sharon Stack, M

2010-01-01

Epithelial ovarian carcinoma (EOC) is a leading cause of death from gynecologic malignancy, due mainly to the prevalence of undetected metastatic disease. The process of cell invasion during intra-peritoneal anchoring of metastatic lesions requires concerted regulation of many processes, including modulation of adhesion to the extracellular matrix and localized invasion. Exploratory cDNA microarray analysis of early response genes (altered after 4 hours of 3-dimensional collagen culture) coupled with confirmatory real-time RT-PCR, multiple three-dimensional cell culture matrices, Western blot, immunostaining, adhesion, migration, and invasion assays were used to identify modulators of adhesion pertinent to EOC progression and metastasis. cDNA microarray analysis indicated a dramatic downregulation of connective tissue growth factor (CTGF) in EOC cells placed in invasion-mimicking conditions (3-dimensional type I collagen). Examination of human EOC specimens revealed that CTGF expression was absent in 46% of the tested samples (n=41), but was present in 100% of normal ovarian epithelium samples (n=7). Reduced CTGF expression occurs in many types of cells and may be a general phenomenon displayed by cells encountering a 3D environment. CTGF levels were inversely correlated with invasion such that downregulation of CTGF increased, while its upregulation reduced, collagen invasion. Cells adhered preferentially to a surface comprised of both collagen I and CTGF relative to either component alone using α6β1 and α3β1 integrins. Together these data suggest that downregulation of CTGF in EOC cells may be important for cell invasion through modulation of cell-matrix adhesion. PMID:19382180

Optimum conditions for extracting collagen from the tunica albuginea of immunologically castrated pig testes and the functional properties of the isolated collagen.

PubMed

Simões, Gislaine Silveira; Silveira, Expedito Tadeu Facco; de Oliveira, Simone Raymundo; Poleze, Evandro; Allison, Jim R D; Ida, Elza Iouko; Shimokomaki, Massami

2014-04-01

This study evaluated alternative methods for extracting collagen from the tunica albuginea of pig testes and characterized the functional properties of the isolated collagen. Using the statistical tools of factorial design (2⁴⁻¹) and a central composite rotatable design (2³), it was concluded that the best conditions were 0.83 mol L⁻¹ acetic acid, 0.24% pepsin and 28 h of hydrolysis to isolate 82.54 g of collagen per 100g of sample. This purified collagen had improved functional properties in relation to bovine skin collagen, including water solubility, water-holding capacity, emulsifying capacity and emulsion stability. These results suggest that isolated collagen from the tunica albuginea can be used in pharmaceutical and food products. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Advances in research and application of beta-tricalcium phosphate, collagen and beta-tricalcium phosphate/collagen composite in bone tissue engineering].

PubMed

Han, Xiang-Yong; Fu, Yuan-Fei; Zhang, Fu-Qiang

2007-02-01

Bone defects in oral and maxillofacial region was a common problem. To repair the defect, bone grafts including autograft, allograft and artificial bone graft were used in clinic despite of their disadvantages. Nowadays, bone tissue engineering has become a commonly used method to repair bone defect. This paper reviewed the application of beta-TCP, collagen and beta-TCP/collagen composite in bone tissue engineering. It was concluded that beta-TCP/collagen composite was a promising materials in bone tissue engineering.

Defining the hierarchical organisation of collagen VI microfibrils at nanometre to micrometre length scales.

PubMed

Godwin, Alan R F; Starborg, Tobias; Sherratt, Michael J; Roseman, Alan M; Baldock, Clair

2017-04-01

Extracellular matrix microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. Collagen VI is a heteromeric network-forming collagen which is expressed in tissues such as skin, lung, blood vessels and articular cartilage where it anchors cells into the matrix allowing for transduction of biochemical and mechanical signals. It is not understood how collagen VI is arranged into microfibrils or how these microfibrils are arranged into tissues. Therefore we have characterised the hierarchical organisation of collagen VI across multiple length scales. The frozen hydrated nanostructure of purified collagen VI microfibrils was reconstructed using cryo-TEM. The bead region has a compact hollow head and flexible tail regions linked by the collagenous interbead region. Serial block face SEM imaging coupled with electron tomography of the pericellular matrix (PCM) of murine articular cartilage revealed that the PCM has a meshwork-like organisation formed from globular densities ∼30nm in diameter. These approaches can characterise structures spanning nanometer to millimeter length scales to define the nanostructure of individual collagen VI microfibrils and the micro-structural organisation of these fibrils within tissues to help in the future design of better mimetics for tissue engineering. Cartilage is a connective tissue rich in extracellular matrix molecules and is tough and compressive to cushion the bones of joints. However, in adults cartilage is poorly repaired after injury and so this is an important target for tissue engineering. Many connective tissues contain collagen VI, which forms microfibrils and networks but we understand very little about these assemblies or the tissue structures they form. Therefore, we have use complementary imaging techniques to image collagen VI microfibrils from the nano-scale to the micro-scale in order to understand the structure and the assemblies it forms. These findings will help to inform the future design of scaffolds to mimic connective tissues in regenerative medicine applications. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Physiologically Distributed Loading Patterns Drive the Formation of Zonally Organized Collagen Structures in Tissue-Engineered Meniscus.

PubMed

Puetzer, Jennifer L; Bonassar, Lawrence J

2016-07-01

The meniscus is a dense fibrocartilage tissue that withstands the complex loads of the knee via a unique organization of collagen fibers. Attempts to condition engineered menisci with compression or tensile loading alone have failed to reproduce complex structure on the microscale or anatomic scale. Here we show that axial loading of anatomically shaped tissue-engineered meniscus constructs produced spatial distributions of local strain similar to those seen in the meniscus when the knee is loaded at full extension. Such loading drove formation of tissue with large organized collagen fibers, levels of mechanical anisotropy, and compressive moduli that match native tissue. Loading accelerated the development of native-sized and aligned circumferential and radial collagen fibers. These loading patterns contained both tensile and compressive components that enhanced the major biochemical and functional properties of the meniscus, with loading significantly improved glycosaminoglycan (GAG) accumulation 200-250%, collagen accumulation 40-55%, equilibrium modulus 1000-1800%, and tensile moduli 500-1200% (radial and circumferential). Furthermore, this study demonstrates local changes in mechanical environment drive heterogeneous tissue development and organization within individual constructs, highlighting the importance of recapitulating native loading environments. Loaded menisci developed cartilage-like tissue with rounded cells, a dense collagen matrix, and increased GAG accumulation in the more compressively loaded horns, and fibrous collagen-rich tissue in the more tensile loaded outer 2/3, similar to native menisci. Loaded constructs reached a level of organization not seen in any previous engineered menisci and demonstrate great promise as meniscal replacements.

Electrospun Collagen/Silk Tissue Engineering Scaffolds: Fiber Fabrication, Post-Treatment Optimization, and Application in Neural Differentiation of Stem Cells

NASA Astrophysics Data System (ADS)

Zhu, Bofan

Biocompatible scaffolds mimicking the locally aligned fibrous structure of native extracellular matrix (ECM) are in high demand in tissue engineering. In this thesis research, unidirectionally aligned fibers were generated via a home-built electrospinning system. Collagen type I, as a major ECM component, was chosen in this study due to its support of cell proliferation and promotion of neuroectodermal commitment in stem cell differentiation. Synthetic dragline silk proteins, as biopolymers with remarkable tensile strength and superior elasticity, were also used as a model material. Good alignment, controllable fiber size and morphology, as well as a desirable deposition density of fibers were achieved via the optimization of solution and electrospinning parameters. The incorporation of silk proteins into collagen was found to significantly enhance mechanical properties and stability of electrospun fibers. Glutaraldehyde (GA) vapor post-treatment was demonstrated as a simple and effective way to tune the properties of collagen/silk fibers without changing their chemical composition. With 6-12 hours GA treatment, electrospun collagen/silk fibers were not only biocompatible, but could also effectively induce the polarization and neural commitment of stem cells, which were optimized on collagen rich fibers due to the unique combination of biochemical and biophysical cues imposed to cells. Taken together, electrospun collagen rich composite fibers are mechanically strong, stable and provide excellent cell adhesion. The unidirectionally aligned fibers can accelerate neural differentiation of stem cells, representing a promising therapy for neural tissue degenerative diseases and nerve injuries.

Age-related accumulation of pentosidine in aggrecan and collagen from normal and degenerate human intervertebral discs

PubMed Central

Sivan, Sarit Sara; Tsitron, Eve; Wachtel, Ellen; Roughley, Peter; Sakkee, Nico; van der Ham, Frits; Degroot, Jeroen; Maroudas, Alice

2006-01-01

During aging and degeneration, many changes occur in the structure and composition of human cartilaginous tissues, which include the accumulation of the AGE (advanced glycation end-product), pentosidine, in long-lived proteins. In the present study, we investigated the accumulation of pentosidine in constituents of the human IVD (intervertebral disc), i.e. collagen, aggrecan-derived PG (proteoglycan) (A1) and its fractions (A1D1–A1D6) in health and pathology. We found that, after maturity, pentosidine accumulates with age. Over the age range studied, a linear 6-fold increase was observed in pentosidine accumulation for A1 and collagen with respective rates of 0.12 and 0.66 nmol·(g of protein)−1·year−1. Using previously reported protein turnover rate constants (kT) obtained from measurements of the D-isomer of aspartic residue in collagen and aggrecan of human IVD, we could calculate the pentosidine formation rate constants (kF) for these constituents [Sivan, Tsitron, Wachtel, Roughley, Sakkee, van der Ham, DeGroot, Roberts and Maroudas (2006) J. Biol. Chem. 281, 13009–13014; Tsitron (2006) MSc Thesis, Technion-Israel Institute of Technology, Haifa, Israel]. In spite of the comparable formation rate constants obtained for A1D1 and collagen [1.81±0.25 compared with 3.71±0.26 μmol of pentosidine·(mol of lysine)−1·year−1 respectively], the higher pentosidine accumulation in collagen is consistent with its slower turnover (0.005 year−1 compared with 0.134 year−1 for A1D1). Pentosidine accumulation increased with decreasing buoyant density and decreasing turnover of the proteins from the most glycosaminoglycan-rich PG components (A1D1) to the least (A1D6), with respective kF values of 1.81±0.25 and 3.18±0.37 μmol of pentosidine·(mol of lysine)−1·year−1. We concluded that protein turnover is an important determinant of pentosidine accumulation in aggrecan and collagen of human IVD, as was found for articular cartilage. Correlation of pentosidine accumulation with protein half-life in both normal and degenerate discs further supports this finding. PMID:16787390

Production and Assessment of Decellularized Pig and Human Lung Scaffolds

PubMed Central

Niles, Jean; Riddle, Michael; Vargas, Gracie; Schilagard, Tuya; Ma, Liang; Edward, Kert; La Francesca, Saverio; Sakamoto, Jason; Vega, Stephanie; Ogadegbe, Marie; Mlcak, Ronald; Deyo, Donald; Woodson, Lee; McQuitty, Christopher; Lick, Scott; Beckles, Daniel; Melo, Esther; Cortiella, Joaquin

2013-01-01

The authors have previously shown that acellular (AC) trachea-lung scaffolds can (1) be produced from natural rat lungs, (2) retain critical components of the extracellular matrix (ECM) such as collagen-1 and elastin, and (3) be used to produce lung tissue after recellularization with murine embryonic stem cells. The aim of this study was to produce large (porcine or human) AC lung scaffolds to determine the feasibility of producing scaffolds with potential clinical applicability. We report here the first attempt to produce AC pig or human trachea-lung scaffold. Using a combination of freezing and sodium dodecyl sulfate washes, pig trachea-lungs and human trachea-lungs were decellularized. Once decellularization was complete we evaluated the structural integrity of the AC lung scaffolds using bronchoscopy, multiphoton microscopy (MPM), assessment of the ECM utilizing immunocytochemistry and evaluation of mechanics through the use of pulmonary function tests (PFTs). Immunocytochemistry indicated that there was loss of collagen type IV and laminin in the AC lung scaffold, but retention of collagen-1, elastin, and fibronectin in some regions. MPM scoring was also used to examine the AC lung scaffold ECM structure and to evaluate the amount of collagen I in normal and AC lung. MPM was used to examine the physical arrangement of collagen-1 and elastin in the pleura, distal lung, lung borders, and trachea or bronchi. MPM and bronchoscopy of trachea and lung tissues showed that no cells or cell debris remained in the AC scaffolds. PFT measurements of the trachea-lungs showed no relevant differences in peak pressure, dynamic or static compliance, and a nonrestricted flow pattern in AC compared to normal lungs. Although there were changes in content of collagen I and elastin this did not affect the mechanics of lung function as evidenced by normal PFT values. When repopulated with a variety of stem or adult cells including human adult primary alveolar epithelial type II cells both pig and human AC scaffolds supported cell attachment and cell viability. Examination of scaffolds produced using a variety of detergents indicated that detergent choice influenced human immune response in terms of T cell activation and chemokine production. PMID:23638920

Production and assessment of decellularized pig and human lung scaffolds.

PubMed

Nichols, Joan E; Niles, Jean; Riddle, Michael; Vargas, Gracie; Schilagard, Tuya; Ma, Liang; Edward, Kert; La Francesca, Saverio; Sakamoto, Jason; Vega, Stephanie; Ogadegbe, Marie; Mlcak, Ronald; Deyo, Donald; Woodson, Lee; McQuitty, Christopher; Lick, Scott; Beckles, Daniel; Melo, Esther; Cortiella, Joaquin

2013-09-01

The authors have previously shown that acellular (AC) trachea-lung scaffolds can (1) be produced from natural rat lungs, (2) retain critical components of the extracellular matrix (ECM) such as collagen-1 and elastin, and (3) be used to produce lung tissue after recellularization with murine embryonic stem cells. The aim of this study was to produce large (porcine or human) AC lung scaffolds to determine the feasibility of producing scaffolds with potential clinical applicability. We report here the first attempt to produce AC pig or human trachea-lung scaffold. Using a combination of freezing and sodium dodecyl sulfate washes, pig trachea-lungs and human trachea-lungs were decellularized. Once decellularization was complete we evaluated the structural integrity of the AC lung scaffolds using bronchoscopy, multiphoton microscopy (MPM), assessment of the ECM utilizing immunocytochemistry and evaluation of mechanics through the use of pulmonary function tests (PFTs). Immunocytochemistry indicated that there was loss of collagen type IV and laminin in the AC lung scaffold, but retention of collagen-1, elastin, and fibronectin in some regions. MPM scoring was also used to examine the AC lung scaffold ECM structure and to evaluate the amount of collagen I in normal and AC lung. MPM was used to examine the physical arrangement of collagen-1 and elastin in the pleura, distal lung, lung borders, and trachea or bronchi. MPM and bronchoscopy of trachea and lung tissues showed that no cells or cell debris remained in the AC scaffolds. PFT measurements of the trachea-lungs showed no relevant differences in peak pressure, dynamic or static compliance, and a nonrestricted flow pattern in AC compared to normal lungs. Although there were changes in content of collagen I and elastin this did not affect the mechanics of lung function as evidenced by normal PFT values. When repopulated with a variety of stem or adult cells including human adult primary alveolar epithelial type II cells both pig and human AC scaffolds supported cell attachment and cell viability. Examination of scaffolds produced using a variety of detergents indicated that detergent choice influenced human immune response in terms of T cell activation and chemokine production.

Comparative immunolocalisation of perlecan with collagen II and aggrecan in human foetal, newborn and adult ovine joint tissues demonstrates perlecan as an early developmental chondrogenic marker.

PubMed

Smith, Susan M; Shu, Cindy; Melrose, James

2010-09-01

We undertook a comparative immunolocalisation study on type II collagen, aggrecan and perlecan in a number of 12- to 14-week-old human foetal and postnatal (7-19 months) ovine joints including finger, toe, knee, elbow, hip and shoulder. This demonstrated that perlecan followed a virtually identical immunolocalisation pattern to that of type II collagen in the foetal tissues, but a slightly divergent localisation pattern in adult tissues. Aggrecan was also localised in the cartilaginous joint tissues, which were clearly delineated by toluidine blue staining and the type II collagen immunolocalisations. It was also present in the capsular joint tissues and in ligaments and tendons in the joint, which stained poorly or not at all with toluidine blue. In higher power microscopic views, antibodies to perlecan also stained small blood vessels in the synovial lining tissues of the joint capsule; however, this was not discernable in low power macroscopic views where the immunolocalisation of perlecan to pericellular regions of cells within the cartilaginous rudiments was a predominant feature. Perlecan was also evident in small blood vessels in stromal connective tissues associated with the cartilage rudiments and with occasional nerves in the vicinity of the joint tissues. Perlecan was expressed by rounded cells in the enthesis attachment points of tendons to bone and in rounded cells in the inner third of the meniscus, which stained prominently with type II collagen and aggrecan identifying the chondrogenic background of these cells and local compressive loads. Flattened cells within the tendon and in the surface laminas of articular cartilages and the meniscus did not express perlecan. Collected evidence presented herein, therefore, indicates that besides being a basement membrane component, perlecan is also a marker of chondrogenic cells in prenatal cartilages. In postnatal cartilages, perlecan displayed a pericellular localisation pattern rather than the territorial or interterritorial localisation it displayed in foetal cartilages. This may reflect processing of extracellular perlecan presumably as a consequence of intrinsic biomechanical loading on these tissues or to divergent functions for perlecan and type II collagen in adult compared to prenatal tissues.

Removing Cross-Linked Telopeptides Enhances the Production of Low-Molecular-Weight Collagen Peptides from Spent Hens.

PubMed

Hong, Hui; Chaplot, Shreyak; Chalamaiah, Meram; Roy, Bimol C; Bruce, Heather L; Wu, Jianping

2017-08-30

The low-molecular-weight (LMW) peptides derived from collagen have shown a potential for various nutritional and pharmaceutical applications. However, production of LMW peptides from vertebrate collagen remains a challenge. Herein, we report a new method to produce LMW collagen peptides using pepsin pretreatment that removed cross-linked telopeptides in collagen molecules. After the pretreatment, the proportion of LMW collagen peptides (<1.4 kDa) that were obtained from pepsin-soluble collagen increased to 32.59% compared to heat-soluble collagen peptides (16.10%). Fourier transform infrared spectroscopy results indicated that telopeptide cleavage retained the triple-helical conformation of collagen. Liquid chromatography-tandem mass spectrometry analysis suggested that Gly-X-Y (X is often proline, while Y is either hydroxyproline or hydroxylysine) repeats were not the main factors that hindered the enzymatic hydrolysis of collagen molecules. However, cross-link quantification demonstrated that trivalent cross-links that included pyridinolines and pyrroles were the primary obstacles to producing small peptides from collagen of spent hens. This study demonstrated for the first time that removing cross-linked telopeptides could enhance the production of LMW peptides from spent hen collagen, which is also of interest to manufacturers who produce LMW collagen peptides from other vertebrate animals, such as bovids and porcids.

Collagen scaffolds derived from a marine source and their biocompatibility.

PubMed

Song, Eun; Yeon Kim, So; Chun, Taehoon; Byun, Hyun-Jung; Lee, Young Moo

2006-05-01

The primary sources of industrial collagens are calf skin and bone. However, these carry a high risk of bovine spongiform encephalopathy or transmissible spongiform encephalopathy. In this study, a novel form of acid-soluble collagen was extracted from jellyfish in an effort to obtain an alternative and safer collagen. Porous scaffolds composed of jellyfish collagen were prepared by freeze-drying and cross-linking with 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide to be used in tissue engineering applications. Enzymatic degradation kinetics of jellyfish collagen scaffolds were controlled by EDC/NHS-cross-linking density. Results from an MTT assay indicated that jellyfish collagen exhibited higher cell viability than other naturally derived biomaterials, including bovine collagen, gelatin, hyaluronic acid, and glucan. Jellyfish collagen scaffolds also had a highly porous and interconnected pore structure, which is useful for an high-density cell seeding, an efficient nutrient and an oxygen supply to the cells cultured in the three-dimensional matrices. To determine whether jellyfish collagen evokes any specific inflammatory response compared to that induced by bovine collagen or gelatin, we measured the levels of pro-inflammatory cytokines and antibody secretions and monitored the population changes of immune cells after in vivo implantation. Jellyfish collagen was found to induce an immune response at least comparable to those caused by bovine collagen and gelatin.

The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

PubMed

Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

2006-05-01

Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

Distribution of Basement Membrane Molecules, Laminin and Collagen Type IV, in Normal and Degenerated Cartilage Tissues

PubMed Central

Toh, Wei Seong; Gomoll, Andreas H.; Olsen, Bjørn Reino; Spector, Myron

2014-01-01

Objective: The objective of the present study was to investigate the presence and distribution of 2 basement membrane (BM) molecules, laminin and collagen type IV, in healthy and degenerative cartilage tissues. Design: Normal and degenerated tissues were obtained from goats and humans, including articular knee cartilage, the intervertebral disc, and meniscus. Normal tissue was also obtained from patella-tibial enthesis in goats. Immunohistochemical analysis was performed using anti-laminin and anti–collagen type IV antibodies. Human and goat skin were used as positive controls. The percentage of cells displaying the pericellular presence of the protein was graded semiquantitatively. Results: When present, laminin and collagen type IV were exclusively found in the pericellular matrix, and in a discrete layer on the articulating surface of normal articular cartilage. In normal articular (hyaline) cartilage in the human and goat, the proteins were found co-localized pericellularly. In contrast, in human osteoarthritic articular cartilage, collagen type IV but not laminin was found in the pericellular region. Nonpathological fibrocartilaginous tissues from the goat, including the menisci and the enthesis, were also positive for both laminin and collagen type IV pericellularly. In degenerated fibrocartilage, including intervertebral disc, as in degenerated hyaline cartilage only collagen type IV was found pericellularly around chondrocytes but with less intense staining than in non-degenerated tissue. In calcified cartilage, some cells were positive for laminin but not type IV collagen. Conclusions: We report differences in expression of the BM molecules, laminin and collagen type IV, in normal and degenerative cartilaginous tissues from adult humans and goats. In degenerative tissues laminin is depleted from the pericellular matrix before collagen type IV. The findings may inform future studies of the processes underlying cartilage degeneration and the functional roles of these 2 extracellular matrix proteins, normally associated with BM. PMID:26069692

Aerosolized polymerized type I collagen reduces airway inflammation and remodelling in a guinea pig model of allergic asthma.

PubMed

Moreno-Alvarez, Paola; Sánchez-Guerrero, Edgar; Martínez-Cordero, Erasmo; Hernández-Pando, Rogelio; Campos, María G; Cetina, Lucely; Bazán-Perkins, Blanca

2010-04-01

Collagen-polyvinylpyrrolidone (Collagen-PVP) has been demonstrated to elicit immunomodulatory properties in different chronic inflammatory diseases. Nevertheless, its effects on asthma are still unknown. We have evaluated whether collagen-PVP could modulate airway inflammation and remodelling in a guinea pig model of allergic asthma. Sensitized guinea pigs were challenged with the allergen (ovalbumin) six times (at 10-day intervals). From the third challenge on, animals were treated every 5 days with saline aerosols containing 0.16, 0.33, or 0.66 mg/ml of collagen-PVP (n = 5, respectively). Some guinea pigs, sensitized and challenged with saline as well as treated with 0 or 0.66 mg/ml collagen-PVP, were included in the study as control (n = 7) and sham groups (n = 5), respectively. From the first challenge on, ovalbumin induced a transient airway obstruction, measured by barometric plethysmography, which was not modified by collagen-PVP treatments. After the last allergen challenge, guinea pigs were anesthetized to obtain bronchoalveolar lavage (BAL) and the left lung caudal lobe. As expected, BAL cell count from allergen-challenged guinea pigs showed abundant neutrophils and eosinophils, as well as numerous tumor necrosis factor (TNF)-alpha-expressing granulocytes and macrophages in airway wall (determined by immunohistochemical assay). Neutrophilia and TNF-alpha-expressing leukocytes, from collagen-PVP treated animals, diminished from 0.16 mg/ml, and eosinophilia from 0.66 mg/ml of collagen-PVP doses. Histological changes induced by allergen challenges include thickening of connective tissue below airway epithelium and vascular wall widening of airway adjacent vessels; these changes were reduced by collagen-PVP treatment. Collagen-PVP seems to have anti-inflammatory and antifibrotic properties in this guinea pig asthma model.

Salivary stones: symptoms, aetiology, biochemical composition and treatment.

PubMed

Kraaij, S; Karagozoglu, K H; Forouzanfar, T; Veerman, E C I; Brand, H S

2014-12-05

Salivary stones, also known as sialoliths, are calcified concrements in the salivary glands. Sialoliths are more frequently located in the submandibular gland (84%), than in the parotid gland (13%). The majority of the submandibular stones are located in Wharton's duct (90%), whereas parotid stones are more often located in the gland itself. Salivary stones consist of an amorphous mineralised nucleus, surrounded by concentric laminated layers of organic and inorganic substances. The organic components of salivary stones include collagen, glycoproteins, amino acids and carbohydrates. The major inorganic components are hydroxyapatite, carbonate apatite, whitlockite and brushite. The management of salivary stones is focused on removing the salivary stones and preservation of salivary gland function which depends on the size and location of the stone. Conservative management of salivary stones consists of salivary gland massage and the use of sialogogues. Other therapeutic options include removal of the stone or in some cases surgical removal of the whole salivary gland.

Imaging of cardiovascular structures using near-infrared femtosecond multiphoton laser scanning microscopy.

PubMed

Schenke-Layland, Katja; Riemann, Iris; Stock, Ulrich A; König, Karsten

2005-01-01

Multiphoton imaging represents a novel and very promising medical diagnostic technology for the high-resolution analysis of living biological tissues. We performed multiphoton imaging to analyzed structural features of extracellular matrix (ECM) components, e.g., collagen and elastin, of vital pulmonary and aortic heart valves. High-resolution autofluorescence images of collagenous and elastic fibers were demonstrated using multifluorophore, multiphoton excitation at two different wavelengths and optical sectioning, without the requirement of embedding, fixation, or staining. Collagenous structures were selectively imaged by detection of second harmonic generation (SHG). Additionally, routine histology and electron microscopy were integrated to verify the observed results. In comparison with pulmonary tissues, aortic heart valve specimens show very similar matrix formations. The quality of the resulting three-dimensional (3-D) images enabled the differentiation between collagenous and elastic fibers. These experimental results indicate that multiphoton imaging with near-infrared (NIR) femtosecond laser pulses may prove to be a useful tool for the nondestructive monitoring and characterization of cardiovascular structures. Copyright 2005 Society of Photo-Optical Instrumentation Engineers.

A randomized controlled trial on the efficacy and safety of a food ingredient, collagen hydrolysate, for improving joint comfort.

PubMed

Benito-Ruiz, P; Camacho-Zambrano, M M; Carrillo-Arcentales, J N; Mestanza-Peralta, M A; Vallejo-Flores, C A; Vargas-López, S V; Villacís-Tamayo, R A; Zurita-Gavilanes, L A

2009-01-01

Current options to promote joint comfort are limited to medicines that can reduce pain but can also have adverse effects. Collagen, a major component of joint cartilage, is found in the diet, particularly in meat. Its hydrolysed form, collagen hydrolysate (CH), is well absorbed. CH may stimulate the joint matrix cells to synthesize collagen, so helping to maintain the structure of the joint and potentially to aid joint comfort. In a randomized, double-blind, controlled multicentre trial, 250 subjects with primary osteoarthritis of the knee were given 10 g CH daily for 6 months. There was a significant improvement in knee joint comfort as assessed by visual analogue scales to assess pain and the Womac pain subscale. Subjects with the greatest joint deterioration, and with least intake of meat protein in their habitual diets, benefited most. CH is safe and effective and warrants further consideration as a food ingredient.

Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure

PubMed Central

Chen, Xiyi; Nadiarynkh, Oleg; Plotnikov, Sergey; Campagnola, Paul J

2013-01-01

Second-harmonic generation (SHG) microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. Because of its underlying physical origin, it is highly sensitive to the collagen fibril/fiber structure, and, importantly, to changes that occur in diseases such as cancer, fibrosis and connective tissue disorders. We discuss how SHG can be used to obtain more structural information on the assembly of collagen in tissues than is possible by other microscopy techniques. We first provide an overview of the state of the art and the physical background of SHG microscopy, and then describe the optical modifications that need to be made to a laser-scanning microscope to enable the measurements. Crucial aspects for biomedical applications are the capabilities and limitations of the different experimental configurations. We estimate that the setup and calibration of the SHG instrument from its component parts will require 2–4 weeks, depending on the level of the user’s experience. PMID:22402635

IgE reactivity to alpha1 and alpha2 chains of bovine type 1 collagen in children with bovine gelatin allergy.

PubMed

Sakaguchi, M; Hori, H; Hattori, S; Irie, S; Imai, A; Yanagida, M; Miyazawa, H; Toda, M; Inouye, S

1999-09-01

Anaphylactic reactions to measles, mumps, and rubella vaccines, including gelatin as a stabilizer, have been reported. It had been found that most of these reactions to live vaccines are caused by the bovine gelatin included in these vaccines. Gelatin mainly includes denatured type I collagen, which consists of alpha1 and alpha2 chains. The current study was designed to investigate the IgE reactivity to alpha1 and alpha2 chains of bovine type I collagen in gelatin-sensitive children. Serum samples were taken from 10 children who had anaphylaxis to the vaccines and high levels of specific IgE to bovine gelatin. Bovine type I collagen was isolated from bovine skin and then separated to alpha1 and alpha2 chains by column chromatography. IgE reactivity to denatured type I collagen and its alpha1 and alpha2 chains was analyzed by immunoblotting, ELISA, and histamine release from the mast cells passive sensitized with IgE antibodies in pooled serum of the children. All children had specific IgE to bovine type I collagen. Furthermore, IgE antibodies in their sera reacted with the alpha;2 chain but not with the alpha1 chain. Similarly, the mast cells sensitized with pooled sera in the children showed alpha2 chain-specific histamine release but not alpha1 chain-specific histamine release. In gelatin allergy denatured bovine type I collagen is a major allergen and IgE-binding sites exist in the alpha2 chain of type I collagen.

Structure of collagen-glycosaminoglycan matrix and the influence to its integrity and stability.

PubMed

Bi, Yuying; Patra, Prabir; Faezipour, Miad

2014-01-01

Glycosaminoglycan (GAG) is a chain-like disaccharide that is linked to polypeptide core to connect two collagen fibrils/fibers and provide the intermolecular force in Collagen-GAG matrix (C-G matrix). Thus, the distribution of GAG in C-G matrix contributes to the integrity and mechanical properties of the matrix and related tissue. This paper analyzes the transverse isotropic distribution of GAG in C-G matrix. The angle of GAGs related to collagen fibrils is used as parameters to qualify the GAGs isotropic characteristic in both 3D and 2D rendering. Statistical results included that over one third of GAGs were perpendicular directed to collagen fibril with symmetrical distribution for both 3D matrix and 2D plane cross through collagen fibrils. The three factors tested in this paper: collagen radius, collagen distribution, and GAGs density, were not statistically significant for the strength of Collagen-GAG matrix in 3D rendering. However in 2D rendering, a significant factor found was the radius of collagen in matrix for the GAGs directed to orthogonal plane of Collagen-GAG matrix. Between two cross-section selected from Collagen-GAG matrix model, the plane cross through collagen fibrils was symmetrically distributed but the total percentage of perpendicular directed GAG was deducted by decreasing collagen radius. There were some symmetry features of GAGs angle distribution in selected 2D plane that passed through space between collagen fibrils, but most models showed multiple peaks in GAGs angle distribution. With less GAGs directed to perpendicular of collagen fibril, strength in collagen cross-section weakened. Collagen distribution was also a factor that influences GAGs angle distribution in 2D rendering. True hexagonal collagen packaging is reported in this paper to have less strength at collagen cross-section compared to quasi-hexagonal collagen arrangement. In this work focus is on GAGs matrix within the collagen and its relevance to anisotropy.

Imaging Collagen in Scar Tissue: Developments in Second Harmonic Generation Microscopy for Biomedical Applications

PubMed Central

Mostaço-Guidolin, Leila; Rosin, Nicole L.; Hackett, Tillie-Louise

2017-01-01

The ability to respond to injury with tissue repair is a fundamental property of all multicellular organisms. The extracellular matrix (ECM), composed of fibrillar collagens as well as a number of other components is dis-regulated during repair in many organs. In many tissues, scaring results when the balance is lost between ECM synthesis and degradation. Investigating what disrupts this balance and what effect this can have on tissue function remains an active area of research. Recent advances in the imaging of fibrillar collagen using second harmonic generation (SHG) imaging have proven useful in enhancing our understanding of the supramolecular changes that occur during scar formation and disease progression. Here, we review the physical properties of SHG, and the current nonlinear optical microscopy imaging (NLOM) systems that are used for SHG imaging. We provide an extensive review of studies that have used SHG in skin, lung, cardiovascular, tendon and ligaments, and eye tissue to understand alterations in fibrillar collagens in scar tissue. Lastly, we review the current methods of image analysis that are used to extract important information about the role of fibrillar collagens in scar formation. PMID:28809791

The anti-photoaging effect of antioxidant collagen peptides from silver carp (Hypophthalmichthys molitrix) skin is preferable to tea polyphenols and casein peptides.

