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Sample records for human tissue engineered

  1. Tissue engineering a human phalanx.

    PubMed

    Landis, W J; Chubinskaya, S; Tokui, T; Wada, Y; Isogai, N; Jacquet, R

    2016-03-21

    A principal purpose of tissue engineering is the augmentation, repair or replacement of diseased or injured human tissue. This study was undertaken to determine whether human biopsies as a cell source could be utilized for successful engineering of human phalanges consisting of both bone and cartilage. This paper reports the use of cadaveric human chondrocytes and periosteum as a model for the development of phalanx constructs. Two factors, osteogenic protein-1 [OP-1/bone morphogenetic protein-7 (BMP7)], alone or combined with insulin-like growth factor (IGF-1), were examined for their potential enhancement of chondrocytes and their secreted extracellular matrices. Design of the study included culture of chondrocytes and periosteum on biodegradable polyglycolic acid (PGA) and poly-l-lactic acid (PLLA)-poly-ε-caprolactone (PCL) scaffolds and subsequent implantation in athymic nu/nu (nude) mice for 5, 20, 40 and 60 weeks. Engineered constructs retrieved from mice were characterized with regard to genotype and phenotype as a function of developmental (implantation) time. Assessments included gross observation, X-ray radiography or microcomputed tomography, histology and gene expression. The resulting data showed that human cell-scaffold constructs could be successfully developed over 60 weeks, despite variability in donor age. Cartilage formation of the distal phalanx models enhanced with both OP-1 and IGF-1 yielded more cells and extracellular matrix (collagen and proteoglycans) than control chondrocytes without added factors. Summary data demonstrated that human distal phalanx models utilizing cadaveric chondrocytes and periosteum were successfully fabricated and OP-1 and OP-1/IGF-1 accelerated construct development and mineralization. The results suggest that similar engineering and transplantation of human autologous tissues in patients are clinically feasible. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Tissue Engineered Human Skin Equivalents

    PubMed Central

    Zhang, Zheng; Michniak-Kohn, Bozena B.

    2012-01-01

    Human skin not only serves as an important barrier against the penetration of exogenous substances into the body, but also provides a potential avenue for the transport of functional active drugs/reagents/ingredients into the skin (topical delivery) and/or the body (transdermal delivery). In the past three decades, research and development in human skin equivalents have advanced in parallel with those in tissue engineering and regenerative medicine. The human skin equivalents are used commercially as clinical skin substitutes and as models for permeation and toxicity screening. Several academic laboratories have developed their own human skin equivalent models and applied these models for studying skin permeation, corrosivity and irritation, compound toxicity, biochemistry, metabolism and cellular pharmacology. Various aspects of the state of the art of human skin equivalents are reviewed and discussed. PMID:24300178

  3. Bone tissue engineering with human stem cells

    PubMed Central

    2010-01-01

    Treatment of extensive bone defects requires autologous bone grafting or implantation of bone substitute materials. An attractive alternative has been to engineer fully viable, biological bone grafts in vitro by culturing osteogenic cells within three-dimensional scaffolds, under conditions supporting bone formation. Such grafts could be used for implantation, but also as physiologically relevant models in basic and translational studies of bone development, disease and drug discovery. A source of human cells that can be derived in large numbers from a small initial harvest and predictably differentiated into bone forming cells is critically important for engineering human bone grafts. We discuss the characteristics and limitations of various types of human embryonic and adult stem cells, and their utility for bone tissue engineering. PMID:20637059

  4. Variation in tissue outcome of ovine and human engineered heart valve constructs: relevance for tissue engineering.

    PubMed

    van Geemen, Daphne; Driessen-Mol, Anita; Grootzwagers, Leonie G M; Soekhradj-Soechit, R Sarita; Riem Vis, Paul W; Baaijens, Frank P T; Bouten, Carlijn V C

    2012-01-01

    Clinical application of tissue engineered heart valves requires precise control of the tissue culture process to predict tissue composition and mechanical properties prior to implantation, and to understand the variation in tissue outcome. To this end we investigated cellular phenotype and tissue properties of ovine (n = 8) and human (n = 7) tissue engineered heart valve constructs to quantify variations in tissue outcome within species, study the differences between species and determine possible indicators of tissue outcome. Tissue constructs consisted of polyglycolic acid/poly-4-hydroxybutyrate scaffolds, seeded with myofibroblasts obtained from the jugular vein (sheep) or the saphenous vein (from humans undergoing cardiac surgery) and cultured under static conditions. Prior to seeding, protein expression of α-smooth muscle actin, vimentin, nonmuscle myosin heavy chain and heat shock protein 47 were determined to identify differences at an early stage of the tissue engineering process. After 4 weeks of culture, tissue composition and mechanical properties were quantified as indicators of tissue outcome. After 4 weeks of tissue culture, tissue properties of all ovine constructs were comparable, while there was a larger variation in the properties of the human constructs, especially the elastic modulus and collagen content. In addition, ovine constructs differed in composition from the human constructs. An increased number of α-smooth muscle actin-positive cells before seeding was correlated with the collagen content in the engineered heart valve constructs. Moreover, tissue stiffness increased with increasing collagen content. The results suggest that the culture process of ovine tissues can be controlled, whereas the mechanical properties, and hence functionality, of tissues originating from human material are more difficult to control. On-line evaluation of tissue properties during culture or more early cellular markers to predict the properties of autologous

  5. Macrophages modulate engineered human tissues for enhanced vascularization and healing

    PubMed Central

    Spiller, Kara L.; Freytes, Donald O.; Vunjak-Novakovic, Gordana

    2014-01-01

    Tissue engineering is increasingly based on recapitulating human physiology, through integration of biological principles into engineering designs. In spite of all progress in engineering functional human tissues, we are just beginning to develop effective methods for establishing blood perfusion and controlling the inflammatory factors following implantation into the host. Functional vasculature largely determines tissue survival and function in vivo. The inflammatory response is a major regulator of vascularization and overall functionality of engineered tissues, through the activity of different types of macrophages and the cytokines they secrete. We discuss cell-scaffold-bioreactor systems for harnessing the inflammatory response for enhanced tissue vascularization and healing. To this end, inert scaffolds that have been considered for many decades a “gold standard” in regenerative medicine are beginning to be replaced by a new generation of “smart” tissue engineering systems designed to actively mediate tissue survival and function. PMID:25331098

  6. Tissue Engineering

    NASA Astrophysics Data System (ADS)

    Langer, Robert; Vacanti, Joseph P.

    1993-05-01

    The loss or failure of an organ or tissue is one of the most frequent, devastating, and costly problems in human health care. A new field, tissue engineering, applies the principles of biology and engineering to the development of functional substitutes for damaged tissue. This article discusses the foundations and challenges of this interdisciplinary field and its attempts to provide solutions to tissue creation and repair.

  7. Plant-derived human collagen scaffolds for skin tissue engineering.

    PubMed

    Willard, James J; Drexler, Jason W; Das, Amitava; Roy, Sashwati; Shilo, Shani; Shoseyov, Oded; Powell, Heather M

    2013-07-01

    Tissue engineering scaffolds are commonly formed using proteins extracted from animal tissues, such as bovine hide. Risks associated with the use of these materials include hypersensitivity and pathogenic contamination. Human-derived proteins lower the risk of hypersensitivity, but possess the risk of disease transmission. Methods engineering recombinant human proteins using plant material provide an alternate source of these materials without the risk of disease transmission or concerns regarding variability. To investigate the utility of plant-derived human collagen (PDHC) in the development of engineered skin (ES), PDHC and bovine hide collagen were formed into tissue engineering scaffolds using electrospinning or freeze-drying. Both raw materials were easily formed into two common scaffold types, electrospun nonwoven scaffolds and lyophilized sponges, with similar architectures. The processing time, however, was significantly lower with PDHC. PDHC scaffolds supported primary human cell attachment and proliferation at an equivalent or higher level than the bovine material. Interleukin-1 beta production was significantly lower when activated THP-1 macrophages where exposed to PDHC electrospun scaffolds compared to bovine collagen. Both materials promoted proper maturation and differentiation of ES. These data suggest that PDHC may provide a novel source of raw material for tissue engineering with low risk of allergic response or disease transmission.

  8. Plant-Derived Human Collagen Scaffolds for Skin Tissue Engineering

    PubMed Central

    Willard, James J.; Drexler, Jason W.; Das, Amitava; Roy, Sashwati; Shilo, Shani; Shoseyov, Oded

    2013-01-01

    Tissue engineering scaffolds are commonly formed using proteins extracted from animal tissues, such as bovine hide. Risks associated with the use of these materials include hypersensitivity and pathogenic contamination. Human-derived proteins lower the risk of hypersensitivity, but possess the risk of disease transmission. Methods engineering recombinant human proteins using plant material provide an alternate source of these materials without the risk of disease transmission or concerns regarding variability. To investigate the utility of plant-derived human collagen (PDHC) in the development of engineered skin (ES), PDHC and bovine hide collagen were formed into tissue engineering scaffolds using electrospinning or freeze-drying. Both raw materials were easily formed into two common scaffold types, electrospun nonwoven scaffolds and lyophilized sponges, with similar architectures. The processing time, however, was significantly lower with PDHC. PDHC scaffolds supported primary human cell attachment and proliferation at an equivalent or higher level than the bovine material. Interleukin-1 beta production was significantly lower when activated THP-1 macrophages where exposed to PDHC electrospun scaffolds compared to bovine collagen. Both materials promoted proper maturation and differentiation of ES. These data suggest that PDHC may provide a novel source of raw material for tissue engineering with low risk of allergic response or disease transmission. PMID:23298216

  9. Osseous differentiation of human fat tissue grafts: From tissue engineering to tissue differentiation

    PubMed Central

    Bondarava, Maryna; Cattaneo, Chiara; Ren, Bin; Thasler, Wolfgang E.; Jansson, Volkmar; Müller, Peter E.; Betz, Oliver B.

    2017-01-01

    Conventional bone tissue engineering approaches require isolation and in vitro propagation of autologous cells, followed by seeding on a variety of scaffolds. Those protracted procedures impede the clinical applications. Here we report the transdifferentiation of human fat tissue fragments retrieved from subcutaneous fat into tissue with bone characteristics in vitro without prior cell isolation and propagation. 3D collagen-I cultures of human fat tissue were cultivated either in growth medium or in osteogenic medium (OM) with or without addition of Bone Morphogenetic Proteins (BMPs) BMP-2, BMP-7 or BMP-9. Ca2+ depositions were observed after two weeks of osteogenic induction which visibly increased when either type of BMP was added. mRNA levels of alkaline phosphatase (ALP) and osteocalcin (OCN) increased when cultured in OM alone but addition of BMP-2, BMP-7 or BMP-9 caused significantly higher expression levels of ALP and OCN. Immunofluorescent staining for OCN, osteopontin and sclerostin supported the observed real-time-PCR data. BMP-9 was the most effective osteogenic inducer in this system. Our findings reveal that tissue regeneration can be remarkably simplified by omitting prior cell isolation and propagation, therefore removing significant obstacles on the way to clinical applications of much needed regeneration treatments. PMID:28054585

  10. Advancing biomaterials of human origin for tissue engineering

    PubMed Central

    Chen, Fa-Ming; Liu, Xiaohua

    2015-01-01

    Biomaterials have played an increasingly prominent role in the success of biomedical devices and in the development of tissue engineering, which seeks to unlock the regenerative potential innate to human tissues/organs in a state of deterioration and to restore or reestablish normal bodily function. Advances in our understanding of regenerative biomaterials and their roles in new tissue formation can potentially open a new frontier in the fast-growing field of regenerative medicine. Taking inspiration from the role and multi-component construction of native extracellular matrices (ECMs) for cell accommodation, the synthetic biomaterials produced today routinely incorporate biologically active components to define an artificial in vivo milieu with complex and dynamic interactions that foster and regulate stem cells, similar to the events occurring in a natural cellular microenvironment. The range and degree of biomaterial sophistication have also dramatically increased as more knowledge has accumulated through materials science, matrix biology and tissue engineering. However, achieving clinical translation and commercial success requires regenerative biomaterials to be not only efficacious and safe but also cost-effective and convenient for use and production. Utilizing biomaterials of human origin as building blocks for therapeutic purposes has provided a facilitated approach that closely mimics the critical aspects of natural tissue with regard to its physical and chemical properties for the orchestration of wound healing and tissue regeneration. In addition to directly using tissue transfers and transplants for repair, new applications of human-derived biomaterials are now focusing on the use of naturally occurring biomacromolecules, decellularized ECM scaffolds and autologous preparations rich in growth factors/non-expanded stem cells to either target acceleration/magnification of the body's own repair capacity or use nature's paradigms to create new tissues for

  11. Advancing biomaterials of human origin for tissue engineering.

    PubMed

    Chen, Fa-Ming; Liu, Xiaohua

    2016-02-01

    Biomaterials have played an increasingly prominent role in the success of biomedical devices and in the development of tissue engineering, which seeks to unlock the regenerative potential innate to human tissues/organs in a state of deterioration and to restore or reestablish normal bodily function. Advances in our understanding of regenerative biomaterials and their roles in new tissue formation can potentially open a new frontier in the fast-growing field of regenerative medicine. Taking inspiration from the role and multi-component construction of native extracellular matrices (ECMs) for cell accommodation, the synthetic biomaterials produced today routinely incorporate biologically active components to define an artificial in vivo milieu with complex and dynamic interactions that foster and regulate stem cells, similar to the events occurring in a natural cellular microenvironment. The range and degree of biomaterial sophistication have also dramatically increased as more knowledge has accumulated through materials science, matrix biology and tissue engineering. However, achieving clinical translation and commercial success requires regenerative biomaterials to be not only efficacious and safe but also cost-effective and convenient for use and production. Utilizing biomaterials of human origin as building blocks for therapeutic purposes has provided a facilitated approach that closely mimics the critical aspects of natural tissue with regard to its physical and chemical properties for the orchestration of wound healing and tissue regeneration. In addition to directly using tissue transfers and transplants for repair, new applications of human-derived biomaterials are now focusing on the use of naturally occurring biomacromolecules, decellularized ECM scaffolds and autologous preparations rich in growth factors/non-expanded stem cells to either target acceleration/magnification of the body's own repair capacity or use nature's paradigms to create new tissues for

  12. Relevance and safety of telomerase for human tissue engineering

    PubMed Central

    Klinger, Rebecca Y.; Blum, Juliana L.; Hearn, Bevin; Lebow, Benjamin; Niklason, Laura E.

    2006-01-01

    Tissue engineering holds the promise of replacing damaged or diseased tissues and organs. The use of autologous donor cells is often not feasible because of the limited replicative lifespan of cells, particularly those derived from elderly patients. Proliferative arrest can be overcome by the ectopic expression of telomerase via human telomerase reverse transcriptase (hTERT) gene transfection. To study the efficacy and safety of this potentially valuable technology, we used differentiated vascular smooth muscle cells (SMC) and vascular tissue engineering as a model system. Although we previously demonstrated that vessels engineered with telomerase-expressing SMC had improved mechanics over those grown with control cells, it is critical to assess the phenotypic impact of telomerase expression in donor cells, because telomerase up-regulation is observed in >95% of human malignancies. To study the impact of telomerase in tissue engineering, expression of hTERT was retrovirally induced in SMC from eight elderly patients and one young donor. In hTERT SMC, significant lifespan extension beyond that of control was achieved without population doubling time acceleration. Karyotype changes were seen in both control and hTERT SMC but were not clonal nor representative of cancerous change. hTERT cells also failed to show evidence of neoplastic transformation in functional assays of tumorigenicity. In addition, the impact of donor age on cellular behavior, particularly the synthetic capability of SMC, was not affected by hTERT expression. Hence, this tissue engineering model system highlights the impact of donor age on cellular synthetic function that appears to be independent of lifespan extension by hTERT. PMID:16477025

  13. New dimensions in tissue engineering: possible models for human physiology.

    PubMed

    Baar, Keith

    2005-11-01

    Tissue engineering is a discipline of great promise. In some areas, such as the cornea, tissues engineered in the laboratory are already in clinical use. In other areas, where the tissue architecture is more complex, there are a number of obstacles to manoeuvre before clinically relevant tissues can be produced. However, even in areas where clinically relevant tissues are decades away, the tissues being produced at the moment provide powerful new models to aid the understanding of complex physiological processes. This article provides a personal view of the role of tissue engineering in advancing our understanding of physiology, with specific attention being paid to musculoskeletal tissues.

  14. Engineering of human hepatic tissue with functional vascular networks.

    PubMed

    Takebe, Takanori; Koike, Naoto; Sekine, Keisuke; Fujiwara, Ryoji; Amiya, Takeru; Zheng, Yun-Wen; Taniguchi, Hideki

    2014-01-01

    Although absolute organ shortage highlights the needs of alternative organ sources for regenerative medicine, the generation of a three-dimensional (3D) and complex vital organ, such as well-vascularized liver, remains a challenge. To this end, tissue engineering holds great promise; however, this approach is significantly limited by the failure of early vascularization in vivo after implantation. Here, we established a stable 3D in vitro pre-vascularization platform to generate human hepatic tissue after implantation in vivo. Human fetal liver cells (hFLCs) were mixed with human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (hMSCs) and were implanted into a collagen/fibronectin matrix composite that was used as a 3-D carrier. After a couple of days, the fluorescent HUVECs developed premature vascular networks in vitro, which were stabilized by hMSCs. The establishment of functional vessels inside the pre-vascularized constructs was proven using dextran infusion studies after implantation under a transparency cranial window. Furthermore, dynamic morphological changes during embryonic liver cell maturation were intravitaly quantified with high-resolution confocal microscope analysis. The engineered human hepatic tissue demonstrated multiple liver-specific features, both structural and functional. Our new techniques discussed here can be implemented in future clinical uses and industrial uses, such as drug testing.

  15. Mechanical stimulation improves tissue-engineered human skeletal muscle

    NASA Technical Reports Server (NTRS)

    Powell, Courtney A.; Smiley, Beth L.; Mills, John; Vandenburgh, Herman H.

    2002-01-01

    Human bioartificial muscles (HBAMs) are tissue engineered by suspending muscle cells in collagen/MATRIGEL, casting in a silicone mold containing end attachment sites, and allowing the cells to differentiate for 8 to 16 days. The resulting HBAMs are representative of skeletal muscle in that they contain parallel arrays of postmitotic myofibers; however, they differ in many other morphological characteristics. To engineer improved HBAMs, i.e., more in vivo-like, we developed Mechanical Cell Stimulator (MCS) hardware to apply in vivo-like forces directly to the engineered tissue. A sensitive force transducer attached to the HBAM measured real-time, internally generated, as well as externally applied, forces. The muscle cells generated increasing internal forces during formation which were inhibitable with a cytoskeleton depolymerizer. Repetitive stretch/relaxation for 8 days increased the HBAM elasticity two- to threefold, mean myofiber diameter 12%, and myofiber area percent 40%. This system allows engineering of improved skeletal muscle analogs as well as a nondestructive method to determine passive force and viscoelastic properties of the resulting tissue.

  16. Mechanical stimulation improves tissue-engineered human skeletal muscle.

    PubMed

    Powell, Courtney A; Smiley, Beth L; Mills, John; Vandenburgh, Herman H

    2002-11-01

    Human bioartificial muscles (HBAMs) are tissue engineered by suspending muscle cells in collagen/MATRIGEL, casting in a silicone mold containing end attachment sites, and allowing the cells to differentiate for 8 to 16 days. The resulting HBAMs are representative of skeletal muscle in that they contain parallel arrays of postmitotic myofibers; however, they differ in many other morphological characteristics. To engineer improved HBAMs, i.e., more in vivo-like, we developed Mechanical Cell Stimulator (MCS) hardware to apply in vivo-like forces directly to the engineered tissue. A sensitive force transducer attached to the HBAM measured real-time, internally generated, as well as externally applied, forces. The muscle cells generated increasing internal forces during formation which were inhibitable with a cytoskeleton depolymerizer. Repetitive stretch/relaxation for 8 days increased the HBAM elasticity two- to threefold, mean myofiber diameter 12%, and myofiber area percent 40%. This system allows engineering of improved skeletal muscle analogs as well as a nondestructive method to determine passive force and viscoelastic properties of the resulting tissue.

  17. Mechanical stimulation improves tissue-engineered human skeletal muscle

    NASA Technical Reports Server (NTRS)

    Powell, Courtney A.; Smiley, Beth L.; Mills, John; Vandenburgh, Herman H.

    2002-01-01

    Human bioartificial muscles (HBAMs) are tissue engineered by suspending muscle cells in collagen/MATRIGEL, casting in a silicone mold containing end attachment sites, and allowing the cells to differentiate for 8 to 16 days. The resulting HBAMs are representative of skeletal muscle in that they contain parallel arrays of postmitotic myofibers; however, they differ in many other morphological characteristics. To engineer improved HBAMs, i.e., more in vivo-like, we developed Mechanical Cell Stimulator (MCS) hardware to apply in vivo-like forces directly to the engineered tissue. A sensitive force transducer attached to the HBAM measured real-time, internally generated, as well as externally applied, forces. The muscle cells generated increasing internal forces during formation which were inhibitable with a cytoskeleton depolymerizer. Repetitive stretch/relaxation for 8 days increased the HBAM elasticity two- to threefold, mean myofiber diameter 12%, and myofiber area percent 40%. This system allows engineering of improved skeletal muscle analogs as well as a nondestructive method to determine passive force and viscoelastic properties of the resulting tissue.

  18. Decellularization of human and porcine lung tissues for pulmonary tissue engineering.

    PubMed

    O'Neill, John D; Anfang, Rachel; Anandappa, Annabelle; Costa, Joseph; Javidfar, Jeffrey; Wobma, Holly M; Singh, Gopal; Freytes, Donald O; Bacchetta, Matthew D; Sonett, Joshua R; Vunjak-Novakovic, Gordana

    2013-09-01

    The only definitive treatment for end-stage organ failure is orthotopic transplantation. Lung extracellular matrix (LECM) holds great potential as a scaffold for lung tissue engineering because it retains the complex architecture, biomechanics, and topologic specificity of the lung. Decellularization of human lungs rejected from transplantation could provide "ideal" biologic scaffolds for lung tissue engineering, but the availability of such lungs remains limited. The present study was designed to determine whether porcine lung could serve as a suitable substitute for human lung to study tissue engineering therapies. Human and porcine lungs were procured, sliced into sheets, and decellularized by three different methods. Compositional, ultrastructural, and biomechanical changes to the LECM were characterized. The suitability of LECM for cellular repopulation was evaluated by assessing the viability, growth, and metabolic activity of human lung fibroblasts, human small airway epithelial cells, and human adipose-derived mesenchymal stem cells over a period of 7 days. Decellularization with 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) showed the best maintenance of both human and porcine LECM, with similar retention of LECM proteins except for elastin. Human and porcine LECM supported the cultivation of pulmonary cells in a similar way, except that the human LECM was stiffer and resulted in higher metabolic activity of the cells than porcine LECM. Porcine lungs can be decellularized with CHAPS to produce LECM scaffolds with properties resembling those of human lungs, for pulmonary tissue engineering. We propose that porcine LECM can be an excellent screening platform for the envisioned human tissue engineering applications of decellularized lungs. Copyright © 2013 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

  19. Characterization of human myoblast cultures for tissue engineering.

    PubMed

    Stern-Straeter, Jens; Bran, Gregor; Riedel, Frank; Sauter, Alexander; Hörmann, Karl; Goessler, Ulrich Reinhart

    2008-01-01

    Skeletal muscle tissue engineering, a promising specialty, aims at the reconstruction of skeletal muscle loss. In vitro tissue engineering attempts to achieve this goal by creating differentiated, functional muscle tissue through a process in which stem cells are extracted from the patient, e.g. by muscle biopsies, expanded and differentiated in a controlled environment, and subsequently re-implanted. A prerequisite for this undertaking is the ability to cultivate and differentiate human skeletal muscle cell cultures. Evidently, optimal culture conditions must be investigated for later clinical utilization. We therefore analysed the proliferation of human cells in different environments and evaluated the differentiation potential of different culture media. It was shown that human myoblasts have a higher rate of proliferation in the alamarBlue assay when cultured on gelatin-coated culture flasks rather than polystyrene-coated flasks. We also demonstrated that myoblasts treated with a culture medium with a high concentration of growth factors [growth medium (GM)] showed a higher proliferation compared to cultures treated with a culture medium with lower amounts of growth factors [differentiation medium (DM)]. Differentiation of human myoblast cell cultures treated with GM and DM was analysed until day 16 and myogenesis was verified by expression of MyoD, myogenin, alpha-sarcomeric actin and myosin heavy chain by semi-quantitative RT-PCR. Immunohistochemical staining for desmin, Myf-5 and alpha-sarcomeric actin was performed to verify the myogenic phenotype of extracted satellite cells and to prove the maturation of cells. Cultures treated with DM showed positive staining for alpha-sarcomeric actin. Notably, markers of differentiation were also detected in cultures treated with GM, but there was no formation of myotubes. In the enzymatic assay of creatine phosphokinase, cultures treated with DM showed a higher activity, evidencing a higher degree of differentiation

  20. Engineering bone tissue substitutes from human induced pluripotent stem cells.

    PubMed

    de Peppo, Giuseppe Maria; Marcos-Campos, Iván; Kahler, David John; Alsalman, Dana; Shang, Linshan; Vunjak-Novakovic, Gordana; Marolt, Darja

    2013-05-21

    Congenital defects, trauma, and disease can compromise the integrity and functionality of the skeletal system to the extent requiring implantation of bone grafts. Engineering of viable bone substitutes that can be personalized to meet specific clinical needs represents a promising therapeutic alternative. The aim of our study was to evaluate the utility of human-induced pluripotent stem cells (hiPSCs) for bone tissue engineering. We first induced three hiPSC lines with different tissue and reprogramming backgrounds into the mesenchymal lineages and used a combination of differentiation assays, surface antigen profiling, and global gene expression analysis to identify the lines exhibiting strong osteogenic differentiation potential. We then engineered functional bone substitutes by culturing hiPSC-derived mesenchymal progenitors on osteoconductive scaffolds in perfusion bioreactors and confirmed their phenotype stability in a subcutaneous implantation model for 12 wk. Molecular analysis confirmed that the maturation of bone substitutes in perfusion bioreactors results in global repression of cell proliferation and an increased expression of lineage-specific genes. These results pave the way for growing patient-specific bone substitutes for reconstructive treatments of the skeletal system and for constructing qualified experimental models of development and disease.

  1. Tissue engineered human tracheas for in vivo implantation.

    PubMed

    Baiguera, Silvia; Jungebluth, Phillip; Burns, Alan; Mavilia, Carmelo; Haag, Johannes; De Coppi, Paolo; Macchiarini, Paolo

    2010-12-01

    Two years ago we performed the first clinical successful transplantation of a fully tissue engineered trachea. Despite the clinically positive outcome, the graft production took almost 3 months, a not feasible period of time for patients with the need of an urgent transplantation. We have then improved decellularization process and herein, for the first time, we completely describe and characterize the obtainment of human tracheal bioactive supports. Histological and molecular biology analysis demonstrated that all cellular components and nuclear material were removed and quantitative PCR confirmed it. SEM analysis revealed that the decellularized matrices retained the hierarchical structures of native trachea, and biomechanical tests showed that decellularization approach did not led to any influence on tracheal morphological and mechanical properties. Moreover immunohistological staining showed the preservation of angiogenic factors and angiogenic assays demonstrated that acellular human tracheal scaffolds exert an in vitro chemo-active action and induce strong in vivo angiogenic response (CAM analysis). We are now able to obtained, in a short and clinically useful time (approximately 3 weeks), a bioengineered trachea that is structurally and mechanically similar to native trachea, which exert chemotactive and pro-angiogenic properties and which could be successfully used for clinical tissue engineered airway clinical replacements.

  2. Characteristics of tissue-engineered cartilage from human auricular chondrocytes.

    PubMed

    Park, Stephen S; Jin, Hong Ryul; Chi, David H; Taylor, Ray S

    2004-05-01

    This study was done to define the mechanical and histological properties of tissue-engineered cartilage (TEC) derived from human chondrocytes and to compare these findings with those of native cartilage. Chondrocytes were obtained from 10 human auricular cartilages and seeded onto a biodegradable template of polyglycolic acid and poly L-lactic acid. Each template was shaped into a 1 cm x 2 cm rectangle. The templates were implanted in athymic mice for 8 weeks. Eight human auricular cartilages were used for comparison. Mechanical analysis with a tensile testing device provided values of ultimate tensile strength (UTS), stiffness, and resilience. Statistical analysis was performed with the Student's t-test. Histological assessment was done with hematoxylin-eosin staining along with other special stains. The TEC had UTS of 2.07 MPa, stiffness of 3.7 MPa, and resilience of 0.37 J/m3. The control specimens had UTS of 2.18 MPa, stiffness of 5.11 MPa, and resilience of 0.42 J/m3. No statistical difference was found between the experimental and control groups for each of the three parameters. Histological analysis showed mature cartilage with characteristic collagen, glycosaminoglycans, and elastin in the TEC. The neo-cartilage showed slightly smaller size and more irregular distribution of chondrocytes and unique fibrous capsule formation with peripheral infiltration of fibrous tissue. This study showed that the mechanical qualities of TEC from human chondrocytes are similar to those of native auricular cartilage. It suggests that the engineered cartilage from human chondrocytes may have sufficient strength and durability for clinical uses. The histological findings revealed some differences with neo-cartilage.

  3. Decellularized extracellular matrix derived from human adipose tissue as a potential scaffold for allograft tissue engineering.

    PubMed

    Choi, Ji Suk; Kim, Beob Soo; Kim, Jun Young; Kim, Jae Dong; Choi, Young Chan; Yang, Hyun-Jin; Park, Kinam; Lee, Hee Young; Cho, Yong Woo

    2011-06-01

    Decellularized tissues composed of extracellular matrix (ECM) have been clinically used to support the regeneration of various human tissues and organs. Most decellularized tissues so far have been derived from animals or cadavers. Therefore, despite the many advantages of decellularized tissue, there are concerns about the potential for immunogenicity and the possible presence of infectious agents. Herein, we present a biomaterial composed of ECM derived from human adipose tissue, the most prevalent, expendable, and safely harvested tissue in the human body. The ECM was extracted by successive physical, chemical, and enzymatic treatments of human adipose tissue isolated by liposuction. Cellular components including nucleic acids were effectively removed without significant disruption of the morphology or structure of the ECM. Major ECM components were quantified, including acid/pepsin-soluble collagen, sulfated glycosaminoglycan (GAG), and soluble elastin. In an in vivo experiment using mice, the decellularized ECM graft exhibited good compatibility to surrounding tissues. Overall results suggest that the decellularized ECM containing biological and chemical cues of native human ECM could be an ideal scaffold material not only for autologous but also for allograft tissue engineering.

  4. Magnetic Resonance Imaging of Human Tissue-Engineered Adipose Substitutes

    PubMed Central

    Proulx, Maryse; Aubin, Kim; Lagueux, Jean; Audet, Pierre; Auger, Michèle

    2015-01-01

    Adipose tissue (AT) substitutes are being developed to answer the strong demand in reconstructive surgery. To facilitate the validation of their functional performance in vivo, and to avoid resorting to excessive number of animals, it is crucial at this stage to develop biomedical imaging methodologies, enabling the follow-up of reconstructed AT substitutes. Until now, biomedical imaging of AT substitutes has scarcely been reported in the literature. Therefore, the optimal parameters enabling good resolution, appropriate contrast, and graft delineation, as well as blood perfusion validation, must be studied and reported. In this study, human adipose substitutes produced from adipose-derived stem/stromal cells using the self-assembly approach of tissue engineering were implanted into athymic mice. The fate of the reconstructed AT substitutes implanted in vivo was successfully followed by magnetic resonance imaging (MRI), which is the imaging modality of choice for visualizing soft ATs. T1-weighted images allowed clear delineation of the grafts, followed by volume integration. The magnetic resonance (MR) signal of reconstructed AT was studied in vitro by proton nuclear magnetic resonance (1H-NMR). This confirmed the presence of a strong triglyceride peak of short longitudinal proton relaxation time (T1) values (200±53 ms) in reconstructed AT substitutes (total T1=813±76 ms), which establishes a clear signal difference between adjacent muscle, connective tissue, and native fat (total T1 ∼300 ms). Graft volume retention was followed up to 6 weeks after implantation, revealing a gradual resorption rate averaging at 44% of initial substitute's volume. In addition, vascular perfusion measured by dynamic contrast-enhanced-MRI confirmed the graft's vascularization postimplantation (14 and 21 days after grafting). Histological analysis of the grafted tissues revealed the persistence of numerous adipocytes without evidence of cysts or tissue necrosis. This study

  5. Human stem cells and articular cartilage tissue engineering.

    PubMed

    Stoltz, J-F; Huselstein, C; Schiavi, J; Li, Y Y; Bensoussan, D; Decot, V; De Isla, N

    2012-12-01

    Injuries to articular cartilage are one of the most challenging issues of musculoskeletal medicine due to the poor intrinsic ability of this tissue for repair. Despite progress in orthopaedic surgery, cell-based surgical therapies such as autologous chondrocyte transplantation (ACT) have been in clinical use for cartilage repair for over a decade but this approach has shown mixed results. Moreover, the lack of efficient modalities of treatment for large chondral defects has prompted research on cartilage tissue engineering combining cells, scaffold materials and environmental factors. This paper focuses on the main parameters in tissue engineering and in particular, on the potential of mesenchymal stem cells (MSCs) as an alternative to cells derived from patient tissues in autologous transplantation and tissue engineering. We discussed the prospects of using autologous chondrocytes or MSCs in regenerative medicine and summarized the advantages and disadvantages of these cells in articular cartilage engineering.

  6. Engineering human neo-tendon tissue in vitro with human dermal fibroblasts under static mechanical strain.

    PubMed

    Deng, Dan; Liu, Wei; Xu, Feng; Yang, Yang; Zhou, Guangdong; Zhang, Wen Jie; Cui, Lei; Cao, Yilin

    2009-12-01

    Proper cell source is one of the key issues for tendon engineering. Our previous study showed that dermal fibroblasts could be used to successfully engineer tendon in vivo and tenocytes could engineer neo-tendon in vitro with static strain. This study further investigated the possibility of engineering human neo-tendon tissue in vitro using dermal fibroblasts. Human dermal fibroblasts were seeded on polyglycolic acid (PGA) fibers pre-fixed on a U-shape as a mechanical loading group, or simply cultured in a dish as a tension-free group. In addition, human tenocytes were also seeded on PGA fibers with tension as a comparison to human dermal fibroblasts. The results showed that human neo-tendon tissue could be generated using dermal fibroblasts during in vitro culture under static strain and the tissue structure became more mature with the increase of culture time. Longitudinally aligned collagen fibers and spindle shape cells were observed histologically and collagen fibril diameter and tensile strength increased with time and reached a peak at 14 weeks. In contrast, the dermal fibroblast-PGA constructs failed to form neo-tendon, but formed disorganized fibrous tissue in tension-free condition with significantly weaker strength and poor collagen fiber formation. Interestingly, neo-tendon tissues generated with human dermal fibroblasts were indistinguishable from the counterpart engineered with human tenocytes, which supports the viewpoint that human dermal fibroblasts is likely to replace tenocytes for future tendon graft development in vitro with dynamic mechanical loading in a bioreactor system.

  7. [Tissue engineering and construction of human skin in vitro].

    PubMed

    Arvelo, Francisco

    2007-09-01

    Tissue engineering is the new science that has come to make possible the growth of new organ tissue from small fragments of healthy tissue, thus partially or totally restoring the lost functions of ill tissues or organs, as shown by the achievements made with the culture of skin, cornea or cartilage. Thus far, this new science is able to ensure the recovery of lost functions and, doubtlessly, in a near future will be capable of developing tissues and organs not unlike natural ones. In our laboratory we have began the development of tissue engineering techniques for the successful construction of in vitro skin with the aim at mid term of producing cornea and cartilage. In a first clinical trial, these techniques were applied in the treatment of chronic skin lesions and the advantages and reach of these new tools were demonstrated for the effective solution of problems with would otherwise not be easily solved through the use of conventional treatments.

  8. Translational issues for human corneal endothelial tissue engineering.

    PubMed

    Soh, Yu Qiang; Peh, Gary S L; Mehta, Jodhbir S

    2017-09-01

    Corneal endothelial disorders collectively represent a significant healthcare burden in most developed nations, and corneal transplantation is currently the only treatment available for patients with poor visual acuity and corneal blindness secondary to endothelial failure. Although vision in these patients can be restored by transplantation, the global demand for donor human corneas is far in excess of what can be provided for by eye banks around the world, and this deficit is set to increase with an ageing global population. As such, there has been a pressing need to explore novel and more sustainable options for the treatment of corneal endothelial diseases. In recent years, significant progress has been made not only in the development of corneal endothelial cell culture techniques, but also in the exploration of various translational strategies. Considered together, we are now much closer to attaining success in the treatment of corneal endothelial diseases via a cell-based, tissue-engineering approach. The aim of this review article is to provide an update of the translational issues currently facing human corneal endothelial cell therapy. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Musculoskeletal tissue engineering with human umbilical cord mesenchymal stromal cells

    PubMed Central

    Wang, Limin; Ott, Lindsey; Seshareddy, Kiran; Weiss, Mark L; Detamore, Michael S

    2011-01-01

    Multipotent mesenchymal stromal cells (MSCs) hold tremendous promise for tissue engineering and regenerative medicine, yet with so many sources of MSCs, what are the primary criteria for selecting leading candidates? Ideally, the cells will be multipotent, inexpensive, lack donor site morbidity, donor materials should be readily available in large numbers, immunocompatible, politically benign and expandable in vitro for several passages. Bone marrow MSCs do not meet all of these criteria and neither do embryonic stem cells. However, a promising new cell source is emerging in tissue engineering that appears to meet these criteria: MSCs derived from Wharton’s jelly of umbilical cord MSCs. Exposed to appropriate conditions, umbilical cord MSCs can differentiate in vitro along several cell lineages such as the chondrocyte, osteoblast, adipocyte, myocyte, neuronal, pancreatic or hepatocyte lineages. In animal models, umbilical cord MSCs have demonstrated in vivo differentiation ability and promising immunocompatibility with host organs/tissues, even in xenotransplantation. In this article, we address their cellular characteristics, multipotent differentiation ability and potential for tissue engineering with an emphasis on musculoskeletal tissue engineering. PMID:21175290

  10. Tissue engineered humanized bone supports human hematopoiesis in vivo.

    PubMed

    Holzapfel, Boris M; Hutmacher, Dietmar W; Nowlan, Bianca; Barbier, Valerie; Thibaudeau, Laure; Theodoropoulos, Christina; Hooper, John D; Loessner, Daniela; Clements, Judith A; Russell, Pamela J; Pettit, Allison R; Winkler, Ingrid G; Levesque, Jean-Pierre

    2015-08-01

    Advances in tissue-engineering have resulted in a versatile tool-box to specifically design a tailored microenvironment for hematopoietic stem cells (HSCs) in order to study diseases that develop within this setting. However, most current in vivo models fail to recapitulate the biological processes seen in humans. Here we describe a highly reproducible method to engineer humanized bone constructs that are able to recapitulate the morphological features and biological functions of the HSC niches. Ectopic implantation of biodegradable composite scaffolds cultured for 4 weeks with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone organ including a large number of human mesenchymal cells which were shown to be metabolically active and capable of establishing a humanized microenvironment supportive of the homing and maintenance of human HSCs. A syngeneic mouse-to-mouse transplantation assay was used to prove the functionality of the tissue-engineered ossicles. We predict that the ability to tissue engineer a morphologically intact and functional large-volume bone organ with a humanized bone marrow compartment will help to further elucidate physiological or pathological interactions between human HSCs and their native niches. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  11. Computer-aided tissue engineering of a human vertebral body.