PubMed

Zhang, Ling; Zheng, Yayao; Cheng, Xiaofeng; Meng, Mengfei; Luo, Yongkang; Li, Bo

2017-04-19

Applying antioxidants to attenuate skin photoaging has received great attention. In this study, antioxidant collagen peptides (ACPs) with different activities were prepared using different proteases, called high (HCP), medium (MCP) and low antioxidant collagen peptides (LCP). The effects of ACPs, tea polyphenols (TP) and casein peptides (CP) on the photoaged skin of mice were evaluated and compared. Ingestion of ACPs significantly alleviated UV-induced abnormal alterations of skin components and antioxidative indicators in both serum and skin (p < 0.05). In addition, HCP had the best effect on protecting skin from photoaging among the three collagen peptides, with no significant differences between MCP and LCP (p > 0.05). TP and CP, with higher antioxidant activity in vitro, only significantly increased hydroxyproline content (only in the TP group) and CAT activity and decreased protein carbonyl content at week 2, showing a much weaker effect than that of the ACP groups. The histological analysis result further demonstrates that ACPs exerted a stronger beneficial effect on normalizing skin structure and collagen arrangement than TP and CP. Accordingly, ACPs have potential for nutraceuticals as anti-skin-photoaging ingredients.

Cytocompatibility and biologic characteristics of synthetic scaffold materials of rabbit acellular vascular matrix combining with human-like collagen I.

PubMed

Liu, Xuqian; Wang, Jie; Dong, Fusheng; Song, Peng; Tian, Songbo; Li, Hexiang; Hou, Yali

2017-10-01

Scaffold material provides a three-dimensional growing environment for seed cells in the research field of tissue engineering. In the present study, rabbit arterial blood vessel cells were chemically removed with trypsin and Triton X-100 to prepare rabbit acellular vascular matrix scaffold material. Observation by He&Masson staining revealed that no cellular components or nuclei existed in the vascular intima and media after decellularization. Human-like collagen I was combined with acellular vascular matrix by freeze-drying to prepare an acellular vascular matrix-0.25% human-like collagen I scaffold to compensate for the extracellular matrix loss during the decellularization process. We next performed a series of experiments to test the water absorbing quality, biomechanics, pressure resistance, cytotoxicity, and ultra-micro structure of the acellular vascular matrix composite material and natural rabbit artery and found that the acellular vascular matrix-0.25% human-like collagen I material behaved similarly to natural rabbit artery. In conclusion, the acellular vascular matrix-0.25% human-like collagen I composite material provides a new approach and lays the foundation for novel scaffold material research into tissue engineering of blood vessels.

Implications of obesity for tendon structure, ultrastructure and biochemistry: a study on Zucker rats.

PubMed

Biancalana, Adriano; Velloso, Lício Augusto; Taboga, Sebastião Roberto; Gomes, Laurecir

2012-02-01

The extracellular matrix consists of collagen, proteoglycans and non-collagen proteins. The incidence of obesity and associated diseases is currently increasing in developed countries. Obesity is considered to be a disease of modern times, and genes predisposing to the disease have been identified in humans and animals. The objective of the present study was to compare the morphological and biochemical aspects of the deep digital flexor tendon of lean (Fa/Fa or Fa/fa) and genetically obese (fa/fa) Zucker rats. Ultrastructural analysis showed the presence of lipid droplets in both groups, whereas disorganized collagen fibril bundles were observed in obese animals. Lean animals presented a larger amount of non-collagen proteins and glycosaminoglycans than obese rats. We propose that the overweight and lesser physical activity in obese animals may have provoked the alterations in the composition and organization of extracellular matrix components but a genetic mechanism cannot be excluded. These alterations might be related to organizational and structural modifications in the collagen bundles that influence the mechanical properties of tendons and the progression to a pathological state. Copyright © 2011 Elsevier Ltd. All rights reserved.

Role of Collagen and Inorganic Components in Electrical Polarizability of Bone

PubMed Central

HIRATAI, Rumi; NAKAMURA, Miho; YAMASHITA, Kimihiro

2013-01-01

ABSTRACT Hydroxyapatite (HA) has polarization capability and is able to accumulate electrical storage in bone. Experiments were conducted to measure the polarization capability of rabbit femurs. After preparing and polarizing bone samples using 2% KOH treatment (denoted 2% koh), 2% KOH and baking (2% koh+bake) and decalcification (decalcification) as well as untreated bone (untreated), stored charges were quantitatively determined using thermally stimulated depolarization current (TSDC) measurements. In TSDC spectra, untreated and 2% koh samples showed peaks at 100 and 500°C, while 2% koh+bake showed one peak at 580°C and decalcification one peak around 100°C. These evidences indicated that collagen and inorganic components play a major role in polarization of the bone at different temperature conditions. PMID:24141277

Regeneration of cervix after excisional treatment for cervical intraepithelial neoplasia: a study of collagen distribution.

PubMed

Phadnis, S V; Atilade, A; Bowring, J; Kyrgiou, M; Young, M P A; Evans, H; Paraskevaidis, E; Walker, P

2011-12-01

To study the distribution of collagen in the regenerated cervical tissue after excisional treatment for cervical intraepithelial neoplasia (CIN). Cohort study. A large tertiary teaching hospital in London. Women who underwent repeat excisional treatment for treatment failure or persistent CIN. Eligible women who underwent a repeat excisional treatment for treatment failure, including hysterectomy, between January 2002 and December 2007 in our colposcopy unit were identified by the Infoflex(®) database and SNOMED encoded histopathology database. Collagen expression was assessed using picro-Sirius red stain and the intensity of staining was compared in paired specimens from the first and second treatments. Differences in collagen expression were examined in the paired excisional treatment specimens. A total of 17 women were included. Increased collagen expression in the regenerated cervical tissue of the second cone compared with the first cone was noted in six women, decreased expression was noted in five women, and the pattern of collagen distribution was equivocal in six women. There is no overall change in collagen distribution during regeneration following excisional treatment for CIN. © 2011 The Authors BJOG An International Journal of Obstetrics and Gynaecology © 2011 RCOG.

Tunable Collagen I Hydrogels for Engineered Physiological Tissue Micro-Environments

PubMed Central

Antoine, Elizabeth E.; Vlachos, Pavlos P.; Rylander, Marissa N.

2015-01-01

Collagen I hydrogels are commonly used to mimic the extracellular matrix (ECM) for tissue engineering applications. However, the ability to design collagen I hydrogels similar to the properties of physiological tissues has been elusive. This is primarily due to the lack of quantitative correlations between multiple fabrication parameters and resulting material properties. This study aims to enable informed design and fabrication of collagen hydrogels in order to reliably and reproducibly mimic a variety of soft tissues. We developed empirical predictive models relating fabrication parameters with material and transport properties. These models were obtained through extensive experimental characterization of these properties, which include compression modulus, pore and fiber diameter, and diffusivity. Fabrication parameters were varied within biologically relevant ranges and included collagen concentration, polymerization pH, and polymerization temperature. The data obtained from this study elucidates previously unknown fabrication-property relationships, while the resulting equations facilitate informed a priori design of collagen hydrogels with prescribed properties. By enabling hydrogel fabrication by design, this study has the potential to greatly enhance the utility and relevance of collagen hydrogels in order to develop physiological tissue microenvironments for a wide range of tissue engineering applications. PMID:25822731

Assessing the age of animal bones and ivories by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Sharikova, Anna; Peerzada, Lubna; Khmaladze, Alexander

2018-02-01

A fast, convenient way to determine the age of bones and ivories is important both in forensics and for classifying art objects in collections of art experts, restorers, art galleries and museums. Knowing the age of elephant tusks is also essential because there are many date-specific regulations of ivory trade. Radiocarbon dating is the standard method used to determine the age of organic materials, but it is expensive, time consuming, and damages the sample in the process. Raman spectroscopy is sensitive to rotational and vibrational molecular transitions, and also intermolecular vibrations. Therefore, it can provide information about sample make up, such as proteins and minerals, as well as detect spectral signatures associated with structural changes in molecules. Since Raman spectroscopy identifies the molecular bonds present in a sample, it is often used to determine its chemical composition. Bones and ivories contain two primary components: collagen and bioapatite. As the protein collagen deteriorates with time, its Raman signal decreases. The ratio of collagen-to-bioapatite peaks, therefore, is smaller in the older samples compared to the younger ones, providing a basis for sample dating. We employed Raman spectroscopy to non-destructively determine the age of several elephant tusk fragments. We have also used it to identify ivory imitations made of vegetable and plastic materials. Such materials have entirely different chemical composition, and their spectra are easily distinguished from those of bone and ivory. Peak fitting was employed to determine collagen and bioapatite components.

A Structurally and Functionally Biomimetic Biphasic Scaffold for Intervertebral Disc Tissue Engineering

PubMed Central

Choy, Andrew Tsz Hang; Chan, Barbara Pui

2015-01-01

Tissue engineering offers high hopes for the treatment of intervertebral disc (IVD) degeneration. Whereas scaffolds of the disc nucleus and annulus have been extensively studied, a truly biomimetic and mechanically functional biphasic scaffold using naturally occurring extracellular matrix is yet to be developed. Here, a biphasic scaffold was fabricated with collagen and glycosaminoglycans (GAGs), two of the most abundant extracellular matrix components in the IVD. Following fabrication, the scaffold was characterized and benchmarked against native disc. The biphasic scaffold was composed of a collagen-GAG co-precipitate making up the nucleus pulposus-like core, and this was encapsulated in multiple lamellae of photochemically crosslinked collagen membranes comprising the annulus fibrosus-like lamellae. On mechanical testing, the height of our engineered disc recovered by ~82-89% in an annulus-independent manner, when compared with the 99% recovery exhibited by native disc. The annulus-independent nature of disc height recovery suggests that the fluid replacement function of the engineered nucleus pulposus core might mimic this hitherto unique feature of native disc. Biphasic scaffolds comprised of 10 annulus fibrosus-like lamellae had the best overall mechanical performance among the various designs owing to their similarity to native disc in most aspects, including elastic compliance during creep and recovery, and viscous compliance during recovery. However, the dynamic mechanical performance (including dynamic stiffness and damping factor) of all the biphasic scaffolds was similar to that of the native discs. This study contributes to the rationalized design and development of a biomimetic and mechanically viable biphasic scaffold for IVD tissue engineering. PMID:26115332

FK506-binding protein 10 (FKBP10) regulates lung fibroblast migration via collagen VI synthesis.

PubMed

Knüppel, Larissa; Heinzelmann, Katharina; Lindner, Michael; Hatz, Rudolf; Behr, Jürgen; Eickelberg, Oliver; Staab-Weijnitz, Claudia A

2018-04-19

In idiopathic pulmonary fibrosis (IPF), fibroblasts gain a more migratory phenotype and excessively secrete extracellular matrix (ECM), ultimately leading to alveolar scarring and progressive dyspnea. Here, we analyzed the effects of deficiency of FK506-binding protein 10 (FKBP10), a potential IPF drug target, on primary human lung fibroblast (phLF) adhesion and migration. Using siRNA, FKBP10 expression was inhibited in phLF in absence or presence of 2ng/ml transforming growth factor-β1 (TGF-β1) and 0.1mM 2-phosphoascorbate. Effects on cell adhesion and migration were monitored by an immunofluorescence (IF)-based attachment assay, a conventional scratch assay, and single cell tracking by time-lapse microscopy. Effects on expression of key players in adhesion dynamics and migration were analyzed by qPCR and Western Blot. Colocalization was evaluated by IF microscopy and by proximity ligation assays. FKBP10 knockdown significantly attenuated adhesion and migration of phLF. Expression of collagen VI was decreased, while expression of key components of the focal adhesion complex was mostly upregulated. The effects on migration were 2-phosphoascorbate-dependent, suggesting collagen synthesis as the underlying mechanism. FKBP10 colocalized with collagen VI and coating culture dishes with collagen VI, and to a lesser extent with collagen I, abolished the effect of FKBP10 deficiency on migration. These findings show, to our knowledge for the first time, that FKBP10 interacts with collagen VI and that deficiency of FKBP10 reduces phLF migration mainly by downregulation of collagen VI synthesis. The results strengthen FKBP10 as an important intracellular regulator of ECM remodeling and support the concept of FKBP10 as drug target in IPF.

Compression-induced structural and mechanical changes of fibrin-collagen composites.

PubMed

Kim, O V; Litvinov, R I; Chen, J; Chen, D Z; Weisel, J W; Alber, M S

2017-07-01

Fibrin and collagen as well as their combinations play an important biological role in tissue regeneration and are widely employed in surgery as fleeces or sealants and in bioengineering as tissue scaffolds. Earlier studies demonstrated that fibrin-collagen composite networks displayed improved tensile mechanical properties compared to the isolated protein matrices. Unlike previous studies, here unconfined compression was applied to a fibrin-collagen filamentous polymer composite matrix to study its structural and mechanical responses to compressive deformation. Combining collagen with fibrin resulted in formation of a composite hydrogel exhibiting synergistic mechanical properties compared to the isolated fibrin and collagen matrices. Specifically, the composite matrix revealed a one order of magnitude increase in the shear storage modulus at compressive strains>0.8 in response to compression compared to the mechanical features of individual components. These material enhancements were attributed to the observed structural alterations, such as network density changes, an increase in connectivity along with criss-crossing, and bundling of fibers. In addition, the compressed composite collagen/fibrin networks revealed a non-linear transformation of their viscoelastic properties with softening and stiffening regimes. These transitions were shown to depend on protein concentrations. Namely, a decrease in protein content drastically affected the mechanical response of the networks to compression by shifting the onset of stiffening to higher degrees of compression. Since both natural and artificially composed extracellular matrices experience compression in various (patho)physiological conditions, our results provide new insights into the structural biomechanics of the polymeric composite matrix that can help to create fibrin-collagen sealants, sponges, and tissue scaffolds with tunable and predictable mechanical properties. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation effect of low level Helium-Neon laser and Iranian propolis extract on Collagen Type I gene expression by human gingival fibroblasts: an in vitro study.

PubMed

Eslami, Hosein; Motahari, Paria; Safari, Ebrahim; Seyyedi, Maryam

2017-06-30

production of collagen by fibroblast cells is a key component in wound healing. Several studies have shown that low level laser therapy (LLLT) and propolis extract stimulate collagen Type I production. The aim of this study is to evaluation the combined effect of LLL helium neon (632.8 nm) and Iranian propolis extract on collagen Type I gene expression by human gingival fibroblasts (HGF3-PI 53). Human gingival fibroblasts after culturing divided into six experimental groups: G1-control group, which received no irradiation and propolis extract, G2-irradiated at1.5 J/cm 2 , G3-irradiated at 0.15 J/cm 2 , G4-recived extract of propolis, G5- combined extract of propolis and 1.5 J/cm 2 laser irradiation and G6- combined extract of propolis and 0.15 J/cm 2 laser irradiation. The experiments were conducted in triplicate. After 24 hour, the total RNA was extracted and cDNA synthesis was performed. Type I collagen mRNA expression was determined with real time PCR. The obtained results illustrated a statistically significant difference between G3 (0.15 J/cm 2 ) and G1 (control group) in levels of collagen Type I messenger RNA (mRNA) expression (p<0.05). The irradiated cells showed a 1.4 times increase in mRNA expression of the collagen Type I gene. Expression of this gene decreases in other groups that this difference was statistically significant. LLLT in different dosage and propolis extract may result in decreased or increased collagen type I gene expression. However this effect should be investigated in clinical studies.

Evaluation effect of low level Helium-Neon laser and Iranian propolis extract on Collagen Type I gene expression by human gingival fibroblasts: an in vitro study

PubMed Central

Eslami, Hosein; Motahari, Paria; Safari, Ebrahim; Seyyedi, Maryam

2017-01-01

Back ground and aim production of collagen by fibroblast cells is a key component in wound healing. Several studies have shown that low level laser therapy (LLLT) and propolis extract stimulate collagen Type I production. The aim of this study is to evaluation the combined effect of LLL helium neon (632.8 nm) and Iranian propolis extract on collagen Type I gene expression by human gingival fibroblasts (HGF3-PI 53). Methods and materials Human gingival fibroblasts after culturing divided into six experimental groups: G1-control group, which received no irradiation and propolis extract, G2-irradiated at1.5 J/cm2, G3-irradiated at 0.15 J/cm2, G4-recived extract of propolis, G5- combined extract of propolis and 1.5 J/cm2 laser irradiation and G6- combined extract of propolis and 0.15 J/cm2 laser irradiation. The experiments were conducted in triplicate. After 24 hour, the total RNA was extracted and cDNA synthesis was performed. Type I collagen mRNA expression was determined with real time PCR. Results The obtained results illustrated a statistically significant difference between G3 (0.15 J/cm2) and G1 (control group) in levels of collagen Type I messenger RNA (mRNA) expression (p<0.05). The irradiated cells showed a 1.4 times increase in mRNA expression of the collagen Type I gene. Expression of this gene decreases in other groups that this difference was statistically significant. Conclusion LLLT in different dosage and propolis extract may result in decreased or increased collagen type I gene expression. However this effect should be investigated in clinical studies. PMID:28785130

CARS and SHG microscopy to follow collagen production in living human corneal fibroblasts and mesenchymal stem cells in fibrin hydrogel 3D cultures

NASA Astrophysics Data System (ADS)

Mortati, L.; Divieto, C.; Sassi, M. P.

2012-05-01

Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with second harmonic generation (SHG) technique in order to follow the early stage of stem cell differentiation within a 3D scaffold. CARS microscopy can detect lipid membranes and droplet compartments in living cells and SHG microscopy enables a strong imaging contrast for molecules with a non-centrosymmetric ordered structure like collagen. One of the first evidence of hMSCs differentiation is the formation of an extracellular matrix (ECM) where the collagen protein is its main component. This work demonstrated the multimodal CARS and SHG microscopy as a powerful non-invasive label free technique to investigate the collagen production dynamic in living cell 3D cultures. Its ability to image the cell morphology and the produced collagen distribution on a long term (4 weeks) experiment allowed to obtain important information about the cell-scaffold interaction and the ECM production. The very low limit reached in detecting collagen has permitted to map even the small amount of collagen produced by the cells in few hours of culture. This demonstrates multimodal CARS and SHG microscopy as a novel method to follow cells collagen production and cells differentiation process. In addition the experiment shows that the technique is a powerful tool for imaging of very thick sections (about 4 mm). The study conducted on mesenchymal stem cell in fibrin gel cultures confirmed that differentiation stimulus is induced by the scaffold. The monitoring of stem cell differentiation within a scaffold in a non-destructive way will be an important advantage in regenerative medicine and tissue engineering field.

Extracellular post-translational modifications of collagen are major determinants of biomechanical properties of fetal bovine cortical bone.

PubMed

Garnero, Patrick; Borel, Olivier; Gineyts, Evelyne; Duboeuf, Francois; Solberg, Helene; Bouxsein, Mary L; Christiansen, Claus; Delmas, Pierre D

2006-03-01

Mechanical behavior of bone depends on its mass and architecture, and on the material properties of the matrix, which is composed of a mineral phase and an organic component mainly constituted of type I collagen. Mineral accounts largely for the stiffness of bone, whereas type I collagen provides bone its ductility and toughness, i.e., its ability to undergo deformation and absorb energy after it begins to yield. The molecular mechanisms underlying the effect of alterations in type I collagen on bone mechanical properties are unclear. We used an in vitro model of fetal bovine cortical bone specimens (n = 44), where the extent of type I collagen cross-linking was modified by incubation at 37 degrees C for 0, 60, 90 and 120 days, keeping constant the architecture and the mineral content. At each incubation time, the following parameters were determined: (1) the bone concentration of enzymatic (pyridinoline; PYD and deoxypyridinoline, DPD) and non-enzymatic (pentosidine) crosslinks by HPLC, (2) the extent of aspartic acid isomerization of the type I collagen C-telopeptide (CTX) by ELISA of native (alpha CTX) and isomerized (beta CTX) forms, (3) the mineral density by DXA, (4) the porosity by micro-computed tomography and (5) the bending and compressive mechanical properties. Incubation of bone specimens at 37 degrees C for 60 days increased the level (per molecule of collagen) of PYD (+98%, P = 0.005), DPD (+42%, P = 0.013), pentosidine (+55-fold, P = 0.005), and the degree of type I collagen C-telopeptide isomerization (+4.9-fold, P = 0.005). These biochemical changes of collagen were associated with a 30% decrease in bending and compressive yield stress and a 2.5-fold increase in compressive post-yield energy absorption (P < 0.02 for all), with no significant change of bone stiffness. In multivariate analyses, the level of collagen cross-linking was associated with yield stress and post-yield energy absorption independently of bone mineral density, explaining up to 25% of their variance. We conclude that the extent and nature of collagen cross-linking contribute to the mechanical properties of fetal bovine cortical bone independently of bone mineral density.

Platelet-Rich Plasma for the Treatment of Photodamage of the Skin of the Hands.

PubMed

Cabrera-Ramírez, J O; Puebla-Mora, A G; González-Ojeda, A; García-Martínez, D; Cortés-Lares, J A; Márquez-Valdés, A R; Contreras-Hernández, G I; Bracamontes-Blanco, J; Saucedo Ortiz, J A; Fuentes-Orozco, C

2017-10-01

Platelet-rich plasma (PRP) provides growth factors that stimulate fibroblast activation and induce the synthesis of collagen and other components of the extracellular matrix. The objective of this study was to evaluate the effect of PRP in the treatment of photodamage of the skin of the hands. Experimental study enrolling persons with photoaged skin on the dorsum of the hands (Glogau photoaging scale, type III, or Fitzpatrick wrinkle classification, type II) were included between August 2012 and January 2013. A histological comparison was made of skin biopsies taken before and after the application of PRP to the skin of the dorsum of the hands. The mean (SD) age of the 18 women enrolled was 47.9 (4.3) years. Histological analysis showed an increase in the number of fibroblasts (P<.001), number of vessels (P<.001), and collagen density (P=.27). These changes produced significant improvements in the Fitzpatrick wrinkle and elastosis scale (P<.001) and in the Glogau photoaging scale (P=.01). PRP induced a reduction in the manifestations of skin aging, including an improvement in wrinkles and elastosis. Copyright © 2017 AEDV. Publicado por Elsevier España, S.L.U. All rights reserved.

The Relation Between Collagen Fibril Kinematics and Mechanical Properties in the Mitral Valve Anterior Leaflet

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liao,J.; Yang, L.; Grashow, J.

2007-01-01

We have recently demonstrated that the mitral valve anterior leaflet (MVAL) exhibited minimal hysteresis, no strain rate sensitivity, stress relaxation but not creep (Grashow et al., 2006, Ann Biomed Eng., 34(2), pp. 315-325; Grashow et al., 2006, Ann Biomed. Eng., 34(10), pp. 1509-1518). However, the underlying structural basis for this unique quasi-elastic mechanical behavior is presently unknown. As collagen is the major structural component of the MVAL, we investigated the relation between collagen fibril kinematics (rotation and stretch) and tissue-level mechanical properties in the MVAL under biaxial loading using small angle X-ray scattering. A novel device was developed and utilizedmore » to perform simultaneous measurements of tissue level forces and strain under a planar biaxial loading state. Collagen fibril D-period strain ({epsilon}{sub D}) and the fibrillar angular distribution were measured under equibiaxial tension, creep, and stress relaxation to a peak tension of 90 N/m. Results indicated that, under equibiaxial tension, collagen fibril straining did not initiate until the end of the nonlinear region of the tissue-level stress-strain curve. At higher tissue tension levels, {epsilon}{sub D} increased linearly with increasing tension. Changes in the angular distribution of the collagen fibrils mainly occurred in the tissue toe region. Using {epsilon}{sub D}, the tangent modulus of collagen fibrils was estimated to be 95.5{+-}25.5 MPa, which was {approx}27 times higher than the tissue tensile tangent modulus of 3.58{+-}1.83 MPa. In creep tests performed at 90 N/m equibiaxial tension for 60 min, both tissue strain and D remained constant with no observable changes over the test length. In contrast, in stress relaxation tests performed for 90 min {epsilon}{sub D} was found to rapidly decrease in the first 10 min followed by a slower decay rate for the remainder of the test. Using a single exponential model, the time constant for the reduction in collagen fibril strain was 8.3 min, which was smaller than the tissue-level stress relaxation time constants of 22.0 and 16.9 min in the circumferential and radial directions, respectively. Moreover, there was no change in the fibril angular distribution under both creep and stress relaxation over the test period. Our results suggest that (1) the MVAL collagen fibrils do not exhibit intrinsic viscoelastic behavior, (2) tissue relaxation results from the removal of stress from the fibrils, possibly by a slipping mechanism modulated by noncollagenous components (e.g. proteoglycans), and (3) the lack of creep but the occurrence of stress relaxation suggests a 'load-locking' behavior under maintained loading conditions. These unique mechanical characteristics are likely necessary for normal valvular function.« less

Region-specific role of water in collagen unwinding and assembly.

PubMed

Ravikumar, Krishnakumar M; Hwang, Wonmuk

2008-09-01

Conformational stability of the collagen triple helix affects its turnover and determines tissue homeostasis. Although it is known that the presence of imino acids (prolines or hydroxyprolines) confer stability to the molecule, little is known regarding the stability of the imino-poor region lacking imino acids, which plays a key role in collagen cleavage. In particular, there have been continuing debates about the role of water in collagen stability. We addressed these issues using molecular dynamics simulations on 30-residue long collagen triple helices, including a structure that has a biologically relevant 9-residue imino-poor region from type III collagen (PDB ID: 1BKV). A torsional map approach was used to characterize the conformational motion of the molecule that differ between imino-rich and imino-poor regions. At temperatures 300 K and above, unwinding initiates at a common cleavage site, the glycine-isoleucine bond in the imino-poor region. This provides a linkage between previous observations that unwinding of the imino-poor region is a requirement for collagenase cleavage, and that isolated collagen molecules are unstable at body temperature. We found that unwinding of the imino-poor region is controlled by dynamic water bridges between backbone atoms with average lifetimes on the order of a few picoseconds, as the degree of unwinding strongly correlated with the loss of water bridges, and unwinding could be either prevented or enhanced, respectively by enforcing or forbidding water bridge formation. While individual water bridges were short-lived in the imino-poor region, the hydration shell surrounding the entire molecule was stable even at 330 K. The diameter of the hydrated collagen including the first hydration shell was about 14 A, in good agreement with the experimentally measured inter-collagen distances. These results elucidate the general role of water in collagen turnover: water not only affects collagen cleavage by controlling its torsional motion, but it also forms a larger-scale lubrication layer mediating collagen self-assembly. 2008 Wiley-Liss, Inc.

Novel synthesis strategy for composite hydrogel of collagen/hydroxyapatite-microsphere originating from conversion of CaCO3 templates.

PubMed

Wei, Qingrong; Lu, Jian; Wang, Qiaoying; Fan, Hongsong; Zhang, Xingdong

2015-03-20

Inspired by coralline-derived hydroxyapatite, we designed a methodological route to synthesize carbonated-hydroxyapatite microspheres from the conversion of CaCO3 spherulite templates within a collagen matrix under mild conditions and thus constructed the composite hydrogel of collagen/hydroxyapatite-microspheres. Fourier transform infrared spectroscopy (FTIR) and x-ray diffraction (XRD) were employed to confirm the successful generation of the carbonated hydroxyapatite phase originating from CaCO3, and the ratios of calcium to phosphate were tracked over time. Variations in the weight portion of the components in the hybrid gels before and after the phase transformation of the CaCO3 templates were identified via thermogravimetric analysis (TGA). Scanning electron microscopy (SEM) shows these composite hydrogels have a unique multiscale microstructure consisting of a collagen nanofibril network and hydroxyapatite microspheres. The relationship between the hydroxyapatite nanocrystals and the collagen fibrils was revealed by transmission electron microscopy (TEM) in detail, and the selected area electron diffraction (SAED) pattern further confirmed the results of the XRD analyses which show the typical low crystallinity of the generated hydroxyapatite. This smart synthesis strategy achieved the simultaneous construction of microscale hydroxyapatite particles and collagen fibrillar hydrogel, and appears to provide a novel route to explore an advanced functional hydrogel materials with promising potentials for applications in bone tissue engineering and reconstruction medicine.

Astragaloside IV controls collagen reduction in photoaging skin by improving transforming growth factor-β/Smad signaling suppression and inhibiting matrix metalloproteinase-1.

PubMed

Chen, Bin; Li, Ran; Yan, Ning; Chen, Gang; Qian, Wen; Jiang, Hui-Li; Ji, Chao; Bi, Zhi-Gang

2015-05-01

Exposure to ultraviolet (UV) light reduces levels of type I collagen in the dermis and results in human skin damage and premature skin aging (photoaging). This leads to a wrinkled appearance through the inhibition of transforming growth factor‑β (TGF‑β)/Smad signaling. UV irradiation increases type I collagen degradation through upregulating matrix metalloproteinase (MMP) expression. Astragaloside IV (AST) is one of the major active components extracted from Astragalus membranaceus. However, its multiple anti‑photoaging effects remain to be elucidated. In the present study, the effects of AST against collagen reduction in UV‑induced skin aging in human skin fibroblasts were investigated. The expression of type I procollagen (COL1), MMP‑1, TGF‑βRⅡ and Smad7 were determined using reverse transcription‑polymerase chain reaction, western blotting and ELISA, respectively. UV irradiation inhibits type I collagen production by suppressing the TGF‑β/Smad signaling pathway and increasing COL1 degradation by inducing MMP‑1 expression. Transforming growth factor‑β type II protein and COL1 mRNA decreased but MMP‑1 and Smad7 levels increased in the photoaging model group, which was reversed by topical application of AST. AST prevents collagen reduction from UV irradiation in photoaging skin by improving TGF‑β/Smad signaling suppression and inhibiting MMP‑1, thus AST may be a potential agent against skin photoaging.

Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum 91 in adhesion and its anti-adhesion potential against gut pathogen.

PubMed

Yadav, Ashok Kumar; Tyagi, Ashish; Kaushik, Jai Kumar; Saklani, Asha Chandola; Grover, Sunita; Batish, Virender Kumar

2013-12-14

Human feacal isolates were ascertain as genus Lactobacillus using specific primer LbLMA1/R16-1 and further identified as Lactobacillus plantarum with species specific primers Lpl-3/Lpl-2. 25 L. plantarum strains were further assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to immobilized human collagen type-1. Surface proteins were isolated from selected L. plantarum 91(Lp91) strain. The purified collagen binding protein (Cbp) protein was assessed for its anti-adhesion activity against enteric Escherichia coli 0157:H7 pathogen on immobilized collagen. Four L. plantarum strains displayed high degree of hydrophobicity and significant adhesion to collagen. A 72 kDa protein was purified which reduced 59.71% adhesion of E. coli 0157:H7 on immobilized collagen as compared to control well during adhesion assay. Cbp protein is the major influencing factor in inhibition of E. coli 0157:H7 adhesion with extracellular matrix (ECM) components. Hydrophobicity and adhesion potential are closely linked attributes precipitating in better colonization potential of the lactobacillus strains. Cbp is substantiated as a crucial surface protein contributing in adhesion of lactobacillus strains. The study can very well be the platform for commercialization of indigenous probiotic strain once their functional attributes are clinically explored. Copyright © 2013 Elsevier GmbH. All rights reserved.

3D bioprinting of BMSC-laden methacrylamide gelatin scaffolds with CBD-BMP2-collagen microfibers.

PubMed

Du, Mingchun; Chen, Bing; Meng, Qingyuan; Liu, Sumei; Zheng, Xiongfei; Zhang, Cheng; Wang, Heran; Li, Hongyi; Wang, Nuo; Dai, Jianwu

2015-12-18

Three-dimensional (3D) bioprinting combines biomaterials, cells and functional components into complex living tissues. Herein, we assembled function-control modules into cell-laden scaffolds using 3D bioprinting. A customized 3D printer was able to tune the microstructure of printed bone mesenchymal stem cell (BMSC)-laden methacrylamide gelatin scaffolds at the micrometer scale. For example, the pore size was adjusted to 282 ± 32 μm and 363 ± 60 μm. To match the requirements of the printing nozzle, collagen microfibers with a length of 22 ± 13 μm were prepared with a high-speed crusher. Collagen microfibers bound bone morphogenetic protein 2 (BMP2) with a collagen binding domain (CBD) as differentiation-control module, from which BMP2 was able to be controllably released. The differentiation behaviors of BMSCs in the printed scaffolds were compared in three microenvironments: samples without CBD-BMP2-collagen microfibers in the growth medium, samples without microfibers in the osteogenic medium and samples with microfibers in the growth medium. The results indicated that BMSCs showed high cell viability (>90%) during printing; CBD-BMP2-collagen microfibers induced BMSC differentiation into osteocytes within 14 days more efficiently than the osteogenic medium. Our studies suggest that these function-control modules are attractive biomaterials and have potential applications in 3D bioprinting.