    PubMed

    Wettergreen, M A; Bucklen, B S; Sun, W; Liebschner, M A K

    2005-10-01

    Tissue engineering is developing into a less speculative science involving the careful interplay of numerous design parameters and multidisciplinary professionals. Problem solving abilities and state of the art research tools are required to develop solutions for a wide variety of clinical issues. One area of particular interest is orthopedic biomechanics, a field that is responsible for the treatment of over 700,000 vertebral fractures in the United States alone last year. Engineers are currently lacking the technology and knowledge required to govern the subsistence of cells in vivo, let alone the knowledge to create a functional tissue replacement for a whole organ. Despite this, advances in computer-aided tissue engineering are continually growing. Using a combinatory approach to scaffold design, patient-specific implants may be constructed. Computer-aided design, optimization of geometry using voxel finite element models or other optimization routines, creation of a library of architectures with specific material properties, rapid prototyping, and determination of a defect site using imaging modalities highlight the current availability of design resources. This study proposes a novel methodology from start to finish which could, in the future, be used to design a tissue-engineered construct for the replacement of an entire vertebral body.

  12. Three-Dimensionally Engineered Normal Human Lung Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.; McCarthy, M.; Lin, Y-H.; Deatly, A. M.

    2008-01-01

    In vitro three-dimensional (3D) human lung epithelio-mesenchymal tissue-like assemblies (3D hLEM TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and the detection of membrane bound glycoproteins over time confirm productive infection with the virus. Therefore, we assert TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host s immune system.

  13. Three-Dimensionally Engineered Normal Human Broncho-epithelial Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

    NASA Technical Reports Server (NTRS)

    Goodwin, T. J.; McCarthy, M.; Lin, Y-H

    2006-01-01

    In vitro three-dimensional (3D) human broncho-epithelial (HBE) tissue-like assemblies (3D HBE TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and parainfluenza virus type 3 (wtPIV3 JS) and the detection of membrane bound glycoproteins over time confirm productive infections with both viruses. Therefore, TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host's immune system.

  14. Three-Dimensionally Engineered Normal Human Broncho-epithelial Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

    NASA Technical Reports Server (NTRS)

    Goodwin, T. J.; McCarthy, M.; Lin, Y-H

    2006-01-01

    In vitro three-dimensional (3D) human broncho-epithelial (HBE) tissue-like assemblies (3D HBE TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and parainfluenza virus type 3 (wtPIV3 JS) and the detection of membrane bound glycoproteins over time confirm productive infections with both viruses. Therefore, TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host's immune system.

  15. Collagen in Human Tissues: Structure, Function, and Biomedical Implications from a Tissue Engineering Perspective

    NASA Astrophysics Data System (ADS)

    Balasubramanian, Preethi; Prabhakaran, Molamma P.; Sireesha, Merum; Ramakrishna, Seeram

    The extracellular matrix is a complex biological structure encoded with various proteins, among which the collagen family is the most significant and abundant of all, contributing 30-35% of the whole-body protein. "Collagen" is a generic term for proteins that forms a triple-helical structure with three polypeptide chains, and around 29 types of collagen have been identified up to now. Although most of the members of the collagen family form such supramolecular structures, extensive diversity exists between each type of collagen. The diversity is not only based on the molecular assembly and supramolecular structures of collagen types but is also observed within its tissue distribution, function, and pathology. Collagens possess complex hierarchical structures and are present in various forms such as collagen fibrils (1.5-3.5 nm wide), collagen fibers (50-70 nm wide), and collagen bundles (150-250 nm wide), with distinct properties characteristic of each tissue providing elasticity to skin, softness of the cartilage, stiffness of the bone and tendon, transparency of the cornea, opaqueness of the sclera, etc. There exists an exclusive relation between the structural features of collagen in human tissues (such as the collagen composition, collagen fibril length and diameter, collagen distribution, and collagen fiber orientation) and its tissue-specific mechanical properties. In bone, a transverse collagen fiber orientation prevails in regions of higher compressive stress whereas longitudinally oriented collagen fibers correlate to higher tensile stress. The immense versatility of collagen compels a thorough understanding of the collagen types and this review discusses the major types of collagen found in different human tissues, highlighting their tissue-specific uniqueness based on their structure and mechanical function. The changes in collagen during a specific tissue damage or injury are discussed further, focusing on the many tissue engineering applications for

  16. Engineered human broncho-epithelial tissue-like assemblies

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor)

    2012-01-01

    Three-dimensional human broncho-epithelial tissue-like assemblies (TLAs) are produced in a rotating wall vessel (RWV) with microcarriers by coculturing mesenchymal bronchial-tracheal cells (BTC) and bronchial epithelium cells (BEC). These TLAs display structural characteristics and express markers of in vivo respiratory epithelia. TLAs are useful for screening compounds active in lung tissues such as antiviral compounds, cystic fibrosis treatments, allergens, and cytotoxic compounds.

  17. Engineering musculoskeletal tissues with human embryonic germ cell derivatives.

    PubMed

    Varghese, Shyni; Hwang, Nathaniel S; Ferran, Angela; Hillel, Alexander; Theprungsirikul, Parnduangjai; Canver, Adam C; Zhang, Zijun; Gearhart, John; Elisseeff, Jennifer

    2010-04-01

    The cells derived from differentiating embryoid bodies of human embryonic germ (hEG) cells express a broad spectrum of gene markers and have been induced toward ecto- and endodermal lineages. We describe here in vitro and in vivo differentiation of hEG-derived cells (LVEC line) toward mesenchymal tissues. The LVEC cells express many surface marker proteins characteristic of mesenchymal stem cells and differentiated into cartilage, bone, and fat. Homogenous hyaline cartilage was generated from cells after 63 population doublings. In vivo results demonstrate cell survival, differentiation, and tissue formation. The high proliferative capacity of hEG-derived cells and their ability to differentiate and form three-dimensional mesenchymal tissues without teratoma formation underscores their significant potential for regenerative medicine. The adopted coculture system also provides new insights into how a microenvironment comprised of extracellular and cellular components may be harnessed to generate hierarchically complex tissues from pluripotent cells.

  18. Elastic, permeability and swelling properties of human intervertebral disc tissues: A benchmark for tissue engineering.

    PubMed

    Cortes, Daniel H; Jacobs, Nathan T; DeLucca, John F; Elliott, Dawn M

    2014-06-27

    The aim of functional tissue engineering is to repair and replace tissues that have a biomechanical function, i.e., connective orthopaedic tissues. To do this, it is necessary to have accurate benchmarks for the elastic, permeability, and swelling (i.e., biphasic-swelling) properties of native tissues. However, in the case of the intervertebral disc, the biphasic-swelling properties of individual tissues reported in the literature exhibit great variation and even span several orders of magnitude. This variation is probably caused by differences in the testing protocols and the constitutive models used to analyze the data. Therefore, the objective of this study was to measure the human lumbar disc annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplates (CEP) biphasic-swelling properties using a consistent experimental protocol and analyses. The testing protocol was composed of a swelling period followed by multiple confined compression ramps. To analyze the confined compression data, the tissues were modeled using a biphasic-swelling model, which augments the standard biphasic model through the addition of a deformation-dependent osmotic pressure term. This model allows considering the swelling deformations and the contribution of osmotic pressure in the analysis of the experimental data. The swelling stretch was not different between the disc regions (AF: 1.28±0.16; NP: 1.73±0.74; CEP: 1.29±0.26), with a total average of 1.42. The aggregate modulus (Ha) of the extra-fibrillar matrix was higher in the CEP (390kPa) compared to the NP (100kPa) or AF (30kPa). The permeability was very different across tissue regions, with the AF permeability (64 E(-16)m(4)/Ns) higher than the NP and CEP (~5.5 E(-16)m(4)/Ns). Additionally, a normalized time-constant (3000s) for the stress relaxation was similar for all the disc tissues. The properties measured in this study are important as benchmarks for tissue engineering and for modeling the disc's mechanical

  19. Creation of a Large Adipose Tissue Construct in Humans Using a Tissue-engineering Chamber: A Step Forward in the Clinical Application of Soft Tissue Engineering.

    PubMed

    Morrison, Wayne A; Marre, Diego; Grinsell, Damien; Batty, Andrew; Trost, Nicholas; O'Connor, Andrea J

    2016-04-01

    Tissue engineering is currently exploring new and exciting avenues for the repair of soft tissue and organ defects. Adipose tissue engineering using the tissue engineering chamber (TEC) model has yielded promising results in animals; however, to date, there have been no reports on the use of this device in humans. Five female post mastectomy patients ranging from 35 to 49years old were recruited and a pedicled thoracodorsal artery perforator fat flap ranging from 6 to 50ml was harvested, transposed onto the chest wall and covered by an acrylic perforated dome-shaped chamber ranging from 140 to 350cm(3). Magnetic resonance evaluation was performed at three and six months after chamber implantation. Chambers were removed at six months and samples were obtained for histological analysis. In one patient, newly formed tissue to a volume of 210ml was generated inside the chamber. One patient was unable to complete the trial and the other three failed to develop significant enlargement of the original fat flap, which, at the time of chamber explantation, was encased in a thick fibrous capsule. Our study provides evidence that generation of large well-vascularized tissue engineered constructs using the TEC is feasible in humans.

  20. Rolling the Human Amnion to Engineer Laminated Vascular Tissues

    PubMed Central

    Amensag, Salma

    2012-01-01

    The prevalence of cardiovascular disease and the limited availability of suitable autologous transplant vessels for coronary and peripheral bypass surgeries is a significant clinical problem. A great deal of progress has been made over recent years to develop biodegradable materials with the potential to remodel and regenerate vascular tissues. However, the creation of functional biological scaffolds capable of withstanding vascular stress within a clinically relevant time frame has proved to be a challenging proposition. As an alternative approach, we report the use of a multilaminate rolling approach using the human amnion to generate a tubular construct for blood vessel regeneration. The human amniotic membrane was decellularized by agitation in 0.03% (w/v) sodium dodecyl sulfate to generate an immune compliant material. The adhesion of human umbilical vein endothelial cells (EC) and human vascular smooth muscle cells (SMC) was assessed to determine initial binding and biocompatibility (monocultures). Extended cultures were either assessed as flat membranes, or rolled to form concentric multilayered conduits. Results showed positive EC adhesion and a progressive repopulation by SMC. Functional changes in SMC gene expression and the constructs' bulk mechanical properties were concomitant with vessel remodeling as assessed over a 40-day culture period. A significant advantage with this approach is the ability to rapidly produce a cell-dense construct with an extracellular matrix similar in architecture and composition to natural vessels. The capacity to control physical parameters such as vessel diameter, wall thickness, shape, and length are critical to match vessel compliance and tailor vessel specifications to distinct anatomical locations. As such, this approach opens new avenues in a range of tissue regenerative applications that may have a much wider clinical impact. PMID:22616610

  1. Rolling the human amnion to engineer laminated vascular tissues.

    PubMed

    Amensag, Salma; McFetridge, Peter S

    2012-11-01

    The prevalence of cardiovascular disease and the limited availability of suitable autologous transplant vessels for coronary and peripheral bypass surgeries is a significant clinical problem. A great deal of progress has been made over recent years to develop biodegradable materials with the potential to remodel and regenerate vascular tissues. However, the creation of functional biological scaffolds capable of withstanding vascular stress within a clinically relevant time frame has proved to be a challenging proposition. As an alternative approach, we report the use of a multilaminate rolling approach using the human amnion to generate a tubular construct for blood vessel regeneration. The human amniotic membrane was decellularized by agitation in 0.03% (w/v) sodium dodecyl sulfate to generate an immune compliant material. The adhesion of human umbilical vein endothelial cells (EC) and human vascular smooth muscle cells (SMC) was assessed to determine initial binding and biocompatibility (monocultures). Extended cultures were either assessed as flat membranes, or rolled to form concentric multilayered conduits. Results showed positive EC adhesion and a progressive repopulation by SMC. Functional changes in SMC gene expression and the constructs' bulk mechanical properties were concomitant with vessel remodeling as assessed over a 40-day culture period. A significant advantage with this approach is the ability to rapidly produce a cell-dense construct with an extracellular matrix similar in architecture and composition to natural vessels. The capacity to control physical parameters such as vessel diameter, wall thickness, shape, and length are critical to match vessel compliance and tailor vessel specifications to distinct anatomical locations. As such, this approach opens new avenues in a range of tissue regenerative applications that may have a much wider clinical impact.

  2. Challenges in tissue engineering

    PubMed Central

    Ikada, Yoshito

    2006-01-01

    Almost 30 years have passed since a term ‘tissue engineering’ was created to represent a new concept that focuses on regeneration of neotissues from cells with the support of biomaterials and growth factors. This interdisciplinary engineering has attracted much attention as a new therapeutic means that may overcome the drawbacks involved in the current artificial organs and organ transplantation that have been also aiming at replacing lost or severely damaged tissues or organs. However, the tissues regenerated by this tissue engineering and widely applied to patients are still very limited, including skin, bone, cartilage, capillary and periodontal tissues. What are the reasons for such slow advances in clinical applications of tissue engineering? This article gives the brief overview on the current tissue engineering, covering the fundamentals and applications. The fundamentals of tissue engineering involve the cell sources, scaffolds for cell expansion and differentiation and carriers for growth factors. Animal and human trials are the major part of the applications. Based on these results, some critical problems to be resolved for the advances of tissue engineering are addressed from the engineering point of view, emphasizing the close collaboration between medical doctors and biomaterials scientists. PMID:16971328

  3. Design Principles for Engineering of Tissues from Human Pluripotent Stem Cells

    PubMed Central

    Matthys, Oriane B.; Hookway, Tracy A.; McDevitt, Todd C.

    2016-01-01

    Recent advances in human pluripotent stem cell (hPSC) technologies have enabled the engineering of human tissue constructs for developmental studies, disease modeling, and drug screening platforms. In vitro tissue formation can be generally described at three levels of cellular organization. Multicellular hPSC constructs are initially formed either with polymeric scaffold materials or simply via self-assembly, adhesive mechanisms. Heterotypic interactions within hPSC tissue constructs can be achieved by physically mixing independently differentiated cell populations or coaxed to simultaneously co-emerge from a common population of undifferentiated cells. Higher order tissue architecture can be engineered by imposing external spatial constraints, such as molds and scaffolds, or depend upon cell-driven organization that exploits endogenous innate developmental mechanisms. The multicellular, heterogeneous, and highly organized structure of hPSC constructs ultimately dictates the resulting form and function of in vitro engineered human tissue models. PMID:27330934

  4. Volume Expansion of Tissue Engineered Human Nasal Septal Cartilage

    PubMed Central

    Reuther, Marsha S; Briggs, Kristen K; Neuman, Monica K; Masuda, Koichi; Sah, Robert L; Watson, Deborah

    2014-01-01

    Importance Cartilaginous craniofacial defects range in size and autologous cartilaginous tissue is preferred for repair of these defects. Therefore, it is important to have the ability to produce large size cartilaginous constructs for repair of cartilaginous abnormalities. Objectives To produce autologous human septal neocartilage constructs substantially larger in size than previously produced constructs To demonstrate that volume expanded neocartilage constructs possess comparable histological and biochemical properties to standard size constructs To show that volume expanded neocartilage constructs retain similar biomechanical properties to standard size constructs Design Prospective, basic science Setting Laboratory Participants The study used remnant human septal specimens removed during routine surgery at the University of California, San Diego Medical Center or San Diego Veterans Affairs Medical Center. Cartilage from a total of 8 donors was collected. Main Outcomes Measured Human septal chondrocytes from 8 donors were used to create 12mm and 24mm neocartilage constructs. These were cultured for a total of 10 weeks. Photo documentation, histological, biochemical, and biomechanical properties were measured and compared. Results The 24mm diameter constructs were qualitatively similar to the 12mm constructs. They possessed adequate strength and durability to be manually manipulated. Histological analysis of the constructs demonstrated similar staining patterns in standard and volume expanded constructs. Proliferation, as measured by DNA content, was similar in 24mm and 12mm constructs. Additionally, glycosaminoglycan (GAG) and total collagen content did not significantly differ between the two construct sizes. Biomechanical analysis of the 24mm and 12mm constructs demonstrated comparable compressive and tensile properties. Conclusion and Relevance Volume expanded human septal neocartilage constructs are qualitatively and histologically similar to standard 12mm

  5. Construction of tissue engineered skin with human amniotic mesenchymal stem cells and human amniotic epithelial cells.

    PubMed

    Yu, S-C; Xu, Y-Y; Li, Y; Xu, B; Sun, Q; Li, F; Zhang, X-G

    2015-12-01

    To establish a new model for construction of tissue engineered skin with human amniotic mesenchymal stem cells (hAMSCs) and human amniotic epithelial cells (hAECs). hAMSCs and hAECs were isolated from amniotic membrane. The morphology and phenotype of hAMSCs and hAECs were confirmed by microscope and flow cytometry, respectively. Then, we performed RT-PCR and immunofluorescence staining to assess the expression of stem cells and keratinocyte markers. Moreover, cell co-culture was performed to observe the growth and phenotype characteristics of hAMSCs and hAECs in vitro. In addition, tissue engineered skin with hAMSCs and hAECs was constructed and assessed with histological methods. hAMSCs and hAECs were successfully isolated, exhibiting fibroblast-like morphous and cobblestone-shape epithelial morphous, respectively. The surface biomarker analysis showed that hAMSCs and hAECs were both positive for CD73, CD90 and CD105, and negative for CD34 and HLA-DR. The RT-PCR showed that hAMSCs expressed stem cell marker Nanog and c-MYC, and keratinocyte marker K19, β1 integrin and K8, whereas hAECs expressed stem cell marker KLF4 and c-MYC, and keratinocyte marker K19, β1 integrin, K5 and K8. The expression of keratinocyte proliferation antigen K14 was also found on hAECs. Furthermore, we found co-culture has no impact on the phenotype of hAMSCs and hAECs, but increased the proliferation activity of hAECs and decreased the proliferation activity of hAMSCs. Finally, the histological analysis showed that the tissue engineered skin exhibited similar structure as normal skin. Tissue engineered skin with hAMSCs and hAECs was successfully constructed and shown a similar feature as a skin equivalent. The tissue engineered skin might have good application prospects in regenerative medicine.

  6. Noninvasive PET Imaging and Tracking of Engineered Human Muscle Precursor Cells for Skeletal Muscle Tissue Engineering.