Collagenase Santyl ointment: a selective agent for wound debridement.

PubMed

Shi, Lei; Carson, Dennis

2009-01-01

Enzymatic debridement is a frequently used technique for removal of necrotic tissue from wounds. Proteases with specificity to break down the collagenous materials in necrotic tissues can achieve selective debridement, digesting denatured collagen in eschar while sparing nonnecrotic tissues. This article provides information about the selectivity of a collagenase-based debriding agent, including evidence of its safe and efficacious uses. Recent research has been conducted, investigating the chemical and biological properties of collagenase ointment, including healing in animal models, digestion power on different collagen types, cell migration activity from collagen degradation products, and compatibility with various wound dressings and metal ions. Evidence presented demonstrates that collagenase ointment is an effective, selective, and safe wound debriding agent.

Collagen implants equipped with 'fish scale'-like nanoreservoirs of growth factors for bone regeneration.

PubMed

Eap, Sandy; Ferrand, Alice; Schiavi, Jessica; Keller, Laetitia; Kokten, Tunay; Fioretti, Florence; Mainard, Didier; Ladam, Guy; Benkirane-Jessel, Nadia

2014-01-01

Implants triggering rapid, robust and durable tissue regeneration are needed to shorten recovery times and decrease risks of postoperative complications for patients. Here, we describe active living collagen implants with highly promising bone regenerative properties. Bioactivity of the implants is obtained through the protective and stabilizing layer-by-layer immobilization of a protein growth factor in association with a polysaccharide (chitosan), within the form of nanocontainers decorating the collagen nanofibers. All components of the implants are US FDA approved. From both in vitro and in vivo evaluations, the sophisticated strategy described here should enhance, at a reduced cost, the safety and efficacy of the therapeutic implants in terms of large bone defects repair compared with current simplistic approaches based on the soaking of the implants with protein growth factor.

Strategies for Directing the Structure and Function of 3D Collagen Biomaterials across Length Scales

PubMed Central

Walters, Brandan D.; Stegemann, Jan P.

2013-01-01

Collagen type I is a widely used natural biomaterial that has found utility in a variety of biological and medical applications. Its well characterized structure and role as an extracellular matrix protein make it a highly relevant material for controlling cell function and mimicking tissue properties. Collagen type I is abundant in a number of tissues, and can be isolated as a purified protein. This review focuses on hydrogel biomaterials made by reconstituting collagen type I from a solubilized form, with an emphasis on in vitro studies in which collagen structure can be controlled. The hierarchical structure of collagen from the nanoscale to the macroscale is described, with an emphasis on how structure is related to function across scales. Methods of reconstituting collagen into hydrogel materials are presented, including molding of macroscopic constructs, creation of microscale modules, and electrospinning of nanoscale fibers. The modification of collagen biomaterials to achieve desired structures and functions is also addressed, with particular emphasis on mechanical control of collagen structure, creation of collagen composite materials, and crosslinking of collagenous matrices. Biomaterials scientists have made remarkable progress in rationally designing collagen-based biomaterials and in applying them to both the study of biology and for therapeutic benefit. This broad review illustrates recent examples of techniques used to control collagen structure, and to thereby direct its biological and mechanical functions. PMID:24012608

Collagen reconstitution is inversely correlated with induction of limb regeneration in Ambystoma mexicanum.

PubMed

Satoh, Akira; Hirata, Ayako; Makanae, Aki

2012-03-01

Amphibians can regenerate missing body parts, including limbs. The regulation of collagen has been considered to be important in limb regeneration. Collagen deposition is suppressed during limb regeneration, so we investigated collagen deposition and apical epithelial cap (AEC) formation during axolotl limb regeneration. The accessory limb model (ALM) has been developed as an alternative model for studying limb regeneration. Using this model, we investigated the relationship between nerves, epidermis, and collagen deposition. We found that Sp-9, an AEC marker gene, was upregulated by direct interaction between nerves and epidermis. However, collagen deposition hindered this interaction, and resulted in the failure of limb regeneration. During wound healing, an increase in deposition of collagen caused a decrease in the blastema induction rate in ALM. Wound healing and limb regeneration are alternate processes.

Core-shell hydrogel beads with extracellular matrix for tumor spheroid formation.

PubMed

Yu, L; Grist, S M; Nasseri, S S; Cheng, E; Hwang, Y-C E; Ni, C; Cheung, K C

2015-03-01

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture.

Osteoblast Differentiation on Collagen Scaffold with Immobilized Alkaline Phosphatase.

PubMed

Jafary, F; Hanachi, P; Gorjipour, K

2017-01-01

In tissue engineering, scaffold characteristics play an important role in the biological interactions between cells and the scaffold. Cell adhesion, proliferation, and activation depend on material properties used for the fabrication of scaffolds. In the present investigation, we used collagen with proper characteristics including mechanically stability, biodegradability and low antigenicity. Optimization of the scaffold was done by immobilization of alkaline phosphatase on the collagen surface via cross-linking method, because this enzyme is one of the most important markers of osteoblast, which increases inorganic phosphate concentration and promote mineralization of bone formation. Alkaline phosphatase was immobilized on a collagen surface by 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride, as a reagent. Then, rat mesenchymal stem cells were cultured in osteogenic medium in control and treated groups. The osteogenesis-related genes were compared between treatments (differentiated cells with immobilized alkaline phosphatase/collagen scaffold) and control groups (differentiated cells on collagen surface without alkaline phosphatase) on days 3 and 7 by quantitative real-time PCR (QIAGEN software). Several genes, including alkaline phosphatase, collagen type I and osteocalcine associated with calcium binding and mineralization, showed upregulation in expression during the first 3 days, whereas tumor necrosis factor-α, acting as an inhibitor of differentiation, was down-regulated during osteogenesis. Collagen scaffold with immobilized alkaline phosphatase can be utilized as a good candidate for enhancing the differentiation of osteoblasts from mesenchymal stem cells.

Cardiac fibrosis and dysfunction in experimental diabetic cardiomyopathy are ameliorated by alpha-lipoic acid.

PubMed

Li, Chun-jun; Lv, Lin; Li, Hui; Yu, De-min

2012-06-19

Alpha-lipoic acid (ALA), a naturally occurring compound, exerts powerful protective effects in various cardiovascular disease models. However, its role in protecting against diabetic cardiomyopathy (DCM) has not been elucidated. In this study, we have investigated the effects of ALA on cardiac dysfunction, mitochondrial oxidative stress (MOS), extracellular matrix (ECM) remodeling and interrelated signaling pathways in a diabetic rat model. Diabetes was induced in rats by I.V. injection of streptozotocin (STZ) at 45 mg/kg. The animals were randomly divided into 4 groups: normal groups with or without ALA treatment, and diabetes groups with or without ALA treatment. All studies were carried out 11 weeks after induction of diabetes. Cardiac catheterization was performed to evaluate cardiac function. Mitochondrial oxidative biochemical parameters were measured by spectophotometeric assays. Extracellular matrix content (total collagen, type I and III collagen) was assessed by staining with Sirius Red. Gelatinolytic activity of Pro- and active matrix metalloproteinase-2 (MMP-2) levels were analyzed by a zymogram. Cardiac fibroblasts differentiation to myofibroblasts was evaluated by Western blot measuring smooth muscle actin (α-SMA) and transforming growth factor-β (TGF-β). Key components of underlying signaling pathways including the phosphorylation of c-Jun N-terminal kinase (JNK), p38 MAPK and ERK were also assayed by Western blot. DCM was successfully induced by the injection of STZ as evidenced by abnormal heart mass and cardiac function, as well as the imbalance of ECM homeostasis. After administration of ALA, left ventricular dysfunction greatly improved; interstitial fibrosis also notably ameliorated indicated by decreased collagen deposition, ECM synthesis as well as enhanced ECM degradation. To further assess the underlying mechanism of improved DCM by ALA, redox status and cardiac remodeling associated signaling pathway components were evaluated. It was shown that redox homeostasis was disturbed and MAPK signaling pathway components activated in STZ-induced DCM animals. While ALA treatment favorably shifted redox homeostasis and suppressed JNK and p38 MAPK activation. These results, coupled with the excellent safety and tolerability profile of ALA in humans, demonstrate that ALA may have therapeutic potential in the treatment of DCM by attenuating MOS, ECM remodeling and JNK, p38 MAPK activation.

Collagen-Like Proteins in Pathogenic E. coli Strains

PubMed Central

Ghosh, Neelanjana; McKillop, Thomas J.; Jowitt, Thomas A.; Howard, Marjorie; Davies, Heather; Holmes, David F.; Roberts, Ian S.; Bella, Jordi

2012-01-01

The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages. PMID:22701585

Nanoscale Structure of Type I Collagen Fibrils: Quantitative Measurement of D-spacing

PubMed Central

Erickson, Blake; Fang, Ming; Wallace, Joseph M.; Orr, Bradford G.; Les, Clifford M.; Holl, Mark M. Banaszak

2012-01-01

This paper details a quantitative method to measure the D-periodic spacing of Type I collagen fibrils using Atomic Force Microscopy coupled with analysis using a 2D Fast Fourier Transform approach. Instrument calibration, data sampling and data analysis are all discussed and comparisons of the data to the complementary methods of electron microscopy and X-ray scattering are made. Examples of the application of this new approach to the analysis of Type I collagen morphology in disease models of estrogen depletion and Osteogenesis Imperfecta are provided. We demonstrate that it is the D-spacing distribution, not the D-spacing mean, that showed statistically significant differences in estrogen depletion associated with early stage Osteoporosis and Osteogenesis Imperfecta. The ability to quantitatively characterize nanoscale morphological features of Type I collagen fibrils will provide important structural information regarding Type I collagen in many research areas, including tissue aging and disease, tissue engineering, and gene knock out studies. Furthermore, we also envision potential clinical applications including evaluation of tissue collagen integrity under the impact of diseases or drug treatments. PMID:23027700

Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis.

PubMed

McKleroy, William; Lee, Ting-Hein; Atabai, Kamran

2013-06-01

Pulmonary fibrosis is a vexing clinical problem with no proven therapeutic options. In the normal lung there is continuous collagen synthesis and collagen degradation, and these two processes are precisely balanced to maintain normal tissue architecture. With lung injury there is an increase in the rate of both collagen production and collagen degradation. The increase in collagen degradation is critical in preventing the formation of permanent scar tissue each time the lung is exposed to injury. In pulmonary fibrosis, collagen degradation does not keep pace with collagen production, resulting in extracellular accumulation of fibrillar collagen. Collagen degradation occurs through both extracellular and intracellular pathways. The extracellular pathway involves cleavage of collagen fibrils by proteolytic enzyme including the metalloproteinases. The less-well-described intracellular pathway involves binding and uptake of collagen fragments by fibroblasts and macrophages for lysosomal degradation. The relationship between these two pathways and their relevance to the development of fibrosis is complex. Fibrosis in the lung, liver, and skin has been associated with an impaired degradative environment. Much of the current scientific effort in fibrosis is focused on understanding the pathways that regulate increased collagen production. However, recent reports suggest an important role for collagen turnover and degradation in regulating the severity of tissue fibrosis. The objective of this review is to evaluate the roles of the extracellular and intracellular collagen degradation pathways in the development of fibrosis and to examine whether pulmonary fibrosis can be viewed as a disease of impaired matrix degradation rather than a disease of increased matrix production.

Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis

PubMed Central

McKleroy, William; Lee, Ting-Hein

2013-01-01

Pulmonary fibrosis is a vexing clinical problem with no proven therapeutic options. In the normal lung there is continuous collagen synthesis and collagen degradation, and these two processes are precisely balanced to maintain normal tissue architecture. With lung injury there is an increase in the rate of both collagen production and collagen degradation. The increase in collagen degradation is critical in preventing the formation of permanent scar tissue each time the lung is exposed to injury. In pulmonary fibrosis, collagen degradation does not keep pace with collagen production, resulting in extracellular accumulation of fibrillar collagen. Collagen degradation occurs through both extracellular and intracellular pathways. The extracellular pathway involves cleavage of collagen fibrils by proteolytic enzyme including the metalloproteinases. The less-well-described intracellular pathway involves binding and uptake of collagen fragments by fibroblasts and macrophages for lysosomal degradation. The relationship between these two pathways and their relevance to the development of fibrosis is complex. Fibrosis in the lung, liver, and skin has been associated with an impaired degradative environment. Much of the current scientific effort in fibrosis is focused on understanding the pathways that regulate increased collagen production. However, recent reports suggest an important role for collagen turnover and degradation in regulating the severity of tissue fibrosis. The objective of this review is to evaluate the roles of the extracellular and intracellular collagen degradation pathways in the development of fibrosis and to examine whether pulmonary fibrosis can be viewed as a disease of impaired matrix degradation rather than a disease of increased matrix production. PMID:23564511

Contribution of collagen fiber undulation to regional biomechanical properties along porcine thoracic aorta.

PubMed

Zeinali-Davarani, Shahrokh; Wang, Yunjie; Chow, Ming-Jay; Turcotte, Raphaël; Zhang, Yanhang

2015-05-01

As major extracellular matrix components, elastin, and collagen play crucial roles in regulating the mechanical properties of the aortic wall and, thus, the normal cardiovascular function. The mechanical properties of aorta, known to vary with age and multitude of diseases as well as the proximity to the heart, have been attributed to the variations in the content and architecture of wall constituents. This study is focused on the role of layer-specific collagen undulation in the variation of mechanical properties along the porcine descending thoracic aorta. Planar biaxial tensile tests are performed to characterize the hyperelastic anisotropic mechanical behavior of tissues dissected from four locations along the thoracic aorta. Multiphoton microscopy is used to image the associated regional microstructure. Exponential-based and recruitment-based constitutive models are used to account for the observed mechanical behavior while considering the aortic wall as a composite of two layers with independent properties. An elevated stiffness is observed in distal regions compared to proximal regions of thoracic aorta, consistent with sharper and earlier collagen recruitment estimated for medial and adventitial layers in the models. Multiphoton images further support our prediction that higher stiffness in distal regions is associated with less undulation in collagen fibers. Recruitment-based models further reveal that regardless of the location, collagen in the media is recruited from the onset of stretching, whereas adventitial collagen starts to engage with a delay. A parameter sensitivity analysis is performed to discriminate between the models in terms of the confidence in the estimated model parameters.

Biosensor studies of collagen and laminin binding with immobilized Escherichia coli O157:H7 and inhibition with naturally occurring food additives

NASA Astrophysics Data System (ADS)

Medina, Marjorie B.

1999-01-01

Escherichia coli O157:H7 outbreaks were mostly due to consumption of undercooked contaminated beef which resulted in severe illness and several fatalities. Recalls of contaminated meat are costly for the meat industry. Our research attempts to understand the mechanisms of bacterial adhesion on animal carcass in order to eliminate or reduce pathogens in foods. We have reported the interactions of immobilized E. coli O157:H7 cells with extracellular matrix (ECM) components using a surface plasmon resonance biosensor (BIAcore). These studies showed that immobilized bacterial cells allowed the study of real-time binding interactions of bacterial surface with the ECM compounds, collagen I, laminin and fibronectin. Collagen I and laminin bound to the E. coli sensor surface with dissociation and association rates ranging from 106 to 109. Binding of collagen I and laminin mixture resulted in synergistic binding signals. An inhibition model was derived using collagen-laminin as the ligand which binds with E. coli sensor. A select group of naturally occurring food additives was evaluated by determining their effectivity in inhibiting the collagen-laminin binding to the bacterial sensor. Bound collagen-laminin was detached from the E. coli sensor surface with the aid of an organic acid. The biosensor results were verified with cell aggregation assays which were observed with optical and electron microscopes. These biosensor studies provided understanding of bacterial adhesion to connective tissue macromolecules. It also provided a model system for the rapid assessment of potential inhibitors that can be used in carcass treatment to inhibit or reduce bacterial contamination.

Embryonic ablation of osteoblast Smad4 interrupts matrix synthesis in response to canonical Wnt signaling and causes an osteogenesis-imperfecta-like phenotype

PubMed Central

Salazar, Valerie S.; Zarkadis, Nicholas; Huang, Lisa; Norris, Jin; Grimston, Susan K.; Mbalaviele, Gabriel; Civitelli, Roberto

2013-01-01

Summary To examine interactions between bone morphogenic protein (BMP) and canonical Wnt signaling during skeletal growth, we ablated Smad4, a key component of the TGF-β–BMP pathway, in Osx1+ cells in mice. We show that loss of Smad4 causes stunted growth, spontaneous fractures and a combination of features seen in osteogenesis imperfecta, cleidocranial dysplasia and Wnt-deficiency syndromes. Bones of Smad4 mutant mice exhibited markers of fully differentiated osteoblasts but lacked multiple collagen-processing enzymes, including lysyl oxidase (Lox), a BMP2-responsive gene regulated by Smad4 and Runx2. Accordingly, the collagen matrix in Smad4 mutants was disorganized, but also hypomineralized. Primary osteoblasts from these mutants did not mineralize in vitro in the presence of BMP2 or Wnt3a, and Smad4 mutant mice failed to accrue new bone following systemic inhibition of the Dickkopf homolog Dkk1. Consistent with impaired biological responses to canonical Wnt, ablation of Smad4 causes cleavage of β-catenin and depletion of the low density lipoprotein receptor Lrp5, subsequent to increased caspase-3 activity and apoptosis. In summary, Smad4 regulates maturation of skeletal collagen and osteoblast survival, and is required for matrix-forming responses to both BMP2 and canonical Wnt. PMID:24006258

Mathematical Modeling of Cancer Invasion: The Role of Membrane-Bound Matrix Metalloproteinases

PubMed Central

Deakin, Niall E.; Chaplain, Mark A. J.

2013-01-01

One of the hallmarks of cancer growth and metastatic spread is the process of local invasion of the surrounding tissue. Cancer cells achieve protease-dependent invasion by the secretion of enzymes involved in proteolysis. These overly expressed proteolytic enzymes then proceed to degrade the host tissue allowing the cancer cells to disseminate throughout the microenvironment by active migration and interaction with components of the extracellular matrix (ECM) such as collagen. In this paper we develop a mathematical model of cancer invasion which consider the role of matrix metalloproteinases (MMPs). Specifically our model will focus on two distinct types of MMP, i.e., soluble, diffusible MMPs (e.g., MMP-2) and membrane-bound MMPs (e.g., MT1-MMP), and the roles each of these plays in cancer invasion. The implications of MMP-2 activation by MMP-14 and the tissue inhibitor of metalloproteinases-2 are considered alongside the effect the architecture of the matrix may have when applied to a model of cancer invasion. Elements of the ECM architecture investigated include pore size of the matrix, since in some highly dense collagen structures such as breast tissue, the cancer cells are unable to physically fit through a porous region, and the crosslinking of collagen fibers. In this scenario, cancer cells rely on membrane-bound MMPs to forge a path through which degradation by other MMPs and movement of cancer cells becomes possible. PMID:23565505

Prelude to corneal tissue engineering – Gaining control of collagen organization

PubMed Central

Ruberti, Jeffrey W.; Zieske, James D.

2012-01-01

By most standard engineering practice principles, it is premature to credibly discuss the “engineering” of a human cornea. A professional design engineer would assert that we still do not know what a cornea is (and correctly so), therefore we cannot possibly build one. The proof resides in the fact that there are no clinically viable corneas based on classical tissue engineering methods available. This is possibly because tissue engineering in the classical sense (seeding a degradable scaffolding with a population synthetically active cells) does not produce conditions which support the generation of organized tissue. Alternative approaches to the problem are in their infancy and include the methods which attempt to recapitulate development or to produce corneal stromal analogs de novo which require minimal remodeling. Nonetheless, tissue engineering efforts, which have been focused on producing the fundamental functional component of a cornea (organized alternating arrays of collagen or “lamellae”) may have already provided valuable new insights and tools relevant to development, growth, remodeling and pathologies associated with connective tissue in general. This is because engineers ask a fundamentally different question (How can that be done?) than do biological scientists (How is that done?). The difference in inquiry has prompted us to closely examine (and to mimic) development as well as investigate collagen physicochemical behavior so that we may exert control over organization both in cell-culture (in vitro) and on the benchtop (de novo). Our initial results indicate that reproducing corneal stroma-like local and long-range organization of collagen may be simpler than we anticipated while controlling spacing and fibril morphology remains difficult, but perhaps not impossible in the (reasonably) near term. PMID:18775789

The study of optical properties and proteoglycan content of tendons by PS-OCT

NASA Astrophysics Data System (ADS)

Yang, Ying; Rupani, Asha; Weightman, Alan; Wimpenny, Ian; Bagnaninchi, Pierre; Ahearne, Mark

2011-03-01

Tendons are load-bearing collagenous tissues consisting mainly of type I collagen and various proteoglycans (PGs) including decorin and versican. It is widely accepted that highly orientated collagen fibers in tendons a play critical role for transferring tensile stress and demonstrate birefringent optical properties. However, the influence that proteoglycans have on the optical properties of tendons is yet to be fully elucidated. Tendinopathy (defined as a syndrome of tendon pain, tenderness and swelling that affects the normal function of the tissue) is a common disease associated with sporting injuries or degeneration. PG's are the essential components of the tendon extracellular matrix; changes in their quantities and compositions have been associated with tendinopathy. In this study, polarization sensitive optical coherence tomography (PS-OCT) has been used to reveal the relationship between proteoglycan content/location and birefringent properties of tendons. Tendons dissected from freshly slaughtered chickens were imaged at regular intervals by PS-OCT and polarizing light microscope during the extraction of PGs or glycosaminoglycans using established protocols (guanidine hydrochloride (GuHCl) or proteinase K solution). The macroscopic and microscopic time lapsed images are complimentary; mutually demonstrating that there was a higher concentration of PG's in the outer sheath region than in the fascicles; and the integrity of the sheath affected extraction process and the OCT birefringence bands. Extraction of PGs using GuHCl disturbed the organization of local collagen bundles, which corresponded to a reduction in the frequency of birefringence bands and the band width by PS-OCT. The feature of OCT penetration depth helped us to define the heterogeneous distribution of PG's in tendon, which was complimented by polarizing light microscopy. The results provide new insight of tendon structure and also demonstrate a great potential for using PS-OCT as a diagnostic tool to examine tendon pathology.

Minor Type IV Collagen α5 Chain Promotes Cancer Progression through Discoidin Domain Receptor-1

PubMed Central

Xiao, Qian; Jiang, Yan; Liu, Qingbo; Yue, Jiao; Liu, Chunying; Zhao, Xiaotong; Qiao, Yuemei; Ji, Hongbin; Chen, Jianfeng; Ge, Gaoxiang

2015-01-01

Type IV collagens (Col IV), components of basement membrane, are essential in the maintenance of tissue integrity and proper function. Alteration of Col IV is related to developmental defects and diseases, including cancer. Col IV α chains form α1α1α2, α3α4α5 and α5α5α6 protomers that further form collagen networks. Despite knowledge on the functions of major Col IV (α1α1α2), little is known whether minor Col IV (α3α4α5 and α5α5α6) plays a role in cancer. It also remains to be elucidated whether major and minor Col IV are functionally redundant. We show that minor Col IV α5 chain is indispensable in cancer development by using α5(IV)-deficient mouse model. Ablation of α5(IV) significantly impeded the development of KrasG12D-driven lung cancer without affecting major Col IV expression. Epithelial α5(IV) supports cancer cell proliferation, while endothelial α5(IV) is essential for efficient tumor angiogenesis. α5(IV), but not α1(IV), ablation impaired expression of non-integrin collagen receptor discoidin domain receptor-1 (DDR1) and downstream ERK activation in lung cancer cells and endothelial cells. Knockdown of DDR1 in lung cancer cells and endothelial cells phenocopied the cells deficient of α5(IV). Constitutively active DDR1 or MEK1 rescued the defects of α5(IV)-ablated cells. Thus, minor Col IV α5(IV) chain supports lung cancer progression via DDR1-mediated cancer cell autonomous and non-autonomous mechanisms. Minor Col IV can not be functionally compensated by abundant major Col IV. PMID:25992553

Matrix Metalloproteinase 9 (MMP-9) Regulates Vein Wall Biomechanics in Murine Thrombus Resolution

PubMed Central

Nguyen, Khanh P.; McGilvray, Kirk C.; Puttlitz, Christian M.; Mukhopadhyay, Subhradip; Chabasse, Christine; Sarkar, Rajabrata

2015-01-01

Objective Deep venous thrombosis is a common vascular problem with long-term complications including post-thrombotic syndrome. Post-thrombotic syndrome consists of leg pain, swelling and ulceration that is related to incomplete or maladaptive resolution of the venous thrombus as well as loss of compliance of the vein wall. We examine the role of metalloproteinase-9 (MMP-9), a gene important in extracellular remodeling in other vascular diseases, in mediating thrombus resolution and biomechanical changes of the vein wall. Methods and Results The effects of targeted deletion of MMP-9 were studied in an in vivo murine model of thrombus resolution using the FVB strain of mice. MMP-9 expression and activity significantly increased on day 3 after DVT. The lack of MMP-9 impaired thrombus resolution by 27% and this phenotype was rescued by the transplantation of wildtype bone marrow cells. Using novel biomechanical techniques, we demonstrated that the lack of MMP-9 significantly decreased thrombus-induced loss of vein wall compliance. Biomechanical analysis of the contribution of individual structural components showed that MMP-9 affected the elasticity of the extracellular matrix and collagen-elastin fibers. Biochemical and histological analyses correlated with these biomechanical effects as thrombi of mice lacking MMP-9 had significantly fewer macrophages and collagen as compared to those of wildtype mice. Conclusions MMP-9 mediates thrombus-induced loss of vein wall compliance by increasing stiffness of the extracellular matrix and collagen-elastin fibers during thrombus resolution. MMP-9 also mediates macrophage and collagen content of the resolving thrombus and bone-marrow derived MMP-9 plays a role in resolution of thrombus mass. These disparate effects of MMP-9 on various aspects of thrombus illustrate the complexity of individual protease function on biomechanical and morphometric aspects of thrombus resolution. PMID:26406902

Periodontal plastic surgery: thermal effect analysis using Erbium:YAG Kesler's handpiece. Histochemical evaluation by Picrosirius red stain and polarization microscopy for collagen determination: in

NASA Astrophysics Data System (ADS)

Kesler, Gavriel; Koren, Rumelia; Kesler, Anat; Kristt, Don; Gal, Rivka

2000-03-01

Recent technological advances lead to an increase in the options for the treatment of the periodontal diseases. Lasers utilized for gingival soft tissue resurfacing mainly for esthetics purposes, require careful histopathological evaluation of the effects on tissue. Up to date no comparative clinical or histological studies have been performed, aiming at demonstration of the effects of laser irradiation on connective tissue, especially its most important component -- the collagen fibers. The alteration in the structures of this tissue plays the most important role in the healing process. The aim of the present study is to evaluate the influence of Erbium: YAG - Kesler's hand piece on gingival tissue. This handpiece is designed for gingival resurfacing, in cases of 'Gummy smile' and gingival pigmentation. The following irradiation parameters were used: energy per pulse -- 500 mJ, repetition rate 10 pps, spot size 3 mm. Gingival biopsies specimens of 10 patients, 6 with 'Gummy smile' and 4 with gingival pigmentation were examined before laser treatment, and at 7 and 14 days after laser treatment. The tissues were fixed in LNRS, embedded in paraffin, and sectioned into 5 micrometer thickness, dewaxed in xylol and stained with H&E and Picrosirius Red (PSR). The sections were examined by polarization microscopy. PSR is a collagen stain that differentiates collagen fiber density by the range of colors from green through yellow to red, and/or fiber size. This was utilized in the present study to evaluate the hypothesis that Erbium -- YAG (Er: YAG) laser energy is capable of remodeling the collagen fibers in the gingival connective tissue through a photothermal process. We found a significant difference between the structures of collagen fibers at the first week and at 14 days post treatment. In the normal gingiva the predominant polarization colors were in the red-orange range, signifying tightly packed, mature collagen. During the first postoperative week, collagen fibers exhibit mainly polarization colors in the green to green-yellow range implying less packed intermediate collagen fibers. After two weeks, collagen fibers required their preoperative polarizing texture. These findings strongly suggest that the 'quality' rather than the quantity of gingival collagen fibers play an important role in order for collagen shrinkage and reproduction to occur.

Proportion of collagen type II in the extracellular matrix promotes the differentiation of human adipose-derived mesenchymal stem cells into nucleus pulposus cells.

PubMed

Tao, Yiqing; Zhou, Xiaopeng; Liu, Dongyu; Li, Hao; Liang, Chengzhen; Li, Fangcai; Chen, Qixin

2016-01-01

During degeneration process, the catabolism of collagen type II and anabolism of collagen type I in nucleus pulposus (NP) may influence the bioactivity of transplanted cells. Human adipose-derived mesenchymal stem cells (hADMSCs) were cultured as a micromass or in a series of gradual proportion hydrogels of a mix of collagen types I and II. Cell proliferation and cytotoxicity were detected using CCK-8 and LDH assays respectively. The expression of differentiation-related genes and proteins, including SOX9, aggrecan, collagen type I, and collagen type II, was examined using RT-qPCR and Western blotting. Novel phenotypic genes were also detected by RT-qPCR and western blotting. Alcian blue and dimethylmethylene blue assays were used to investigate sulfate proteoglycan expression, and PI3K/AKT, MAPK/ERK, and Smad signaling pathways were examined by Western blotting. The results showed collagen hydrogels have good biocompatibility, and cell proliferation increased after collagen type II treatment. Expressions of SOX9, aggrecan, and collagen type II were increased in a collagen type II dependent manner. Sulfate proteoglycan synthesis increased in proportion to collagen type II concentration. Only hADMSCs highly expressed NP cell marker KRT19 in collagen type II culture. Additionally, phosphorylated Smad3, which is associated with phosphorylated ERK, was increased after collagen type II-stimulation. The concentration and type of collagen affect hADMSC differentiation into NP cells. Collagen type II significantly ameliorates hADMSC differentiation into NP cells and promotes extracellular matrix synthesis. Therefore, anabolism of collagen type I and catabolism of type II may attenuate the differentiation and biosynthesis of transplanted stem cells. © 2016 International Union of Biochemistry and Molecular Biology.

Biomimetic hydrogels direct spinal progenitor cell differentiation and promote functional recovery after spinal cord injury.

PubMed

Geissler, Sydney A; Sabin, Alexandra L; Besser, Rachel R; Gooden, Olivia M; Shirk, Bryce D; Nguyen, Quan M; Khaing, Zin Z; Schmidt, Christine E

2018-04-01

Demyelination that results from disease or traumatic injury, such as spinal cord injury (SCI), can have a devastating effect on neural function and recovery. Many researchers are examining treatments to minimize demyelination by improving oligodendrocyte availability in vivo. Transplantation of stem and oligodendrocyte progenitor cells is a promising option, however, trials are plagued by undirected differentiation. Here we introduce a biomaterial that has been optimized to direct the differentiation of neural progenitor cells (NPCs) toward oligodendrocytes as a cell delivery vehicle after SCI. A collagen-based hydrogel was modified to mimic the mechanical properties of the neonatal spinal cord, and components present in the developing extracellular matrix were included to provide appropriate chemical cues to the NPCs to direct their differentiation toward oligodendrocytes. The hydrogel with cells was then transplanted into a unilateral cervical contusion model of SCI to examine the functional recovery with this treatment. Six behavioral tests and histological assessment were performed to examine the in vivo response to this treatment. Our results demonstrate that we can achieve a significant increase in oligodendrocyte differentiation of NPCs compared to standard culture conditions using a three-component biomaterial composed of collagen, hyaluronic acid, and laminin that has mechanical properties matched to those of neonatal neural tissue. Additionally, SCI rats with hydrogel transplants, with and without NPCs, showed functional recovery. Animals transplanted with hydrogels with NPCs showed significantly increased functional recovery over six weeks compared to the media control group. The three-component hydrogel presented here has the potential to provide cues to direct differentiation in vivo to encourage regeneration of the central nervous system.

Col-F, a fluorescent probe for ex vivo confocal imaging of collagen and elastin in animal tissues.

PubMed

Biela, Ewa; Galas, Jerzy; Lee, Brian; Johnson, Gary L; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W

2013-06-01

A new low-molecular-weight fluorescent probe, Col-F, that exhibits affinity to collagen and elastin, was used successfully in imaging of extracellular matrix in freshly excised animal tissues. Col-F readily penetrates between live cells into tissues and binds to fibers of collagen and elastin by a noncovalent mechanism. Fibers of collagen and elastin have been stained in a variety of tissues, including tendon, skeletal muscle, connective tissue, and arteries. Cells migrating in a Col-F-stained collagenous biomaterial were also imaged. No phototoxic effects were detected when live keratocytes were imaged in the in vitro culture in the presence of Col-F. In conclusion, Col-F provides a simple and convenient tool for fluorescence three-dimensional imaging of intricate collagenous and elastic structures in live and fixed animal tissues, as well as in collagen-containing biomaterials. Copyright © 2013 International Society for Advancement of Cytometry.