    PubMed

    Haralampieva, Deana; Betzel, Thomas; Dinulovic, Ivana; Salemi, Souzan; Stoelting, Meline; Krämer, Stefanie D; Schibli, Roger; Sulser, Tullio; Handschin, Christoph; Eberli, Daniel; Ametamey, Simon M

    2016-09-01

    Transplantation of human muscle precursor cells (hMPCs) is envisioned for the treatment of various muscle diseases. However, a feasible noninvasive tool to monitor cell survival, migration, and integration into the host tissue is still missing. In this study, we designed an adenoviral delivery system to genetically modify hMPCs to express a signaling-deficient form of human dopamine D2 receptor (hD2R). The gene expression levels of the receptor were evaluated by reverse transcriptase polymerase chain reaction, and infection efficiency was evaluated by fluorescent microscopy. The viability, proliferation, and differentiation capacity of the transduced cells, as well as their myogenic phenotype, were determined by flow cytometry analysis and fluorescent microscopy. (18)F-fallypride and (18)F-fluoromisonidazole, two well-established PET radioligands, were assessed for their potential to image engineered hMPCs in a mouse model and their uptakes were evaluated at different time points after cell inoculation in vivo. Biodistribution studies, autoradiography, and PET experiments were performed to determine the extent of signal specificity. To address feasibility for tracking hMPCs in an in vivo model, the safety of the adenoviral gene delivery was evaluated. Finally, the harvested tissues were histologically examined to determine whether survival of the transplanted cells was sustained at different time points. Adenoviral gene delivery was shown to be safe, with no detrimental effects on the primary human cells. The viability, proliferation, and differentiation capacity of the transduced cells were confirmed, and flow cytometry analysis and fluorescent microscopy showed that their myogenic phenotype was sustained. (18)F-fallypride and (18)F-fluoromisonidazole were successfully synthesized. Specific binding of (18)F-fallypride to hD2R hMPCs was demonstrated in vitro and in vivo. Furthermore, the (18)F-fluoromisonidazole signal was high at the early stages. Finally

  7. Normal human epithelial cells regulate the size and morphology of tissue-engineered capillaries.

    PubMed

    Rochon, Marie-Hélène; Fradette, Julie; Fortin, Véronique; Tomasetig, Florence; Roberge, Charles J; Baker, Kathleen; Berthod, François; Auger, François A; Germain, Lucie

    2010-05-01

    The survival of thick tissues/organs produced by tissue engineering requires rapid revascularization after grafting. Although capillary-like structures have been reconstituted in some engineered tissues, little is known about the interaction between normal epithelial cells and endothelial cells involved in the in vitro angiogenic process. In the present study, we used the self-assembly approach of tissue engineering to examine this relationship. An endothelialized tissue-engineered dermal substitute was produced by adding endothelial cells to the tissue-engineered dermal substitute produced by the self-assembly approach. The latter consists in culturing fibroblasts in the medium supplemented with serum and ascorbic acid. A network of tissue-engineered capillaries (TECs) formed within the human extracellular matrix produced by dermal fibroblasts. To determine whether epithelial cells modify TECs, the size and form of TECs were studied in the endothelialized tissue-engineered dermal substitute cultured in the presence or absence of epithelial cells. In the presence of normal keratinocytes from skin, cornea or uterine cervix, endothelial cells formed small TECs (cross-sectional area estimated at less than 50 microm(2)) reminiscent of capillaries found in the skin's microcirculation. In contrast, TECs grown in the absence of epithelial cells presented variable sizes (larger than 50 microm(2)), but the addition of keratinocyte-conditioned media or exogenous vascular endothelial growth factor induced their normalization toward a smaller size. Vascular endothelial growth factor neutralization inhibited the effect of keratinocyte-conditioned media. These results provide new direct evidence that normal human epithelial cells play a role in the regulation of the underlying TEC network, and advance our knowledge in tissue engineering for the production of TEC networks in vitro.

  8. Adipose Tissue Engineering for Soft Tissue Regeneration

    PubMed Central

    Choi, Jennifer H.; Gimble, Jeffrey M.; Lee, Kyongbum; Marra, Kacey G.; Rubin, J. Peter; Yoo, James J.; Vunjak-Novakovic, Gordana

    2010-01-01

    Current treatment modalities for soft tissue defects caused by various pathologies and trauma include autologous grafting and commercially available fillers. However, these treatment methods present a number of challenges and limitations, such as donor-site morbidity and volume loss over time. As such, improved therapeutic modalities need to be developed. Tissue engineering techniques offer novel solutions to these problems through development of bioactive tissue constructs that can regenerate adipose tissue in both structure and function. Recently, a number of studies have been designed to explore various methods to engineer human adipose tissue. This review will focus on these developments in the area of adipose tissue engineering for soft tissue replacement. The physiology of adipose tissue and current surgical therapies used to replace lost tissue volume, specifically in breast tissue, are introduced, and current biomaterials, cell sources, and tissue culture strategies are discussed. We discuss future areas of study in adipose tissue engineering. PMID:20166810

  9. Human urinary bladder regeneration through tissue engineering - an analysis of 131 clinical cases.

    PubMed

    Pokrywczynska, Marta; Adamowicz, Jan; Sharma, Arun K; Drewa, Tomasz

    2014-03-01

    Replacement of urinary bladder tissue with functional equivalents remains one of the most challenging problems of reconstructive urology over the last several decades. The gold standard treatment for urinary diversion after radical cystectomy is the ileal conduit or neobladder; however, this technique is associated with numerous complications including electrolyte imbalances, mucus production, and the potential for malignant transformation. Tissue engineering techniques provide the impetus to construct functional bladder substitutes de novo. Within this review, we have thoroughly perused the literature utilizing PubMed in order to identify clinical studies involving bladder reconstruction utilizing tissue engineering methodologies. The idea of urinary bladder regeneration through tissue engineering dates back to the 1950s. Many natural and synthetic biomaterials such as plastic mold, gelatin sponge, Japanese paper, preserved dog bladder, lyophilized human dura, bovine pericardium, small intestinal submucosa, bladder acellular matrix, or composite of collagen and polyglycolic acid were used for urinary bladder regeneration with a wide range of outcomes. Recent progress in the tissue engineering field suggest that in vitro engineered bladder wall substitutes may have expanded clinical applicability in near future but preclinical investigations on large animal models with defective bladders are necessary to optimize the methods of bladder reconstruction by tissue engineering in humans.

  10. Fetal tissue engineering.

    PubMed

    Turner, Christopher G B; Fauza, Dario O

    2009-06-01

    Attempts at harnessing the prospective benefits of the therapeutic use of fetal cells or tissues date many decades before the modern era of transplantation. The first reported transplantation of human fetal tissue took place in 1922. Fetal cells or tissues also have been used as helpful investigational tools since the 1930s. Still, it was only in the last three decades that fetal tissue transplantation in people has started to lead to favorable outcomes, yet by and large anecdotally. This article offers an outlook on a relatively new dimension in fetal cell-based therapies, namely the engineering of tissues in the laboratory, along with its prospective applications.

  11. Effects of mechanical loading on human mesenchymal stem cells for cartilage tissue engineering.

    PubMed

    Choi, Jane Ru; Yong, Kar Wey; Choi, Jean Yu

    2017-05-19

    Today, articular cartilage damage is a major health problem, affecting people of all ages. The existing conventional articular cartilage repair techniques, such as autologous chondrocyte implantation (ACI), microfracture, and mosaicplasty, have many shortcomings which negatively affect their clinical outcomes. Therefore, it is essential to develop an alternative and efficient articular repair technique that can address those shortcomings. Cartilage tissue engineering, which aims to create a tissue-engineered cartilage derived from human mesenchymal stem cells (MSCs), shows great promise for improving articular cartilage defect therapy. However, the use of tissue-engineered cartilage for the clinical therapy of articular cartilage defect still remains challenging. Despite the importance of mechanical loading to create a functional cartilage has been well demonstrated, the specific type of mechanical loading and its optimal loading regime is still under investigation. This review summarizes the most recent advances in the effects of mechanical loading on human MSCs. First, the existing conventional articular repair techniques and their shortcomings are highlighted. The important parameters for the evaluation of the tissue-engineered cartilage, including chondrogenic and hypertrophic differentiation of human MSCs are briefly discussed. The influence of mechanical loading on human MSCs is subsequently reviewed and the possible mechanotransduction signaling is highlighted. The development of non-hypertrophic chondrogenesis in response to the changing mechanical microenvironment will aid in the establishment of a tissue-engineered cartilage for efficient articular cartilage repair. © 2017 Wiley Periodicals, Inc.

  12. High and low mammographic density human breast tissues maintain histological differential in murine tissue engineering chambers.

    PubMed

    Chew, G L; Huang, D; Lin, S J; Huo, C; Blick, T; Henderson, M A; Hill, P; Cawson, J; Morrison, W A; Campbell, I G; Hopper, J L; Southey, M C; Haviv, I; Thompson, E W

    2012-08-01

    Mammographic density (MD) is the area of breast tissue that appears radiologically white on mammography. Although high MD is a strong risk factor for breast cancer, independent of BRCA1/2 mutation status, the molecular basis of high MD and its associated breast cancer risk is poorly understood. MD studies will benefit from an animal model, where hormonal, gene and drug perturbations on MD can be measured in a preclinical context. High and low MD tissues were selectively sampled by stereotactic biopsy from operative specimens of high-risk women undergoing prophylactic mastectomy. The high and low MD tissues were transferred into separate vascularised biochambers in the groins of SCID mice. Chamber material was harvested after 6 weeks for histological analyses and immunohistochemistry for cytokeratins, vimentin and a human-specific mitochondrial antigen. Within-individual analysis was performed in replicate mice, eliminating confounding by age, body mass index and process-related factors, and comparisons were made to the parental human tissue. Maintenance of differential MD post-propagation was assessed radiographically. Immunohistochemical staining confirmed the preservation of human glandular and stromal components in the murine biochambers, with maintenance of radiographic MD differential. Propagated high MD regions had higher stromal (p = 0.0002) and lower adipose (p = 0.0006) composition, reflecting the findings in the original human breast tissue, although glands appeared small and non-complex in both high and low MD groups. No significant differences were observed in glandular area (p = 0.4) or count (p = 0.4) between high and low MD biochamber tissues. Human mammary glandular and stromal tissues were viably maintained in murine biochambers, with preservation of differential radiographic density and histological features. Our study provides a murine model for future studies into the biomolecular basis of MD as a risk factor for breast cancer.

  13. Scaffold-free cartilage tissue engineering with a small population of human nasoseptal chondrocytes.

    PubMed

    Chiu, Loraine L Y; To, William T H; Lee, John M; Waldman, Stephen D

    2017-03-01

    Cartilage tissue engineering is a promising approach to provide suitable materials for nasal reconstruction; however, it typically requires large numbers of cells. We have previously shown that a small number of chondrocytes cultivated within a continuous flow bioreactor can elicit substantial tissue growth, but translation to human chondrocytes is not trivial. Here, we aimed to demonstrate the application of the bioreactor to generate large-sized tissues from a small population of primary human nasoseptal chondrocytes. Experimental study. Chondrocytes were cultured in the bioreactor using different medium compositions, with varying amounts of serum and with or without growth factors. Resulting engineered tissues were analyzed for physical properties, biochemical composition, tissue microstructure, and protein localization. Bioreactor-cultivated constructs grown with serum and growth factors (basic fibroblast growth factor and transforming growth factor beta 2) had greater thickness, as well as DNA and glycosaminoglycan (GAG) contents, compared to low serum and no growth factor controls. These constructs also showed the most intense proteoglycan and collagen II staining. The combination of bioreactor conditions, serum, and growth factors allowed the generation of large, thick scaffold-free human cartilaginous tissues that resembled the native nasoseptal cartilage. There also may be implications for patient selection in future clinical applications of these engineered tissues because their GAG content decreased with donor age. NA. Laryngoscope, 127:E91-E99, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

  14. A tissue engineered human endometrial stroma that responds to cues for secretory differentiation, decidualization and menstruation

    PubMed Central

    Schutte, Stacey C.; Taylor, Robert N.

    2012-01-01

    Objective To show the responsiveness of a tissue engineered human endometrial stroma to combinations of hormones mimicking the secretory and menstrual phases of the cycle. Design In vitro experimental study Setting University uterine biology research laboratory Cells Telomerase immortalized human endometrial stromal cells Interventions The stromal cells were cultured in monolayers (2D) or encapsulated in a collagen I hydrogel (3D) to create a simplified tissue engineered stroma. The cells and tissues were exposed to hormone treatments mimicking early and late secretory phases, decidualization and steroid withdrawal conditions to recapitulate menstruation. Main Outcome Measure(s) Morphological and biochemical markers of decidualization and collagenase activity Result(s) The 3D tissue is capable of manifesting changes in morphology and biochemical markers of decidualization similar to 2D culture and characteristic of endometrial stroma in vivo. Unlike 2D culture, the 3D tissue responded to steroid withdrawal by increased collagenase activity and tissue breakdown. Conclusion(s) 3D tissue engineered endometrial stroma can mimic secretory and menstrual phases of the cycle and may be useful for studying uterine receptivity and menstruation in a physiological endocrine environment. PMID:22306710

  15. Decellularization of human stromal refractive lenticules for corneal tissue engineering

    PubMed Central

    Yam, Gary Hin-Fai; Yusoff, Nur Zahirah Binte M.; Goh, Tze-Wei; Setiawan, Melina; Lee, Xiao-Wen; Liu, Yu-Chi; Mehta, Jodhbir S.

    2016-01-01

    Small incision lenticule extraction (SMILE) becomes a procedure to correct myopia. The extracted lenticule can be used for other clinical scenarios. To prepare for allogeneic implantation, lenticule decellularization with preserved optical property, stromal architecture and chemistry would be necessary. We evaluated different methods to decellularize thin human corneal stromal lenticules created by femtosecond laser. Treatment with 0.1% sodium dodecylsulfate (SDS) followed by extensive washes was the most efficient protocol to remove cellular and nuclear materials. Empty cell space was found inside the stroma, which displayed aligned collagen fibril architecture similar to native stroma. The SDS-based method was superior to other treatments with hyperosmotic 1.5 M sodium chloride, 0.1% Triton X-100 and nucleases (from 2 to 10 U/ml DNase and RNase) in preserving extracellular matrix content (collagens, glycoproteins and glycosaminoglycans). The stromal transparency and light transmittance was indifferent to untreated lenticules. In vitro recellularization showed that the SDS-treated lenticules supported corneal stromal fibroblast growth. In vivo re-implantation into a rabbit stromal pocket further revealed the safety and biocompatibility of SDS-decellularized lenticules without short- and long-term rejection risk. Our results concluded that femtosecond laser-derived human stromal lenticules decellularized by 0.1% SDS could generate a transplantable bioscaffold with native-like stromal architecture and chemistry. PMID:27210519

  16. [The application progress of human urine derived stem cells in bone tissue engineering].

    PubMed

    Gao, Peng; Jiang, Dapeng; Li, Zhaozhu

    2016-04-01

    The research of bone tissue engineering bases on three basic directions of seed cells, scaffold materials and growth information. Stem cells have been widely studied as seed cells. Human urine-derived stem cell (hUSC) is extracted from urine and described to be adhesion growth, cloning, expression of the majority of mesenchymal stem cell markers and peripheral cell markers, multi-potential and no tumor but stable karyotype with passaging many times. Some researches proposed that hUSC might be a new source of seed cells in tissue engineering because of their invasive and convenient obtention, stable culture and multiple differentiation potential.

  17. Human flexor tendon tissue engineering: decellularization of human flexor tendons reduces immunogenicity in vivo.

    PubMed

    Raghavan, Shyam S; Woon, Colin Y L; Kraus, Armin; Megerle, Kai; Choi, Matthew S S; Pridgen, Brian C; Pham, Hung; Chang, James

    2012-04-01

    In mutilating hand injuries, tissue engineered tendon grafts may provide a reconstructive solution. We have previously described a method to decellularize cadaveric human flexor tendons while preserving mechanical properties and biocompatibility. The purpose of this study is to evaluate the immunogenicity and strength of these grafts when implanted into an immunocompetent rat model. Cadaveric human flexor tendons were divided into two groups. Group 1 was untreated, and Group 2 was decellularized by treatment with sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and peracetic acid (PAA). Both groups were then analyzed for the presence of major histocompatibility complexes by immunohistochemistry (IHC). Pair-matched tendons from each group were then placed into the dorsal subcutaneous tissue and anchored to the spinal ligaments of Wistar rats for 2 or 4 weeks, and harvested. The infiltration of B-cells and macrophages was determined using IHC. The explants where then subjected to mechanical testing to determine the ultimate tensile stress (UTS) and elastic modulus (EM). Statistical analysis was performed using a paired Student's t-test. The decellularization protocol successfully removed cells and MHC-1 complexes. At 2 weeks after implantation, there was increased infiltration of B-cells in Group 1 (untreated) compared with Group 2 (acellular), both in the capsule and tendon substance. There was improved ultimate tensile stress (UTS, 42.7 ± 8.3 vs. 22.8 ± 7.8 MPa, p<0.05) and EM (830.2 ± 206.7 vs. 421.2 ± 171.3 MPa, p<0.05) in tendons that were decellularized. At 4 weeks, there was continued B-cell infiltration in Group 1 (untreated) compared with Group 2 (acellular). There was no appreciable difference in macrophage infiltration at both time points. At 4 weeks Group 2 (acellular) demonstrated persistently greater UTS (40.5 ± 9.1 vs. 14.6 ± 4.2 MPa, p<0.05) and EM (454.05 ± 101.5 vs. 204.6 ± 91.3 MPa, p<0.05) compared with Group 1

  18. The Case for Applying Tissue Engineering Methodologies to Instruct Human Organoid Morphogenesis.

    PubMed

    Marti-Figueroa, Carlos R; Ashton, Randolph S

    2017-03-15

    Three-dimensional organoids derived from human pluripotent stem cell (hPSC) derivatives have become widely used in vitro models for studying development and disease. Their ability to recapitulate facets of normal human development during in vitro morphogenesis produces tissue structures with unprecedented biomimicry. Current organoid derivation protocols primarily rely on spontaneous morphogenesis processes to occur within 3-D spherical cell aggregates with minimal to no exogenous control. This yields organoids containing microscale regions of biomimetic tissues, but at the macroscale (i.e. 100's of microns to millimeters), the organoids' morphology, cytoarchitecture, and cellular composition are non-biomimetic and variable. The current lack of control over in vitro organoid morphogenesis at the microscale induces aberrations at the macroscale, which impedes realization of the technology's potential to reproducibly form anatomically correct human tissue units that could serve as optimal human in vitro models and even transplants. Here, we review tissue engineering methodologies that could be used to develop powerful approaches for instructing multiscale, 3-D human organoid morphogenesis. Such technological mergers are critically needed to harness organoid morphogenesis as a tool for engineering functional human tissues with biomimetic anatomy and physiology.

  19. [Obtention of human skin sheets by means of tissue engineering].

    PubMed

    Arvelo, Francisco; Pérez, Pedro; Cotte, Carlos

    2004-01-01

    The aim of this "in vitro" study was to develop a new system for keratinocyte culture on a dermal equivalent that enables treatment of different skin injuries. The keratinocyte where obtained from primary cell cultures derived from skin biopsies, seeded over a fibrin matrix enhanced with live human fibroblast. Cells growing over the dermal equivalent, rapidly confluences and a stratified epithelium was obtained within 20-25 days culture. Detachment of composite culture from flask is a simple and quick procedure with no need for chemical or enzyme treatments. The method described provides a number of advantages which include the large expansion of keratinocyte from the primary cell cultures without the need of a feeder layer, the availability of plasma from blood banks, and the versatile and safe manipulation of composite obtained "in vitro". All these facts allow to assure that this system could result very efficient for the treatment of all type of skin injuries.

  20. Tissue engineering potential of human dermis-isolated adult stem cells from multiple anatomical locations.

    PubMed

    Kwon, Heenam; Haudenschild, Anne K; Brown, Wendy E; Vapniarsky, Natalia; Paschos, Nikolaos K; Arzi, Boaz; Hu, Jerry C; Athanasiou, Kyriacos A

    2017-01-01

    Abundance and accessibility render skin-derived stem cells an attractive cell source for tissue engineering applications. Toward assessing their utility, the variability of constructs engineered from human dermis-isolated adult stem (hDIAS) cells was examined with respect to different anatomical locations (foreskin, breast, and abdominal skin), both in vitro and in a subcutaneous, athymic mouse model. All anatomical locations yielded hDIAS cells with multi-lineage differentiation potentials, though adipogenesis was not seen for foreskin-derived hDIAS cells. Using engineered cartilage as a model, tissue engineered constructs from hDIAS cells were compared. Construct morphology differed by location. The mechanical properties of human foreskin- and abdominal skin-derived constructs were similar at implantation, remaining comparable after 4 additional weeks of culture in vivo. Breast skin-derived constructs were not mechanically testable. For all groups, no signs of abnormality were observed in the host. Addition of aggregate redifferentiation culture prior to construct formation improved chondrogenic differentiation of foreskin-derived hDIAS cells, as evident by increases in glycosaminoglycan and collagen contents. More robust Alcian blue staining and homogeneous cell populations were also observed compared to controls. Human DIAS cells elicited no adverse host responses, reacted positively to chondrogenic regimens, and possessed multi-lineage differentiation potential with the caveat that efficacy may differ by anatomical origin of the skin. Taken together, these results suggest that hDIAS cells hold promise as a potential cell source for a number of tissue engineering applications.

  1. Stromal Cells in Dense Collagen Promote Cardiomyocyte and Microvascular Patterning in Engineered Human Heart Tissue.

    PubMed

    Roberts, Meredith A; Tran, Dominic; Coulombe, Kareen L K; Razumova, Maria; Regnier, Michael; Murry, Charles E; Zheng, Ying

    2016-04-01

    Cardiac tissue engineering is a strategy to replace damaged contractile tissue and model cardiac diseases to discover therapies. Current cardiac and vascular engineering approaches independently create aligned contractile tissue or perfusable vasculature, but a combined vascularized cardiac tissue remains to be achieved. Here, we sought to incorporate a patterned microvasculature into engineered heart tissue, which balances the competing demands from cardiomyocytes to contract the matrix versus the vascular lumens that need structural support. Low-density collagen hydrogels (1.25 mg/mL) permit human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to form a dense contractile tissue but cannot support a patterned microvasculature. Conversely, high collagen concentrations (density ≥6 mg/mL) support a patterned microvasculature, but the hESC-CMs lack cell-cell contact, limiting their electrical communication, structural maturation, and tissue-level contractile function. When cocultured with matrix remodeling stromal cells, however, hESC-CMs structurally mature and form anisotropic constructs in high-density collagen. Remodeling requires the stromal cells to be in proximity with hESC-CMs. In addition, cocultured cardiac constructs in dense collagen generate measurable active contractions (on the order of 0.1 mN/mm(2)) and can be paced up to 2 Hz. Patterned microvascular networks in these high-density cocultured cardiac constructs remain patent through 2 weeks of culture, and hESC-CMs show electrical synchronization. The ability to maintain microstructural control within engineered heart tissue enables generation of more complex features, such as cellular alignment and a vasculature. Successful incorporation of these features paves the way for the use of large scale engineered tissues for myocardial regeneration and cardiac disease modeling.

  2. Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

    NASA Technical Reports Server (NTRS)

    Powell, C.; Shansky, J.; Del Tatto, M.; Forman, D. E.; Hennessey, J.; Sullivan, K.; Zielinski, B. A.; Vandenburgh, H. H.

    1999-01-01

    Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

  3. Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

    NASA Technical Reports Server (NTRS)

    Powell, C.; Shansky, J.; Del Tatto, M.; Forman, D. E.; Hennessey, J.; Sullivan, K.; Zielinski, B. A.; Vandenburgh, H. H.

    1999-01-01

    Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

  4. Factors affecting the structure and maturation of human tissue engineered skeletal muscle.

    PubMed

    Martin, Neil R W; Passey, Samantha L; Player, Darren J; Khodabukus, Alastair; Ferguson, Richard A; Sharples, Adam P; Mudera, Vivek; Baar, Keith; Lewis, Mark P

    2013-07-01

    Tissue engineered skeletal muscle has great utility in experimental studies of physiology, clinical testing and its potential for transplantation to replace damaged tissue. Despite recent work in rodent tissue or cell lines, there is a paucity of literature concerned with the culture of human muscle derived cells (MDCs) in engineered constructs. Here we aimed to tissue engineer for the first time in the literature human skeletal muscle in self-assembling fibrin hydrogels and determine the effect of MDC seeding density and myogenic proportion on the structure and maturation of the constructs. Constructs seeded with 4 × 10(5) MDCs assembled to a greater extent than those at 1 × 10(5) or 2 × 10(5), and immunostaining revealed a higher fusion index and a higher density of myotubes within the constructs, showing greater structural semblance to in vivo tissue. These constructs primarily expressed perinatal and slow type I myosin heavy chain mRNA after 21 days in culture. In subsequent experiments MACS(®) technology was used to separate myogenic and non-myogenic cells from their heterogeneous parent population and these cells were seeded at varying myogenic (desmin +) proportions in fibrin based constructs. Only in the constructs seeded with 75% desmin + cells was there evidence of striations when immunostained for slow myosin heavy chain compared with constructs seeded with 10 or 50% desmin + cells. Overall, this work reveals the importance of cell number and myogenic proportions in tissue engineering human skeletal muscle with structural resemblance to in vivo tissue.

  5. Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function.

    PubMed

    Grant, Christa N; Mojica, Salvador Garcia; Sala, Frederic G; Hill, J Ryan; Levin, Daniel E; Speer, Allison L; Barthel, Erik R; Shimada, Hiroyuki; Zachos, Nicholas C; Grikscheit, Tracy C

    2015-04-15

    Short bowel syndrome (SBS) is a devastating condition in which insufficient small intestinal surface area results in malnutrition and dependence on intravenous parenteral nutrition. There is an increasing incidence of SBS, particularly in premature babies and newborns with congenital intestinal anomalies. Tissue-engineered small intestine (TESI) offers a therapeutic alternative to the current standard treatment, intestinal transplantation, and has the potential to solve its biggest challenges, namely donor shortage and life-long immunosuppression. We have previously demonstrated that TESI can be generated from mouse and human small intestine and histologically replicates key components of native intestine. We hypothesized that TESI also recapitulates native small intestine function. Organoid units were generated from mouse or human donor intestine and implanted into genetically identical or immunodeficient host mice. After 4 wk, TESI was harvested and either fixed and paraffin embedded or immediately subjected to assays to illustrate function. We demonstrated that both mouse and human tissue-engineered small intestine grew into an appropriately polarized sphere of intact epithelium facing a lumen, contiguous with supporting mesenchyme, muscle, and stem/progenitor cells. The epithelium demonstrated major ultrastructural components, including tight junctions and microvilli, transporters, and functional brush-border and digestive enzymes. This study demonstrates that tissue-engineered small intestine possesses a well-differentiated epithelium with intact ion transporters/channels, functional brush-border enzymes, and similar ultrastructural components to native tissue, including progenitor cells, whether derived from mouse or human cells.

  6. Comparison of therapeutic antibiotic treatments on tissue-engineered human skin substitutes.

    PubMed

    Gibson, Angela L; Schurr, Michael J; Schlosser, Sandy J; Comer, Allen R; Allen-Hoffmann, B Lynn

    2008-05-01

    For regenerative medicine to gain clinical acceptance, the effects of commonly used treatment regimens on bioengineered organs must be considered. The antibiotics mafenide acetate (mafenide) and neomycin plus polymyxin (neo/poly) are routinely used to irrigate postoperative skin grafts on contaminated wounds. The effects of these clinically used antibiotics were investigated using tissue-engineered human skin substitutes generated with primary human keratinocytes or the near-diploid human keratinocyte cell line, Near-diploid Immortal Keratinocytes. Following topical or dermal treatment, the skin substitutes were assayed for viability, tissue morphology, glycogen content, and the expression of active caspase 3. Mafenide, but not neo/poly, induced morphological and biochemical changes in tissue-engineered skin substitutes. Keratinocytes in all histological layers of mafenide-treated skin substitutes exhibited ballooning degeneration and glycogen depletion. Mafenide-treatment also triggered separation of basal keratinocytes from the underlying dermis. None of the antibiotic treatments induced apoptosis, as measured by active caspase 3 immunostaining. The results demonstrate that mafenide, but not neo/poly, is detrimental to the viability and structural integrity of tissue-engineered human skin substitutes. These findings highlight the need to identify treatment regimens that are compatible with and hence enable the therapeutic efficacy of first-generation bioengineered organs such as skin.

  7. Tissue engineering of human nasal alar cartilage precisely by using three-dimensional printing.

    PubMed

    Xu, Yihao; Fan, Fei; Kang, Ning; Wang, Sheng; You, Jianjun; Wang, Huan; Zhang, Bo

    2015-02-01

    Tissue engineering strategies hold promise for the restoration of damaged cartilage. However, the results of most studies report irregularly shaped beads of cartilage, which are not precise enough. Thus, a precise shape of cartilage graft must be taken into consideration. The goal of this study was to develop a simple method of creating a precisely predetermined nasal alar shape with the aid of three-dimensional printing. Lower lateral cartilage from cadavers was observed and scanned by computed tomography. Molds of the lower lateral cartilage were achieved by using three-dimensional printing. Human nasal cartilage was obtained and chondrocytes were harvested. Then, the mixture of cells and poly(glycolic acid)/poly-L-lactic acid was cultured in vitro and implanted into the subcutaneous tissue of nude mice. After subcutaneous implantation, the length and width of the samples were measured, and the results were not statistically significantly different from the native lower lateral cartilage (p > 0.05). Their thickness was measured and the results were statistically different from the native lower lateral cartilage (p < 0.05). Histologic examination of the engineered constructs revealed that both the cell and tissue morphologic features of engineered cartilage were similar to those of native lower lateral cartilage. The biomechanical properties of the engineered cartilage exceeded those of native cartilage. This study demonstrates that three-dimensional printing-aided tissue engineering can achieve precise three-dimensional shapes of human nasal alar cartilage. To our knowledge, this is the first reported creation of a precise nasal alar cartilage with a tissue-engineering strategy and three-dimensional printing technique.

  8. Development and characterization of decellularized human nasoseptal cartilage matrix for use in tissue engineering.

    PubMed

    Graham, M Elise; Gratzer, Paul F; Bezuhly, Michael; Hong, Paul

    2016-10-01

    Reconstruction of cartilage defects in the head and neck can require harvesting of autologous cartilage grafts, which can be associated with donor site morbidity. To overcome this limitation, tissue-engineering approaches may be used to generate cartilage grafts. The objective of this study was to decellularize and characterize human nasoseptal cartilage with the aim of generating a biological scaffold for cartilage tissue engineering. Laboratory study using nasoseptal cartilage. Remnant human nasoseptal cartilage specimens were collected and subjected to a novel decellularization treatment. The decellularization process involved several cycles of enzymatic detergent treatments. For characterization, decellularized and fresh (control) specimens underwent histological, biochemical, and mechanical analyses. Scanning electron microscopy and biocompatibility assay were also performed. The decellularization process had minimal effect on glycosaminoglycan content of the cartilage extracellular matrix. Deoxyribonucleic acid (DNA) analysis revealed the near-complete removal of genomic DNA from decellularized tissues. The effectiveness of the decellularization process was also confirmed on histological and scanning electron microscopic analyses. Mechanical testing results showed that the structural integrity of the decellularized tissue was maintained, and biocompatibility was confirmed. Overall, the current decellularization treatment resulted in significant reduction of genetic/cellular material with preservation of the underlying extracellular matrix structure. This decellularized material may serve as a potential scaffold for cartilage tissue engineering. N/A. Laryngoscope, 126:2226-2231, 2016. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

  9. Vascularized subcutaneous human liver tissue from engineered hepatocyte/fibroblast sheets in mice.

    PubMed

    Sakai, Yusuke; Yamanouchi, Kosho; Ohashi, Kazuo; Koike, Makiko; Utoh, Rie; Hasegawa, Hideko; Muraoka, Izumi; Suematsu, Takashi; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Kuroki, Tamotsu; Eguchi, Susumu

    2015-10-01

    Subcutaneous liver tissue engineering is an attractive and minimally invasive approach used to curative treat hepatic failure and inherited liver diseases. However, graft failure occurs frequently due to insufficient infiltration of blood vessels (neoangiogenesis), while the maintenance of hepatocyte phenotype and function requires in vivo development of the complex cellular organization of the hepatic lobule. Here we describe a subcutaneous human liver construction allowing for rapidly vascularized grafts by transplanting engineered cellular sheets consisting of human primary hepatocytes adhered onto a fibroblast layer. The engineered hepatocyte/fibroblast sheets (EHFSs) showed superior expression levels of vascularization-associated growth factors (vascular endothelial growth factor, transforming growth factor beta 1, and hepatocyte growth factor) in vitro. EHFSs developed into vascularized subcutaneous human liver tissues contained glycogen stores, synthesized coagulation factor IX, and showed significantly higher synthesis rates of liver-specific proteins (albumin and alpha 1 anti-trypsin) in vivo than tissues from hepatocyte-only sheets. The present study describes a new approach for vascularized human liver organogenesis under mouse skin. This approach could prove valuable for establishing novel cell therapies for liver diseases.

  10. Advanced imaging and tissue engineering of the human limbal epithelial stem cell niche.

    PubMed

    Massie, Isobel; Dziasko, Marc; Kureshi, Alvena; Levis, Hannah J; Morgan, Louise; Neale, Michael; Sheth, Radhika; Tovell, Victoria E; Vernon, Amanda J; Funderburgh, James L; Daniels, Julie T

    2015-01-01

    The limbal epithelial stem cell niche provides a unique, physically protective environment in which limbal epithelial stem cells reside in close proximity with accessory cell types and their secreted factors. The use of advanced imaging techniques is described to visualize the niche in three dimensions in native human corneal tissue. In addition, a protocol is provided for the isolation and culture of three different cell types, including human limbal epithelial stem cells from the limbal niche of human donor tissue. Finally, the process of incorporating these cells within plastic compressed collagen constructs to form a tissue-engineered corneal limbus is described and how immunohistochemical techniques may be applied to characterize cell phenotype therein.

  11. Advanced Imaging and Tissue Engineering of the Human Limbal Epithelial Stem Cell Niche

    PubMed Central

    Massie, Isobel; Dziasko, Marc; Kureshi, Alvena; Levis, Hannah J.; Morgan, Louise; Neale, Michael; Sheth, Radhika; Tovell, Victoria E.; Vernon, Amanda J.; Funderburgh, James L.; Daniels, Julie T.

    2015-01-01

    The limbal epithelial stem cell niche provides a unique, physically protective environment in which limbal epithelial stem cells reside in close proximity with accessory cell types and their secreted factors. The use of advanced imaging techniques is described to visualize the niche in three dimensions in native human corneal tissue. In addition, a protocol is provided for the isolation and culture of three different cell types, including human limbal epithelial stem cells from the limbal niche of human donor tissue. Finally, the process of incorporating these cells within plastic compressed collagen constructs to form a tissue-engineered corneal limbus is described and how immunohistochemical techniques may be applied to characterize cell phenotype therein. PMID:25388395

  12. Tissue engineering for clinical applications.

    PubMed

    Bhatia, Sujata K

    2010-12-01

    Tissue engineering is increasingly being recognized as a beneficial means for lessening the global disease burden. One strategy of tissue engineering is to replace lost tissues or organs with polymeric scaffolds that contain specialized populations of living cells, with the goal of regenerating tissues to restore normal function. Typical constructs for tissue engineering employ biocompatible and degradable polymers, along with organ-specific and tissue-specific cells. Once implanted, the construct guides the growth and development of new tissues; the polymer scaffold degrades away to be replaced by healthy functioning tissue. The ideal biomaterial for tissue engineering not only defends against disease and supports weakened tissues or organs, it also provides the elements required for healing and repair, stimulates the body's intrinsic immunological and regenerative capacities, and seamlessly interacts with the living body. Tissue engineering has been investigated for virtually every organ system in the human body. This review describes the potential of tissue engineering to alleviate disease, as well as the latest advances in tissue regeneration. The discussion focuses on three specific clinical applications of tissue engineering: cardiac tissue regeneration for treatment of heart failure; nerve regeneration for treatment of stroke; and lung regeneration for treatment of chronic obstructive pulmonary disease. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Creation of a Large Adipose Tissue Construct in Humans Using a Tissue-engineering Chamber: A Step Forward in the Clinical Application of Soft Tissue Engineering☆

    PubMed Central

    Morrison, Wayne A.; Marre, Diego; Grinsell, Damien; Batty, Andrew; Trost, Nicholas; O'Connor, Andrea J.

    2016-01-01

    Tissue engineering is currently exploring new and exciting avenues for the repair of soft tissue and organ defects. Adipose tissue engineering using the tissue engineering chamber (TEC) model has yielded promising results in animals; however, to date, there have been no reports on the use of this device in humans. Five female post mastectomy patients ranging from 35 to 49 years old were recruited and a pedicled thoracodorsal artery perforator fat flap ranging from 6 to 50 ml was harvested, transposed onto the chest wall and covered by an acrylic perforated dome-shaped chamber ranging from 140 to 350 cm3. Magnetic resonance evaluation was performed at three and six months after chamber implantation. Chambers were removed at six months and samples were obtained for histological analysis. In one patient, newly formed tissue to a volume of 210 ml was generated inside the chamber. One patient was unable to complete the trial and the other three failed to develop significant enlargement of the original fat flap, which, at the time of chamber explantation, was encased in a thick fibrous capsule. Our study provides evidence that generation of large well-vascularized tissue engineered constructs using the TEC is feasible in humans. PMID:27211566

  14. Tissue engineering potential of human dermis-isolated adult stem cells from multiple anatomical locations

    PubMed Central

    Brown, Wendy E.; Vapniarsky, Natalia; Paschos, Nikolaos K.; Arzi, Boaz; Hu, Jerry C.

    2017-01-01

    Abundance and accessibility render skin-derived stem cells an attractive cell source for tissue engineering applications. Toward assessing their utility, the variability of constructs engineered from human dermis-isolated adult stem (hDIAS) cells was examined with respect to different anatomical locations (foreskin, breast, and abdominal skin), both in vitro and in a subcutaneous, athymic mouse model. All anatomical locations yielded hDIAS cells with multi-lineage differentiation potentials, though adipogenesis was not seen for foreskin-derived hDIAS cells. Using engineered cartilage as a model, tissue engineered constructs from hDIAS cells were compared. Construct morphology differed by location. The mechanical properties of human foreskin- and abdominal skin-derived constructs were similar at implantation, remaining comparable after 4 additional weeks of culture in vivo. Breast skin-derived constructs were not mechanically testable. For all groups, no signs of abnormality were observed in the host. Addition of aggregate redifferentiation culture prior to construct formation improved chondrogenic differentiation of foreskin-derived hDIAS cells, as evident by increases in glycosaminoglycan and collagen contents. More robust Alcian blue staining and homogeneous cell populations were also observed compared to controls. Human DIAS cells elicited no adverse host responses, reacted positively to chondrogenic regimens, and possessed multi-lineage differentiation potential with the caveat that efficacy may differ by anatomical origin of the skin. Taken together, these results suggest that hDIAS cells hold promise as a potential cell source for a number of tissue engineering applications. PMID:28767737

  15. Human Corneal Endothelial Cells Expanded In Vitro Are a Powerful Resource for Tissue Engineering.

    PubMed

    Liu, Yongsong; Sun, Hong; Hu, Min; Zhu, Min; Tighe, Sean; Chen, Shuangling; Zhang, Yuan; Su, Chenwei; Cai, Subo; Guo, Ping

    2017-01-01

    Human corneal endothelial cells have two major functions: barrier function mediated by proteins such as ZO-1 and pump function mediated by Na-K-ATPase which help to maintain visual function. However, human corneal endothelial cells are notorious for their limited proliferative capability in vivo and are therefore prone to corneal endothelial dysfunction that eventually may lead to blindness. At present, the only method to cure corneal endothelial dysfunction is by transplantation of a cadaver donor cornea with normal corneal endothelial cells. Due to the global shortage of donor corneas, it is vital to engineer corneal tissue in vitro that could potentially be transplanted clinically. In this review, we summarize the advances in understanding the behavior of human corneal endothelial cells, their current engineering strategy in vitro and their potential applications.