An age-related study of morphology and cross-link composition of collagen fibrils in the digital flexor tendons of young thoroughbred horses.

PubMed

Patterson-Kane, J C; Parry, D A; Birch, H L; Goodship, A E; Firth, E C

1997-01-01

The superficial digital flexor tendon is the most commonly injured tendon in the racing Thoroughbred. Despite the clinical significance of this structure, only limited data exist regarding normal age-related morphology of the tensile units, the collagen fibrils. The age at which these collagen fibrils become mature in composition and structure may be of importance. Consequently, the association of age and collagen fibril crosslink composition, diameter distribution and crimp morphology in the superficial and deep digital flexor tendons of Thoroughbreds up to and including three years of age has been studied. Replacement of immature crosslinks, peaking of the collagen fibril mass-average diameter and collagen fibril index, and stabilization of collagen crimp morphology changes supported the hypothesis that both digital flexor tendons become mature in structure by two years of age.

Characterizing intestinal strictures with acoustic resolution photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Lei, Hao; Xu, Guan; Liu, Shengchun; Johnson, Laura A.; Moons, David S.; Higgins, Peter D. R.; Rice, Michael D.; Ni, Jun; Wang, Xueding

2016-03-01

Crohn's disease (CD) is an autoimmune disease, which may cause obstructing intestinal strictures due to inflammation, fibrosis (deposition of collagen), or a combination of both. Identifying the different stages of the disease progression is still challenging. In this work, we indicated the feasibility of non-invasively characterizing intestinal strictures using photoacoustic imaging (PAI), utilizing the uniquely optical absorption of hemoglobin and collagen. Surgically removed human intestinal stricture specimens were investigated with a prototype PAI system. 2D PA images with acoustic resolution at wavelength 532, 1210 and 1310 nm were formulated, and furthermore, the PA histochemical components images which show the microscopic distributions of histochemical components were solved. Imaging experiments on surgically removed human intestinal specimens has demonstrated the solved PA images were significantly different associated with the presence of fibrosis, which could be applied to characterize the intestinal strictures for given specimens.

Bone embrittlement and collagen modifications due to high-dose gamma-irradiation sterilization.

PubMed

Burton, Brianne; Gaspar, Anne; Josey, David; Tupy, Jindra; Grynpas, Marc D; Willett, Thomas L

2014-04-01

Bone allografts are often used in orthopedic reconstruction of skeletal defects resulting from trauma, bone cancer or revision of joint arthroplasty. γ-Irradiation sterilization is a widely-used biological safety measure; however it is known to embrittle bone. Irradiation has been shown to affect the post-yield properties, which are attributed to the collagen component of bone. In order to find a solution to the loss of toughness in irradiated bone allografts, it is important to fully understand the effects of irradiation on bone collagen. The objective of this study was to evaluate changes in the structure and integrity of bone collagen as a result of γ-irradiation, with the hypothesis that irradiation fragments collagen molecules leading to a loss of collagen network connectivity and therefore loss of toughness. Using cortical bone from bovine tibiae, sample beams irradiated at 33kGy on dry ice were compared to native bone beams (paired controls). All beams were subjected to three-point bend testing to failure followed by characterization of the decalcified bone collagen, using differential scanning calorimetry (DSC), hydrothermal isometric tension testing (HIT), high performance liquid chromatography (HPLC) and gel electrophoresis (SDS-PAGE). The carbonyl content of demineralized bone collagen was also measured chemically to assess oxidative damage. Barium sulfate staining after single edge notch bending (SEN(B)) fracture testing was also performed on bovine tibia bone beams with a machined and sharpened notch to evaluate the fracture toughness and ability of irradiated bone to form micro-damage during fracture. Irradiation resulted in a 62% loss of work-to-fracture (p≤0.001). There was significantly less micro-damage formed during fracture propagation in the irradiated bone. HPLC showed no significant effect on pentosidine, pyridinoline, or hydroxypyridinoline levels suggesting that the loss of toughness is not due to changes in these stable crosslinks. For DSC, there was a 20% decrease in thermal stability (p<0.001) with a 100% increase (p<0.001) in enthalpy of denaturation (melting). HIT testing also showed a decrease in thermal stability (20% lower denaturation temperature, p<0.001) and greatly reduced measures of collagen network connectivity (p<0.001). Interestingly, the increase in enthalpy of denaturation suggests that irradiated collagen requires more energy to denature (melt), perhaps a result of alterations in the hydrogen bonding sites (increased carbonyl content detected in the insoluble collagen) on the irradiated bone collagen. Altogether, this new data strongly indicates that a large loss of overall collagen connectivity due to collagen fragmentation resulting from γ-irradiation sterilization leads to inferior cortical bone toughness. In addition, notable changes in the thermal denaturation of the bone collagen along with chemical indicators of oxidative modification of the bone collagen indicate that the embrittlement may be a function not only of collagen fragmentation but also of changes in bonding. Copyright © 2014 Elsevier Inc. All rights reserved.

Lipo-PGE1 suppresses collagen production in human dermal fibroblasts via the ERK/Ets-1 signaling pathway.

PubMed

Yang, Yoolhee; Kim, Hee Jung; Woo, Kyong-Je; Cho, Daeho; Bang, Sa Ik

2017-01-01

Dysregulation of collagen production contributes to various pathological processes, including tissue fibrosis as well as impaired wound healing. Lipo-prostaglandin E1 (Lipo-PGE1), a lipid microsphere-incorporated prostaglandin E1, is used as a vasodilator for the treatment of peripheral vascular diseases. Lipo-PGE1 was recently shown to enhance human dermal fibroblast (HDF) migration and in vivo wound healing. No published study has characterized the role of Lipo-PGE1 in collagen regulation in HDFs. Here, we investigated the cellular signaling mechanism by which Lipo-PGE1 regulates collagen in HDFs. Collagen production was evaluated by the Sircol collagen assay, Western blot analysis of type I collagen and real time PCR. Unexpectedly, Lipo-PGE1 decreased mRNA expression of collagen 1A1, 1A2, and 3A1. Lipo-PGE1 markedly inhibited type I collagen and total soluble collagen production. In addition, Lipo-PGE1 inhibited transforming growth factor-β-induced collagen expression via Smad2 phosphorylation. To further investigate whether extracellular signal-regulated kinase (ERK)/Ets-1 signaling, a crucial pathway in collagen regulation, is involved in Lipo-PGE1-inhibited collagen production, cells were pretreated with an ERK-specific inhibitor, PD98059, prior to the addition of Lipo-PGE1. Lipo-PGE1-inhibited collagen mRNA expression and total soluble collagen production were recovered by pretreatment with PD98059. Moreover, Lipo-PGE1 directly induced the phosphorylation of ERK. Furthermore, silencing of Ets-1 recovered Lipo-PGE1-inhibited collagen production and PD98059 blocked Lipo-PGE1-enhanced Ets-1 expression. The present study reveals an important role for Lipo-PGE1 as a negative regulator of collagen gene expression and production via ERK/Ets-1 signaling. These results suggest that Lipo-PGE1 could potentially be a therapeutic target in diseases with deregulated collagen turnover.

Metabolic Effects of Avocado/Soy Unsaponifiables on Articular Chondrocytes

PubMed Central

Nardo, Joseph V.; Harlan, Robert; Chiou, Tiffany

2008-01-01

Avocado/soy unsaponifiable (ASU) components are reported to have a chondroprotective effect by virtue of anti-inflammatory and proanabolic effects on articular chondrocytes. The identity of the active component(s) remains unknown. In general, sterols, the major component of unsaponifiable plant material have been demonstrated to be anti-inflammatory in vitro and in animal models. These studies were designed to clarify whether the sterol content of ASU preparations were the primary contributors to biological activity in articular chondrocytes. ASU samples were analyzed by high pressure liquid chromatography (HPLC) and GC mass spectrometry. The sterol content was normalized between diverse samples prior to in vitro testing on bovine chondrocytes. Anabolic activity was monitored by uptake of 35-sulfate into proteoglycans and quantitation of labeled hydroxyproline and proline content after incubation with labeled proline. Anti-inflammatory activity was assayed by measuring reduction of interleukin-1 (IL-1)-induced synthesis of PGE2 and metalloproteases and release of label from tissue prelabeled with S-35.All ASU samples exerted a similar time-dependent up-regulation of 35-sulfate uptake in bovine cells reaching a maximum of greater than 100% after 72 h at sterol doses of 1–10 μg/ml. Non-collagenous protein (NCP) and collagen synthesis were similarly up-regulated. All ASU were equally effective in dose dependently inhibiting IL-1-induced MMP-3 activity (23–37%), labeled sulfate release (15–23%) and PGE2 synthesis (45–58%). Up-regulation of glycosaminoglycan and collagen synthesis and reduction of IL-1 effects in cartilage are consistent with chondroprotective activity. The similarity of activity of ASU from diverse sources when tested at equal sterol levels suggests sterols are important for biologic effects in articular chondrocytes. PMID:18604259

Telemetric ambulatory arterial stiffness index, a predictor of cardio-cerebro-vascular mortality, is associated with aortic stiffness-determining factors.

PubMed

Li, Zhi-Yong; Xu, Tian-Ying; Zhang, Sai-Long; Zhou, Xiao-Ming; Xu, Xue-Wen; Guan, Yun-Feng; Lo, Ming; Miao, Chao-Yu

2013-09-01

Ambulatory arterial stiffness index (AASI) has been proposed as a new measure of arterial stiffness for predicting cardio-cerebro-vascular morbidity and mortality. However, there has been no research on the direct relationships between AASI and arterial stiffness-determining factors. We utilized beat-to-beat intra-aortic blood pressure (BP) telemetry to characterize AASI in Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). By determination of aortic structural components and analysis of their correlations with AASI, we provided the first direct evidence for the associations between AASI and arterial stiffness-determining factors including the collagen content and collagen/elastin. Ambulatory arterial stiffness index was positively correlated with pulse pressure in both WKY and SHR, less dependent on BP and BP variability than pulse pressure, and relatively stable, especially the number of BP readings not less than ~36. The correlations between AASI and aortic components were comparable for various AASI values derived from BP readings not less than ~36. Not only AASI but also BP variability and pulse pressure demonstrated a direct relationship with arterial stiffness. These findings indicate AASI may become a routine measure in human arterial stiffness assessment. It is recommended to use a cluster of parameters such as AASI, BP variability, and pulse pressure for evaluating arterial stiffness. © 2013 John Wiley & Sons Ltd.

Relative ratios of collagen composition of periarticular tissue of joints of the upper limb.

PubMed

Cheah, A; Harris, A; Le, W; Huang, Y; Yao, J

2017-07-01

We investigated the relative ratios of collagen composition of periarticular tissue of the elbow, wrist, metacarpophalangeal, proximal and distal interphalangeal joints. Periarticulat tissue, which we defined as the ligaments, palmar plate and capsule, was harvested from ten fresh-frozen cadaveric upper limbs, yielding 50 samples. The mean paired differences (95% confidence interval) of the relative ratios of collagen between the five different joints were estimated using mRNA expression of collagen in the periarticular tissue. We found that the relative collagen composition of the elbow was not significantly different to that of the proximal interphalangeal joint, nor between the proximal interphalangeal joint and distal interphalangeal joint, whereas the differences in collagen composition between all the other paired comparisons of the joints had confidence intervals that did not include zero.

The effect of collagen fibril orientation on the biphasic mechanics of articular cartilage.

PubMed

Meng, Qingen; An, Shuqiang; Damion, Robin A; Jin, Zhongmin; Wilcox, Ruth; Fisher, John; Jones, Alison

2017-01-01

The highly inhomogeneous distribution of collagen fibrils may have important effects on the biphasic mechanics of articular cartilage. However, the effect of the inhomogeneity of collagen fibrils has mainly been investigated using simplified three-layered models, which may have underestimated the effect of collagen fibrils by neglecting their realistic orientation. The aim of this study was to investigate the effect of the realistic orientation of collagen fibrils on the biphasic mechanics of articular cartilage. Five biphasic material models, each of which included a different level of complexity of fibril reinforcement, were solved using two different finite element software packages (Abaqus and FEBio). Model 1 considered the realistic orientation of fibrils, which was derived from diffusion tensor magnetic resonance images. The simplified three-layered orientation was used for Model 2. Models 3-5 were three control models. The realistic collagen orientations obtained in this study were consistent with the literature. Results from the two finite element implementations were in agreement for each of the conditions modelled. The comparison between the control models confirmed some functions of collagen fibrils. The comparison between Models 1 and 2 showed that the widely-used three-layered inhomogeneous model can produce similar fluid load support to the model including the realistic fibril orientation; however, an accurate prediction of the other mechanical parameters requires the inclusion of the realistic orientation of collagen fibrils. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Diagnosis Applications of Non-Crystalline Diffraction of Collagen Fibres: Breast Cancer and Skin Diseases

NASA Astrophysics Data System (ADS)

Costa, M.; Benseny-Cases, N.; Cócera, M.; Teixeira, C. V.; Alsina, M.; Cladera, J.; López, O.; Fernández, M.; Sabés, M.

In previous chapters, the basis of SAXS for the study of biological systems like proteins in solution have been presented. The SAXS patterns of proteins in solution present, in general, broad dependences with the scattering vector, and the interpretation requires a huge component of modelling. In this chapter and in the following one, it is shown how SAXS technique can be used to study biological systems that are partially crystalline and with a large crystalline cells. This is done by analysing the diffraction obtained from these systems at small angles. In this chapter, a new approach to the application of small-angle X-ray scattering (SAXS) for diagnosis using the diffraction pattern of collagen is presented. This chapter shows the development of a new strategy in the preventive diagnosis of breast cancer following changes on collagen from breast connective tissue. SAXS profiles are related to different features in cutaneous preparations and to the supra-molecular arrangement of skin layers (stratum corneum, epidermis and dermis), in order to introduce objective values on the diagnosis of different skin pathologies. Working parameters (size, thickness) and methods (freezing, paraffin embedment) have been established. The results suggest that collagen diffraction patterns could be used as diagnostic indicators; especially for breast cancer and preliminary results obtained with skin collagen are promising too.

Novel synthesis and characterization of a collagen-based biopolymer initiated by hydroxyapatite nanoparticles.

PubMed

Bhuiyan, D; Jablonsky, M J; Kolesov, I; Middleton, J; Wick, T M; Tannenbaum, R

2015-03-01

In this study, we developed a novel synthesis method to create a complex collagen-based biopolymer that promises to possess the necessary material properties for a bone graft substitute. The synthesis was carried out in several steps. In the first step, a ring-opening polymerization reaction initiated by hydroxyapatite nanoparticles was used to polymerize d,l-lactide and glycolide monomers to form poly(lactide-co-glycolide) co-polymer. In the second step, the polymerization product was coupled with succinic anhydride, and subsequently was reacted with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide as the cross-linking agent, in order to activate the co-polymer for collagen attachment. In the third and final step, the activated co-polymer was attached to calf skin collagen type I, in hydrochloric acid/phosphate buffer solution and the precipitated co-polymer with attached collagen was isolated. The synthesis was monitored by proton nuclear magnetic resonance, infrared and Raman spectroscopies, and the products after each step were characterized by thermal and mechanical analysis. Calculations of the relative amounts of the various components, coupled with initial dynamic mechanical analysis testing of the resulting biopolymer, afforded a preliminary assessment of the structure of the complex biomaterial formed by this novel polymerization process. Copyright © 2015. Published by Elsevier Ltd.

Assessment of a new biomimetic scaffold and its effects on bone formation by OCT

NASA Astrophysics Data System (ADS)

Yang, Ying; Aydin, Halil M.; Piskin, Erhan; El Haj, Alicia J.

2009-02-01

The ultimate target of bone tissue engineering is to generate functional load bearing bone. By nature, the porous volume in the trabecular bone is occupied by osseous medulla. The natural bone matrix consists of hydroxyapatite (HA) crystals precipitated along the collagen type I fibres. The mineral phase renders bone strength while collagen provides flexibility. Without mineral component, bone is very flexible and can not bear loads, whereas it is brittle in the case of mineral phase without the collagen presence. In this study, we designed and prepared a new type of scaffold which mimics the features of natural bone. The scaffold consists of three different components, a biphasic polymeric base composed of two different biodegradable polymers prepared by using dual porogen approach and bioactive agents, i.e., collagen and HA particles which are distributed throughout the matrix only in the pore surfaces. Interaction of the bioactive scaffolds possessing very high porosity and interconnected pore structures with cells were investigated in a prolonged culture period by using an osteoblastic cell line. The mineral HA particles have a slight different refractive index from the other elements such as polymeric scaffolds and cell/matrix in a tissue engineering constructs, exhibiting brighter images in OCT. Thus, OCT renders a convenient means to assess the morphology and architecture of the blank biomimetic scaffolds. This study also takes a close observation of OCT images for the cultured cell-scaffold constructs in order to assess neo-formed minerals and matrix. The OCT assessments have been compared with the results from confocal and SEM analysis.

miRNA-29a targets COL3A1 to regulate the level of type III collagen in pig.

PubMed

Chuan-Hao, Li; Wei, Chen; Jia-Qing, Hu; Yan-Dong, Wang; Shou-Dong, Wang; Yong-Qing, Zeng; Hui, Wang

2016-10-30

COL3A1 encodes the protein, collagen type III alpha 1, which is an important component of collagen. Collagen can have a considerable effect on the processing quality of meat, and is nutritious. Bioinformatic analysis using Targetscan showed that COL3A1 could be a target gene of miRNA-29a. Moreover, we found that Laiwu pigs have higher levels of type III collagen and lower levels of miRNA-29a than Landrace pigs. Therefore, we hypothesized that miRNA-29a suppresses the expression of COL3A1 by targeting its 3'-UTR. miRNA-29a appears to play an inhibitory role in the regulation of COL3A1 in PK15 cells because of the following: (1) overexpression of miRNA-29a resulted in a significant down-regulation of COL3A1 protein levels (2) overexpression of miRNA-29a significantly decreased the level of COL3A1 mRNA. (3) The activity of a COL3A1 luciferase reporter was significant reduced by miRNA-29a. Furthermore, the levels of miRNA-29a and collagen type III in four tissues in Laiwu and Landrace pigs were consistent with the above observations. In this study, we identified COL3A1 as a direct target for miRNA-29a, which will inform further studies of meat quality. Copyright © 2016 Elsevier B.V. All rights reserved.

Specific Accumulation of Tumor-Derived Adhesion Factor in Tumor Blood Vessels and in Capillary Tube-Like Structures of Cultured Vascular Endothelial Cells

NASA Astrophysics Data System (ADS)

Akaogi, Kotaro; Okabe, Yukie; Sato, Junji; Nagashima, Yoji; Yasumitsu, Hidetaro; Sugahara, Kazuyuki; Miyazaki, Kaoru

1996-08-01

Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding.

PubMed

Benny, Paula; Badowski, Cedric; Lane, E Birgitte; Raghunath, Michael

2016-08-22

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls. The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as "bio-scaffolds" for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications. In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

The Extracellular Matrix of the Lateral Pharyngeal Wall in Obstructive Sleep Apnea

PubMed Central

Dantas, Danielle Andrade da Silva; Mauad, Thais; Silva, Luiz F. F.; Lorenzi-Filho, Geraldo; Formigoni, Gilberto G. S.; Cahali, Michel B.

2012-01-01

Study Objectives: To compare the components of the extracellular matrix in the lateral pharyngeal muscular wall in patients with and without obstructive sleep apnea (OSA). This may help to explain the origin of the increased collapsibility of the pharynx in patients with OSA. Design: Specimens from the superior pharyngeal constrictor muscle, obtained during pharyngeal surgeries, were evaluated using histochemical and immunohistochemical analyses to determine the fractional area of collagen types I and III, elastic fibers, versican, fibronectin, and matrix metalloproteinases 1 and 2 in the endomysium. Setting: Academic tertiary center. Patiens: A total of 51 nonobese adult patients, divided into 38 patients with OSA and 13 nonsnoring control subjects without OSA. Interventions: Postintervention study performed on tissues from patients after elective surgery. Measurements and Results: Pharyngeal muscles of patients with OSA had significantly more collagen type I than pharyngeal muscles in control subjects. Collagen type I was correlated positively and independently with age. The other tested components of the extracellular matrix did not differ significantly between groups. In a logistic regression, an additive effect of both the increase of collagen type I and the increase in age with the presence of OSA was observed (odds ratio (OR), 2.06; 95% confidence interval (CI), 1.17-3.63), when compared with the effect of increased age alone (OR, 1.11; 95% CI, 1.03-1.20). Conclusion: Collagen type I in the superior pharyngeal constrictor muscle was more prevalent in patients with OSA and also increased with age. It was hypothesized that this increase could delay contractile-relaxant responses in the superior pharyngeal constrictor muscle at the expiratory-inspiratory phase transition, thus increasing pharyngeal collapsibility. Citation: Dantas DAS; Mauad T; Silva LFF; Lorenzi-Filho G; Formigoni GGS; Cahali MB. The extracellular matrix of the lateral pharyngeal wall in obstructive sleep apnea. SLEEP 2012;35(4):483-490. PMID:22467986

Collagen matrix as an inlay in endoscopic skull base reconstruction.

PubMed

Oakley, G M; Christensen, J M; Winder, M; Jonker, B P; Davidson, A; Steel, T; Teo, C; Harvey, R J

2018-03-01

Multi-layer reconstruction has become standard in endoscopic skull base surgery. The inlay component used can vary among autografts, allografts, xenografts and synthetics, primarily based on surgeon preference. The short- and long-term outcomes of collagen matrix in skull base reconstruction are described. A case series of patients who underwent endoscopic skull base reconstruction with collagen matrix inlay were assessed. Immediate peri-operative outcomes (cerebrospinal fluid leak, meningitis, ventriculitis, intracranial bleeding, epistaxis, seizures) and delayed complications (delayed healing, meningoencephalocele, prolapse of reconstruction, delayed cerebrospinal fluid leak, ascending meningitis) were examined. Of 120 patients (51.0 ± 17.5 years, 41.7 per cent female), peri-operative complications totalled 12.7 per cent (cerebrospinal fluid leak, 3.3 per cent; meningitis, 3.3 per cent; other intracranial infections, 2.5 per cent; intracranial bleeding, 1.7 per cent; epistaxis, 1.7 per cent; and seizures, 0 per cent). Delayed complications did not occur in any patients. Collagen matrix is an effective inlay material. It provides robust long-term separation between sinus and cranial cavities, and avoids donor site morbidity, but carries additional cost.

A unique evolution of the kidney phenotype in a patient with autosomal recessive Alport syndrome.

PubMed

Vischini, Gisella; Kapp, Meghan E; Wheeler, Ferrin C; Hopp, Laszlo; Fogo, Agnes B

2018-03-09

Alport syndrome is due to mutations in one of the genes encoding (α3,4,5) type IV collagen resulting in defective type IV collagen, a key component of the glomerular basement membrane (GBM). The GBM is initially thin, and with ongoing remodeling, develops a thickened basket-woven appearance. We report a unique case of a 9-year-old boy who was biopsied for hematuria and proteinuria, diagnosed as IgA nephropathy, with normal GBM appearance and thickness. Due to a family history of hematuria and chronic kidney disease, he subsequently underwent genetic evaluation and a mutation of α3 type IV collagen (COL4A3) was detected. Additional studies of the initial biopsy demonstrated abnormal type IV collagen immunostaining. A repeat biopsy 4years later showed characteristic glomerular basement membrane morphology of Alport syndrome, and scarring consistent with sequelae of IgA nephropathy. This is the first description of this unusual transition from an initial normal appearance of the glomerular basement membrane to the classic Alport phenotype. Copyright © 2018. Published by Elsevier Inc.

Proteinases secreted by Fasciola hepatica degrade extracellular matrix and basement membrane components.

PubMed

Berasaín, P; Goñi, F; McGonigle, S; Dowd, A; Dalton, J P; Frangione, B; Carmona, C

1997-02-01

The invasive stages of the parasitic trematode Fasciola hepatica release proteinases into the medium in which they are maintained. In this study, we investigated the interaction of F. hepatica excretory/secretory (E/S) products and 2 cysteine proteinases (CL1 and CL2) purified from these products with extracellular matrix and basement membrane macromolecules. Fasciola hepatica E/S products contained collagenolytic activity on fibrillar types I and III collagen as well as basement membrane type IV collagen. CL1 and CL2 were capable of degrading acid-soluble type III and type IV collagen but not insoluble type I collagen. In contrast, neither the E/S products nor the purified CL1 and CL2 showed elastinolytic activity. Fibronectin and laminin were degraded by E/S products and by CL1 and CL2. Sequence analysis of fibronectin degradation products showed that the fragments obtained corresponded to complete biologically active domains. These results indicate that the cysteine proteinases secreted by F. hepatica may be involved in the process of tissue invasion by the parasite.

Differential expression of extracellular matrix molecules and the alpha 6-integrins in the normal and neoplastic prostate.

PubMed Central

Knox, J. D.; Cress, A. E.; Clark, V.; Manriquez, L.; Affinito, K. S.; Dalkin, B. L.; Nagle, R. B.

1994-01-01

The epithelial basal lamina composition and integrin expression profile of normal and neoplastic human prostate was characterized using immunohistochemical analysis of frozen samples. The major components of the basal lamina surrounding normal acini were laminin, type IV collagen, entactin, and type VII collagen with variable amounts of tenascin. The basal lamina of neoplastic acini had a similar composition, except for the loss of type VII collagen, which was observed in all grades of carcinoma. The basal cells of the normal prostate express the alpha 6-, beta 1-, and beta 4-integrin subunits, suggesting that both the alpha 6 beta 1- and alpha 6 beta 4-integrin complexes are formed. In prostate carcinoma there is a complete loss of beta 4 expression and the alpha 6- and beta 1-integrin subunits, which are restricted to the basal and basal lateral surfaces of basal cells, are distributed diffusely throughout the cytoplasmic membrane. The differential expression of type VII collagen and beta 4 are discussed in relationship to their possible role in tumor progression. Images Figure 1 Figure 2 Figure 3 PMID:8030747

Collagenous gastritis.

PubMed

Jin, Xiaoyi; Koike, Tomoyuki; Chiba, Takashi; Kondo, Yutaka; Ara, Nobuyuki; Uno, Kaname; Asano, Naoki; Iijima, Katsunori; Imatani, Akira; Watanabe, Mika; Shirane, Akio; Shimosegawa, Tooru

2013-09-01

In the present paper, we report a case of rare collagenous gastritis. The patient was a 25-year-old man who had experienced nausea, abdominal distention and epigastralgia since 2005. Esophagogastroduodenoscopy (EGD) carried out at initial examination by the patient's local doctor revealed an extensively discolored depression from the upper gastric body to the lower gastric body, mainly including the greater curvature, accompanied by residual mucosa with multiple islands and nodularity with a cobblestone appearance. Initial biopsies sampled from the nodules and accompanying atrophic mucosa were diagnosed as chronic gastritis. In August, 2011, the patient was referred to Tohoku University Hospital for observation and treatment. EGD at our hospital showed the same findings as those by the patient's local doctor. Pathological findings included a membranous collagen band in the superficial layer area of the gastric mucosa, which led to a diagnosis of collagenous gastritis. Collagenous gastritis is an extremely rare disease, but it is important to recognize its characteristic endoscopic findings to make a diagnosis. © 2012 The Authors. Digestive Endoscopy © 2012 Japan Gastroenterological Endoscopy Society.

Accumulation of worn-out GBM material substantially contributes to mesangial matrix expansion in diabetic nephropathy.

PubMed

Kriz, Wilhelm; Löwen, Jana; Federico, Giuseppina; van den Born, Jacob; Gröne, Elisabeth; Gröne, Hermann Josef

2017-06-01

Thickening of the glomerular basement membrane (GBM) and expansion of the mesangial matrix are hallmarks of diabetic nephropathy (DN), generally considered to emerge from different sites of overproduction: GBM components from podocytes and mesangial matrix from mesangial cells. Reevaluation of 918 biopsies with DN revealed strong evidence that these mechanisms are connected to each other, wherein excess GBM components fail to undergo degradation and are deposited in the mesangium. These data do not exclude that mesangial cells also synthesize components that contribute to the accumulation of matrix in the mesangium. Light, electron microscopic, immunofluorescence, and in situ hybridization studies clearly show that the thickening of the GBM is due not only to overproduction of components of the mature GBM (α3 and α5 chains of collagen IV and agrin) by podocytes but also to resumed increased synthesis of the α1 chain of collagen IV and of perlecan by endothelial cells usually seen during embryonic development. We hypothesize that these abnormal production mechanisms are caused by different processes: overproduction of mature GBM-components by the diabetic milieu and regression of endothelial cells to an embryonic production mode by decreased availability of mediators from podocytes. Copyright © 2017 the American Physiological Society.

The importance of size-exclusion characteristics of type I collagen in bonding to dentin matrices

PubMed Central

M, Takahashi; M, Nakajima; J, Tagami; DLS, Scheffel; RM, Carvalho; A, Mazzoni; M, Carrilho; A, Tezvergil-Mutluay; L, Breschi; L, Tjäderhane; SS, Jang; FR, Tay; KA, Agee; DH, Pashley

2013-01-01

The mineral phase of dentin is located primarily within collagen fibrils. During development, bone or dentin collagen fibrils are formed first and then water within the fibril is replaced with apatite crystallites. Mineralized collagen contains very little water. During dentin bonding, acid-etching of mineralized dentin solubilizes the mineral crystallites and replaces them with water. During the infiltration phase of dentin bonding, adhesive comonomers are supposed to replace all of the collagen water with adhesive monomers that are then polymerized into copolymers. The authors of a recently published review suggested that dental monomers were too large to enter and displace water from collagen fibrils. If that were true, the endogenous proteases bound to dentin collagen could be responsible for unimpeded collagen degradation that is responsible for the poor durability of resin-dentin bonds. The current work studied the size-exclusion characteristics of dentin collagen, using a gel-filtration-like column chromatography technique, using dentin powder instead of Sephadex. The elution volumes of test molecules, including adhesive monomers, revealed that adhesive monomers smaller than about 1000 Da can freely diffuse into collagen water, while molecules of 10,000 Da begin to be excluded, and bovine serum albumin (66,000 Da) was fully excluded. These results validate the concept that dental monomers can permeate between collagen molecules during infiltration by etch-and-rinse adhesives. PMID:23928333

Influence of functionalized nanoparticles on conformational stability of type I collagen for possible biomedical applications.

PubMed

Kandamchira, Aswathy; Selvam, Sangeetha; Marimuthu, Nidhin; Janardhanan, Sreeram Kalarical; Fathima, Nishter Nishad

2013-12-01

Collagen-nanoparticle interactions are vital for many biomedical applications including drug delivery and tissue engineering applications. Iron oxide nanoparticles synthesized using starch template according to our earlier reported procedures were functionalized by treating them with Gum Arabic (GA), a biocompatible polysaccharide, so as to enhance the interaction between nanoparticle surfaces and collagen. Viscosity, circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) techniques have been used to study the collagen-nanoparticle interactions. The relative viscosity for collagen-nanoparticle conjugate was found to increase with increase in concentration of the nanoparticle within the concentration range investigated, which is due to the aggregation of protein onto the surface of nanoparticle. The CD spectra for the collagen-nanoparticle at different concentration ratios do not have much variation in the Rpn values (ratio of positive peak intensity over negative peak intensity) after functionalization with GA. The variation of molar ellipticity values for collagen-nanoparticle is due to the glycoprotein present in GA. The collagen triple helical structure is maintained after interaction with nanoparticles. The FTIR spectra of native collagen, Coll-Fs (nanoparticle without functionalization) and Coll-FsG (nanoparticle functionalized with GA) show clearly the amide I, II, III bands, with respect to collagen. The ability of polysaccharide stabilized/functionalized nanoparticles to maintain the collagen properties would help in its biomedical applications. © 2013.

Collagen content in the bladder of men with LUTS undergoing open prostatectomy: A pilot study.

PubMed

Averbeck, Marcio A; De Lima, Nelson G; Motta, Gabriela A; Beltrao, Lauro F; Abboud Filho, Nury J; Rigotti, Clarice P; Dos Santos, William N; Dos Santos, Steven K J; Da Silva, Luis F B; Rhoden, Ernani L

2018-03-01

To evaluate the collagen content in the bladder wall of men undergoing open prostate surgery. From July 2014 to August 2016, men aged ≥ 50 years, presenting LUTS and undergoing open prostate surgery due to benign prostatic enlargement (BPE) or prostate cancer were prospectively enrolled. Preoperative assessment included validated questionnaires (IPSS and OAB-V8), lower urinary tract ultrasound, and urodynamics. Bladder biopsies were obtained during open prostatectomy for determination of collagen content (sirius red-picric acid stain; polarized light analysis). Collagen to smooth muscle ratio (C/M) in the detrusor was measured and its relationship with preoperative parameters was investigated. The level of significance was P < 0.05. Thirty-eight consecutive patients were included in this pilot study. Mean age was 66.36 ± 6.44 years and mean IPSS was 11.05 ± 8.72 points. Men diagnosed with diabetes mellitus (DM2) were found to have higher collagen content in the bladder wall when compared to non-diabetic patients (17.71 ± 6.82% vs 12.46 ± 5.2%, respectively; P = 0.024). Reduced bladder compliance was also marker for higher collagen content (P = 0.042). Bladder outlet obstruction (BOO) was not a predictor of increased collagen deposition in the bladder wall (P = 0.75). Patients with PVR ≥ 200 mL showed a higher collagen to smooth muscle ratio in the bladder wall (P = 0.036). DM2 and urodynamic parameters, such as increased PVR and reduced bladder compliance, were associated with higher collagen content in the bladder wall of men with LUTS. © 2017 Wiley Periodicals, Inc.

The impact of atherosclerosis and vascular collagen on energy-based vessel sealing.

PubMed

Martin, Kimberly; Krugman, Kimberly; Latimer, Cassandra; Moore, Camille

2013-12-01

Bipolar energy ligation of vessels in surgery is common. Although rare, serious failures occur. Atherosclerosis may contribute to seal failures by altering vascular compressibility and collagen content; however, no data exist. Femoral and iliac arteries of six Yucatan swine with an identified genetic locus predisposing them to atherosclerosis were denuded with a Fogarty catheter. Animals were fed a high-fat diet for 28 wk. A Yorkshire pig was used as a normal control and fed a standard diet. At 28 wk, arteries were measured for their diameters, sealed, and divided in vivo with LigaSure. The sealed artery sections were excised and subjected to burst pressure testing. Half of the seal distal to the aorta was kept intact for histology and collagen and elastin quantification. A multiple linear regression model was used to assess variables contributing to burst pressure. Covariates included were vessel diameter, degree of atherosclerosis, and collagen content. Experimental animals were hypercholesterolemic. Atherosclerosis occurred in 90% of seals in induced animals, with severe atherosclerosis in 62% of seals. There was site-selective deposition of atherosclerotic plaques in larger diameter iliac vessels. A model including collagen and size best predicted burst pressure. Every 10-U increase in collagen resulted in 15% increase in burst pressure (95% confidence interval = 0.2%-32%, P = 0.047, R(2) = 0.36). Atherosclerosis was unrelated to burst pressure controlling for collagen and size. Collagen and size provide the best model fit for predicting burst pressure. Quantitative research in human vasculature is warranted to better understand the influence of atherosclerosis and collagen content on seal failures. Copyright © 2013 Elsevier Inc. All rights reserved.

Collagen from Marine Biological Sources and Medical Applications.

PubMed

Felician, Fatuma Felix; Xia, Chunlei; Qi, Weiyan; Xu, Hanmei

2018-05-01

Collagen is the most studied protein with a wide range of applications including pharmaceutical, biomedical, cosmetics, leather, and film industries due to its special characteristics that are high biocompatibility, good bioactivity, and weak antigenicity. Although collagen sources are abundant, the outbreak of varied diseases among land animals posed threat to its utilization in our daily life. Thus, a probe for an alternative source began, which in turn revealed the immense untapped marine sources, such as fish, jellyfish, and some marine Mammals. The present article deals with a brief description of collagen, its characteristics, marine sources, extraction, collagen peptides and their biological activities, potential use and application in various field. © 2018 Wiley-VHCA AG, Zurich, Switzerland.

Distinct characteristics of mandibular bone collagen relative to long bone collagen: relevance to clinical dentistry.

PubMed

Matsuura, Takashi; Tokutomi, Kentaro; Sasaki, Michiko; Katafuchi, Michitsuna; Mizumachi, Emiri; Sato, Hironobu

2014-01-01

Bone undergoes constant remodeling throughout life. The cellular and biochemical mechanisms of bone remodeling vary in a region-specific manner. There are a number of notable differences between the mandible and long bones, including developmental origin, osteogenic potential of mesenchymal stem cells, and the rate of bone turnover. Collagen, the most abundant matrix protein in bone, is responsible for determining the relative strength of particular bones. Posttranslational modifications of collagen, such as intermolecular crosslinking and lysine hydroxylation, are the most essential determinants of bone strength, although the amount of collagen is also important. In comparison to long bones, the mandible has greater collagen content, a lower amount of mature crosslinks, and a lower extent of lysine hydroxylation. The great abundance of immature crosslinks in mandibular collagen suggests that there is a lower rate of cross-link maturation. This means that mandibular collagen is relatively immature and thus more readily undergoes degradation and turnover. The greater rate of remodeling in mandibular collagen likely renders more flexibility to the bone and leaves it more suited to constant exercise. As reviewed here, it is important in clinical dentistry to understand the distinctive features of the bones of the jaw.

Distinct Characteristics of Mandibular Bone Collagen Relative to Long Bone Collagen: Relevance to Clinical Dentistry

PubMed Central

Tokutomi, Kentaro; Sasaki, Michiko; Katafuchi, Michitsuna; Mizumachi, Emiri; Sato, Hironobu

2014-01-01

Bone undergoes constant remodeling throughout life. The cellular and biochemical mechanisms of bone remodeling vary in a region-specific manner. There are a number of notable differences between the mandible and long bones, including developmental origin, osteogenic potential of mesenchymal stem cells, and the rate of bone turnover. Collagen, the most abundant matrix protein in bone, is responsible for determining the relative strength of particular bones. Posttranslational modifications of collagen, such as intermolecular crosslinking and lysine hydroxylation, are the most essential determinants of bone strength, although the amount of collagen is also important. In comparison to long bones, the mandible has greater collagen content, a lower amount of mature crosslinks, and a lower extent of lysine hydroxylation. The great abundance of immature crosslinks in mandibular collagen suggests that there is a lower rate of cross-link maturation. This means that mandibular collagen is relatively immature and thus more readily undergoes degradation and turnover. The greater rate of remodeling in mandibular collagen likely renders more flexibility to the bone and leaves it more suited to constant exercise. As reviewed here, it is important in clinical dentistry to understand the distinctive features of the bones of the jaw. PMID:24818151

Collagen based polyurethanes—A review of recent advances and perspective.

PubMed

Zuber, Mohammad; Zia, Fatima; Zia, Khalid Mahmood; Tabasum, Shazia; Salman, Mahwish; Sultan, Neelam

2015-09-01

Collagen is mostly found in fibrous tissues such as tendons, ligaments and skin. Collagen makes up approximately 30% of the proteins within the body. These are tough and strong structures found all over the body: in bones, tendons and ligaments. Collagen being the most abundant protein provides tensile strength via cell matrix interactions to tissue architecture. Biomimetic materials of collagen origin gained wide spread acceptance in clinical applications. Vitamin C deficiency causes scurvy a serious and painful disease in which defective collagen prevents the formation of strong connective tissue, gums deteriorate and bleed, with loss of teeth; skin discolors, and wounds do not heal. Effective collagens prevent the manifestation of such disorders. Polyurethanes on the other hand are frequently used for various applications as they offered in wide-ranging of compositions, properties and complex structures. Collagen/PU bio-composites have potential array for biomedical applications. Considering versatile properties of the elongated fibrils and wide industrial and biomedical applications including biocompatibility of polyurethane, this review shed a light on collagen based polyurethane materials with their potential applications especially focusing the bio-medical field. Copyright © 2015 Elsevier B.V. All rights reserved.

'Universal' microstructural patterns in cortical and trabecular, extracellular and extravascular bone materials: micromechanics-based prediction of anisotropic elasticity.

PubMed

Fritsch, Andreas; Hellmich, Christian

2007-02-21

Bone materials are characterized by an astonishing variability and diversity. Still, because of 'architectural constraints' due to once chosen material constituents and their physical interaction, the fundamental hierarchical organization or basic building plans of bone materials remain largely unchanged during biological evolution. Such universal patterns of microstructural organization govern the mechanical interaction of the elementary components of bone (hydroxyapatite, collagen, water; with directly measurable tissue-independent elastic properties), which are here quantified through a multiscale homogenization scheme delivering effective elastic properties of bone materials: at a scale of 10nm, long cylindrical collagen molecules, attached to each other at their ends by approximately 1.5nm long crosslinks and hosting intermolecular water inbetween, form a contiguous matrix called wet collagen. At a scale of several hundred nanometers, wet collagen and mineral crystal agglomerations interpenetrate each other, forming the mineralized fibril. At a scale of 5-10microm, the extracellular solid bone matrix is represented as collagen fibril inclusions embedded in a foam of largely disordered (extrafibrillar) mineral crystals. At a scale above the ultrastructure, where lacunae are embedded in extracellular bone matrix, the extravascular bone material is observed. Model estimates predicted from tissue-specific composition data gained from a multitude of chemical and physical tests agree remarkably well with corresponding acoustic stiffness experiments across a variety of cortical and trabecular, extracellular and extravascular materials. Besides from reconciling the well-documented, seemingly opposed concepts of 'mineral-reinforced collagen matrix' and 'collagen-reinforced mineral matrix' for bone ultrastructure, this approach opens new possibilities in the exploitation of computer tomographic data for nano-to-macro mechanics of bone organs.

Optimizing dentin bond durability: strategies to prevent hydrolytic degradation of the hybrid layer

PubMed Central

Tjäderhane, Leo; Nascimento, Fabio D.; Breschi, Lorenzo; Mazzoni, Annalisa; Tersariol, Ivarne L.S.; Geraldeli, Saulo; Tezvergil-Mutluay, Arzu; Carrilho, Marcela; Carvalho, Ricardo M.; Tay, Franklin R.; Pashley, David H.

2014-01-01

Objectives Endogenous dentin collagenolytic enzymes, matrix metalloproteinases (MMPs) and cysteine cathepsins, are responsible for the time-related hydrolysis of collagen matrix of the hybrid layers. As the integrity of the collagen matrix is essential for the preservation of long-term dentin bond strength, inhibition or inactivation of endogenous dentin proteases is necessary for durable resin-bonded composite resin restorations. Methods Dentin contains collagenolytic enzymes, matrix metalloproteinases (MMPs) and cysteine cathepsins, which are responsible for the hydrolytic degradation of collagen matrix in the bonded interface. Several tentative approaches to prevent enzyme function either directly or indirectly have been proposed in the literature. Results Chlorhexidine, a general inhibitor of both MMPs and cysteine cathepsins, applied before primer/adhesive application is the most tested method. In general, these experiments have shown that enzyme inhibition is a promising scheme to improve hybrid layer preservation and bond strength durability. Other enzyme inhibitors, e.g. enzyme-inhibiting monomers and antimicrobial compounds, may be considered promising alternatives that would allow more simple clinical application than chlorhexidine. Cross-linking collagen and/or dentin organic matrix-bound enzymes could render hybrid layer organic matrix resistant to degradation, and complete removal of water from the hybrid layer with ethanol wet bonding or biomimetic remineralization should eliminate hydrolysis of both collagen and resin components. Significance Identification of the enzymes responsible for the hydrolysis of hybrid layer collagen and understanding their function has prompted several innovative approaches to retain the hybrid layer integrity and strong dentin bonding. The ultimate goal, prevention of collagen matrix degradation with techniques and commercially available materials that are simple and effective in clinical settings may be achievable in several ways, and will likely become reality in the near future. PMID:23953737

Characterising human atherosclerotic carotid plaque tissue composition and morphology using combined spectroscopic and imaging modalities.

PubMed

Barrett, Hilary E; Mulvihill, John J; Cunnane, Eoghan M; Walsh, Michael T

2015-01-01

Calcification is a marked pathological component in carotid artery plaque. Studies have suggested that calcification may induce regions of high stress concentrations therefore increasing the potential for rupture. However, the mechanical behaviour of the plaque under the influence of calcification is not fully understood. A method of accurately characterising the calcification coupled with the associated mechanical plaque properties is needed to better understand the impact of calcification on the mechanical behaviour of the plaque during minimally invasive treatments. This study proposes a comparison of biochemical and structural characterisation methods of the calcification in carotid plaque specimens to identify plaque mechanical behaviour. Biochemical analysis, by Fourier Transform Infrared (FTIR) spectroscopy, was used to identify the key components, including calcification, in each plaque sample. However, FTIR has a finite penetration depth which may limit the accuracy of the calcification measurement. Therefore, this FTIR analysis was coupled with the identification of the calcification inclusions located internally in the plaque specimen using micro x-ray computed tomography (μX-CT) which measures the calcification volume fraction (CVF) to total tissue content. The tissue characterisation processes were then applied to the mechanical material plaque properties acquired from experimental circumferential loading of human carotid plaque specimen for comparison of the methods. FTIR characterised the degree of plaque progression by identifying the functional groups associated with lipid, collagen and calcification in each specimen. This identified a negative relationship between stiffness and 'lipid to collagen' and 'calcification to collagen' ratios. However, μX-CT results suggest that CVF measurements relate to overall mechanical stiffness, while peak circumferential strength values may be dependent on specific calcification geometries. This study demonstrates the need to fully characterise the calcification structure of the plaque tissue and that a combination of FTIR and μX-CT provides the necessary information to fully understand the mechanical behaviour of the plaque tissue.

Identification of Collagen-Binding Proteins in Lactobacillus spp. with Surface-Enhanced Laser Desorption/Ionization–Time of Flight ProteinChip Technology

PubMed Central

Howard, Jeffrey C.; Heinemann, Christine; Thatcher, Bradley J.; Martin, Brian; Gan, Bing Siang; Reid, Gregor

2000-01-01

Biosurfactants produced by Lactobacillus fermentum RC-14, L. rhamnosus GR-1 and 36, and L. casei Shirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)–time of flight mass spectrometry. Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins. Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form). Although biosurfactants isolated from L. casei Shirota and L. rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein. Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions. PMID:11010889

Structure to function: Spider silk and human collagen

NASA Astrophysics Data System (ADS)

Rabotyagova, Olena S.

Nature has the ability to assemble a variety of simple molecules into complex functional structures with diverse properties. Collagens, silks and muscles fibers are some examples of fibrous proteins with self-assembling properties. One of the great challenges facing Science is to mimic these designs in Nature to find a way to construct molecules that are capable of organizing into functional supra-structures by self-assembly. In order to do so, a construction kit consisting of molecular building blocks along with a complete understanding on how to form functional materials is required. In this current research, the focus is on spider silk and collagen as fibrous protein-based biopolymers that can shed light on how to generate nanostructures through the complex process of self-assembly. Spider silk in fiber form offers a unique combination of high elasticity, toughness, and mechanical strength, along with biological compatibility and biodegrability. Spider silk is an example of a natural block copolymer, in which hydrophobic and hydrophilic blocks are linked together generating polymers that organize into functional materials with extraordinary properties. Since silks resemble synthetic block copolymer systems, we adopted the principles of block copolymer design from the synthetic polymer literature to build block copolymers based on spider silk sequences. Moreover, we consider spider silk to be an important model with which to study the relationships between structure and properties in our system. Thus, the first part of this work was dedicated to a novel family of spider silk block copolymers, where we generated a new family of functional spider silk-like block copolymers through recombinant DNA technology. To provide fundamental insight into relationships between peptide primary sequence, block composition, and block length and observed morphological and structural features, we used these bioengineered spider silk block copolymers to study secondary structure, morphological features and assembly. Aside from fundamental perspectives, we anticipate that these results will provide a blueprint for the design of precise materials for a range of potential applications such as controlled release devices, functional coatings, components of tissue regeneration materials and environmentally friendly polymers in future studies. In the second part of this work, human collagen type I was studied as another representative of the family of fibrous proteins. Collagen type I is the most abundant extracellular matrix protein in the human body, providing the basis for tissue structure and directing cellular functions. Collagen has a complex structural hierarchy, organized at different length scales, including the characteristic triple helical feature. In the present study we assessed the relationship between collagen structure (native vs. denatured) and sensitivity to UV radiation with a focus on changes in the primary structure, conformation, microstructure and material properties. Free radical reactions are involved in collagen degradation and a mechanism for UV-induced collagen degradation related to structure was proposed. The results from this study demonstrated the role of collagen supramolecular organization (triple helix) in the context of the effects of electromagnetic radiation on extracellular matrices. Owing to the fact that both silks and collagens are proteins that have found widespread interest for biomaterial related needs, we anticipate that the current studies will serve as a foundation for future biomaterial designs with controlled properties. Furthermore, fundamental insight into self-assembly and environmentally-2mediated degradation, will build a foundation for fundamental understanding of the remodeling and functions of these types of fibrous proteins in vivo and in vitro. This type of insight is essential for many areas of scientific inquiry, from drug delivery, to scaffolds for tissue engineering, and to the stability of materials in space.

A novel electrospun nerve conduit enhanced by carbon nanotubes for peripheral nerve regeneration

NASA Astrophysics Data System (ADS)

Yu, Wenwen; Jiang, Xinquan; Cai, Ming; Zhao, Wen; Ye, Dongxia; Zhou, Yong; Zhu, Chao; Zhang, Xiuli; Lu, Xiaofeng; Zhang, Zhiyuan

2014-04-01

For artificial nerve conduits, great improvements have been achieved in mimicking the structures and components of autologous nerves. However, there are still some problems in conduit construction, especially in terms of mechanical properties, biomimetic surface tomography, electrical conductivity and sustained release of neurotrophic factors or cells. In this study, we designed and fabricated a novel electrospun nerve conduit enhanced by multi-walled carbon nanotubes (MWNTs) on the basis of a collagen/poly(ɛ-caprolactone) (collagen/PCL) fibrous scaffold. Our aim was to provide further knowledge about the mechanical effects and efficacy of MWNTs on nerve conduits as well as the biocompatibility and toxicology of MWNTs when applied in vivo. The results showed that as one component, carboxyl MWNTs could greatly alter the composite scaffold’s hydrophilicity, mechanical properties and degradability. The electrospun fibers enhanced by MWNTs could support Schwann cell adhesion and elongation as a substrate in vitro. In vivo animal studies demonstrated that the MWNT-enhanced collagen/PCL conduit could effectively promote nerve regeneration of sciatic nerve defect in rats and prevent muscle atrophy without invoking body rejection or serious chronic inflammation. All of these results showed that this MWNT-enhanced scaffold possesses good biocompatibility and MWNTs might be excellent candidates as engineered nanocarriers for further neurotrophic factor delivery research.

A novel electrospun nerve conduit enhanced by carbon nanotubes for peripheral nerve regeneration.

PubMed

Yu, Wenwen; Jiang, Xinquan; Cai, Ming; Zhao, Wen; Ye, Dongxia; Zhou, Yong; Zhu, Chao; Zhang, Xiuli; Lu, Xiaofeng; Zhang, Zhiyuan

2014-04-25

For artificial nerve conduits, great improvements have been achieved in mimicking the structures and components of autologous nerves. However, there are still some problems in conduit construction, especially in terms of mechanical properties, biomimetic surface tomography, electrical conductivity and sustained release of neurotrophic factors or cells. In this study, we designed and fabricated a novel electrospun nerve conduit enhanced by multi-walled carbon nanotubes (MWNTs) on the basis of a collagen/poly(ε-caprolactone) (collagen/PCL) fibrous scaffold. Our aim was to provide further knowledge about the mechanical effects and efficacy of MWNTs on nerve conduits as well as the biocompatibility and toxicology of MWNTs when applied in vivo.The results showed that as one component, carboxyl MWNTs could greatly alter the composite scaffold's hydrophilicity, mechanical properties and degradability. The electrospun fibers enhanced by MWNTs could support Schwann cell adhesion and elongation as a substrate in vitro. In vivo animal studies demonstrated that the MWNT-enhanced collagen/PCL conduit could effectively promote nerve regeneration of sciatic nerve defect in rats and prevent muscle atrophy without invoking body rejection or serious chronic inflammation. All of these results showed that this MWNT-enhanced scaffold possesses good biocompatibility and MWNTs might be excellent candidates as engineered nanocarriers for further neurotrophic factor delivery research.

Lumican Deficiency Results In Cardiomyocyte Hypertrophy With Altered Collagen Assembly

PubMed Central

Dupuis, Loren E.; Berger, Matthew G.; Feldman, Samuel; Doucette, Lorna; Fowlkes, Vennece; Chakravarti, Shukti; Thibaudeau, Sarah; Alcala, Nicolas E.; Bradshaw, Amy D.; Kern, Christine B.

2015-01-01

The ability of the heart to adapt to increased stress is dependent on modification of its extracellular matrix (ECM) architecture that is established during postnatal development as cardiomyocytes differentiate, a process that is poorly understood. We hypothesized that the small leucine-rich proteoglycan (SLRP) lumican (LUM), which binds collagen and facilitates collagen assembly in other tissues, may play a critical role in establishing the postnatal murine myocardial ECM. Although previous studies suggest LUM deficient mice (lum−/−) exhibit skin anomalies consistent with Ehlers-Danlos syndrome, lum−/− hearts have not been evaluated. These studies show LUM was immunolocalized to non-cardiomyocytes of the cardiac ventricles and its expression increased throughout development. Lumican deficiency resulted in significant (50%) perinatal death and further examination of the lum−/− neonatal hearts revealed an increase in myocardial tissue without a significant increase in cell proliferation. However cardiomyocytes from surviving postnatal day 0 (P0), 1 month (1 mo) and adult (4 mo) lum−/− hearts were significantly larger than their wild type (WT) littermates. Immunohistochemistry revealed that the increased cardiomyocyte size in the lum−/− hearts correlated with alteration of the cardiomyocyte pericellular ECM components collagenα1(I) and the class I SLRP decorin (DCN). Western blot analysis demonstrated that the ratio of glycosaminoglycan (GAG) decorated DCN to core DCN was reduced in P0 and 1 mo lum−/− hearts. There was also a reduction in the β and γ forms of collagenα1(I) in lum−/− hearts. While the total insoluble collagen content was significantly reduced, the fibril size was increased in lum−/− hearts, indicating LUM may play a role in collagen fiber stability and lateral fibril assembly. These results suggest that LUM controls cardiomyocyte growth by regulating the pericellular ECM and also indicates that LUM may coordinate multiple factors of collagen assembly in the murine heart. Further investigation into the role of LUM may yield novel therapeutic targets and/or biomarkers for patients with cardiovascular disease. PMID:25886697

Quantitative changes in human epithelial cancers and osteogenesis imperfecta disease detected using nonlinear multicontrast microscopy

NASA Astrophysics Data System (ADS)

Adur, Javier; Pelegati, Vitor B.; de Thomaz, Andre A.; D'Souza-Li, Lilia; Assunção, Maria do Carmo; Bottcher-Luiz, Fátima; Andrade, Liliana A. L. A.; Cesar, Carlos L.

2012-08-01

We show that combined multimodal nonlinear optical (NLO) microscopies, including two-photon excitation fluorescence, second-harmonic generation (SHG), third harmonic generation, and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation during the progression of cancer and osteogenesis imperfecta (OI) disease. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for different types of human breast cancer, mucinous ovarian tumors, and skin dermis of patients with OI. Using a set of scoring methods (anisotropy, correlation, uniformity, entropy, and lifetime components), we found significant differences in the content, distribution and organization of collagen fibrils in the stroma of breast and ovary as well as in the dermis of skin. We suggest that our results provide a framework for using NLO techniques as a clinical diagnostic tool for human cancer and OI. We further suggest that the SHG and FLIM metrics described could be applied to other connective or epithelial tissue disorders that are characterized by abnormal cells proliferation and collagen assembly.

Hypericin-mediated selective photomodification of connective tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hovhannisyan, V., E-mail: hovv@phys.ntu.edu.tw; Guo, H. W.; Chen, Y. F., E-mail: yfchen@phys.ntu.edu.tw

2014-12-29

Controllable modification of biological molecules and supramolecular components of connective tissue are important for biophysical and biomedical applications. Through the use of second harmonic generation imaging, two-photon fluorescence microscopy, and spectrofluorimetry, we found that hypericin, a natural pigment, induces photosensitized destruction of collagen fibers but does not affect elastic fibers and lipids in chicken tendon, skin, and blood vessels. We demonstrated the dynamics and efficiency of collagen photomodification and investigated mechanisms of this processes. Our results suggest that hypericin–mediated photoprocesses in biological tissues may be useful in biomedical applications that require selective modification of connective tissues.

A Model for the Ultrastructure of Bone Based on Electron Microscopy of Ion-Milled Sections

PubMed Central

McNally, Elizabeth A.; Schwarcz, Henry P.; Botton, Gianluigi A.; Arsenault, A. Larry

2012-01-01

The relationship between the mineral component of bone and associated collagen has been a matter of continued dispute. We use transmission electron microscopy (TEM) of cryogenically ion milled sections of fully-mineralized cortical bone to study the spatial and topological relationship between mineral and collagen. We observe that hydroxyapatite (HA) occurs largely as elongated plate-like structures which are external to and oriented parallel to the collagen fibrils. Dark field images suggest that the structures (“mineral structures”) are polycrystalline. They are approximately 5 nm thick, 70 nm wide and several hundred nm long. Using energy-dispersive X-ray analysis we show that approximately 70% of the HA occurs as mineral structures external to the fibrils. The remainder is found constrained to the gap zones. Comparative studies of other species suggest that this structural motif is ubiquitous in all vertebrates. PMID:22272230

A one-step method to fabricate PLLA scaffolds with deposition of bioactive hydroxyapatite and collagen using ice-based microporogens

PubMed Central

Li, Jiashen; Chen, Yun; Mak, Arthur F.T.; Tuan, Rocky S.; Li, Lin; Li, Yi

2010-01-01

Porous poly(L-lactic acid) (PLLA) scaffolds with bioactive coatings were prepared by a novel one-step method. In this process, ice-based microporogens containing bioactive molecules, such as hydroxyapatite (HA) and collagen, served as both porogens to form the porous structure and vehicles to transfer the bioactive molecules to the inside of PLLA scaffolds in a single step. Based on scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis, the bioactive components were found to be transferred successfully from the porogens to PLLA scaffolds evenly. Osteoblast cells were used to evaluate the cellular behaviors of the composite scaffolds. After 8 days culturing, MTT assay and alkaline phosphatase (ALP) activity results suggested that HA/collagen could improve the interactions between osteoblast cells and the polymeric scaffold. PMID:20004261

Hyperspectral vibrational photoacoustic imaging of lipids and collagen

NASA Astrophysics Data System (ADS)

Wang, Pu; Wang, Ping; Wang, Han-Wei; Cheng, Ji-Xin

2012-02-01

The recently developed vibrational photoacoustic (VPA) microscopy allows bond-selective imaging of deep tissues by taking advantage of intrinsic contrast from harmonic vibration of C-H bonds. Due to the spectral similarity of molecules in the overtone vibration region, the compositional information is not available from VPA images acquired by single wavelength excitation. Here we demonstrate that lipids and collagen, two critical markers in many kinds of diseases, can be distinguished by hyperspectral VPA imaging. A phantom consisted of rat tail tendon (collagen) and fat tissue (lipids) was constructed. Wavelengths between 1650 and 1850 nm were scanned to excite the first overtone/combination vibration of C-H bond. B-scan hyperspectral VPA images, in which each pixel contains a spectrum, was analyzed by a Multivariate Curve Resolution - Alternating Least Squares (MCR-ALS) algorism to recover the spatial distribution of two chemical components in the phantom.

Mapping lipid and collagen by multispectral photoacoustic imaging of chemical bond vibration

NASA Astrophysics Data System (ADS)

Wang, Pu; Wang, Ping; Wang, Han-Wei; Cheng, Ji-Xin

2012-09-01

Photoacoustic microscopy using vibrational overtone absorption as a contrast mechanism allows bond-selective imaging of deep tissues. Due to the spectral similarity of molecules in the region of overtone vibration, it is difficult to interrogate chemical components using photoacoustic signal at single excitation wavelength. Here we demonstrate that lipids and collagen, two critical markers for many kinds of diseases, can be distinguished by multispectral photoacoustic imaging of the first overtone of C-H bond. A phantom consisting of rat-tail tendon and fat was constructed to demonstrate this technique. Wavelengths between 1650 and 1850 nm were scanned to excite both the first overtone and combination bands of C-H bonds. B-scan multispectral photoacoustic images, in which each pixel contains a spectrum, were analyzed by a multivariate curve resolution-alternating least squares algorithm to recover the spatial distribution of collagen and lipids in the phantom.

Mapping lipid and collagen by multispectral photoacoustic imaging of chemical bond vibration.

PubMed

Wang, Pu; Wang, Ping; Wang, Han-Wei; Cheng, Ji-Xin

2012-09-01

Photoacoustic microscopy using vibrational overtone absorption as a contrast mechanism allows bond-selective imaging of deep tissues. Due to the spectral similarity of molecules in the region of overtone vibration, it is difficult to interrogate chemical components using photoacoustic signal at single excitation wavelength. Here we demonstrate that lipids and collagen, two critical markers for many kinds of diseases, can be distinguished by multispectral photoacoustic imaging of the first overtone of C-H bond. A phantom consisting of rat-tail tendon and fat was constructed to demonstrate this technique. Wavelengths between 1650 and 1850 nm were scanned to excite both the first overtone and combination bands of C-H bonds. B-scan multispectral photoacoustic images, in which each pixel contains a spectrum, were analyzed by a multivariate curve resolution-alternating least squares algorithm to recover the spatial distribution of collagen and lipids in the phantom.

Stability of silk and collagen protein materials in space.

PubMed

Hu, Xiao; Raja, Waseem K; An, Bo; Tokareva, Olena; Cebe, Peggy; Kaplan, David L

2013-12-05

Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered.

Stability of Silk and Collagen Protein Materials in Space

PubMed Central

Hu, Xiao; Raja, Waseem K.; An, Bo; Tokareva, Olena; Cebe, Peggy; Kaplan, David L.

2013-01-01

Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered. PMID:24305951

The role of the extracellular matrix in tissue distribution of macromolecules in normal and pathological tissues: potential therapeutic consequences.

PubMed

Wiig, Helge; Gyenge, Christina; Iversen, Per Ole; Gullberg, Donald; Tenstad, Olav

2008-05-01

The interstitial space is a dynamic microenvironment that consists of interstitial fluid and structural molecules of the extracellular matrix, such as glycosaminoglycans (hyaluronan and proteoglycans) and collagen. Macromolecules can distribute in the interstitium only in those spaces unoccupied by structural components, a phenomenon called interstitial exclusion. The exclusion phenomenon has direct consequences for plasma volume regulation. Early studies have assigned a major role to collagen as an excluding agent that accounts for the sterical (geometrical) exclusion. More recently, it has been shown that the contribution of negatively charged glycosaminoglycans might also be significant, resulting in an additional electrostatical exclusion effect. This charge effect may be of importance for drug uptake and suggests that either the glycosaminoglycans or the net charge of macromolecular substances to be delivered may be targeted to increase the available volume and uptake of macromolecular therapeutic agents in tumor tissue. Here, we provide an overview of the structural components of the interstitium and discuss the importance the sterical and electrostatical components have on the dynamics of transcapillary fluid exchange.

β-Catenin Serves as a Clutch between Low and High Intercellular E-Cadherin Bond Strengths

PubMed Central

Bajpai, Saumendra; Feng, Yunfeng; Wirtz, Denis; Longmore, Gregory D.

2013-01-01

A wide range of invasive pathological outcomes originate from the loss of epithelial phenotype and involve either loss of function or downregulation of transmembrane adhesive receptor complexes, including Ecadherin (Ecad) and binding partners β-catenin and α-catenin at adherens junctions. Cellular pathways regulating wild-type β-catenin level, or direct mutations in β-catenin that affect the turnover of the protein have been shown to contribute to cancer development, through induction of uncontrolled proliferation of transformed tumor cells, particularly in colon cancer. Using single-molecule force spectroscopy, we show that depletion of β-catenin or the prominent cancer-related S45 deletion mutation in β-catenin present in human colon cancers both weaken tumor intercellular Ecad/Ecad bond strength and diminishes the capacity of specific extracellular matrix proteins—including collagen I, collagen IV, and laminin V—to modulate intercellular Ecad/Ecad bond strength through α-catenin and the kinase activity of glycogen synthase kinase 3 (GSK-3β). Thus, in addition to regulating tumor cell proliferation, cancer-related mutations in β-catenin can influence tumor progression by weakening the adhesion of tumor cells to one another through reduced individual Ecad/Ecad bond strength and cellular adhesion to specific components of the extracellular matrix and the basement membrane. PMID:24268141

Modern collagen wound dressings: function and purpose.