  16. Human Corneal Endothelial Cells Expanded In Vitro Are a Powerful Resource for Tissue Engineering

    PubMed Central

    Liu, Yongsong; Sun, Hong; Hu, Min; Zhu, Min; Tighe, Sean; Chen, Shuangling; Zhang, Yuan; Su, Chenwei; Cai, Subo; Guo, Ping

    2017-01-01

    Human corneal endothelial cells have two major functions: barrier function mediated by proteins such as ZO-1 and pump function mediated by Na-K-ATPase which help to maintain visual function. However, human corneal endothelial cells are notorious for their limited proliferative capability in vivo and are therefore prone to corneal endothelial dysfunction that eventually may lead to blindness. At present, the only method to cure corneal endothelial dysfunction is by transplantation of a cadaver donor cornea with normal corneal endothelial cells. Due to the global shortage of donor corneas, it is vital to engineer corneal tissue in vitro that could potentially be transplanted clinically. In this review, we summarize the advances in understanding the behavior of human corneal endothelial cells, their current engineering strategy in vitro and their potential applications. PMID:28260988

  17. Engineering complex tissues.

    PubMed

    Atala, Anthony; Kasper, F Kurtis; Mikos, Antonios G

    2012-11-14

    Tissue engineering has emerged at the intersection of numerous disciplines to meet a global clinical need for technologies to promote the regeneration of functional living tissues and organs. The complexity of many tissues and organs, coupled with confounding factors that may be associated with the injury or disease underlying the need for repair, is a challenge to traditional engineering approaches. Biomaterials, cells, and other factors are needed to design these constructs, but not all tissues are created equal. Flat tissues (skin); tubular structures (urethra); hollow, nontubular, viscus organs (vagina); and complex solid organs (liver) all present unique challenges in tissue engineering. This review highlights advances in tissue engineering technologies to enable regeneration of complex tissues and organs and to discuss how such innovative, engineered tissues can affect the clinic.

  18. In vitro cytokeratin expression profiling of human oral mucosa substitutes developed by tissue engineering.

    PubMed

    Garzon, Ingrid; Serrato, Deyanira; Roda, Olga; Del Carmen Sanchez-Quevedo, Maria; Gonzales-Jaranay, Maximino; Moreu, Gerardo; Nieto-Aguilar, Renato; Alaminos, Miguel; Campos, Antonio

    2009-10-01

    In this work we performed a study of cytokeratin (CK) expression profiling on human artificial oral mucosa developed in vitro by tissue engineering at different stages of maturation (from immature to well-developed stages) at the protein and mRNA levels. Human artificial oral mucosa was generated in the laboratory using fibrin-agarose biomaterials. As controls, we used human native normal oral mucosa and embryonic oral tissues. Our results demonstrated that human embryonic oral tissues tended to express CK8 and CK19. In contrast, monolayered bioengineered oral mucosa did not show any CK expression by immunohistochemistry, whereas bilayered and multilayered artificial oral mucosa showed several markers of stratified epithelia, but did not express CK10. These results suggest that the CK expression pattern is strongly dependent on the maturation state of the artificial tissues and that the CK expression profile of our model of artificial oral mucosa was partially similar to that of the non-keratinized human adult oral mucosa. However, the expression of CK8 by the artificial oral mucosa suggests that these samples correspond to an early stage of development while kept in vitro.

  19. Tissue bionics: examples in biomimetic tissue engineering.

    PubMed

    Green, David W

    2008-09-01

    Many important lessons can be learnt from the study of biological form and the functional design of organisms as design criteria for the development of tissue engineering products. This merging of biomimetics and regenerative medicine is termed 'tissue bionics'. Clinically useful analogues can be generated by appropriating, modifying and mimicking structures from a diversity of natural biomatrices ranging from marine plankton shells to sea urchin spines. Methods in biomimetic materials chemistry can also be used to fabricate tissue engineering scaffolds with added functional utility that promise human tissues fit for the clinic.

  20. Mesenchymal Stromal Cells Derived from Human Umbilical Cord Tissues: Primitive Cells with Potential for Clinical and Tissue Engineering Applications

    NASA Astrophysics Data System (ADS)

    Moretti, Pierre; Hatlapatka, Tim; Marten, Dana; Lavrentieva, Antonina; Majore, Ingrida; Hass, Ralf; Kasper, Cornelia

    Mesenchymal stem or stromal cells (MSCs) have a high potential for cell-based therapies as well as for tissue engineering applications. Since Friedenstein first isolated stem or precursor cells from the human bone marrow (BM) stroma that were capable of osteogenesis, BM is currently the most common source for MSCs. However, BM presents several disadvantages, namely low frequency of MSCs, high donor-dependent variations in quality, and painful invasive intervention. Thus, tremendous research efforts have been observed during recent years to find alternative sources for MSCs.

  1. Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system

    PubMed Central

    Workman, Michael J; Mahe, Maxime M; Trisno, Stephen; Poling, Holly M; Watson, Carey L; Sundaram, Nambirajan; Chang, Ching-Fang; Schiesser, Jacqueline; Aubert, Philippe; Stanley, Edouard G; Elefanty, Andrew G; Miyaoka, Yuichiro; Mandegar, Mohammad A; Conklin, Bruce R; Neunlist, Michel; Brugmann, Samantha A; Helmrath, Michael A; Wells, James M

    2017-01-01

    The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract. PMID:27869805

  2. Electrospun multifunctional tissue engineering scaffolds

    NASA Astrophysics Data System (ADS)

    Wang, Chong; Wang, Min

    2014-03-01

    Tissue engineering holds great promises in providing successful treatments of human body tissue loss that current methods are unable to treat or unable to achieve satisfactory clinical outcomes. In scaffold-based tissue engineering, a highperformance scaffold underpins the success of a tissue engineering strategy and a major direction in the field is to create multifunctional tissue engineering scaffolds for enhanced biological performance and for regenerating complex body tissues. Electrospinning can produce nanofibrous scaffolds that are highly desirable for tissue engineering. The enormous interest in electrospinning and electrospun fibrous structures by the science, engineering and medical communities has led to various developments of the electrospinning technology and wide investigations of electrospun products in many industries, including biomedical engineering, over the past two decades. It is now possible to create novel, multicomponent tissue engineering scaffolds with multiple functions. This article provides a concise review of recent advances in the R & D of electrospun multifunctional tissue engineering scaffolds. It also presents our philosophy and research in the designing and fabrication of electrospun multicomponent scaffolds with multiple functions.

  3. Using human umbilical cord cells for tissue engineering: a comparison with skin cells.

    PubMed

    Hayward, Cindy J; Fradette, Julie; Morissette Martin, Pascal; Guignard, Rina; Germain, Lucie; Auger, François A

    2014-01-01

    The epithelial cells and Wharton׳s jelly cells (WJC) from the human umbilical cord have yet to be extensively studied in respect to their capacity to generate tissue-engineered substitutes for clinical applications. Our reconstruction strategy, based on the self-assembly approach of tissue engineering, allows the production of various types of living human tissues such as skin and cornea from a wide range of cell types originating from post-natal tissue sources. Here we placed epithelial cells and WJC from the umbilical cord in the context of a reconstructed skin substitute in combination with skin keratinocytes and fibroblasts. We compared the ability of the epithelial cells from both sources to generate a stratified, differentiated skin-like epithelium upon exposure to air when cultured on the two stromal cell types. Conversely, the ability of the WJC to behave as dermal fibroblasts, producing extracellular matrix and supporting the formation of a differentiated epithelium for both types of epithelial cells, was also investigated. Of the four types of constructs produced, the combination of WJC and keratinocytes was the most similar to skin engineered from dermal fibroblasts and keratinocytes. When cultured on dermal fibroblasts, the cord epithelial cells were able to differentiate in vitro into a stratified multilayered epithelium expressing molecules characteristic of keratinocyte differentiation after exposure to air, and maintaining the expression of keratins K18 and K19, typical of the umbilical cord epithelium. WJC were able to support the growth and differentiation of keratinocytes, especially at the early stages of air-liquid culture. In contrast, cord epithelial cells cultured on WJC did not form a differentiated epidermis when exposed to air. These results support the premise that the tissue from which cells originate can largely affect the properties and homoeostasis of reconstructed substitutes featuring both epithelial and stromal compartments

  4. The Use of Human Wharton's Jelly Cells for Cochlear Tissue Engineering.

    PubMed

    Mellott, Adam J; Detamore, Michael S; Staecker, Hinrich

    2016-01-01

    Tissue engineering focuses on three primary components: stem cells, biomaterials, and growth factors. Together, the combination of these components is used to regrow and repair damaged tissues that normally do not regenerate easily on their own. Much attention has been focused on the use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), due to their broad differentiation potential. However, ESCs and iPSCs require very detailed protocols to differentiate into target tissues, which are not always successful. Furthermore, procurement of ESCs is considered ethically controversial in some regions and procurement of iPSCs requires laborious transformation of adult tissues and characterization. However, mesenchymal stem cells are an adult stem cell population that are not ethically controversial and are readily available for procurement. Furthermore, mesenchymal stem cells exhibit the ability to differentiate into a variety of cell types arising from the mesoderm. In particular, human Wharton's jelly cells (hWJCs) are mesenchymal-type stem cells found in umbilical cords that possess remarkable differentiation potential. hWJCs are a highly desirable stem cell population due to their abundance in supply, high proliferation rates, and ability to differentiate into multiple cell types arising from all three germ layers. hWJCs are used to generate several neurological phenotypes arising from the ectoderm and are considered for engineering mechanosensory hair cells found in the auditory complex. Here, we report the methods for isolating hWJCs from human umbilical cords and non-virally transfected for use in cochlear tissue engineering studies.

  5. Esophageal tissue engineering.

    PubMed

    Luc, Guillaume; Durand, Marlène; Collet, Denis; Guillemot, Fabien; Bordenave, Laurence

    2014-03-01

    Esophageal tissue engineering is still in an early state, and ideal methods have not been developed. Since the beginning of the 20th century, advances have been made in the materials that can be used to produce an esophageal substitute. Three approaches to scaffold-based tissue engineering have yielded good results. The first development concerned non-absorbable constructs based on silicone and collagen. The need to remove the silicone tube is the main disadvantage of this material. Polymeric absorbable scaffolds have been used since the 1990s. The main polymeric material used is poly (glycolic) acid combined with collagen. The problem of stenosis remains prevalent in most studies using an absorbable construct. Finally, decellularized scaffolds have been used since 2000. The promises of this new approach are unfulfilled. Indeed, stenosis occurs when the esophageal defect is circumferential regardless of the scaffold materials. Cell supplementation can decrease the rate of stenosis, but the type(s) of cells and their roles have not been defined. Finally, esophageal tissue engineering cannot provide a functional esophageal substitute, and further development is necessary prior to conducting human clinical studies.

  6. Biomechanical characterisation of the human nasal cartilages; implications for tissue engineering.

    PubMed

    Griffin, M F; Premakumar, Y; Seifalian, A M; Szarko, M; Butler, P E M

    2016-01-01

    Nasal reconstruction is currently performed using autologous grafts provides but is limited by donor site morbidity, tissue availability and potentially graft failure. Additionally, current alternative alloplastic materials are limited by their high extrusion and infection rates. Matching mechanical properties of synthetic materials to the native tissue they are replacing has shown to be important in the biocompatibility of implants. To date the mechanical properties of the human nasal cartilages has not been studied in depth to be able to create tissue-engineered replacements with similar mechanical properties to native tissue. The young's modulus was characterized in compression on fresh-frozen human cadaveric septal, alar, and lateral cartilage. Due to the functional differences experienced by the various aspects of the septal cartilage, 16 regions were evaluated with an average elastic modulus of 2.72 ± 0.63 MPa. Furthermore, the posterior septum was found to be significantly stiffer than the anterior septum (p < 0.01). The medial and lateral alar cartilages were tested at four points with an elastic modulus ranging from 2.09 ± 0.81 MPa, with no significant difference between the cartilages (p < 0.78). The lateral cartilage was tested once in all cadavers with an average elastic modulus of 0.98 ± 0.29 MPa. In conclusion, this study provides new information on the compressive mechanical properties of the human nasal cartilage, allowing surgeons to have a better understanding of the difference between the mechanical properties of the individual nasal cartilages. This study has provided a reference, by which tissue-engineered should be developed for effective cartilage replacements for nasal reconstruction.

  7. Tissue engineering from human mesenchymal amniocytes: a prelude to clinical trials.

    PubMed

    Kunisaki, Shaun M; Armant, Myriam; Kao, Grace S; Stevenson, Kristen; Kim, Haesook; Fauza, Dario O

    2007-06-01

    The surgical treatment of congenital anomalies using tissues engineered from amniotic fluid-derived mesenchymal cells has been validated experimentally. As a prerequisite for testing the clinical feasibility of this therapeutic concept, this study was aimed to expand human mesenchymal amniocytes in the absence of animal products. Human mesenchymal cells were isolated from amniotic fluid samples (n = 12) obtained at 20 to 37 weeks' gestation. Their phenotypic profiles and cell proliferation rates were compared during expansion under 2 different media, containing either fetal bovine serum or allogeneic human AB serum. Statistical analyses were by the 2-sided Wilcoxon signed rank test and linear regression (P < .05). Mesenchymal cells could be isolated and expanded at any gestational age. There was a greater than 9-fold logarithmic expansion of mesenchymal cells, with no significant differences in the overall proliferation rates based on serum type (P = .94), or gestational age (P = .14). At any passage, cells expanded for up to 50 days remained positive for markers consistent with a multipotent mesenchymal progenitor lineage, regardless of the medium used. Human mesenchymal amniocytes retain their progenitor phenotype and can be dependably expanded ex vivo in the absence of animal serum. Clinical trials of amniotic fluid-based tissue engineering are feasible within preferred regulatory guidelines.

  8. Engineering Complex Tissues

    PubMed Central

    MIKOS, ANTONIOS G.; HERRING, SUSAN W.; OCHAREON, PANNEE; ELISSEEFF, JENNIFER; LU, HELEN H.; KANDEL, RITA; SCHOEN, FREDERICK J.; TONER, MEHMET; MOONEY, DAVID; ATALA, ANTHONY; VAN DYKE, MARK E.; KAPLAN, DAVID; VUNJAK-NOVAKOVIC, GORDANA

    2010-01-01

    This article summarizes the views expressed at the third session of the workshop “Tissue Engineering—The Next Generation,” which was devoted to the engineering of complex tissue structures. Antonios Mikos described the engineering of complex oral and craniofacial tissues as a “guided interplay” between biomaterial scaffolds, growth factors, and local cell populations toward the restoration of the original architecture and function of complex tissues. Susan Herring, reviewing osteogenesis and vasculogenesis, explained that the vascular arrangement precedes and dictates the architecture of the new bone, and proposed that engineering of osseous tissues might benefit from preconstruction of an appropriate vasculature. Jennifer Elisseeff explored the formation of complex tissue structures based on the example of stratified cartilage engineered using stem cells and hydrogels. Helen Lu discussed engineering of tissue interfaces, a problem critical for biological fixation of tendons and ligaments, and the development of a new generation of fixation devices. Rita Kandel discussed the challenges related to the re-creation of the cartilage-bone interface, in the context of tissue engineered joint repair. Frederick Schoen emphasized, in the context of heart valve engineering, the need for including the requirements derived from “adult biology” of tissue remodeling and establishing reliable early predictors of success or failure of tissue engineered implants. Mehmet Toner presented a review of biopreservation techniques and stressed that a new breakthrough in this field may be necessary to meet all the needs of tissue engineering. David Mooney described systems providing temporal and spatial regulation of growth factor availability, which may find utility in virtually all tissue engineering and regeneration applications, including directed in vitro and in vivo vascularization of tissues. Anthony Atala offered a clinician’s perspective for functional tissue

  9. Production of human tissue-engineered skin trilayer on a plasma-based hypodermis.

    PubMed

    Monfort, Asun; Soriano-Navarro, Mario; García-Verdugo, José Manuel; Izeta, Ander

    2013-06-01

    Full thickness wounds require a dermal component to achieve functional permanent skin restoration. Currently available tissue-engineered skin substitutes lack a subcutaneous fat layer that would functionally contribute some of the mechanical and thermoregulatory properties of normal skin. To generate a trilayer engineered skin equivalent, we included bone marrow mesenchymal (BM-MSC) or adipose tissue-derived (ASC) stromal cells in a human plasma hydrogel exposed to adipogenic clues for three weeks. Approximately half of the cells differentiated under these conditions into mature adipocytes that survived for two years in culture with minimal medium change. In vitro generation of bona fide fully differentiated adipocytes was assessed by leptin secretion and ultrastructurally demonstrated through semithin to ultrathin sectioning and lipid staining with osmium tetroxide. Furthermore, presence of BM-MSCs or ASCs within the subcutaneous layer contributed to the epidermal differentiation program, with more proliferating basal cells depositing basal membrane proteins and differentiating into mature keratinocytes that were able to generate a pluristratified epithelium. In conclusion, we engineered a fully differentiated human skin trilayer that could present multiple applications such as use for in vitro drug absorption tests and regenerative therapies. Copyright © 2012 John Wiley & Sons, Ltd.

  10. Studying Kidney Disease Using Tissue and Genome Engineering in Human Pluripotent Stem Cells.

    PubMed

    Garreta, Elena; González, Federico; Montserrat, Núria

    2017-10-07

    Kidney morphogenesis and patterning have been extensively studied in animal models such as the mouse and zebrafish. These seminal studies have been key to define the molecular mechanisms underlying this complex multistep process. Based on this knowledge, the last 3 years have witnessed the development of a cohort of protocols allowing efficient differentiation of human pluripotent stem cells (hPSCs) towards defined kidney progenitor populations using two-dimensional (2D) culture systems or through generating organoids. Kidney organoids are three-dimensional (3D) kidney-like tissues, which are able to partially recapitulate kidney structure and function in vitro. The current possibility to combine state-of-the art tissue engineering with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems 9 (Cas9)-mediated genome engineering provides an unprecedented opportunity for studying kidney disease with hPSCs. Recently, hPSCs with genetic mutations introduced through CRISPR/Cas9-mediated genome engineering have shown to produce kidney organoids able to recapitulate phenotypes of polycystic kidney disease and glomerulopathies. This mini review provides an overview of the most recent advances in differentiation of hPSCs into kidney lineages, and the latest implementation of the CRISPR/Cas9 technology in the organoid setting, as promising platforms to study human kidney development and disease. © 2017 S. Karger AG, Basel.

  11. Development of tissue-engineered human periodontal ligament constructs with intrinsic angiogenic potential.

    PubMed

    Nagai, Nobuhiro; Hirakawa, Ayumi; Otani, Nao; Munekata, Masanobu

    2009-01-01

    One approach to treat periodontal diseases is grafting of tissue-engineered periodontal ligaments. Therefore, periodontal ligaments were constructed by layering cell sheets. A cell sheet was prepared by enzymatic digestion of salmon collagen gel on which human periodontal ligament fibroblasts (HPLFs) were co-cultured with or without human umbilical vein endothelial cells (HUVECs). Three cell sheets were layered and then cultured in angiogenic media, in which the HUVECs were found to form capillary-like structures when co-cultured on the HPLFs. The layered HPLFs sheets with HUVEC co-culture (PL-EC construct) demonstrated longer survival, higher alkaline phosphatase activities and lower osteocalcin production than layered HPLFs sheets without HUVEC co-culture (PL construct). Hematoxylin-eosin and Masson's trichrome staining of histological sections showed that cell density, mass and extracellular matrix deposition of the PL-EC construct were higher than those of the PL construct. Furthermore, CD31 immunostaining revealed the formation of capillary-like structures throughout the PL-EC construct. In conclusion, we successfully developed tissue-engineered periodontal ligament constructs with intrinsic angiogenic potential using cell sheet engineering and HUVEC co-culture.