PubMed

Fleck, Cynthia Ann; Simman, Richard

2010-09-01

Collagen, which is produced by fibroblasts, is the most abundant protein in the human body. A natural structural protein, collagen is involved in all 3 phases of the wound-healing cascade. It stimulates cellular migration and contributes to new tissue development. Because of their chemotactic properties on wound fibroblasts, collagen dressings encourage the deposition and organization of newly formed collagen, creating an environment that fosters healing. Collagen-based biomaterials stimulate and recruit specific cells, such as macrophages and fibroblasts, along the healing cascade to enhance and influence wound healing. These biomaterials can provide moisture or absorption, depending on the delivery system. Collagen dressings are easy to apply and remove and are conformable. Collagen dressings are usually formulated with bovine, avian, or porcine collagen. Oxidized regenerated cellulose, a plant-based material, has been combined with collagen to produce a dressing capable of binding to and protecting growth factors by binding and inactivating matrix metalloproteinases in the wound environment. The increased understanding of the biochemical processes involved in chronic wound healing allows the design of wound care products aimed at correcting imbalances in the wound microenvironment. Traditional advanced wound care products tend to address the wound's macroenvironment, including moist wound environment control, fluid management, and controlled transpiration of wound fluids. The newer class of biomaterials and wound-healing agents, such as collagen and growth factors, targets specific defects in the chronic wound environment. In vitro laboratory data point to the possibility that these agents benefit the wound healing process at a biochemical level. Considerable evidence has indicated that collagen-based dressings may be capable of stimulating healing by manipulating wound biochemistry.

Type V Collagen is Persistently Altered after Inguinal Hernia Repair.

PubMed

Lorentzen, L; Henriksen, N A; Juhl, P; Mortensen, J H; Ågren, M S; Karsdal, M A; Jorgensen, L N

2018-04-01

Hernia formation is associated with alterations of collagen metabolism. Collagen synthesis and degradation cause a systemic release of products, which are measurable in serum. Recently, we reported changes in type V and IV collagen metabolisms in patients with inguinal and incisional hernia. The aim of this study was to determine if the altered collagen metabolism was persistent after hernia repair. Patients who had undergone repairs for inguinal hernia (n = 11) or for incisional hernia (n = 17) were included in this study. Patients who had undergone elective cholecystectomy served as controls (n = 10). Whole venous blood was collected 35-55 months after operation. Biomarkers for type V collagen synthesis (Pro-C5) and degradation (C5M) and those for type IV collagen synthesis (P4NP) and degradation (C4M2) were measured by a solid-phase competitive assay. The turnover of type V collagen (Pro-C5/C5M) was slightly higher postoperatively when compared to preoperatively in the inguinal hernia group (P = 0.034). In addition, the results revealed a postoperatively lower type V collagen turnover level in the inguinal hernia group compared to controls (P = 0.012). In the incisional hernia group, the type V collagen turnover was higher after hernia repair (P = 0.004) and the postoperative turnover level was not different from the control group (P = 0.973). Patients with an inguinal hernia demonstrated a systemic and persistent type V collagen turnover alteration. This imbalance of the collagen metabolism may be involved in the development of inguinal hernias.

X-ray crystal structures of Staphylococcus aureus collagen adhesin and sortases

NASA Astrophysics Data System (ADS)

Zong, Yinong

For many gram-positive bacteria, adhesion to host tissues is the first critical step in developing an infection. The adhesion is mediated by a superfamily of bacterial surface proteins, called MSCRAMM (microbial surface components recognizing adhesive matrix molecules), which in most cases are covalently attached to the cell wall peptidoglycan. Collagen adhesin (CNA) from Staphylococcus aureus, one of the MSCRAMMs, is responsible for bacterial binding to collagen molecules. CNA and other MSCRAMMs are anchored to the cell wall by a transpeptidase, sortase. The knowledge about how bacterial surface proteins adhere to host molecules and how they are sorted onto the cell wall is crucial for the design of novel antibiotics against bacterial infections. The crystal structures of CNA31--344 (residue 31 to 334), a truncation of CNA's collagen binding region, and CNA31--344 in complex with a collagen peptide were determined. CNA31--344 contains two domains, and between them is a big hole formed by a loop connecting the two domains. In the structure of CNA31--344-collagen complex, the collagen peptide is locked in the hole formed by the two domains of CNA 31--344. We reason that the two domains of CNA31--344 are open in the physiological condition, and close up when binding to collagen. This binding mechanism may be common for other bacterial collagen adhesins. There are two known sortases in Staphylococcus aureus. Sortase A is responsible for anchoring most MSCRAMMs that have a LPXTG (X represents any amino acid) sorting motif and sortase B for a bacterial ion acquisition protein. The crystal structures of both sortases indicate that they share a common catalytic mechanism. Unlike typical cysteine transpeptidases, sortases may use a novel Cys-Arg catalytic dyad instead of a Cys-His pair. All other sortases found in gram-positive bacteria may have similar active site architecture and employ the same catalytic dyad because the critical residues are all conserved among them. The structures of sortases in complex with their substrates and inhibitors are also useful to explain their substrate specificity and catalytic kinetics.

Effects of collagen and collagen hydrolysate from jellyfish (Rhopilema esculentum) on mice skin photoaging induced by UV irradiation.

PubMed

Zhuang, Yongliang; Hou, Hu; Zhao, Xue; Zhang, Zhaohui; Li, Bafang

2009-08-01

Collagen (JC) was extracted from jellyfish (Rhopilema esculentum) and hydrolyzed to prepare collagen hydrolysate (JCH). The protective effects of JC and JCH against UV-induced damages to mice skin were evaluated and compared in this article. JC and JCH could alleviate the UV-induced abnormal changes of antioxidative indicators, including the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities and the contents of glutathione (GSH) and malondiaidehyde (MDA). JC and JCH could protect skin lipid and collagen from the UV radiation damages. Furthermore, the changes of total ceramide and glycosaminoglycan in skin were recovered significantly by JC and JCH. The action mechanisms mainly involved the antioxidative properties and the repairing to endogenous collagen synthesis of JC and JCH in vivo. JCH with the lower molecular weight showed much higher effects than JC. The results indicated that JCH was a novel antiphotoaging agent from natural resources.

Detection of substrate binding of a collagen-specific molecular chaperone HSP47 in solution using fluorescence correlation spectroscopy.

PubMed

Kitamura, Akira; Ishida, Yoshihito; Kubota, Hiroshi; Pack, Chan-Gi; Homma, Takayuki; Ito, Shinya; Araki, Kazutaka; Kinjo, Masataka; Nagata, Kazuhiro

2018-02-26

Heat shock protein 47 kDa (HSP47), an ER-resident and collagen-specific molecular chaperone, recognizes collagenous hydrophobic amino acid sequences (Gly-Pro-Hyp) and assists in secretion of correctly folded collagen. Elevated collagen production is correlated with HSP47 expression in various diseases, including fibrosis and keloid. HSP47 knockdown ameliorates liver fibrosis by inhibiting collagen secretion, and inhibition of the interaction of HSP47 with procollagen also prevents collagen secretion. Therefore, a high-throughput system for screening of drugs capable of inhibiting the interaction between HSP47 and collagen would aid the development of novel therapies for fibrotic diseases. In this study, we established a straightforward method for rapidly and quantitatively measuring the interaction between HSP47 and collagen in solution using fluorescence correlation spectroscopy (FCS). The diffusion rate of HSP47 labeled with Alexa Fluor 488 (HSP47-AF), a green fluorescent dye, decreased upon addition of type I or III collagen, whereas that of dye-labeled protein disulfide isomerase (PDI) or bovine serum albumin (BSA) did not, indicating that specific binding of HSP47 to collagen could be detected using FCS. Using this method, we calculated the dissociation constant of the interaction between HSP47 and collagen. The binding ratio between HSP47-AF and collagen did not change in the presence of sodium chloride, confirming that the interaction was hydrophobic in nature. In addition, we observed dissociation of collagen from HSP47 at low pH and re-association after recovery to neutral pH. These observations indicate that this system is appropriate for detecting the interaction between HSP47 and collagen, and could be applied to high-throughput screening for drugs capable of suppressing and/or curing fibrosis. Copyright © 2018 Elsevier Inc. All rights reserved.

Using carboxylated cellulose nanofibers to enhance mechanical and barrier properties of collagen fiber film by electrostatic interaction.

PubMed

Wang, Wenhang; Zhang, Xiuling; Li, Cong; Du, Guanhua; Zhang, Hongjie; Ni, Yonghao

2018-06-01

Collagen-based films including casings with a promising application in meat industry are still needed to improve its inferior performance. In the present study, the reinforcement of carboxylated cellulose nanofibers (CNF) for collagen film, based on inter-/intra- molecular electrostatic interaction between cationic acid-swollen collagen fiber and anionic carboxylated CNF, was investigated. Adding CNF decreased the zeta-potential but increased particle size of collagen fiber suspension, with little effect on pH. Furthermore, CNF addition led to a higher tensile strength but a lower elongation, and the water vapor and oxygen barrier properties were improved remarkably. Because the CNF content was 50 g kg -1 or lower, the films had a homogeneous interwoven network, and CNF homogeneously embedded into collagen fiber matrix according to the scanning electron microscopy and atomic force microscopy analysis. Additionally, CNF addition increased film thickness and opacity, as well as swelling rate. The incorporation of CNF endows collagen fiber films good mechanical and barrier properties over a proper concentration range (≤ 50 g kg -1 collagen fiber), which is closely associated with electrostatic reaction of collagen fiber and CNF and, subsequently, the form of the homogenous, compatible spatial network, indicating a potential applications of CNF in collagenous protein films, such as edible casings. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Biomimetic hydrogels direct spinal progenitor cell differentiation and promote functional recovery after spinal cord injury

NASA Astrophysics Data System (ADS)

Geissler, Sydney A.; Sabin, Alexandra L.; Besser, Rachel R.; Gooden, Olivia M.; Shirk, Bryce D.; Nguyen, Quan M.; Khaing, Zin Z.; Schmidt, Christine E.

2018-04-01

Objective. Demyelination that results from disease or traumatic injury, such as spinal cord injury (SCI), can have a devastating effect on neural function and recovery. Many researchers are examining treatments to minimize demyelination by improving oligodendrocyte availability in vivo. Transplantation of stem and oligodendrocyte progenitor cells is a promising option, however, trials are plagued by undirected differentiation. Here we introduce a biomaterial that has been optimized to direct the differentiation of neural progenitor cells (NPCs) toward oligodendrocytes as a cell delivery vehicle after SCI. Approach. A collagen-based hydrogel was modified to mimic the mechanical properties of the neonatal spinal cord, and components present in the developing extracellular matrix were included to provide appropriate chemical cues to the NPCs to direct their differentiation toward oligodendrocytes. The hydrogel with cells was then transplanted into a unilateral cervical contusion model of SCI to examine the functional recovery with this treatment. Six behavioral tests and histological assessment were performed to examine the in vivo response to this treatment. Main results. Our results demonstrate that we can achieve a significant increase in oligodendrocyte differentiation of NPCs compared to standard culture conditions using a three-component biomaterial composed of collagen, hyaluronic acid, and laminin that has mechanical properties matched to those of neonatal neural tissue. Additionally, SCI rats with hydrogel transplants, with and without NPCs, showed functional recovery. Animals transplanted with hydrogels with NPCs showed significantly increased functional recovery over six weeks compared to the media control group. Significance. The three-component hydrogel presented here has the potential to provide cues to direct differentiation in vivo to encourage regeneration of the central nervous system.

The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic therapeutic in murine collagen-induced arthritis

PubMed Central

Malfait, A. M.; Gallily, R.; Sumariwalla, P. F.; Malik, A. S.; Andreakos, E.; Mechoulam, R.; Feldmann, M.

2000-01-01

The therapeutic potential of cannabidiol (CBD), the major nonpsychoactive component of cannabis, was explored in murine collagen-induced arthritis (CIA). CIA was elicited by immunizing DBA/1 mice with type II collagen (CII) in complete Freund's adjuvant. The CII used was either bovine or murine, resulting in classical acute CIA or in chronic relapsing CIA, respectively. CBD was administered after onset of clinical symptoms, and in both models of arthritis the treatment effectively blocked progression of arthritis. CBD was equally effective when administered i.p. or orally. The dose dependency showed a bell-shaped curve, with an optimal effect at 5 mg/kg per day i.p. or 25 mg/kg per day orally. Clinical improvement was associated with protection of the joints against severe damage. Ex vivo, draining lymph node cells from CBD-treated mice showed a diminished CII-specific proliferation and IFN-γ production, as well as a decreased release of tumor necrosis factor by knee synovial cells. In vitro effects of CBD included a dose-dependent suppression of lymphocyte proliferation, both mitogen-stimulated and antigen-specific, and the blockade of the Zymosan-triggered reactive oxygen burst by peritoneal granulocytes. It also was found that CBD administration was capable of blocking the lipopolysaccharide-induced rise in serum tumor necrosis factor in C57/BL mice. Taken together, these data show that CBD, through its combined immunosuppressive and anti-inflammatory actions, has a potent anti-arthritic effect in CIA. PMID:10920191

Elastic fiber-mediated enthesis in the human middle ear

PubMed Central

Kawase, Tetsuaki; Shibata, Shunichi; Katori, Yukio; Ohtsuka, Aiji; Murakami, Gen; Fujimiya, Mineko

2012-01-01

Adaptation to constant vibration (acoustic oscillation) is likely to confer a specific morphology at the bone–tendon and bone–ligament interfaces at the ear ossicles, which therefore represent an exciting target of enthesis research. We histologically examined (i) the bone attachments of the tensor tympani and stapedius muscles and (ii) the annular ligament of the incudostapedial joint obtained from seven elderly donated cadavers. Notably, both aldehyde-fuchsin and elastic-Masson staining demonstrated that the major fibrous component of the entheses was not collagen fibers but mature elastic fibers. The positive controls for elastic fiber staining were the arterial wall elastic laminae included in the temporal bone materials. The elastic fibers were inserted deeply into the type II collagen-poor fibrocartilage covering the ear ossicles. The muscle tendons were composed of an outer thin layer of collagen fibers and an inner thick core of elastic fibers near the malleus or stapes. In the unique elastic fiber-mediated entheses, hyaluronan, versican and fibronectin were expressed strongly along the elastic fibers. The hyaluronan seemed to act as a friction-reducing lubricant for the elastic fibers. Aggrecan was labeled strongly in a disk- or plica-like fibrous mass on the inner side of the elastic fiber-rich ligament, possibly due to compression stress from the ligament. Tenascin-c was not evident in the entheses. The elastic fiber-mediated entheses appeared resistant to tissue destruction in an environment exposed to constant vibration. The morphology was unlikely to be the result of age-related degeneration. PMID:22803514

Potency of Fish Collagen as a Scaffold for Regenerative Medicine

PubMed Central

Yamamoto, Kohei; Yanagiguchi, Kajiro

2014-01-01

Cells, growth factors, and scaffold are the crucial factors for tissue engineering. Recently, scaffolds consisting of natural polymers, such as collagen and gelatin, bioabsorbable synthetic polymers, such as polylactic acid and polyglycolic acid, and inorganic materials, such as hydroxyapatite, as well as composite materials have been rapidly developed. In particular, collagen is the most promising material for tissue engineering due to its biocompatibility and biodegradability. Collagen contains specific cell adhesion domains, including the arginine-glycine-aspartic acid (RGD) motif. After the integrin receptor on the cell surface binds to the RGD motif on the collagen molecule, cell adhesion is actively induced. This interaction contributes to the promotion of cell growth and differentiation and the regulation of various cell functions. However, it is difficult to use a pure collagen scaffold as a tissue engineering material due to its low mechanical strength. In order to make up for this disadvantage, collagen scaffolds are often modified using a cross-linker, such as gamma irradiation and carbodiimide. Taking into account the possibility of zoonosis, a variety of recent reports have been documented using fish collagen scaffolds. We herein review the potency of fish collagen scaffolds as well as associated problems to be addressed for use in regenerative medicine. PMID:24982861

Second harmonic generation microscopy differentiates collagen type I and type III in COPD

NASA Astrophysics Data System (ADS)

Suzuki, Masaru; Kayra, Damian; Elliott, W. Mark; Hogg, James C.; Abraham, Thomas

2012-03-01

The structural remodeling of extracellular matrix proteins in peripheral lung region is an important feature in chronic obstructive pulmonary disease (COPD). Multiphoton microscopy is capable of inducing specific second harmonic generation (SHG) signal from non-centrosymmetric structural proteins such as fibrillar collagens. In this study, SHG microscopy was used to examine structural remodeling of the fibrillar collagens in human lungs undergoing emphysematous destruction (n=2). The SHG signals originating from these diseased lung thin sections from base to apex (n=16) were captured simultaneously in both forward and backward directions. We found that the SHG images detected in the forward direction showed well-developed and well-structured thick collagen fibers while the SHG images detected in the backward direction showed striking different morphological features which included the diffused pattern of forward detected structures plus other forms of collagen structures. Comparison of these images with the wellestablished immunohistochemical staining indicated that the structures detected in the forward direction are primarily the thick collagen type I fibers and the structures identified in the backward direction are diffusive structures of forward detected collagen type I plus collagen type III. In conclusion, we here demonstrate the feasibility of SHG microscopy in differentiating fibrillar collagen subtypes and understanding their remodeling in diseased lung tissues.

Dendritic cells: In vitro culture in two- and three-dimensional collagen systems and expression of collagen receptors in tumors and atherosclerotic microenvironments.

PubMed

Sprague, Leslee; Muccioli, Maria; Pate, Michelle; Singh, Manindra; Xiong, Chengkai; Ostermann, Alexander; Niese, Brandon; Li, Yihan; Li, Yandi; Courreges, Maria Cecilia; Benencia, Fabian

2014-04-15

Dendritic cells (DCs) are immune cells found in the peripheral tissues where they sample the organism for infections or malignancies. There they take up antigens and migrate towards immunological organs to contact and activate T lymphocytes that specifically recognize the antigen presented by these antigen presenting cells. In the steady state there are several types of resident DCs present in various different organs. For example, in the mouse, splenic DC populations characterized by the co-expression of CD11c and CD8 surface markers are specialized in cross-presentation to CD8 T cells, while CD11c/SIRP-1α DCs seem to be dedicated to activating CD4 T cells. On the other hand, DCs have also been associated with the development of various diseases such as cancer, atherosclerosis, or inflammatory conditions. In such disease, DCs can participate by inducing angiogenesis or immunosuppression (tumors), promoting autoimmune responses, or exacerbating inflammation (atherosclerosis). This change in DC biology can be prompted by signals in the microenvironment. We have previously shown that the interaction of DCs with various extracellular matrix components modifies the immune properties and angiogenic potential of these cells. Building on those studies, herewith we analyzed the angiogenic profile of murine myeloid DCs upon interaction with 2D and 3D type-I collagen environments. As determined by PCR array technology and quantitative PCR analysis we observed that interaction with these collagen environments induced the expression of particular angiogenic molecules. In addition, DCs cultured on collagen environments specifically upregulated the expression of CXCL-1 and -2 chemokines. We were also able to establish DC cultures on type-IV collagen environments, a collagen type expressed in pathological conditions such as atherosclerosis. When we examined DC populations in atherosclerotic veins of Apolipoprotein E deficient mice we observed that they expressed adhesion molecules capable of interacting with collagen. Finally, to further investigate the interaction of DCs with collagen in other pathological conditions, we determined that both murine ovarian and breast cancer cells express several collagen molecules that can contribute to shape their particular tumor microenvironment. Consistently, tumor-associated DCs were shown to express adhesion molecules capable of interacting with collagen molecules as determined by flow cytometry analysis. Of particular relevance, tumor-associated DCs expressed high levels of CD305/LAIR-1, an immunosuppressive receptor. This suggests that signaling through this molecule upon interaction with collagen produced by tumor cells might help define the poorly immunogenic status of these cells in the tumor microenvironment. Overall, these studies demonstrate that through interaction with collagen proteins, DCs can be capable of modifying the microenvironments of inflammatory disease such as cancer or atherosclerosis. Copyright © 2014. Published by Elsevier Inc.

Analysis of a collagen II degradation protein C2C and a collagen II formation protein CP II in serum of Asian elephants (Elephas maximus).

PubMed

Kilgallon, Conor P; Larsen, Scott; Wong, Alice; Yellowley, Clare

2015-03-01

Osteoarthritis is a major cause of chronic lameness in Asian elephants (Elephas maximus) in captivity worldwide. Radiology and other imaging technologies are of limited use in the early diagnosis of this condition in elephants. Collagen II is a major component of articular cartilage. The degradation and formation of collagen II can be monitored by the measurement of specific biomarkers in biologic fluids such as serum. It is possible that these biomarkers could also prove useful in identifying disease in elephants. In this study two commercially available immunoassays which measure a marker of collagen II degradation (C2C) and a marker of collagen II formation (CPII) were evaluated in Asian elephants. The ability of the assays to detect and measure C2C and CPII in the serum of Asian elephants was confirmed. Median serum concentration of C2C was 148 ng/L in nonlame elephants (n=33) and 91.2 ng/L in lame elephants (n=7). The difference was statistically significant (P=0.0002). Median serum concentration of CPII was 519.3 ng/L in nonlame elephants and 318.7 ng/L in lame elephants. The difference was also statistically significant (P=0.039). Whereas CPII concentrations in lame elephants mirrored findings from human and animal osteoarthritis studies, C2C concentrations did not. Further studies which evaluate these and other similar biomarkers are necessary to elucidate their usefulness in the diagnosis of osteoarthritis in proboscidae.

Type VII collagen regulates expression of OATP1B3, promotes front-to-rear polarity and increases structural organisation in 3D spheroid cultures of RDEB tumour keratinocytes

PubMed Central

Dayal, Jasbani H. S.; Cole, Clare L.; Pourreyron, Celine; Watt, Stephen A.; Lim, Yok Zuan; Salas-Alanis, Julio C.; Murrell, Dedee F.; McGrath, John A.; Stieger, Bruno; Jahoda, Colin; Leigh, Irene M.; South, Andrew P.

2014-01-01

ABSTRACT Type VII collagen is the main component of anchoring fibrils, structures that are integral to basement membrane homeostasis in skin. Mutations in the gene encoding type VII collagen COL7A1 cause recessive dystrophic epidermolysis bullosa (RDEB) an inherited skin blistering condition complicated by frequent aggressive cutaneous squamous cell carcinoma (cSCC). OATP1B3, which is encoded by the gene SLCO1B3, is a member of the OATP (organic anion transporting polypeptide) superfamily responsible for transporting a wide range of endogenous and xenobiotic compounds. OATP1B3 expression is limited to the liver in healthy tissues, but is frequently detected in multiple cancer types and is reported to be associated with differing clinical outcome. The mechanism and functional significance of tumour-specific expression of OATP1B3 has yet to be determined. Here, we identify SLCO1B3 expression in tumour keratinocytes isolated from RDEB and UV-induced cSCC and demonstrate that SLCO1B3 expression and promoter activity are modulated by type VII collagen. We show that reduction of SLCO1B3 expression upon expression of full-length type VII collagen in RDEB cSCC coincides with acquisition of front-to-rear polarity and increased organisation of 3D spheroid cultures. In addition, we show that type VII collagen positively regulates the abundance of markers implicated in cellular polarity, namely ELMO2, PAR3, E-cadherin, B-catenin, ITGA6 and Ln332. PMID:24357722

Cartilage proteoglycans inhibit fibronectin-mediated adhesion

NASA Astrophysics Data System (ADS)

Rich, A. M.; Pearlstein, E.; Weissmann, G.; Hoffstein, S. T.

1981-09-01

Normal tissues and organs show, on histological examination, a pattern of cellular and acellular zones that is characteristic and unique for each organ or tissue. This pattern is maintained in health but is sometimes destroyed by disease. For example, in mobile joints, the articular surfaces consist of relatively acellular hyaline cartilage, and the joint space is enclosed by a capsule of loose connective tissue with a lining of fibroblasts and macrophages. In the normal joint these cells are confined to the synovial lining and the articular surface remains acellular. In in vitro culture, macrophages and their precursor monocytes are very adhesive, and fibroblasts can migrate and overgrow surfaces such as collagen or plastic used for tissue culture. The fibroblasts adhere to collagen by means of fibronectin, which they synthesize and secrete1. Because the collagen of cartilage is capable of binding serum fibronectin2 and fibronectin is present in cartilage during its development3, these cells should, in theory, slowly migrate from the synovial lining to the articular surface. It is their absence from the articular cartilage in normal circumstances, and then presence in such pathological states as rheumatoid arthritis, that is striking. We therefore set out to determine whether a component of cartilage could prevent fibroblast adherence in a defined adhesion assay. As normal cartilage is composed of 50% proteoglycans and 50% collagen by dry weight4, we tested the possibility that the proteoglycans in cartilage inhibit fibroblast adhesion to collagen. We present here evidence that fibroblast spreading and adhesion to collagenous substrates is inhibited by cartilage proteoglycans.

Micro- and nano-mechanics of osteoarthritic cartilage: The effects of tonicity and disease severity.

PubMed

Moshtagh, P R; Pouran, B; van Tiel, J; Rauker, J; Zuiddam, M R; Arbabi, V; Korthagen, N M; Weinans, H; Zadpoor, A A

2016-06-01

The present study aims to discover the contribution of glycosaminoglycans (GAGs) and collagen fibers to the mechanical properties of the osteoarthritic (OA) cartilage tissue. We used nanoindentation experiments to understand the mechanical behavior of mild and severe osteoarthritic cartilage at micro- and nano-scale at different swelling conditions. Contrast enhanced micro-computed tomography (EPIC-μCT) was used to confirm that mild OA specimens had significantly higher GAGs content compared to severe OA specimens. In micro-scale, the semi-equilibrium modulus of mild OA specimens significantly dropped after immersion in a hypertonic solution and at nano-scale, the histograms of the measured elastic modulus revealed three to four components. Comparing the peaks with those observed for healthy cartilage in a previous study indicated that the first and third peaks represent the mechanical properties of GAGs and the collagen network. The third peak shows considerably stiffer elastic modulus for mild OA samples as compared to the severe OA samples in isotonic conditions. Furthermore, this peak clearly dropped when the tonicity increased, indicating the loss of collagen (pre-) stress in the shrunk specimen. Our observations support the association of the third peak with the collagen network. However, our results did not provide any direct evidence to support the association of the first peak with GAGs. For severe OA specimens, the peak associated with the collagen network did not drop when the tonicity increased, indicating a change in the response of OA cartilage to hypertonicity, likely collagen damage, as the disease progresses to its latest stages. Copyright © 2016 Elsevier Ltd. All rights reserved.

Immunological and Toxinological Responses to Jellyfish Stings

PubMed Central

Tibballs, James; Yanagihara, Angel A.; Turner, Helen C.; Winkel, Ken

2013-01-01

Just over a century ago, animal responses to injections of jellyfish extracts unveiled the phenomenon of anaphylaxis. Yet, until very recently, understanding of jellyfish sting toxicity has remained limited. Upon contact, jellyfish stinging cells discharge complex venoms, through thousands of barbed tubules, into the skin resulting in painful and, potentially, lethal envenomations. This review examines the immunological and toxinological responses to stings by prominent species of jellyfish including Physalia sp. (Portuguese Man-o-War, Blue-bottle), Cubozoan jellyfish including Chironex fleckeri, several Carybdeids including Carybdea arborifera and Alatina moseri, Linuche unguiculta (Thimble jellyfish), a jellyfish responsible for Irukandji syndrome (Carukia barnesi) and Pelagia noctiluca. Jellyfish venoms are composed of potent proteinaceous porins (cellular membrane pore-forming toxins), neurotoxic peptides, bioactive lipids and other small molecules whilst the tubules contain ancient collagens and chitins. We postulate that immunologically, both tubular structural and functional biopolymers as well as venom components can initiate innate, adaptive, as well as immediate and delayed hypersensitivity reactions that may be amenable to topical anti-inflammatory-immunomodifier therapy. The current challenge for immunotoxinologists is to deconstruct the actions of venom components to target therapeutic modalities for sting treatment. PMID:21824077

Anterior Cruciate Ligament Reconstruction in a Rabbit Model Using Silk-Collagen Scaffold and Comparison with Autograft

PubMed Central

Bi, Fanggang; Shi, Zhongli; Liu, An; Guo, Peng; Yan, Shigui

2015-01-01

The objective of the present study was to perform an in vivo assessment of a novel silk-collagen scaffold for anterior cruciate ligament (ACL) reconstruction. First, a silk-collagen scaffold was fabricated by combining sericin-extracted knitted silk fibroin mesh and type I collagen to mimic the components of the ligament. Scaffolds were electron-beam sterilized and rolled up to replace the ACL in 20 rabbits in the scaffold group, and autologous semitendinosus tendons were used to reconstruct the ACL in the autograft control group. At 4 and 16 weeks after surgery, grafts were retrieved and analyzed for neoligament regeneration and tendon-bone healing. To evaluate neoligament regeneration, H&E and immunohistochemical staining was performed, and to assess tendon-bone healing, micro-CT, biomechanical test, H&E and Russell-Movat pentachrome staining were performed. Cell infiltration increased over time in the scaffold group, and abundant fibroblast-like cells were found in the core of the scaffold graft at 16 weeks postoperatively. Tenascin-C was strongly positive in newly regenerated tissue at 4 and 16 weeks postoperatively in the scaffold group, similar to observations in the autograft group. Compared with the autograft group, tendon-bone healing was better in the scaffold group with trabecular bone growth into the scaffold. The results indicate that the silk-collagen scaffold has considerable potential for clinical application. PMID:25938408

Mechanical stiffness of TMJ condylar cartilage increases after artificial aging by ribose.

PubMed

Mirahmadi, Fereshteh; Koolstra, Jan Harm; Lobbezoo, Frank; van Lenthe, G Harry; Ghazanfari, Samaneh; Snabel, Jessica; Stoop, Reinout; Everts, Vincent

2018-03-01

Aging is accompanied by a series of changes in mature tissues that influence their properties and functions. Collagen, as one of the main extracellular components of cartilage, becomes highly crosslinked during aging. In this study, the aim was to examine whether a correlation exists between collagen crosslinking induced by artificial aging and mechanical properties of the temporomandibular joint (TMJ) condyle. To evaluate this hypothesis, collagen crosslinks were induced using ribose incubation. Porcine TMJ condyles were incubated for 7 days with different concentrations of ribose. The compressive modulus and stiffness ratio (incubated versus control) was determined after loading. Glycosaminoglycan and collagen content, and the number of crosslinks were analyzed. Tissue structure was visualized by microscopy using different staining methods. Concomitant with an increasing concentration of ribose, an increase of collagen crosslinks was found. The number of crosslinks increased almost 50 fold after incubation with the highest concentration of ribose. Simultaneously, the stiffness ratio of the samples showed a significant increase after incubation with the ribose. Pearson correlation analyses showed a significant positive correlation between the overall stiffness ratio and the crosslink level; the higher the number of crosslinks the higher the stiffness. The present model, in which ribose was used to mimic certain aspects of age-related changes, can be employed as an in vitro model to study age-related mechanical changes in the TMJ condyle. Copyright © 2017 Elsevier Ltd. All rights reserved.

Alignment hierarchies: engineering architecture from the nanometre to the micrometre scale.

PubMed

Kureshi, Alvena; Cheema, Umber; Alekseeva, Tijna; Cambrey, Alison; Brown, Robert

2010-12-06

Natural tissues are built of metabolites, soluble proteins and solid extracellular matrix components (largely fibrils) together with cells. These are configured in highly organized hierarchies of structure across length scales from nanometre to millimetre, with alignments that are dominated by anisotropies in their fibrillar matrix. If we are to successfully engineer tissues, these hierarchies need to be mimicked with an understanding of the interaction between them. In particular, the movement of different elements of the tissue (e.g. molecules, cells and bulk fluids) is controlled by matrix structures at distinct scales. We present three novel systems to introduce alignment of collagen fibrils, cells and growth factor gradients within a three-dimensional collagen scaffold using fluid flow, embossing and layering of construct. Importantly, these can be seen as different parts of the same hierarchy of three-dimensional structure, as they are all formed into dense collagen gels. Fluid flow aligns collagen fibrils at the nanoscale, embossed topographical features provide alignment cues at the microscale and introducing layered configuration to three-dimensional collagen scaffolds provides microscale- and mesoscale-aligned pathways for protein factor delivery as well as barriers to confine protein diffusion to specific spatial directions. These seemingly separate methods can be employed to increase complexity of simple extracellular matrix scaffolds, providing insight into new approaches to directly fabricate complex physical and chemical cues at different hierarchical scales, similar to those in natural tissues.

Human skeletal muscle biochemical diversity.