  12. Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium

    PubMed Central

    Turnbull, Irene C.; Karakikes, Ioannis; Serrao, Gregory W.; Backeris, Peter; Lee, Jia-Jye; Xie, Chaoqin; Senyei, Grant; Gordon, Ronald E.; Li, Ronald A.; Akar, Fadi G.; Hajjar, Roger J.; Hulot, Jean-Sébastien; Costa, Kevin D.

    2014-01-01

    Cardiac experimental biology and translational research would benefit from an in vitro surrogate for human heart muscle. This study investigated structural and functional properties and interventional responses of human engineered cardiac tissues (hECTs) compared to human myocardium. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs, >90% troponin-positive) were mixed with collagen and cultured on force-sensing elastomer devices. hECTs resembled trabecular muscle and beat spontaneously (1.18±0.48 Hz). Microstructural features and mRNA expression of cardiac-specific genes (α-MHC, SERCA2a, and ACTC1) were comparable to human myocardium. Optical mapping revealed cardiac refractoriness with loss of 1:1 capture above 3 Hz, and cycle length dependence of the action potential duration, recapitulating key features of cardiac electrophysiology. hECTs reconstituted the Frank-Starling mechanism, generating an average maximum twitch stress of 660 μN/mm2 at Lmax, approaching values in newborn human myocardium. Dose-response curves followed exponential pharmacodynamics models for calcium chloride (EC50 1.8 mM) and verapamil (IC50 0.61 μM); isoproterenol elicited a positive chronotropic but negligible inotropic response, suggesting sarcoplasmic reticulum immaturity. hECTs were amenable to gene transfer, demonstrated by successful transduction with Ad.GFP. Such 3-D hECTs recapitulate an early developmental stage of human myocardium and promise to offer an alternative preclinical model for cardiology research.—Turnbull, I. C., Karakikes, I., Serrao, G. W., Backeris, P., Lee, J.-J., Xie, C., Senyei, G., Gordon, R. E., Li, R. A., Akar, F. G., Hajjar, R. J., Hulot, J.-S., Costa, K. D. Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium. PMID:24174427

  13. Chondroprotective supplementation promotes the mechanical properties of injectable scaffold for human nucleus pulposus tissue engineering.

    PubMed

    Foss, Berit L; Maxwell, Thomas W; Deng, Ying

    2014-01-01

    A result of intervertebral disc (IVD) degeneration, the nucleus pulposus (NP) is no longer able to withstand applied load leading to pain and disability. The objective of this study is to fabricate a tissue-engineered injectable scaffold with chondroprotective supplementation in vitro to improve the mechanical properties of a degenerative NP. Tissue-engineered scaffolds were fabricated using different concentrations of alginate and calcium chloride and mechanically evaluated. Fabrication conditions were based on structural and mechanical resemblance to the native NP. Chondroprotective supplementation, glucosamine (GCSN) and chondroitin sulfate (CS), were added to scaffolds at concentrations of 0:0µg/mL (0:0-S), 125:100µg/mL (125:100-S), 250:200µg/mL (250:200-S), and 500:400µg/mL (500:400-S), GCSN and CS, respectively. Scaffolds were used to fabricate tissue-engineered constructs through encapsulation of human nucleus pulposus cells (HNPCs). The tissue-engineered constructs were collected at days 1, 14, and 28 for biochemical and biomechanical evaluations. Confocal microscopy showed HNPC viability and rounded morphology over the 28 day period. MTT analysis resulted in significant increases in cell proliferation for each group. Collagen type II ELISA quantification and compressive aggregate moduli (HA) showed increasing trends for both 250:200-S and the 500:400-S groups on Day 28 with significantly greater HA compared to 0:0-S group. Glycosaminoglycan and water content decreased for all groups. Results indicate the increased mechanical properties of the 250:200-S and the 500:400-S was due to production of a functional matrix. This study demonstrated potential for a chondroprotective supplemented injectable scaffold to restore biomechanical function of a degenerative disc through the production of a mechanically functional matrix. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Cartilage tissue engineering of nasal septal chondrocyte-macroaggregates in human demineralized bone matrix.

    PubMed

    Liese, Juliane; Marzahn, Ulrike; El Sayed, Karym; Pruss, Axel; Haisch, Andreas; Stoelzel, Katharina

    2013-06-01

    Tissue Engineering is an important method for generating cartilage tissue with isolated autologous cells and the support of biomaterials. In contrast to various gel-like biomaterials, human demineralized bone matrix (DBM) guarantees some biomechanical stability for an application in biomechanically loaded regions. The present study combined for the first time the method of seeding chondrocyte-macroaggregates in DBM for the purpose of cartilage tissue engineering. After isolating human nasal chondrocytes and creating a three-dimensional macroaggregate arrangement, the DBM was cultivated in vitro with the macroaggregates. The interaction of the cells within the DBM was analyzed with respect to cell differentiation and the inhibitory effects of chondrocyte proliferation. In contrast to chondrocyte-macroaggregates in the cell-DBM constructs, morphologically modified cells expressing type I collagen dominated. The redifferentiation of chondrocytes, characterized by the expression of type II collagen, was only found in low amounts in the cell-DBM constructs. Furthermore, caspase 3, a marker for apoptosis, was detected in the chondrocyte-DBM constructs. In another experimental setting, the vitality of chondrocytes as related to culture time and the amount of DBM was analyzed with the BrdU assay. Higher amounts of DBM tended to result in significantly higher proliferation rates of the cells within the first 48 h. After 96 h, the vitality decreased in a dose-dependent fashion. In conclusion, this study provides the proof of concept of chondrocyte-macroaggregates with DBM as an interesting method for the tissue engineering of cartilage. The as-yet insufficient redifferentiation of the chondrocytes and the sporadic initiation of apoptosis will require further investigations.

  15. The suitability of human adipose-derived stem cells for the engineering of ligament tissue.

    PubMed

    Eagan, Michael J; Zuk, Patricia A; Zhao, Ke-Wei; Bluth, Benjamin E; Brinkmann, Elyse J; Wu, Benjamin M; McAllister, David R

    2012-10-01

    Rupture of the anterior cruciate ligament (ACL) is the one of the most common sports-related injuries. With its poor healing capacity, surgical reconstruction using either autografts or allografts is currently required to restore function. However, serious complications are associated with graft reconstructions and the number of such reconstructions has steadily risen over the years, necessitating the search for an alternative approach to ACL repair. Such an approach may likely be tissue engineering. Recent engineering approaches using ligament-derived fibroblasts have been promising, but the slow growth rate of such fibroblasts in vitro may limit their practical application. More promising results are being achieved using bone marrow mesenchymal stem cells (MSCs). The adipose-derived stem cell (ASC) is often proposed as an alternative choice to the MSC and, as such, may be a suitable stem cell for ligament engineering. However, the use of ASCs in ligament engineering still remains relatively unexplored. Therefore, in this study, the potential use of human ASCs in ligament tissue engineering was initially explored by examining their ability to express several ligament markers under growth factor treatment. ASC populations treated for up to 4 weeks with TGFβ1 or IGF1 did not show any significant and consistent upregulation in the expression of collagen types 1 and 3, tenascin C and scleraxis. While treatment with EGF or bFGF resulted in increased tenascin C expression, increased expression of collagens 1 and 3 were never observed. Therefore, simple in vitro treatment of human ASC populations with growth factors may not stimulate their ligament differentiative potential. Copyright © 2011 John Wiley & Sons, Ltd.

  16. 3D Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications.

    PubMed

    Markstedt, Kajsa; Mantas, Athanasios; Tournier, Ivan; Martínez Ávila, Héctor; Hägg, Daniel; Gatenholm, Paul

    2015-05-11

    The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine. The 3D bioprinter is able to dispense materials while moving in X, Y, and Z directions, which enables the engineering of complex structures from the bottom up. In this study, a bioink that combines the outstanding shear thinning properties of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate was formulated for the 3D bioprinting of living soft tissue with cells. Printability was evaluated with concern to printer parameters and shape fidelity. The shear thinning behavior of the tested bioinks enabled printing of both 2D gridlike structures as well as 3D constructs. Furthermore, anatomically shaped cartilage structures, such as a human ear and sheep meniscus, were 3D printed using MRI and CT images as blueprints. Human chondrocytes bioprinted in the noncytotoxic, nanocellulose-based bioink exhibited a cell viability of 73% and 86% after 1 and 7 days of 3D culture, respectively. On the basis of these results, we can conclude that the nanocellulose-based bioink is a suitable hydrogel for 3D bioprinting with living cells. This study demonstrates the potential use of nanocellulose for 3D bioprinting of living tissues and organs.

  17. Tissue Engineering of the Penis

    PubMed Central

    Patel, Manish N.; Atala, Anthony

    2011-01-01

    Congenital disorders, cancer, trauma, or other conditions of the genitourinary tract can lead to significant organ damage or loss of function, necessitating eventual reconstruction or replacement of the damaged structures. However, current reconstructive techniques are limited by issues of tissue availability and compatibility. Physicians and scientists have begun to explore tissue engineering and regenerative medicine strategies for repair and reconstruction of the genitourinary tract. Tissue engineering allows the development of biological substitutes which could potentially restore normal function. Tissue engineering efforts designed to treat or replace most organs are currently being undertaken. Most of these efforts have occurred within the past decade. However, before these engineering techniques can be applied to humans, further studies are needed to ensure the safety and efficacy of these new materials. Recent progress suggests that engineered urologic tissues and cell therapy may soon have clinical applicability. PMID:22235188

  18. Natural Scaffolds for Renal Differentiation of Human Embryonic Stem Cells for Kidney Tissue Engineering.

    PubMed

    Batchelder, Cynthia A; Martinez, Michele L; Tarantal, Alice F

    2015-01-01

    Despite the enthusiasm for bioengineering of functional renal tissues for transplantation, many obstacles remain before the potential of this technology can be realized in a clinical setting. Viable tissue engineering strategies for the kidney require identification of the necessary cell populations, efficient scaffolds, and the 3D culture conditions to develop and support the unique architecture and physiological function of this vital organ. Our studies have previously demonstrated that decellularized sections of rhesus monkey kidneys of all age groups provide a natural extracellular matrix (ECM) with sufficient structural properties with spatial and organizational influences on human embryonic stem cell (hESC) migration and differentiation. To further explore the use of decellularized natural kidney scaffolds for renal tissue engineering, pluripotent hESC were seeded in whole- or on sections of kidney ECM and cell migration and phenotype compared with the established differentiation assays for hESC. Results of qPCR and immunohistochemical analyses demonstrated upregulation of renal lineage markers when hESC were cultured in decellularized scaffolds without cytokine or growth factor stimulation, suggesting a role for the ECM in directing renal lineage differentiation. hESC were also differentiated with growth factors and compared when seeded on renal ECM or a new biologically inert polysaccharide scaffold for further maturation. Renal lineage markers were progressively upregulated over time on both scaffolds and hESC were shown to express signature genes of renal progenitor, proximal tubule, endothelial, and collecting duct populations. These findings suggest that natural scaffolds enhance expression of renal lineage markers particularly when compared to embryoid body culture. The results of these studies show the capabilities of a novel polysaccharide scaffold to aid in defining a protocol for renal progenitor differentiation from hESC, and advance the promise

  19. New Methods in Tissue Engineering

    PubMed Central

    Sheahan, Timothy P.; Rice, Charles M.; Bhatia, Sangeeta N.

    2015-01-01

    New insights in the study of virus and host biology in the context of viral infection are made possible by the development of model systems that faithfully recapitulate the in vivo viral life cycle. Standard tissue culture models lack critical emergent properties driven by cellular organization and in vivo–like function, whereas animal models suffer from limited susceptibility to relevant human viruses and make it difficult to perform detailed molecular manipulation and analysis. Tissue engineering techniques may enable virologists to create infection models that combine the facile manipulation and readouts of tissue culture with the virus-relevant complexity of animal models. Here, we review the state of the art in tissue engineering and describe how tissue engineering techniques may alleviate some common shortcomings of existing models of viral infection, with a particular emphasis on hepatotropic viruses. We then discuss possible future applications of tissue engineering to virology, including current challenges and potential solutions. PMID:25893203

  20. Calcium phosphate bioceramics fabricated from extracted human teeth for tooth tissue engineering.

    PubMed

    Lim, Ki-Taek; Suh, Je Duck; Kim, Jangho; Choung, Pill-Hoon; Chung, Jong Hoon

    2011-11-01

    Bioceramic tooth powders were prepared via heat treatment of extracted human teeth using sintering temperatures between 600°C and 1200°C, and their properties were investigated for potential tooth tissue engineering. The sintered human tooth powders were characterized using thermal analysis (thermogravimetric analysis (TG) and differential thermal analysis (DTA)), field emission scanning electron microscopy, X-ray diffraction, energy dispersive X-ray spectroscopy, and Fourier transformed infrared (FTIR) spectroscopy. Additionally, the phase constitutions and chemical homogeneities of the composite samples were examined using a quantitative chemical analysis with inductively coupled plasma spectroscopy. The results revealed that the annealing process produced useful hydroxyapatite-based bioceramic biomaterials when annealed above 1000°C. The FTIR spectra and the TG/DTA thermograms of the tooth powders indicated the presence of organic compounds, which were completely removed after annealing at temperatures above 1000°C. The tooth powders annealed between 1000°C and 1200°C had good characteristics as bioceramic biomaterials. Furthermore, the biocompatibility of each tooth powder was evaluated using in vitro and in vivo techniques; our results indicate that the prepared human tooth powders have great potential for tooth tissue engineering applications.

  1. A Novel Human Tissue-Engineered 3-D Functional Vascularized Cardiac Muscle Construct

    PubMed Central

    Valarmathi, Mani T.; Fuseler, John W.; Davis, Jeffrey M.; Price, Robert L.

    2017-01-01

    Organ tissue engineering, including cardiovascular tissues, has been an area of intense investigation. The major challenge to these approaches has been the inability to vascularize and perfuse the in vitro engineered tissue constructs. Attempts to provide oxygen and nutrients to the cells contained in the biomaterial constructs have had varying degrees of success. The aim of this current study is to develop a three-dimensional (3-D) model of vascularized cardiac tissue to examine the concurrent temporal and spatial regulation of cardiomyogenesis in the context of postnatal de novo vasculogenesis during stem cell cardiac regeneration. In order to achieve the above aim, we have developed an in vitro 3-D functional vascularized cardiac muscle construct using human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-ECMs) and human mesenchymal stem cells (hMSCs). First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured onto a 3-D collagen cell carrier (CCC) for 7 days under vasculogenic culture conditions. In this milieu, hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed extensive plexuses of vascular networks. Next, the hiPSC-ECMs and hMSCs were co-cultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were analyzed at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated cells revealed neo-angiogenesis and neo-cardiomyogenesis. Thus, our unique 3-D co-culture system provided us the apt in vitro functional vascularized 3-D cardiac patch that can be utilized for cellular cardiomyoplasty. PMID:28194397

  2. A Novel Human Tissue-Engineered 3-D Functional Vascularized Cardiac Muscle Construct.

    PubMed

    Valarmathi, Mani T; Fuseler, John W; Davis, Jeffrey M; Price, Robert L

    2017-01-01

    Organ tissue engineering, including cardiovascular tissues, has been an area of intense investigation. The major challenge to these approaches has been the inability to vascularize and perfuse the in vitro engineered tissue constructs. Attempts to provide oxygen and nutrients to the cells contained in the biomaterial constructs have had varying degrees of success. The aim of this current study is to develop a three-dimensional (3-D) model of vascularized cardiac tissue to examine the concurrent temporal and spatial regulation of cardiomyogenesis in the context of postnatal de novo vasculogenesis during stem cell cardiac regeneration. In order to achieve the above aim, we have developed an in vitro 3-D functional vascularized cardiac muscle construct using human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-ECMs) and human mesenchymal stem cells (hMSCs). First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured onto a 3-D collagen cell carrier (CCC) for 7 days under vasculogenic culture conditions. In this milieu, hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed extensive plexuses of vascular networks. Next, the hiPSC-ECMs and hMSCs were co-cultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were analyzed at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated cells revealed neo-angiogenesis and neo-cardiomyogenesis. Thus, our unique 3-D co-culture system provided us the apt in vitro functional vascularized 3-D cardiac patch that can be utilized for cellular cardiomyoplasty.

  3. Brief Communication: Tissue-engineered Microenvironment Systems for Modeling Human Vasculature

    PubMed Central

    Tourovskaia, Anna; Fauver, Mark; Kramer, Gregory; Simonson, Sara; Neumann, Thomas

    2015-01-01

    The high attrition rate of drug candidates late in the development process has led to an increasing demand for test assays that predict clinical outcome better than conventional 2D cell culture systems and animal models. Government agencies, the military, and the pharmaceutical industry have started initiatives for the development of novel in-vitro systems that recapitulate functional units of human tissues and organs. There is growing evidence that 3D cell arrangement, co-culture of different cell types, and physico-chemical cues lead to improved predictive power. A key element of all tissue microenvironments is the vasculature. Beyond transporting blood the microvasculature assumes important organ-specific functions. It is also involved in pathologic conditions, such as inflammation, tumor growth, metastasis, and degenerative diseases. To provide a tool for modeling this important feature of human tissue microenvironments, we developed a microfluidic chip for creating tissue-engineered microenvironment systems (TEMS) composed of tubular cell structures. Our chip design encompasses a small chamber that is filled with an extracellular matrix (ECM) surrounding one or more tubular channels. Endothelial cells seeded into the channels adhere to the ECM walls and grow into perfusable tubular tissue structures that are fluidically connected to upstream and downstream fluid channels in the chip. Using these chips we created models of angiogenesis, the blood-brain-barrier (BBB), and tumor-cell extravasation. Our angiogenesis model recapitulates true angiogenesis, in which sprouting occurs from a “parent” vessel in response to a gradient of growth factors. Our BBB model is composed of a microvessel generated from brain-specific endothelial cells (ECs) within an ECM populated with astrocytes and pericytes. Our tumor-cell extravasation model can be utilized to visualize and measure tumor-cell migration through vessel walls into the surrounding matrix. The described

  4. Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids

    PubMed Central

    Finkbeiner, Stacy R.; Freeman, Jennifer J.; Wieck, Minna M.; El-Nachef, Wael; Altheim, Christopher H.; Tsai, Yu-Hwai; Huang, Sha; Dyal, Rachel; White, Eric S.; Grikscheit, Tracy C.; Teitelbaum, Daniel H.; Spence, Jason R.