PubMed

Tirrell, Timothy F; Cook, Mark S; Carr, J Austin; Lin, Evie; Ward, Samuel R; Lieber, Richard L

2012-08-01

The molecular components largely responsible for muscle attributes such as passive tension development (titin and collagen), active tension development (myosin heavy chain, MHC) and mechanosensitive signaling (titin) have been well studied in animals but less is known about their roles in humans. The purpose of this study was to perform a comprehensive analysis of titin, collagen and MHC isoform distributions in a large number of human muscles, to search for common themes and trends in the muscular organization of the human body. In this study, 599 biopsies were obtained from six human cadaveric donors (mean age 83 years). Three assays were performed on each biopsy - titin molecular mass determination, hydroxyproline content (a surrogate for collagen content) and MHC isoform distribution. Titin molecular mass was increased in more distal muscles of the upper and lower limbs. This trend was also observed for collagen. Percentage MHC-1 data followed a pattern similar to collagen in muscles of the upper extremity but this trend was reversed in the lower extremity. Titin molecular mass was the best predictor of anatomical region and muscle functional group. On average, human muscles had more slow myosin than other mammals. Also, larger titins were generally associated with faster muscles. These trends suggest that distal muscles should have higher passive tension than proximal ones, and that titin size variability may potentially act to 'tune' the protein's mechanotransduction capability.

Human skeletal muscle biochemical diversity

PubMed Central

Tirrell, Timothy F.; Cook, Mark S.; Carr, J. Austin; Lin, Evie; Ward, Samuel R.; Lieber, Richard L.

2012-01-01

SUMMARY The molecular components largely responsible for muscle attributes such as passive tension development (titin and collagen), active tension development (myosin heavy chain, MHC) and mechanosensitive signaling (titin) have been well studied in animals but less is known about their roles in humans. The purpose of this study was to perform a comprehensive analysis of titin, collagen and MHC isoform distributions in a large number of human muscles, to search for common themes and trends in the muscular organization of the human body. In this study, 599 biopsies were obtained from six human cadaveric donors (mean age 83 years). Three assays were performed on each biopsy – titin molecular mass determination, hydroxyproline content (a surrogate for collagen content) and MHC isoform distribution. Titin molecular mass was increased in more distal muscles of the upper and lower limbs. This trend was also observed for collagen. Percentage MHC-1 data followed a pattern similar to collagen in muscles of the upper extremity but this trend was reversed in the lower extremity. Titin molecular mass was the best predictor of anatomical region and muscle functional group. On average, human muscles had more slow myosin than other mammals. Also, larger titins were generally associated with faster muscles. These trends suggest that distal muscles should have higher passive tension than proximal ones, and that titin size variability may potentially act to ‘tune’ the protein's mechanotransduction capability. PMID:22786631

Enriched Astaxanthin Extract from Haematococcus pluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts

PubMed Central

Chou, Hsin-Yu; Lee, Chelsea; Pan, Jian-Liang; Wen, Zhi-Hong; Huang, Shu-Hung; Lan, Chi-Wei John; Liu, Wang-Ta; Hour, Tzyh-Chyuan; Hseu, You-Cheng; Hwang, Byeong Hee; Cheng, Kuo-Chen; Wang, Hui-Min David

2016-01-01

Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE) from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA), and doxycycline. With sirius red staining and quantitative real-time polymerase chain reaction (qRT-PCR), we found that PMA decreased the collagen concentration and production while overall the addition of doxycycline and EAE increased the collagen concentration in a trial experiments. EAE increased collagen contents through inhibited MMP1 and MMP3 mRNA expression and induced TIMP1, the antagonists of MMPs protein, gene expression. As for when tested for various proteins through western blotting, it was seen that the addition of EAE increased the expression of certain proteins that promote cell proliferation. Testing those previous solutions using growth factor assay, it was noticeable that EAE had a positive impact on cell proliferation and vascular endothelial growth factor (VEGF) than doxycycline, indicating that it was a better alternative treatment for collagen production. To sum up, the data confirmed the possible applications as medical cosmetology agentsand food supplements. PMID:27322248

Impact of Chronic Hypoxia on Proximal Pulmonary Artery Wave Propagation and Mechanical Properties in Rats.

PubMed

Su, Junjing; Logan, Charmilie C; Hughes, Alun D; Parker, Kim H; Dhutia, Niti M; Danielsen, Carl Christian; Simonsen, Ulf

2018-03-16

Arterial stiffness and wave reflection are important components of the ventricular afterload. Therefore, we aimed to assess the arterial wave characteristics and mechanical properties of the proximal pulmonary arteries (PAs) in the hypoxic pulmonary hypertensive rat model. After 21 days in normoxic or hypoxic chambers (24 animals in each group), the animals underwent transthoracic echocardiography and pulmonary artery catheterization with a dual-tipped pressure and Doppler flow sensor wire. Wave intensity analysis (WIA) was performed. Artery rings obtained from the pulmonary trunk, right and left PAs and the aorta were subjected to a tensile test to rupture. Collagen and elastin content was determined. In hypoxic rats, proximal PA wall thickness, collagen content, tensile strength per unit collagen, maximal elastic modulus and wall viscosity increased; while the elastin:collagen ratio and arterial distensibility decreased. Arterial pulse wave velocity was also increased and the increase was more prominent in vivo than ex vivo. Wave intensity was similar in the hypoxic and normoxic animals with negligible wave reflection. In contrast, aortic maximal elastic modulus remained unchanged, while the wall viscosity decreased. There was no evidence of altered arterial wave propagation in the proximal PAs of hypoxic rats, while the extracellular matrix protein composition altered and the collagen tensile strength increased. This was accompanied by altered mechanical properties in vivo and ex vivo.

Bone toughness at the molecular scale: A model for fracture toughness using crosslinked osteopontin on synthetic and biogenic mineral substrates.

PubMed

Cavelier, S; Dastjerdi, A K; McKee, M D; Barthelat, F

2018-05-01

The most prominent structural components in bone are collagen and mineral. However, bone additionally contains a substantial amount of noncollagenous proteins (most notably of the SIBLING protein family), some of which may act as cohesive/adhesive "binders" for the composite hybrid collagen/mineral scaffolding, whether in the bulk phase of bone, or at its interfaces. One such noncollagenous protein - osteopontin (OPN) - appears to be critical to the deformability and fracture toughness of bone. In the present study, we used a reconstructed synthetic mineral-OPN-mineral interface, and a biogenic (natural tooth dentin) mineral/collagen-OPN-mineral/collagen interface, to measure the fracture toughness of OPN on mineralized substrates. We used this system to test the hypothesis that OPN crosslinking by the enzyme tissue transglutaminase 2 (TG2) that is found in bone enhances interfacial adhesion to increase the fracture toughness of bone. For this, we prepared double-cantilever beam substrates of synthetic pure hydroxyapatite mineral, and of narwhal dentin, and directly apposed them to one another under different intervening OPN/crosslinking conditions, and fracture toughness was tested using a miniaturized loading stage. The work-of-fracture of the OPN interface was measured for different OPN formulations (monomer vs. polymer), crosslinking states, and substrate composition. Noncrosslinked OPN provided negligible adhesion on pure hydroxyapatite, whereas OPN crosslinking (by the chemical crosslinker glutaraldehyde, and TG2 enzyme) provided strong interfacial adhesion for both hydroxyapatite and dentin using monomeric and polymeric OPN. Pre-coating of the substrate beams with monomeric OPN further improved the adhesive performance of the samples, likely by allowing effective binding of this nascent OPN form to mineral/matrix components, with this pre-attachment providing a protein layer for additional crosslinking between the substrates. Copyright © 2018 Elsevier Inc. All rights reserved.

Role for transforming growth factor-beta1 in alport renal disease progression.

PubMed

Sayers, R; Kalluri, R; Rodgers, K D; Shield, C F; Meehan, D T; Cosgrove, D

1999-11-01

Alport syndrome results from mutations in either the alpha3(IV), alpha4(IV), or alpha5(IV) collagen genes. The disease is characterized by a progressive glomerulonephritis usually associated with a high-frequency sensorineural hearing loss. A mouse model for an autosomal form of Alport syndrome [collagen alpha3(IV) knockout] was produced and characterized. In this study, the model was exploited to demonstrate a potential role for transforming growth factor-beta1 (TGF-beta1) in Alport renal disease pathogenesis. Kidneys from normal and Alport mice, taken at different stages during the course of renal disease progression, were analyzed by Northern blot, in situ hybridization, and immunohistology for expression of TGF-beta1 and components of the extracellular matrix. Normal and Alport human kidney was examined for TGF-beta1 expression using RNase protection. The mRNAs encoding TGF-beta1 (in both mouse and human), entactin, fibronectin, and the collagen alpha1(IV) and alpha2(IV) chains were significantly induced in total kidney as a function of Alport renal disease progression. The induction of these specific mRNAs was observed in the glomerular podocytes of animals with advanced disease. Type IV collagen, laminin-1, and fibronectin were markedly elevated in the tubulointerstitium at 10 weeks, but not at 6 weeks, suggesting that elevated expression of specific mRNAs on Northern blots reflects events associated with tubulointerstitial fibrosis. The concomitant accumulation of mRNAs encoding TGF-beta1 and extracellular matrix components in the podocytes of diseased kidneys may reflect key events in Alport renal disease progression. These data suggest a role for TGF-beta1 in both glomerular and tubulointerstitial damage associated with Alport syndrome.

Mimicking the tumor microenvironment to regulate macrophage phenotype and assessing chemotherapeutic efficacy in embedded cancer cell/macrophage spheroid models.

PubMed

Tevis, Kristie M; Cecchi, Ryan J; Colson, Yolonda L; Grinstaff, Mark W

2017-03-01

Tumor associated macrophages (TAMs) are critical stromal components intimately involved with the progression, invasion, and metastasis of cancer cells. To address the need for an in vitro system that mimics the clinical observations of TAM localizations and subsequent functional performance, a cancer cell/macrophage spheroid model is described. The central component of the model is a triple negative breast cancer spheroid embedded in a three-dimensional collagen gel. Macrophages are incorporated in two different ways. The first is a heterospheroid, a spheroid containing both tumor cells and macrophages. The heterospheroid mimics the population of TAMs infiltrated into the tumor mass, thus being exposed to hypoxia and metabolic gradients. In the second model, macrophages are diffusely seeded in the collagen surrounding the spheroid, thus modeling TAMs in the cancer stroma. The inclusion of macrophages as a heterospheroid changes the metabolic profile, indicative of synergistic growth. In contrast, macrophages diffusely seeded in the collagen bear the same profile regardless of the presence of a tumor cell spheroid. The macrophages in the heterospheroid secrete EGF, a cytokine critical to tumor/macrophage co-migration, and an EGF inhibitor decreases the metabolic activity of the heterospheroid, which is not observed in the other systems. The increased secretion of IL-10 indicates that the heterospheroid macrophages follow an M2/TAM differentiation pathway. Lastly, the heterospheroid exhibits resistance to paclitaxel. In summary, the collagen embedded heterospheroid model promotes TAM-like characteristics, and will be of utility in cancer biology and drug discovery. Two in vitro collagen-embedded multicellular spheroid models are described that mimic the clinical observations of macrophage localization within a tumor. Incorporation of macrophages within a breast cancer spheroid emphasizes cell-cell interactions with subsequent differentiation toward a tumor-promoting TAM phenotype. In contrast, macrophages seeded around the tumor spheroid display decreased interaction with cancer cells and no indication of a TAM phenotype. Finally, the presence of macrophages in the heterospheroid increases resistance to paclitaxel. This study demonstrates that cell-cell interactions and 3D collagen matrix direct macrophage activity, and, thus, highlights the important role the local environment itself plays in macrophage behavior. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Collagen fibril surface displays a constellation of sites capable of promoting fibril assembly, stability, and hemostasis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orgel, J.P.; Antipova, O.; Sagi, I.

Fibrillar collagens form the structural basis of organs and tissues including the vasculature, bone, and tendon. They are also dynamic, organizational scaffolds that present binding and recognition sites for ligands, cells, and platelets. We interpret recently published X-ray diffraction findings and use atomic force microscopy data to illustrate the significance of new insights into the functional organization of the collagen fibril. These data indicate that collagen's most crucial functional domains localize primarily to the overlap region, comprising a constellation of sites we call the 'master control region.' Moreover, the collagen's most exposed aspect contains its most stable part - themore » C-terminal region that controls collagen assembly, cross-linking, and blood clotting. Hidden beneath the fibril surface exists a constellation of 'cryptic' sequences poised to promote hemostasis and cell - collagen interactions in tissue injury and regeneration. These findings begin to address several important, and previously unresolved, questions: How functional domains are organized in the fibril, which domains are accessible, and which require proteolysis or structural trauma to become exposed? Here we speculate as to how collagen fibrillar organization impacts molecular processes relating to tissue growth, development, and repair.« less

Computational segmentation of collagen fibers from second-harmonic generation images of breast cancer

NASA Astrophysics Data System (ADS)

Bredfeldt, Jeremy S.; Liu, Yuming; Pehlke, Carolyn A.; Conklin, Matthew W.; Szulczewski, Joseph M.; Inman, David R.; Keely, Patricia J.; Nowak, Robert D.; Mackie, Thomas R.; Eliceiri, Kevin W.

2014-01-01

Second-harmonic generation (SHG) imaging can help reveal interactions between collagen fibers and cancer cells. Quantitative analysis of SHG images of collagen fibers is challenged by the heterogeneity of collagen structures and low signal-to-noise ratio often found while imaging collagen in tissue. The role of collagen in breast cancer progression can be assessed post acquisition via enhanced computation. To facilitate this, we have implemented and evaluated four algorithms for extracting fiber information, such as number, length, and curvature, from a variety of SHG images of collagen in breast tissue. The image-processing algorithms included a Gaussian filter, SPIRAL-TV filter, Tubeness filter, and curvelet-denoising filter. Fibers are then extracted using an automated tracking algorithm called fiber extraction (FIRE). We evaluated the algorithm performance by comparing length, angle and position of the automatically extracted fibers with those of manually extracted fibers in twenty-five SHG images of breast cancer. We found that the curvelet-denoising filter followed by FIRE, a process we call CT-FIRE, outperforms the other algorithms under investigation. CT-FIRE was then successfully applied to track collagen fiber shape changes over time in an in vivo mouse model for breast cancer.

Enhancement of healing in osteochondral defects by collagen sponge implants.

PubMed

Speer, D P; Chvapil, M; Volz, R G; Holmes, M D

1979-10-01

Implants of porous, highly cross-linked collagen sponge (CS) were tested for their capacity to enhance the healing of osteochondral defects in rabbits. Comparison was made to the healing of similar defects with polyvinyl alcohol sponge (PVAS) implants and with no implants (CONT). Evaluation was carried out up to 44 weeks following implantation and included observation of host cellular response, biodegradability of implant, gross appearance of restored joint surface, collagenous architecture of repair tissue, and properties of the junctions of implants and host articular cartilage, subchondral bone, and medullary bone. Collagen sponge proved most effective in promoting healing of osteochondral defects with fibrous and fibrocartilaginous tissue over restored subchondral bone. Collagen sponge showed many desirable properties as a potential material for biologic resurfacing of damaged joints. These properties included porosity, biodegradability, biocompatability, ability to mechanically protect cells and matrix while directing cell ingrowth, and an available chemical technology for modifying its biomechanical and biological properties. Comparative analysis of results of healing of CS, PVAS, and CONT osteochondral defects suggest rational design criteria for implant materials to improve their effectiveness in restoration of articular surfaces.

Biological safety of fish (tilapia) collagen.

PubMed

Yamamoto, Kohei; Igawa, Kazunari; Sugimoto, Kouji; Yoshizawa, Yuu; Yanagiguchi, Kajiro; Ikeda, Takeshi; Yamada, Shizuka; Hayashi, Yoshihiko

2014-01-01

Marine collagen derived from fish scales, skin, and bone has been widely investigated for application as a scaffold and carrier due to its bioactive properties, including excellent biocompatibility, low antigenicity, and high biodegradability and cell growth potential. Fish type I collagen is an effective material as a biodegradable scaffold or spacer replicating the natural extracellular matrix, which serves to spatially organize cells, providing them with environmental signals and directing site-specific cellular regulation. This study was conducted to confirm the safety of fish (tilapia) atelocollagen for use in clinical application. We performed in vitro and in vivo biological studies of medical materials to investigate the safety of fish collagen. The extract of fish collagen gel was examined to clarify its sterility. All present sterility tests concerning bacteria and viruses (including endotoxin) yielded negative results, and all evaluations of cell toxicity, sensitization, chromosomal aberrations, intracutaneous reactions, acute systemic toxicity, pyrogenic reactions, and hemolysis were negative according to the criteria of the ISO and the Ministry of Health, Labour and Welfare of Japan. The present study demonstrated that atelocollagen prepared from tilapia is a promising biomaterial for use as a scaffold in regenerative medicine.

Endocytosis of collagen by hepatic stellate cells regulates extracellular matrix dynamics

PubMed Central

Bi, Yan; Mukhopadhyay, Dhriti; Drinane, Mary; Ji, Baoan; Li, Xing; Cao, Sheng

2014-01-01

Hepatic stellate cells (HSCs) generate matrix, which in turn may also regulate HSCs function during liver fibrosis. We hypothesized that HSCs may endocytose matrix proteins to sense and respond to changes in microenvironment. Primary human HSCs, LX2, or mouse embryonic fibroblasts (MEFs) [wild-type; c-abl−/−; or Yes, Src, and Fyn knockout mice (YSF−/−)] were incubated with fluorescent-labeled collagen or gelatin. Fluorescence-activated cell sorting analysis and confocal microscopy were used for measuring cellular internalization of matrix proteins. Targeted PCR array and quantitative real-time PCR were used to evaluate gene expression changes. HSCs and LX2 cells endocytose collagens in a concentration- and time-dependent manner. Endocytosed collagen colocalized with Dextran 10K, a marker of macropinocytosis, and 5-ethylisopropyl amiloride, an inhibitor of macropinocytosis, reduced collagen internalization by 46%. Cytochalasin D and ML7 blocked collagen internalization by 47% and 45%, respectively, indicating that actin and myosin are critical for collagen endocytosis. Wortmannin and AKT inhibitor blocked collagen internalization by 70% and 89%, respectively, indicating that matrix macropinocytosis requires phosphoinositide-3-kinase (PI3K)/AKT signaling. Overexpression of dominant-negative dynamin-2 K44A blocked matrix internalization by 77%, indicating a role for dynamin-2 in matrix macropinocytosis. Whereas c-abl−/− MEF showed impaired matrix endocytosis, YSF−/− MEF surprisingly showed increased matrix endocytosis. It was also associated with complex gene regulations that related with matrix dynamics, including increased matrix metalloproteinase 9 (MMP-9) mRNA levels and zymographic activity. HSCs endocytose matrix proteins through macropinocytosis that requires a signaling network composed of PI3K/AKT, dynamin-2, and c-abl. Interaction with extracellular matrix regulates matrix dynamics through modulating multiple gene expressions including MMP-9. PMID:25080486

Endocytosis of collagen by hepatic stellate cells regulates extracellular matrix dynamics.

PubMed

Bi, Yan; Mukhopadhyay, Dhriti; Drinane, Mary; Ji, Baoan; Li, Xing; Cao, Sheng; Shah, Vijay H

2014-10-01

Hepatic stellate cells (HSCs) generate matrix, which in turn may also regulate HSCs function during liver fibrosis. We hypothesized that HSCs may endocytose matrix proteins to sense and respond to changes in microenvironment. Primary human HSCs, LX2, or mouse embryonic fibroblasts (MEFs) [wild-type; c-abl(-/-); or Yes, Src, and Fyn knockout mice (YSF(-/-))] were incubated with fluorescent-labeled collagen or gelatin. Fluorescence-activated cell sorting analysis and confocal microscopy were used for measuring cellular internalization of matrix proteins. Targeted PCR array and quantitative real-time PCR were used to evaluate gene expression changes. HSCs and LX2 cells endocytose collagens in a concentration- and time-dependent manner. Endocytosed collagen colocalized with Dextran 10K, a marker of macropinocytosis, and 5-ethylisopropyl amiloride, an inhibitor of macropinocytosis, reduced collagen internalization by 46%. Cytochalasin D and ML7 blocked collagen internalization by 47% and 45%, respectively, indicating that actin and myosin are critical for collagen endocytosis. Wortmannin and AKT inhibitor blocked collagen internalization by 70% and 89%, respectively, indicating that matrix macropinocytosis requires phosphoinositide-3-kinase (PI3K)/AKT signaling. Overexpression of dominant-negative dynamin-2 K44A blocked matrix internalization by 77%, indicating a role for dynamin-2 in matrix macropinocytosis. Whereas c-abl(-/-) MEF showed impaired matrix endocytosis, YSF(-/-) MEF surprisingly showed increased matrix endocytosis. It was also associated with complex gene regulations that related with matrix dynamics, including increased matrix metalloproteinase 9 (MMP-9) mRNA levels and zymographic activity. HSCs endocytose matrix proteins through macropinocytosis that requires a signaling network composed of PI3K/AKT, dynamin-2, and c-abl. Interaction with extracellular matrix regulates matrix dynamics through modulating multiple gene expressions including MMP-9. Copyright © 2014 the American Physiological Society.

RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

PubMed

Stefanovic, Branko

2013-01-01

Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments. Copyright © 2013 John Wiley & Sons, Ltd.

Osteoinduction and proliferation of bone-marrow stromal cells in three-dimensional poly (ε-caprolactone)/ hydroxyapatite/collagen scaffolds.

PubMed

Wang, Ting; Yang, Xiaoyan; Qi, Xin; Jiang, Chaoyin

2015-05-08

Osteoinduction and proliferation of bone-marrow stromal cells (BMSCs) in three-dimensional (3D) poly(ε-caprolactone) (PCL) scaffolds have not been studied throughly and are technically challenging. This study aimed to optimize nanocomposites of 3D PCL scaffolds to provide superior adhesion, proliferation and differentiation environment for BMSCs in this scenario. BMSCs were isolated and cultured in a novel 3D tissue culture poly(ε-caprolactone) (PCL) scaffold coated with poly-lysine, hydroxyapatite (HAp), collagen and HAp/collagen. Cell morphology was observed and BMSC biomarkers for osteogenesis, osteoblast differentiation and activation were analyzed. Scanning Electron Microscope (SEM) micrographs showed that coating materials were uniformly deposited on the surface of PCL scaffolds and BMSCs grew and aggregated to form clusters during 3D culture. Both mRNA and protein levels of the key players of osteogenesis and osteoblast differentiation and activation, including runt-related transcription factor 2 (Runx2), alkaline phosphates (ALP), osterix, osteocalcin, and RANKL, were significantly higher in BMSCs seeded in PCL scaffolds coated with HAp or HAp/collagen than those seeded in uncoated PCL scaffolds, whereas the expression levels were not significantly different in collagen or poly-lysine coated PCL scaffolds. In addition, poly-lysine, collagen, HAp/collagen, and HAp coated PCL scaffolds had significantly more viable cells than uncoated PCL scaffolds, especially scaffolds with HAp/collagen and collagen-alone coatings. That BMSCs in HAp or HAp/collagen PCL scaffolds had remarkably higher ALP activities than those in collagen-coated alone or uncoated PCL scaffolds indicating higher osteogenic differentiation levels of BMSCs in HAp or HAp/collagen PCL scaffolds. Moreover, morphological changes of BMSCs after four-week of 3D culture confirmed that BMSCs successfully differentiated into osteoblast with spread-out phenotype in HAp/collagen coated PCL scaffolds. This study showed a proof of concept for preparing biomimetic 3D poly (ε-caprolactone)/ hydroxyapatite/collagen scaffolds with excellent osteoinduction and proliferation capacity for bone regeneration.

Extracellular Collagen Promotes Interleukin-1β-Induced Urokinase-Type Plasminogen Activator Production by Human Corneal Fibroblasts.

PubMed

Sugioka, Koji; Kodama-Takahashi, Aya; Yoshida, Koji; Aomatsu, Keiichi; Okada, Kiyotaka; Nishida, Teruo; Shimomura, Yoshikazu

2017-03-01

Keratocytes maintain homeostasis of the corneal stroma through synthesis, secretion, and degradation of collagen fibrils of the extracellular matrix. Given that these cells are essentially embedded in a collagen matrix, keratocyte-collagen interactions may play a key role in regulation of the expression or activation of enzymes responsible for matrix degradation including urokinase-type plasminogen activator (uPA), plasmin, and matrix metalloproteinases (MMPs). We examined the effect of extracellular collagen on the production of uPA by corneal fibroblasts (activated keratocytes) stimulated with the proinflammatory cytokine interleukin-1β (IL-1β). Human corneal fibroblasts were cultured either on plastic or in a three-dimensional gel of type I collagen. Plasminogen activators were detected by fibrin zymography, whereas the IL-1 receptor (IL-1R) and MMPs were detected by immunoblot analysis. Collagen degradation by corneal fibroblasts was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. Collagen and IL-1β synergistically increased the synthesis and secretion of uPA in corneal fibroblasts. Collagen also upregulated IL-1R expression in the cells in a concentration-dependent manner. The conversion of extracellular plasminogen to plasmin, as well as the plasminogen-dependent activation of MMP-1 and MMP-3 and degradation of collagen apparent in three-dimensional cultures of corneal fibroblasts exposed to IL-1β, were all abolished by a selective uPA inhibitor. Collagen and IL-1β cooperate to upregulate uPA production by corneal fibroblasts. Furthermore, IL-1β-induced collagen degradation by these cells appears to be strictly dependent on uPA expression and mediated by a uPA-plasmin-MMP pathway.

Ultrastructure of the extracellular matrix of bovine dura mater, optic nerve sheath and sclera.

PubMed

Raspanti, M; Marchini, M; Della Pasqua, V; Strocchi, R; Ruggeri, A

1992-10-01

The sclera, the outermost sheath of the optic nerve and the dura mater have been investigated histologically and ultrastructurally. Although these tissues appear very similar under the light microscope, being dense connective tissues mainly composed of collagen bundles and a limited amount of cells and elastic fibres, they exhibit subtle differences on electron microscopy. In the dura and sclera collagen appears in the form of large, nonuniform fibrils, similar to those commonly found in tendons, while in the optic nerve sheath the fibrils appear smaller and uniform, similar to those commonly observed in reticular tissues, vessel walls and skin. Freeze-fracture also reveals these fibrils to have different subfibrillar architectures, straight or helical, which correspond to 2 distinct forms of collagen fibril previously described (Raspanti et al. 1989). The other extracellular matrix components also vary with the particular collagen fibril structure. Despite their common embryological derivation, the dura mater, optic nerve sheath and sclera exhibit diversification of their extracellular matrix consistent with the mechanical loads to which these tissues are subjected. Our observations indicate that the outermost sheath of the optic nerve resembles the epineurium of peripheral nerves rather than the dura to which it is commonly likened.

Human dermal and gingival fibroblasts in a three-dimensional culture: a comparative study on matrix remodeling.

PubMed

Chaussain Miller, C; Septier, D; Bonnefoix, M; Lecolle, S; Lebreton-Decoster, C; Coulomb, B; Pellat, B; Godeau, G

2002-03-01

Free-floating collagen lattice is considered a useful tool for assessing wound healing in vitro. This work compared extracellular matrix remodeling in collagen lattices populated by gingival or dermal fibroblasts. For 21 days we followed gel contraction and changes in cell number of collagen lattices seeded with l.5 x 10(5) fibroblasts of each tissue. We also used indirect immunodetection to study extracellular matrix components, metalloproteinases (MMPs), and their tissues inhibitors (TIMPs). In addition, the presence of MMPs and TIMPs in the culture media was analyzed by zymography and western blotting. No significant difference was found concerning gel contraction and changes in cell number. We observed the early expression of fibrillin I and collagen type III, apparently codistributed and at the end of the gel contraction their disappearance. Concomitantly we demonstrated the expression of MMPs and TIMPs, initially localized in cellular cytoplasm, then spreading in the extracellular compartment, and even found in the culture medium. This remodeling was more rapid and intense with gingival fibroblasts than dermal fibroblasts. In conclusion, gingival fibroblasts seem more efficient at remodeling the connective tissue than dermal fibroblasts and could lead to the better wound healing observed in vivo.

3D printed miniaturized spectral system for tissue fluorescence lifetime measurements

NASA Astrophysics Data System (ADS)

Zou, Luwei; Mahmoud, Mohamad; Fahs, Mehdi; Liu, Rui; Lo, Joe F.

2016-04-01

Various types of collagens, e.g. type I and III, represent the main load-bearing components in biological tissues. Their composition changes during processes like wound healing and fibrosis. Collagens exhibit autofluorescence when excited by ultra-violet light, distinguishable by their unique fluorescent lifetimes across a range of emission wavelengths. Therefore, we designed a miniaturized spectral-lifetime detection system for collagens as a non-invasive probe for monitoring tissue in wound healing and scarring applications. A sine modulated LED illumination was applied to enable frequency domain (FD) fluorescence lifetime measurements under different wavelengths bands, separated via a series of longpass dichroics at 387nm, 409nm and 435nm. To achieve the minute scale of optomechanics, we employed a stereolithography based 3D printer with <50 μm resolution to create a custom designed optical mount in a hand-held form factor. We examined the characteristics of the 3D printed optical system with finite element modeling to simulate the effect of thermal (LED) and mechanical (handling) strain on the optical system. Using this device, the phase shift and demodulation of collagen types were measured, where the separate spectral bands enhanced the differentiation of their lifetimes.

Sheep antibodies to soluble rat collagen

PubMed Central

Chidlow, J. W.; Bourne, F. J.; Bailey, A. J.

1974-01-01

Sheep were immunized by multiple injections of acid-extracted rat tail tendon tropocollagen. Antibody activity could be demonstrated by quantitative precipitation and passive haemagglutination against denatured tropocollagen. Immunodiffusion experiments showed strong precipitin lines with denatured tendon tropocollagen, and with peptides obtained by CNBr digestion of whole rat tail tendon. Immunoelectrophoresis showed one line with denatured tropocollagen but four lines with the CNBr digest of whole tendon indicating at least four antigenic determinants. Immunosorbents prepared from antisera raised against tropocollagen readily absorbed labelled peptides from CNBr digests of rat tail tendons reduced with tritiated borohydride. These peptides were recoverable by desorption with 1 M ammonia and had a hydroxyproline and hydroxylysine content typical of collagen but increased tyrosine levels. Presence of the normal reducible components of collagen known to be involved in cross-linking was confirmed by ion-exchange chromatography, and there was an increase in the proportion of fraction C. The majority of the tritium label was found in a cross-linked peptide, or group of peptides, with molecular weight around 60,000. The technique therefore has the potential for further development in the isolation of specific collagen peptides. ImagesFIG. 3FIG. 4 PMID:4140151

Pathogenesis and therapeutic approaches for improved topical treatment in localized scleroderma and systemic sclerosis.

PubMed

Badea, I; Taylor, M; Rosenberg, A; Foldvari, M

2009-03-01

SSc is a chronic progressive disorder of unknown aetiology characterized by excess synthesis and deposition of collagen and other extracellular matrix components in a variety of tissues and organs. Localized scleroderma (LS) differs from SSc in that with LS only skin and occasionally subcutaneous tissues are involved. Although rarely life threatening, LS can be disfiguring and disabling and, consequently, can adversely affect quality of life. There is no known effective treatment for LS, and various options, including, as examples, corticosteroids and other immunomodulatory agents, ultraviolet radiation and vitamin D analogues, are of unproven efficacy. Clinical trials evaluating combination therapy such as corticosteroids with MTX or UVA1 exposure with psoralens have not been established as consistently effective. New immunomodulators such as tacrolimus and thalidomide are also being evaluated. A better understanding of the molecular and cellular mechanisms of LS has led to evaluation of new treatments that modulate profibrotic cytokines such as TGF-beta and IL-4, regulate assembly and deposition of extracellular matrix components, and restore Th1/Th2 immune balance by administering IL-12 or IFN-gamma. IFN-gamma acts by directly inhibiting collagen synthesis and by restoring immune balance. In this review, we evaluate current and future treatment options for LS and cutaneous involvement in SSc. Recent advances in therapy focus mainly on anti-fibrotic agents. Delivery of these drugs into the skin as the target tissue might be a key factor in developing more effective and safer therapy.

Tumor Cell Invasion Can Be Blocked by Modulators of Collagen Fibril Alignment That Control Assembly of the Extracellular Matrix.

PubMed

Grossman, Moran; Ben-Chetrit, Nir; Zhuravlev, Alina; Afik, Ran; Bassat, Elad; Solomonov, Inna; Yarden, Yosef; Sagi, Irit

2016-07-15

Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECM-associated pathologies. Cancer Res; 76(14); 4249-58. ©2016 AACR. ©2016 American Association for Cancer Research.

Minerals and aligned collagen fibrils in tilapia fish scales: structural analysis using dark-field and energy-filtered transmission electron microscopy and electron tomography.

PubMed

Okuda, Mitsuhiro; Ogawa, Nobuhiro; Takeguchi, Masaki; Hashimoto, Ayako; Tagaya, Motohiro; Chen, Song; Hanagata, Nobutaka; Ikoma, Toshiyuki

2011-10-01

The mineralized structure of aligned collagen fibrils in a tilapia fish scale was investigated using transmission electron microscopy (TEM) techniques after a thin sample was prepared using aqueous techniques. Electron diffraction and electron energy loss spectroscopy data indicated that a mineralized internal layer consisting of aligned collagen fibrils contains hydroxyapatite crystals. Bright-field imaging, dark-field imaging, and energy-filtered TEM showed that the hydroxyapatite was mainly distributed in the hole zones of the aligned collagen fibrils structure, while needle-like materials composed of calcium compounds including hydroxyapatite existed in the mineralized internal layer. Dark-field imaging and three-dimensional observation using electron tomography revealed that hydroxyapatite and needle-like materials were mainly found in the matrix between the collagen fibrils. It was observed that hydroxyapatite and needle-like materials were preferentially distributed on the surface of the hole zones in the aligned collagen fibrils structure and in the matrix between the collagen fibrils in the mineralized internal layer of the scale.

Fluorescent nanonetworks: A novel bioalley for collagen scaffolds and Tissue Engineering

PubMed Central

Nidhin, Marimuthu; Vedhanayagam, Mohan; Sangeetha, Selvam; Kiran, Manikantan Syamala; Nazeer, Shaiju S.; Jayasree, Ramapurath S.; Sreeram, Kalarical Janardhanan; Nair, Balachandran Unni

2014-01-01

Native collagen is arranged in bundles of aligned fibrils to withstand in vivo mechanical loads. Reproducing such a process under in vitro conditions has not met with major success. Our approach has been to induce nanolinks, during the self-assembly process, leading to delayed rather than inhibited fibrillogenesis. For this, a designed synthesis of nanoparticles - using starch as a template and a reflux process, which would provide a highly anisotropic (star shaped) nanoparticle, with large surface area was adopted. Anisotropy associated decrease in Morin temperature and superparamagnetic behavior was observed. Polysaccharide on the nanoparticle surface provided aqueous stability and low cytotoxicity. Starch coated nanoparticles was utilized to build polysaccharide - collagen crosslinks, which supplemented natural crosslinks in collagen, without disturbing the conformation of collagen. The resulting fibrillar lamellae showed a striking resemblance to native lamellae, but had a melting and denaturation temperature higher than native collagen. The biocompatibility and superparamagnetism of the nanoparticles also come handy in the development of stable collagen constructs for various biomedical applications, including that of MRI contrast agents. PMID:25095810

The contribution of collagen fibers to the mechanical compressive properties of the temporomandibular joint disc.

PubMed

Fazaeli, S; Ghazanfari, S; Everts, V; Smit, T H; Koolstra, J H

2016-07-01

The Temporomandibular Joint (TMJ) disc is a fibrocartilaginous structure located between the mandibular condyle and the temporal bone, facilitating smooth movements of the jaw. The load-bearing properties of its anisotropic collagenous network have been well characterized under tensile loading conditions. However, recently it has also been speculated that the collagen fibers may contribute dominantly in reinforcing the disc under compression. Therefore, in this study, the structural-functional role of collagen fibers in mechanical compressive properties of TMJ disc was investigated. Intact porcine TMJ discs were enzymatically digested with collagenase to disrupt the collagenous network of the cartilage. The digested and non-digested articular discs were analyzed mechanically, biochemically and histologically in five various regions. These tests included: (1) cyclic compression tests, (2) biochemical quantification of collagen and glycosaminoglycan (GAG) content and (3) visualization of collagen fibers' alignment by polarized light microscopy (PLM). The instantaneous compressive moduli of the articular discs were reduced by as much as 50-90% depending on the region after the collagenase treatment. The energy dissipation properties of the digested discs showed a similar tendency. Biochemical analysis of the digested samples demonstrated an average of 14% and 35% loss in collagen and GAG, respectively. Despite the low reduction of collagen content the PLM images showed considerable perturbation of the collagenous network of the TMJ disc. The results indicated that even mild disruption of collagen fibers can lead to substantial mechanical softening of TMJ disc undermining its reinforcement and mechanical stability under compression. Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Withaferin-A Reduces Type I Collagen Expression In Vitro and Inhibits Development of Myocardial Fibrosis In Vivo

PubMed Central

Challa, Azariyas A.; Vukmirovic, Milica; Blackmon, John; Stefanovic, Branko

2012-01-01

Type I collagen is the most abundant protein in the human body. Its excessive synthesis results in fibrosis of various organs. Fibrosis is a major medical problem without an existing cure. Excessive synthesis of type I collagen in fibrosis is primarily due to stabilization of collagen mRNAs. We recently reported that intermediate filaments composed of vimentin regulate collagen synthesis by stabilizing collagen mRNAs. Vimentin is a primary target of Withaferin-A (WF-A). Therefore, we hypothesized that WF-A may reduce type I collagen production by disrupting vimentin filaments and decreasing the stability of collagen mRNAs. This study is to determine if WF-A exhibits anti-fibrotic properties in vitro and in vivo and to elucidate the molecular mechanisms of its action. In lung, skin and heart fibroblasts WF-A disrupted vimentin filaments at concentrations of 0.5–1.5 µM and reduced 3 fold the half-lives of collagen α1(I) and α2(I) mRNAs and protein expression. In addition, WF-A inhibited TGF-β1 induced phosphorylation of TGF-β1 receptor I, Smad3 phosphorylation and transcription of collagen genes. WF-A also inhibited in vitro activation of primary hepatic stellate cells and decreased their type I collagen expression. In mice, administration of 4 mg/kg WF-A daily for 2 weeks reduced isoproterenol-induced myocardial fibrosis by 50%. Our findings provide strong evidence that Withaferin-A could act as an anti-fibrotic compound against fibroproliferative diseases, including, but not limited to, cardiac interstitial fibrosis. PMID:22900077

Polymerized-type I collagen downregulates inflammation and improves clinical outcomes in patients with symptomatic knee osteoarthritis following arthroscopic lavage: a randomized, double-blind, and placebo-controlled clinical trial.

PubMed

Furuzawa-Carballeda, Janette; Lima, Guadalupe; Llorente, Luis; Nuñez-Álvarez, Carlos; Ruiz-Ordaz, Blanca H; Echevarría-Zuno, Santiago; Hernández-Cuevas, Virgilio

2012-01-01

Polymerized-type I collagen (polymerized collagen) is a downmodulator of inflammation and cartilage regenerator biodrug. To evaluate the effect of intraarticular injections of polymerized collagen after arthroscopic lavage on inflammation and clinical improvement in patients with knee osteoarthritis (OA). Patients (n = 19) were treated with 6 intraarticular injections of 2 mL of polymerized collagen (n = 10) or 2 mL of placebo (n = 9) during 3 months. Followup was 3 months. The primary endpoints included Lequesne index, pain on a visual analogue scale (VAS), WOMAC, analgesic usage, the number of Tregs and proinflammatory/anti-inflammatory cytokine-expressing peripheral cells. Secondary outcomes were Likert score and drug evaluation. Clinical and immunological improvement was determined if the decrease in pain exceeds 20 mm on a VAS, 20% of clinical outcomes, and inflammatory parameters from baseline. Urinary levels of C-terminal crosslinking telopeptide of collagen type II (CTXII) and erythrocyte sedimentation rate (ESR) were determined. Polymerized collagen was safe and well tolerated. Patients had a statistically significant improvement (P < 0.05) from baseline versus polymerized collagen and versus placebo at 6 months on Lequesne index, VAS, ESR, Tregs IL-1β, and IL-10 peripheral-expressing cells. Urinary levels of CTXII were decreased 44% in polymerized collagen versus placebo. No differences were found on incidence of adverse events between groups. Polymerized collagen is safe and effective on downregulation of inflammation in patients with knee OA.

Reprogramming of the Ovarian Tumor Stroma by Activation of a Biomechanical ECM Switch

DTIC Science & Technology

2016-09-01

Denatured collagen was detec- ted with anticollagen antibody (1:1000). For integrin-blocking enzyme -linked immunosorbent assay, wells were coated with...migration on denatured collagen; it failed to reduce cell adhesion. Moreover a peptide antagonist of alpha 10 beta 1 may inhibit ovarian tumor growth in...stromal cell adhesion, migration and proliferation on distinct ECM substrates including native and denatured collagen. 4   D). As outlined in aim 2

Shell extracts from the marine bivalve Pecten maximus regulate the synthesis of extracellular matrix in primary cultured human skin fibroblasts.

PubMed

Latire, Thomas; Legendre, Florence; Bigot, Nicolas; Carduner, Ludovic; Kellouche, Sabrina; Bouyoucef, Mouloud; Carreiras, Franck; Marin, Frédéric; Lebel, Jean-Marc; Galéra, Philippe; Serpentini, Antoine

2014-01-01

Mollusc shells are composed of more than 95% calcium carbonate and less than 5% of an organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. Previous studies have elucidated the biological activities of the shell matrices from bivalve molluscs on skin, especially on the expression of the extracellular matrix components of fibroblasts. In this work, we have investigated the potential biological activities of shell matrix components extracted from the shell of the scallop Pecten maximus on human fibroblasts in primary culture. Firstly, we demonstrated that shell matrix components had different effects on general cellular activities. Secondly, we have shown that the shell matrix components stimulate the synthesis of type I and III collagens, as well as that of sulphated GAGs. The increased expression of type I collagen is likely mediated by the recruitment of transactivating factors (Sp1, Sp3 and human c-Krox) in the -112/-61 bp COL1A1 promoter region. Finally, contrarily to what was obtained in previous works, we demonstrated that the scallop shell extracts have only a small effect on cell migration during in vitro wound tests and have no effect on cell proliferation. Thus, our research emphasizes the potential use of shell matrix of Pecten maximus for dermo-cosmetic applications.

Computational model for the analysis of cartilage and cartilage tissue constructs

PubMed Central

Smith, David W.; Gardiner, Bruce S.; Davidson, John B.; Grodzinsky, Alan J.

2013-01-01

We propose a new non-linear poroelastic model that is suited to the analysis of soft tissues. In this paper the model is tailored to the analysis of cartilage and the engineering design of cartilage constructs. The proposed continuum formulation of the governing equations enables the strain of the individual material components within the extracellular matrix (ECM) to be followed over time, as the individual material components are synthesized, assembled and incorporated within the ECM or lost through passive transport or degradation. The material component analysis developed here naturally captures the effect of time-dependent changes of ECM composition on the deformation and internal stress states of the ECM. For example, it is shown that increased synthesis of aggrecan by chondrocytes embedded within a decellularized cartilage matrix initially devoid of aggrecan results in osmotic expansion of the newly synthesized proteoglycan matrix and tension within the structural collagen network. Specifically, we predict that the collagen network experiences a tensile strain, with a maximum of ~2% at the fixed base of the cartilage. The analysis of an example problem demonstrates the temporal and spatial evolution of the stresses and strains in each component of a self-equilibrating composite tissue construct, and the role played by the flux of water through the tissue. PMID:23784936

Tumor-associated fibrosis as a regulator of tumor immunity and response to immunotherapy.

PubMed

Jiang, Hong; Hegde, Samarth; DeNardo, David G

2017-08-01

Tumor-associated fibrosis is characterized by unchecked pro-fibrotic and pro-inflammatory signaling. The components of fibrosis including significant numbers of cancer-associated fibroblasts, dense collagen deposition, and extracellular matrix stiffness, are well appreciated regulators of tumor progression but may also be critical regulators of immune surveillance. While this suggests that the efficacy of immunotherapy may be limited in highly fibrotic cancers like pancreas, it also suggests a therapeutic opportunity to target fibrosis in these tumor types to reawaken anti-tumor immunity. This review discusses the mechanisms by which fibrosis might subvert tumor immunity and how to overcome these mechanisms.

The 2014International Workshop on Alport Syndrome.

PubMed

Miner, Jeffrey H; Baigent, Colin; Flinter, Frances; Gross, Oliver; Judge, Parminder; Kashtan, Clifford E; Lagas, Sharon; Savige, Judith; Blatt, Dave; Ding, Jie; Gale, Daniel P; Midgley, Julian P; Povey, Sue; Prunotto, Marco; Renault, Daniel; Skelding, Jules; Turner, A Neil; Gear, Susie

2014-10-01

Alport syndrome, historically referred to as hereditary glomerulonephritis with sensorineural deafness and anterior lenticonus, is a genetic disease of collagen α3α4α5(IV) resulting in renal failure. The collagen α3α4α5(IV) heterotrimer forms a network that is a major component of the kidney glomerular basement membrane (GBM) and basement membranes in the cochlea and eye. Alport syndrome, estimated to affect 1 in 5000-10,000 individuals, is caused by mutations in any one of the three genes that encode the α chain components of the collagen α3α4α5(IV) heterotrimer: COL4A3, COL4A4, and COL4A5. Although angiotensin-converting enzyme inhibition is effective in Alport syndrome patients for slowing progression to end-stage renal disease, it is neither a cure nor an adequate long-term protector. The 2014 International Workshop on Alport Syndrome, held in Oxford, UK, from January 3-5, was organized by individuals and families living with Alport syndrome, in concert with international experts in the clinical, genetic, and basic science aspects of the disease. Stakeholders from diverse communities-patient families, physicians, geneticists, researchers, Pharma, and funding organizations-were brought together so that they could meet and learn from each other and establish strategies and collaborations for the future, with the overall aim of discovering much needed new treatments to prolong kidney function.

Pyruvate dehydrogenase subunit β of Lactobacillus plantarum is a collagen adhesin involved in biofilm formation.

PubMed

Salzillo, Marzia; Vastano, Valeria; Capri, Ugo; Muscariello, Lidia; Marasco, Rosangela

2017-04-01

Multi-functional surface proteins have been observed in a variety of pathogenic bacteria, where they mediate host cell adhesion and invasion, as well as in commensal bacterial species, were they mediate positive interaction with the host. Among these proteins, some glycolytic enzymes, expressed on the bacterial cell surface, can bind human extracellular matrix components (ECM). A major target for them is collagen, an abundant glycoprotein of connective tissues. We have previously shown that the enolase EnoA1 of Lactobacillus plantarum, one of the most predominant species in the gut microbiota of healthy individuals, is involved in binding with collagen type I (CnI). In this study, we found that PDHB, a component of the pyruvate dehydrogenase complex, contributes to the L. plantarum LM3 adhesion to CnI. By a cellular adhesion assay to immobilized CnI, we show that LM3-B1 cells, carrying a null mutation in the pdhB gene, bind to CnI - coated surfaces less efficiently than wild-type cells. Moreover, we show that the PDHB-CnI interaction requires a native state for PDHB. We also analyzed the ability to develop biofilm in wild-type and mutant strains and we found that the lack of the PDHB on cell surface generates cells partially impaired in biofilm development. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The 2014 International Workshop on Alport Syndrome

PubMed Central

Miner, Jeffrey H; Baigent, Colin; Flinter, Frances; Gross, Oliver; Judge, Parminder; Kashtan, Clifford E; Lagas, Sharon; Savige, Judith; Blatt, Dave; Ding, Jie; Gale, Daniel P; Midgley, Julian P; Povey, Sue; Prunotto, Marco; Renault, Daniel; Skelding, Jules; Turner, A Neil; Gear, Susie

2014-01-01

Alport syndrome, historically referred to as hereditary glomerulonephritis with sensorineural deafness and anterior lenticonus, is a genetic disease of collagen α3α4α5(IV) resulting in renal failure. The collagen α3α4α5(IV) heterotrimer forms a network that is a major component of the kidney glomerular basement membrane (GBM) and basement membranes in the cochlea and eye. Alport syndrome, estimated to affect 1 in 5000–10,000 individuals, is caused by mutations in any one of the three genes that encode the α chain components of the collagen α3α4α5(IV) heterotrimer: COL4A3, COL4A4, and COL4A5. Although angiotensin-converting enzyme inhibition is effective in Alport syndrome patients for slowing progression to end-stage renal disease, it is neither a cure nor an adequate long-term protector. The 2014 International Workshop on Alport Syndrome, held in Oxford, UK, from January 3–5, was organized by individuals and families living with Alport syndrome, in concert with international experts in the clinical, genetic, and basic science aspects of the disease. Stakeholders from diverse communities—patient families, physicians, geneticists, researchers, Pharma, and funding organizations—were brought together so that they could meet and learn from each other and establish strategies and collaborations for the future, with the overall aim of discovering much needed new treatments to prolong kidney function. PMID:24988067

Co-ordinated expression of MMP-2 and its putative activator, MT1-MMP, in human placentation.

PubMed

Bjørn, S F; Hastrup, N; Lund, L R; Danø, K; Larsen, J F; Pyke, C

1997-08-01

The spatial expression of mRNA for matrix metalloproteinase 2 (MMP-2), its putative activator, the membrane-type 1 matrix metalloproteinase (MT1-MMP), and the MMP-2 substrate type IV collagen was investigated in human placentas of both normal and tubal ectopic pregnancies and in cyclic endometrium using in-situ hybridization. Cytokeratin staining applied to adjacent sections was used to identify epithelial and trophoblast cells. In both normal and tubal pregnancies MT1-MMP, MMP-2 and type IV collagen mRNA were highly expressed and co-localized in the extravillous cytotrophoblasts of anchoring villi, in cytotrophoblasts that had penatrated into the placental bed and in cytotrophoblastic cell islands. In addition, the decidual cells of normal pregnancies in some areas co-expressed MT1-MMP and MMP-2 mRNA, with moderate signals for both components. Fibroblast-like stromal cells in tubal pregnancies were positive for MMP-2 mRNA but generally negative for MT1-MMP mRNA. The consistent co-localization of MT1-MMP with MMP-2 and type IV collagen in the same subset of cytotrophoblasts strongly suggests that all three components co-operate in the tightly regulated fetal invasion process. The co-expression of MT1-MMP and MMP-2 mRNA in some of the decidual cells indicates that these cells are also actively involved in the placentation process.

Use of Mueller matrix polarimetry and optical coherence tomography in the characterization of cervical collagen anisotropy

NASA Astrophysics Data System (ADS)

Chue-Sang, Joseph; Bai, Yuqiang; Stoff, Susan; Gonzalez, Mariacarla; Gomes, Jefferson; Gandjbakhche, Amir; Chernomordik, Viktor V.; Ramella-Roman, Jessica C.

2017-02-01

Preterm birth (PTB) presents a serious medical heath concern throughout the world. There is a high incidence of PTB in both developed and developing countries ranging from 11%-15%, respectively. Studies have shown there may be numerous precursors to PTB including infections, genetic predisposition, nutrition and various other morbidities which all lead to a premature disorganization in the cervical collagen resulting in the weakening of the structure designed to keep the fetus in utero. The changes in cervical collagen orientation and distribution may prove to be a predictor of PTB. Polarization imaging is an effective means to measure optical anisotropy in birefringent materials such as those rich in collagen as the cervix is. Non-invasive, full-field Mueller Matrix polarimetry (MMP) imaging methodologies and ex-vivo second harmonic generation (SHG) imaging were used to assess cervical collagen content and structure in non-pregnant porcine cervices. The SHG microscopy was used to verify the efficacy of the MMP in assessing changes in collagen orientation.

Oxidants produced by methylglyoxal-modified collagen trigger ER stress and apoptosis in skin fibroblasts.

PubMed

Nowotny, Kerstin; Castro, José Pedro; Hugo, Martín; Braune, Sabine; Weber, Daniela; Pignitter, Marc; Somoza, Veronika; Bornhorst, Julia; Schwerdtle, Tanja; Grune, Tilman

2018-05-20

Methylglyoxal (MG), a highly reactive dicarbonyl, interacts with proteins to form advanced glycation end products (AGEs). AGEs include a variety of compounds which were shown to have damaging potential and to accumulate in the course of different conditions such as diabetes mellitus and aging. After confirming collagen as a main target for MG modifications in vivo within the extracellular matrix, we show here that MG-collagen disrupts fibroblast redox homeostasis and induces endoplasmic reticulum (ER) stress and apoptosis. In particular, MG-collagen-induced apoptosis is associated with the activation of the PERK-eIF2α pathway and caspase-12. MG-collagen contributes to altered redox homeostasis by directly generating hydrogen peroxide and oxygen-derived free radicals. The induction of ER stress in human fibroblasts was confirmed using collagen extracts isolated from old mice in which MG-derived AGEs were enriched. In conclusion, MG-derived AGEs represent one factor contributing to diminished fibroblast function during aging. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Collagen IV Diseases: A Focus on the Glomerular Basement Membrane in Alport Syndrome

PubMed Central

Cosgrove, Dominic; Liu, Shiguang

2016-01-01

Alport syndrome is the result of mutations in any of three type IV collagen genes, COL4A3, COL4A4, or COL4A5. Because the three collagen chains form heterotrimers, there is an absence of all three proteins in the basement membranes where they are expressed. In the glomerulus, the mature glomerular basement membrane type IV collagen network, normally comprised of two separate networks, α3(IV)/α4(IV)/α5(IV) and α1(IV)/α2(IV), is comprised entirely of collagen α1(IV)/α2. This review addresses the current state of our knowledge regarding the consequence of this change in basement membrane composition, including both the direct, via collagen receptor binding, and indirect, regarding influences on glomerular biomechanics. The state of our current understanding regarding mechanisms of glomerular disease initiation and progression will be examined, as will the current state of the art regarding emergent therapeutic approaches to slow or arrest glomerular disease in Alport patients. PMID:27576055

Chondrogenically primed tonsil-derived mesenchymal stem cells encapsulated in riboflavin-induced photocrosslinking collagen-hyaluronic acid hydrogel for meniscus tissue repairs.

PubMed

Koh, Rachel H; Jin, Yinji; Kang, Byung-Jae; Hwang, Nathaniel S

2017-04-15

Current meniscus tissue repairing strategies involve partial or total meniscectomy, followed by allograft transplantation or synthetic material implantation. However, allografts and synthetic implants have major drawbacks such as the limited supply of grafts and lack of integration into host tissue, respectively. In this study, we investigated the effects of conditioned medium (CM) from meniscal fibrochondrocytes and TGF-β3 on tonsil-derived mesenchymal stem cells (T-MSCs) for meniscus tissue engineering. CM-expanded T-MSCs were encapsulated in riboflavin-induced photocrosslinked collagen-hyaluronic acid (COL-RF-HA) hydrogels and cultured in chondrogenic medium containing TGF-β3. In vitro results indicate that CM-expanded cells followed by TGF-β3 exposure stimulated the expression of fibrocartilage-related genes (COL2, SOX9, ACAN, COL1) and production of extracellular matrix components. Histological assessment of in vitro and subcutaneously implanted in vivo constructs demonstrated that CM-expanded cells followed by TGF-β3 exposure resulted in highest cell proliferation, GAG accumulation, and collagen deposition. Furthermore, when implanted into meniscus defect model, CM treatment amplified the potential of TGF-β3 and induced complete regeneration. Conditioned medium derived from chondrocytes have been reported to effectively prime mesenchymal stem cells toward chondrogenic lineage. Type I collagen is the main component of meniscus extracellular matrix and hyaluronic acid is known to promote meniscus regeneration. In this manuscript, we investigated the effects of conditioned medium (CM) and transforming growth factor-β3 (TGF-β3) on tonsil-derived mesenchymal stem cells (T-MSCs) encapsulated in riboflavin-induced photocrosslinked collagen-hyaluronic acid (COL-RF-HA) hydrogel. We employed a novel source of conditioned medium, derived from meniscal fibrochondrocytes. Our in vitro and in vivo results collectively illustrate that CM-expanded cells followed by TGF-β3 exposure have the best potential for meniscus regeneration. This manuscript highlights a novel stem cell commitment strategy combined with biomaterials designs for meniscus regeneration. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Higher iron bioavailability of a human-like collagen iron complex.

PubMed

Zhu, Chenhui; Yang, Fan; Fan, Daidi; Wang, Ya; Yu, Yuanyuan

2017-07-01

Iron deficiency remains a public health problem around the world due to low iron intake and/or bioavailability. FeSO 4 , ferrous succinate, and ferrous glycinate chelate are rich in iron but have poor bioavailability. To solve the problem of iron deficiency, following previous research studies, a thiolated human-like collagen-ironcomplex supplement with a high iron content was prepared in an anaerobic workstation. In addition, cell viability tests were evaluated after conducting an MTT assay, and a quantitative analysis of the thiolated human-like collagen-iron digesta samples was performed using the SDS-PAGE method coupled with gel filtration chromatography. The iron bioavailability was assessed using Caco-2 cell monolayers and iron-deficiency anemia mice models. The results showed that (1) one mole of thiolated human-like collagen-iron possessed approximately 35.34 moles of iron; (2) thiolated human-like collagen-iron did not exhibit cytotoxity and (3) thiolated human-like collagen- iron digesta samples had higher bioavailability than other iron supplements, including FeSO 4 , ferrous succinate, ferrous glycine chelate and thiolated human-like collagen-Fe iron. Finally, the iron bioavailability was significantly enhanced by vitamin C. These results indicated that thiolated human-like collagen-iron is a promising iron supplement for use in the future.

Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, P.-F.; Yeh, Alvin T.; Bayless, Kayla J.

The interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are fundamental in mediating various steps of angiogenesis, including cell adhesion, migration and sprout formation. Here, we used a noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique to optically image endothelial sprouting morphogenesis in three-dimensional (3D) collagen matrices. We simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Dynamic 3D imaging revealed EC interactions with collagen fibers along with quantifiable alterations in collagen matrix density elicited by EC movement through and morphogenesis within the matrix. Specifically, we observedmore » increased collagen density in the area between bifurcation points of sprouting structures and anisotropic increases in collagen density around the perimeter of lumenal structures, but not advancing sprout tips. Proteinase inhibition studies revealed membrane-associated matrix metalloproteinase were utilized for sprout advancement and lumen expansion. Rho-associated kinase (p160ROCK) inhibition demonstrated that the generation of cell tension increased collagen matrix alterations. This study followed sprouting ECs within a 3D matrix and revealed that the advancing structures recognize and significantly alter their extracellular environment at the periphery of lumens as they progress.« less

Accumulation of Maillard reaction products in skin collagen in diabetes and aging.

PubMed Central

Dyer, D G; Dunn, J A; Thorpe, S R; Bailie, K E; Lyons, T J; McCance, D R; Baynes, J W

1993-01-01

To investigate the contribution of glycation and oxidation reactions to the modification of insoluble collagen in aging and diabetes, Maillard reaction products were measured in skin collagen from 39 type 1 diabetic patients and 52 nondiabetic control subjects. Compounds studied included fructoselysine (FL), the initial glycation product, and the glycoxidation products, N epsilon-(carboxymethyl) lysine (CML) and pentosidine, formed during later Maillard reactions. Collagen-linked fluorescence was also studied. In nondiabetic subjects, glycation of collagen (FL content) increased only 33% between 20 and 85 yr of age. In contrast, CML, pentosidine and fluorescence increased five-fold, correlating strongly with age. In diabetic patients, collagen FL was increased threefold compared with nondiabetic subjects, correlating strongly with glycated hemoglobin but not with age. Collagen CML, pentosidine and fluorescence were increased up to twofold in diabetic compared with control patients: this could be explained by the increase in glycation alone, without invoking increased oxidative stress. There were strong correlations among CML, pentosidine and fluorescence in both groups, providing evidence for age-dependent chemical modification of collagen via the Maillard reaction, and acceleration of this process in diabetes. These results support the description of diabetes as a disease characterized by accelerated chemical aging of long-lived tissue proteins. PMID:8514858

Evaluation of sericin/collagen membranes as prospective wound dressing biomaterial.

PubMed

Akturk, Omer; Tezcaner, Aysen; Bilgili, Hasan; Deveci, M Salih; Gecit, M Rusen; Keskin, Dilek

2011-09-01

Sericin, a silk protein, has high potential for use in biomedical applications. In this study, wound dressing membranes of Sericin (S) and Collagen (C) were prepared by glutaraldehyde cross-linking at S/C; 2:1, 1:1, 1:2, and 0:1 weight ratios. They were stable in water for 4 weeks. However, increasing the proportion of sericin had decreasing effect on the membrane stability. Water swelling property of membranes was enhanced with sericin. The highest water swelling was obtained in 1:1 group (9.06 g/g), but increasing collagen or sericin content in the membranes had a diminishing effect. Highest water vapor transmission rate was obtained with 1:2 group (1013.80 g/m(2)/day). Oxygen permeability results showed that 1:2 (7.67 mg/L) and 2:1 (7.85 mg/L) S/C groups were better than the other groups. While sericin decreased the tensile strength and elongation of membranes, it increased modulus. Sericin also increased brittleness of membranes, but their UTS range (24.93-44.92 MPa) was still suitable for a wound dressing. Membranes were not penetrable to microorganisms. Cytotoxicity studies showed that fibroblasts and keratinocytes attached and gained their characteristic morphologies. They also proliferated on membranes significantly. After 1 week of subcutaneous implantation, a fibrous capsule formed around all membranes with an acute inflammation. Sericin containing membranes showed signs of degradation (at 2nd week), while collagen only membranes remained largely intact. Eventually, sericin containing membranes degraded in 3 weeks with moderate inflammatory response. Overall results suggest that sericin/collagen membranes would be favorable as wound dressing material when sericin ratio is less than or equal to the collagen component. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Adhesive interactions of human multiple myeloma cell lines with different extracellular matrix molecules.

PubMed

Kibler, C; Schermutzki, F; Waller, H D; Timpl, R; Müller, C A; Klein, G

1998-06-01

Multiple myeloma represents a human B cell malignancy which is characterized by a predominant localization of the malignant cell clone within the bone marrow. With the exception of the terminal stage of the disease the myeloma tumor cells do not circulate in the peripheral blood. The bone marrow microenvironment is believed to play an important role in homing, proliferation and terminal differentiation of myeloma cells. Here we have studied the expression of several extracellular matrix (ECM) molecules in the bone marrow of multiple myeloma patients and analyzed their adhesive capacities with four different human myeloma-derived cell lines. All ECM molecules analyzed (tenascin, laminin, fibronectin, collagen types I, III, V and VI) could be detected in bone marrow cryostat sections of multiple myeloma patients. Adhesion assays showed that only laminin, the microfibrillar collagen type VI and fibronectin were strong adhesive components for the myeloma cell lines U266, IM-9, OPM-2 and NCI-H929. Tenascin and collagen type I were only weak adhesive substrates for these myeloma cells. Adhesion to laminin and fibronectin was beta 1-integrin-mediated since addition of anti-beta 1-integrin antibodies could inhibit the binding of the four different cell types to both matrix molecules. In contrast, integrins do not seem to be involved in binding of the myeloma cells to collagen type VI. Instead, inhibition of binding by heparin suggested that membrane-bound heparan sulfate proteoglycans are responsible ligands for binding to collagen type VI. Adhesion assays with several B-cell lines resembling earlier differentiation stages revealed only weak interactions with tenascin and no interactions with collagen type VI, laminin or fibronectin. In summary, the interactions of human myeloma cells with the extracellular matrix may explain the specific retention of the plasma cells within the bone marrow.

Jararhagin disruption of endothelial cell anchorage is enhanced in collagen enriched matrices.

PubMed

Baldo, C; Lopes, D S; Faquim-Mauro, E L; Jacysyn, J F; Niland, S; Eble, J A; Clissa, P B; Moura-da-Silva, A M

2015-12-15

Hemorrhage is one of the most striking effects of bites by viper snakes resulting in fast bleeding and ischemia in affected tissues. Snake venom metalloproteinases (SVMPs) are responsible for hemorrhagic activity, but the mechanisms involved in SVMP-induced hemorrhage are not entirely understood and the study of such mechanisms greatly depends on in vivo experiments. In vivo, hemorrhagic SVMPs accumulate on basement membrane (BM) of venules and capillary vessels allowing the hydrolysis of collagen IV with consequent weakness and rupture of capillary walls. These effects are not reproducible in vitro with conventional endothelial cell cultures. In this study we used two-dimension (2D) or three-dimension (3D) cultures of HUVECs on matrigel and observed the same characteristics as in ex vivo experiments: only the hemorrhagic toxin was able to localize on surfaces or internalize endothelial cells in 2D cultures or in the surface of tubules formed on 3D cultures. The contribution of matrigel, fibronectin and collagen matrices in jararhagin-induced endothelial cell damage was then analyzed. Collagen and matrigel substrates enhanced the endothelial cell damage induced by jararhagin allowing toxin binding to focal adhesions, disruption of stress fibers, detachment and apoptosis. The higher affinity of jararhagin to collagen than to fibronectin explains the localization of the toxin within BM. Moreover, once located in BM, interactions of jararhagin with α2β1 integrin would favor its localization on focal adhesions, as observed in our study. The accumulation of toxin in focal adhesions, observed only in cells grown in collagen matrices, would explain the enhancement of cell damage in these matrices and reflects the actual interaction among toxin, endothelial cells and BM components that occurs in vivo and results in the hemorrhagic lesions induced by viper venoms. Copyright © 2015 Elsevier Ltd. All rights reserved.