    2015-01-01

    ABSTRACT Short bowel syndrome (SBS) is characterized by poor nutrient absorption due to a deficit of healthy intestine. Current treatment practices rely on providing supportive medical therapy with parenteral nutrition; while life saving, such interventions are not curative and are still associated with significant co-morbidities. As approaches to lengthen remaining intestinal tissue have been met with only limited success and intestinal transplants have poor survival outcomes, new approaches to treating SBS are necessary. Human intestine derived from embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), called human intestinal organoids (HIOs), have the potential to offer a personalized and scalable source of intestine for regenerative therapies. However, given that HIOs are small three-dimensional structures grown in vitro, methods to generate usable HIO-derived constructs are needed. We investigated the ability of hESCs or HIOs to populate acellular porcine intestinal matrices and artificial polyglycolic/poly L lactic acid (PGA/PLLA) scaffolds, and examined the ability of matrix/scaffolds to thrive when transplanted in vivo. Our results demonstrate that the acellular matrix alone is not sufficient to instruct hESC differentiation towards an endodermal or intestinal fate. We observed that while HIOs reseed acellular porcine matrices in vitro, the HIO-reseeded matrices do not thrive when transplanted in vivo. In contrast, HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. Our results suggest that HIO-seeded PGA/PLLA scaffolds are a promising avenue for developing the mucosal component of tissue engineered human small intestine, which need to be explored further to develop them into fully functional tissue. PMID:26459240

  5. Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids.

    PubMed

    Finkbeiner, Stacy R; Freeman, Jennifer J; Wieck, Minna M; El-Nachef, Wael; Altheim, Christopher H; Tsai, Yu-Hwai; Huang, Sha; Dyal, Rachel; White, Eric S; Grikscheit, Tracy C; Teitelbaum, Daniel H; Spence, Jason R

    2015-10-12

    Short bowel syndrome (SBS) is characterized by poor nutrient absorption due to a deficit of healthy intestine. Current treatment practices rely on providing supportive medical therapy with parenteral nutrition; while life saving, such interventions are not curative and are still associated with significant co-morbidities. As approaches to lengthen remaining intestinal tissue have been met with only limited success and intestinal transplants have poor survival outcomes, new approaches to treating SBS are necessary. Human intestine derived from embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), called human intestinal organoids (HIOs), have the potential to offer a personalized and scalable source of intestine for regenerative therapies. However, given that HIOs are small three-dimensional structures grown in vitro, methods to generate usable HIO-derived constructs are needed. We investigated the ability of hESCs or HIOs to populate acellular porcine intestinal matrices and artificial polyglycolic/poly L lactic acid (PGA/PLLA) scaffolds, and examined the ability of matrix/scaffolds to thrive when transplanted in vivo. Our results demonstrate that the acellular matrix alone is not sufficient to instruct hESC differentiation towards an endodermal or intestinal fate. We observed that while HIOs reseed acellular porcine matrices in vitro, the HIO-reseeded matrices do not thrive when transplanted in vivo. In contrast, HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. Our results suggest that HIO-seeded PGA/PLLA scaffolds are a promising avenue for developing the mucosal component of tissue engineered human small intestine, which need to be explored further to develop them into fully functional tissue.

  6. Advancing cardiovascular tissue engineering

    PubMed Central

    Truskey, George A.

    2016-01-01

    Cardiovascular tissue engineering offers the promise of biologically based repair of injured and damaged blood vessels, valves, and cardiac tissue. Major advances in cardiovascular tissue engineering over the past few years involve improved methods to promote the establishment and differentiation of induced pluripotent stem cells (iPSCs), scaffolds from decellularized tissue that may produce more highly differentiated tissues and advance clinical translation, improved methods to promote vascularization, and novel in vitro microphysiological systems to model normal and diseased tissue function. iPSC technology holds great promise, but robust methods are needed to further promote differentiation. Differentiation can be further enhanced with chemical, electrical, or mechanical stimuli. PMID:27303643

  7. Advances in tissue engineering.

    PubMed

    Langer, Robert; Vacanti, Joseph

    2016-01-01

    Nearly 30 years ago, we reported on a concept now known as Tissue Engineering. Here, we report on some of the advances in this now thriving area of research. In particular, significant advances in tissue engineering of skin, liver, spinal cord, blood vessels, and other areas are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Advances in Tissue Engineering

    PubMed Central

    Vacanti, Joseph

    2016-01-01

    Nearly 30 years ago, we reported on a concept now known as Tissue Engineering. Here, we report on some of the advances in this now thriving area of research. In particular, significant advances in tissue engineering of skin, liver, spinal cord, blood vessels, and other areas are discussed. PMID:26711689

  9. Tissue engineering in dentistry.

    PubMed

    Abou Neel, Ensanya Ali; Chrzanowski, Wojciech; Salih, Vehid M; Kim, Hae-Won; Knowles, Jonathan C

    2014-08-01

    of this review is to inform practitioners with the most updated information on tissue engineering and its potential applications in dentistry. The authors used "PUBMED" to find relevant literature written in English and published from the beginning of tissue engineering until today. A combination of keywords was used as the search terms e.g., "tissue engineering", "approaches", "strategies" "dentistry", "dental stem cells", "dentino-pulp complex", "guided tissue regeneration", "whole tooth", "TMJ", "condyle", "salivary glands", and "oral mucosa". Abstracts and full text articles were used to identify causes of craniofacial tissue loss, different approaches for craniofacial reconstructions, how the tissue engineering emerges, different strategies of tissue engineering, biomaterials employed for this purpose, the major attempts to engineer different dental structures, finally challenges and future of tissue engineering in dentistry. Only those articles that dealt with the tissue engineering in dentistry were selected. There have been a recent surge in guided tissue engineering methods to manage periodontal diseases beyond the traditional approaches. However, the predictable reconstruction of the innate organisation and function of whole teeth as well as their periodontal structures remains challenging. Despite some limited progress and minor successes, there remain distinct and important challenges in the development of reproducible and clinically safe approaches for oral tissue repair and regeneration. Clearly, there is a convincing body of evidence which confirms the need for this type of treatment, and public health data worldwide indicates a more than adequate patient resource. The future of these therapies involving more biological approaches and the use of dental tissue stem cells is promising and advancing. Also there may be a significant interest of their application and wider potential to treat disorders beyond the craniofacial region. Considering the

  10. Dental Pulp Tissue Engineering in Full-length Human Root Canals

    PubMed Central

    Rosa, V.; Zhang, Z.; Grande, R.H.M.; Nör, J.E.

    2013-01-01

    The clinical translation of stem-cell-based dental pulp regeneration will require the use of injectable scaffolds. Here, we tested the hypothesis that stem cells from exfoliated deciduous teeth (SHED) can generate a functional dental pulp when injected into full-length root canals. SHED survived and began to express putative markers of odontoblastic differentiation after 7 days when mixed with Puramatrix™ (peptide hydrogel), or after 14 days when mixed with recombinant human Collagen (rhCollagen) type I, and injected into the root canals of human premolars in vitro. Roots of human premolars injected with scaffolds (Puramatrix™ or rhCollagen) containing SHED were implanted subcutaneously into immunodeficient mice (CB-17 SCID). We observed pulp-like tissues with odontoblasts capable of generating new tubular dentin throughout the root canals. Notably, the pulp tissue engineered with SHED injected with either Puramatrix™ or rhCollagen type I presented similar cellularity and vascularization when compared with control human dental pulps. Analysis of these data, collectively, demonstrates that SHED injected into full-length human root canals differentiate into functional odontoblasts, and suggests that such a strategy might facilitate the completion of root formation in necrotic immature permanent teeth. PMID:24056227

  11. Dental pulp tissue engineering in full-length human root canals.

    PubMed

    Rosa, V; Zhang, Z; Grande, R H M; Nör, J E

    2013-11-01

    The clinical translation of stem-cell-based dental pulp regeneration will require the use of injectable scaffolds. Here, we tested the hypothesis that stem cells from exfoliated deciduous teeth (SHED) can generate a functional dental pulp when injected into full-length root canals. SHED survived and began to express putative markers of odontoblastic differentiation after 7 days when mixed with Puramatrix™ (peptide hydrogel), or after 14 days when mixed with recombinant human Collagen (rhCollagen) type I, and injected into the root canals of human premolars in vitro. Roots of human premolars injected with scaffolds (Puramatrix™ or rhCollagen) containing SHED were implanted subcutaneously into immunodeficient mice (CB-17 SCID). We observed pulp-like tissues with odontoblasts capable of generating new tubular dentin throughout the root canals. Notably, the pulp tissue engineered with SHED injected with either Puramatrix™ or rhCollagen type I presented similar cellularity and vascularization when compared with control human dental pulps. Analysis of these data, collectively, demonstrates that SHED injected into full-length human root canals differentiate into functional odontoblasts, and suggests that such a strategy might facilitate the completion of root formation in necrotic immature permanent teeth.

  12. Osteogenic Differentiation Capacity of In Vitro Cultured Human Skeletal Muscle for Expedited Bone Tissue Engineering

    PubMed Central

    Miao, Chunlei; Zhou, Lulu; Tian, Lufeng; Zhang, Yingjie; Zhang, Wei; Yang, Fanghong; Liu, Tianyi

    2017-01-01

    Expedited bone tissue engineering employs the biological stimuli to harness the intrinsic regenerative potential of skeletal muscle to trigger the reparative process in situ to improve or replace biological functions. When genetically modified with adenovirus mediated BMP2 gene transfer, muscle biopsies from animals have demonstrated success in regenerating bone within rat bony defects. However, it is uncertain whether the human adult skeletal muscle displays an osteogenic potential in vitro when a suitable biological trigger is applied. In present study, human skeletal muscle cultured in a standard osteogenic medium supplemented with dexamethasone demonstrated significant increase in alkaline phosphatase activity approximately 24-fold over control at 2-week time point. More interestingly, measurement of mRNA levels revealed the dramatic results for osteoblast transcripts of alkaline phosphatase, bone sialoproteins, transcription factor CBFA1, collagen type I, and osteocalcin. Calcified mineral deposits were demonstrated on superficial layers of muscle discs after an extended 8-week osteogenic induction. Taken together, these are the first data supporting human skeletal muscle tissue as a promising potential target for expedited bone regeneration, which of the technologies is a valuable method for tissue repair, being not only effective but also inexpensive and clinically expeditious. PMID:28210626

  13. Osteogenic Differentiation Capacity of In Vitro Cultured Human Skeletal Muscle for Expedited Bone Tissue Engineering.

    PubMed

    Miao, Chunlei; Zhou, Lulu; Tian, Lufeng; Zhang, Yingjie; Zhang, Wei; Yang, Fanghong; Liu, Tianyi; Tang, Shengjian; Liu, Fangjun

    2017-01-01

    Expedited bone tissue engineering employs the biological stimuli to harness the intrinsic regenerative potential of skeletal muscle to trigger the reparative process in situ to improve or replace biological functions. When genetically modified with adenovirus mediated BMP2 gene transfer, muscle biopsies from animals have demonstrated success in regenerating bone within rat bony defects. However, it is uncertain whether the human adult skeletal muscle displays an osteogenic potential in vitro when a suitable biological trigger is applied. In present study, human skeletal muscle cultured in a standard osteogenic medium supplemented with dexamethasone demonstrated significant increase in alkaline phosphatase activity approximately 24-fold over control at 2-week time point. More interestingly, measurement of mRNA levels revealed the dramatic results for osteoblast transcripts of alkaline phosphatase, bone sialoproteins, transcription factor CBFA1, collagen type I, and osteocalcin. Calcified mineral deposits were demonstrated on superficial layers of muscle discs after an extended 8-week osteogenic induction. Taken together, these are the first data supporting human skeletal muscle tissue as a promising potential target for expedited bone regeneration, which of the technologies is a valuable method for tissue repair, being not only effective but also inexpensive and clinically expeditious.

  14. Engineering functional bladder tissues.

    PubMed

    Horst, Maya; Madduri, Srinivas; Gobet, Rita; Sulser, Tullio; Milleret, Vinzent; Hall, Heike; Atala, Anthony; Eberli, Daniel

    2013-07-01

    End stage bladder disease can seriously affect patient quality of life and often requires surgical reconstruction with bowel tissue, which is associated with numerous complications. Bioengineering of functional bladder tissue using tissue-engineering techniques could provide new functional tissues for reconstruction. In this review, we discuss the current state of this field and address different approaches to enable physiologic voiding in engineered bladder tissues in the near future. In a collaborative effort, we gathered researchers from four institutions to discuss the current state of functional bladder engineering. A MEDLINE® and PubMed® search was conducted for articles related to tissue engineering of the bladder, with special focus on the cells and biomaterials employed as well as the microenvironment, vascularisation and innervation strategies used. Over the last decade, advances in tissue engineering technology have laid the groundwork for the development of a biological substitute for bladder tissue that can support storage of urine and restore physiologic voiding. Although many researchers have been able to demonstrate the formation of engineered tissue with a structure similar to that of native bladder tissue, restoration of physiologic voiding using these constructs has never been demonstrated. The main issues hindering the development of larger contractile tissues that allow physiologic voiding include the development of correct muscle alignment, proper innervation and vascularization. Tissue engineering of a construct that will support the contractile properties that allow physiologic voiding is a complex process. The combination of smart scaffolds with controlled topography, the ability to deliver multiple trophic factors and an optimal cell source will allow for the engineering of functional bladder tissues in the near future. Copyright © 2012 John Wiley & Sons, Ltd.

  15. Generation and characterization of a human acellular meniscus scaffold for tissue engineering.

    PubMed

    Sandmann, G H; Eichhorn, S; Vogt, S; Adamczyk, C; Aryee, S; Hoberg, M; Milz, S; Imhoff, A B; Tischer, T

    2009-11-01

    Meniscus tears are frequent indications for arthroscopic evaluation which can result in partial or total meniscectomy. Allografts or synthetic meniscus scaffolds have been used with varying success to prevent early degenerative joint disease in these cases. Problems related to reduced initial and long-term stability, as well as immunological reactions prevent widespread clinical use so far. Therefore, the aim of this study was to develop a new construct for tissue engineering of the human meniscus based on an acellular meniscus allograft. Human menisci (n = 16) were collected and acellularized using the detergent sodium dodecyl sulfate as the main ingredient or left untreated as control group. These acellularized menisci were characterized biomechanically using a repetitive ball indentation test (Stiffness N/mm, residual force N, relative compression force N) and by histological (hematoxylin-eosin, phase-contrast) as well as immunohistochemical (collagen I, II, VI) investigation. The processed menisci histologically appeared cell-free and had biomechanical properties similar to the intact meniscus samples (p > 0.05). The collagen fiber arrangement was not altered, according to phase-contrast microscopy and immunohistochemical labeling. The removal of the immunogenic cell components combined with the preservation of the mechanically relevant parts of the extracellular matrix could make these scaffolds ideal implants for future tissue engineering of the meniscus.

  16. Identification of a bioactive core sequence from human laminin and its applicability to tissue engineering.

    PubMed

    Yeo, In-Sung; Min, Seung-Ki; Kang, Hyun Ki; Kwon, Taek-Ka; Jung, Sung Youn; Min, Byung-Moo

    2015-12-01

    Finding bioactive short peptides derived from proteins is a critical step to the advancement of tissue engineering and regenerative medicine, because the former maintains the functions of the latter without immunogenicity in biological systems. Here, we discovered a bioactive core nonapeptide sequence, PPFEGCIWN (residues 2678-2686; Ln2-LG3-P2-DN3), from the human laminin α2 chain, and investigated the role of this peptide in binding to transmembrane proteins to promote intracellular events leading to cell functions. This minimum bioactive sequence had neither secondary nor tertiary structures in a computational structure prediction. Nonetheless, Ln2-LG3-P2-DN3 bound to various cell types as actively as laminin in cell adhesion assays. The in vivo healing tests using rats revealed that Ln2-LG3-P2-DN3 promoted bone formation without any recognizable antigenic activity. Ln2-LG3-P2-DN3-treated titanium (Ti) discs and Ti implant surfaces caused the enhancement of bone cell functions in vitro and induced faster osseointegration in vivo, respectively. These findings established a minimum bioactive sequence within human laminin, and its potential application value for regenerative medicine, especially for bone tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Chondrogenic induction of human mesenchymal stem cells using combined growth factors for cartilage tissue engineering.

    PubMed

    Bosetti, Michela; Boccafoschi, Francesca; Leigheb, Massimiliano; Bianchi, Andrea E; Cannas, Mario

    2012-03-01

    The objective of this study was to evaluate whether growth factors (FGF-2, FGF-4 and FGF-6) used alone or in combination with TGFβ2 are able to increase the proliferation and induce the differentiation of human bone marrow mesenchymal stem cells (hMSCs) to chondrocytes, with a view to using them in cartilage tissue engineering. Cells cultured in monolayer, used to test the activity of the growth factors on cell proliferation, showed that a combination of FGFs with TGFβ2 increases cell proliferation compared to cells cultured in control medium or in the presence of growth factors alone. The chondrogenic potential, evaluated in three-dimensional (3D) cell aggregates, showed that FGF-2 and FGF-6, when used in combination with TGFβ2 increased the size and glycosaminoglycan content of the cell aggregates without increasing cell number. Extracellular matrix (ECM) also showed higher collagen type II immunoreactivity, which was particularly evident in an area similar to a germinative pole that was observed only in pellets cultured with FGF-2 and FGF-6 combined with TGFβ2, or in pellets cultured with FGF-2 alone. Moreover, the RT-PCR assay has highlighted an increased expression of collagen type II and Sox9, used as gene markers for chondrogenesis. We can conclude that combinations of FGF-2 or FGF-6 with TGFβ2 may provide a novel tool to induce the differentiation of adult human mesenchymal stem cells for applications in cartilage tissue engineering.

  18. Rheological characterization of human fibrin and fibrin-agarose oral mucosa substitutes generated by tissue engineering.

    PubMed

    Rodríguez, I A; López-López, M T; Oliveira, A C X; Sánchez-Quevedo, M C; Campos, A; Alaminos, M; Durán, J D G

    2012-08-01

    In regenerative medicine, the generation of biocompatible substitutes of tissues by in vitro tissue engineering must fulfil certain requirements. In the case of human oral mucosa, the rheological properties of tissues deserve special attention because of their influence in the acoustics and biomechanics of voice production. This work is devoted to the rheological characterization of substitutes of the connective tissue of the human oral mucosa. Two substitutes, composed of fibrin and fibrin-agarose, were prepared in cell culture for periods in the range 1-21 days. The time evolution of the rheological properties of both substitutes was studied by two different experimental procedures: steady-state and oscillatory measurements. The former allows the plastic behaviour of the substitutes to be characterized by estimating their yield stress; the latter is employed to quantify their viscoelastic responses by obtaining the elastic (G') and viscous (G'') moduli. The results demonstrate that both substitutes are characterized by a predominant elastic response, in which G' (order 100 Pa) is roughly one order of magnitude larger than G'' (order 10 Pa). But the most relevant insight is the stability, throughout the 21 days of culture time, of the rheological quantities in the case of fibrin-agarose, whereas the fibrin substitute shows a significant hardening. This result provides evidence that the addition to fibrin of a small amount of agarose allows the rheological stability of the oral mucosa substitute to be maintained. This feature, together with its viscoelastic similitude with native tissues, makes this biomaterial appropriate for potential use as a scaffold in regenerative therapies of human oral mucosa. Copyright © 2011 John Wiley & Sons, Ltd.

  19. Recombinant human gelatin substitute with photoreactive properties for cell culture and tissue engineering.

    PubMed

    Kitajima, Takashi; Obuse, Sei; Adachi, Takahiro; Tomita, Masahiro; Ito, Yoshihiro

    2011-10-01

    The human recombinant collagen I α1 chain monomer (rh-gelatin) was modified by the incorporation of an azidophenyl group to prepare photoreactive human gelatin (Az-rh-gelatin), with approximately 90% of the lysine residues conjugated with azidobenzoic acid. Slight changes in conformation (circular dichroism spectra) and thermal properties (gelation and melting points) were noticed after modification. Ultraviolet (UV) irradiation could immobilize the Az-rh-gelatin on polymer surfaces, such as polystyrene and polytetrafluoroethylene. Az-rh-gelatin was stably retained on the polymer surfaces, while unmodified gelatin was mostly lost by brief washing. Human mesenchymal cells grew more efficiently on the immobilized surface than on the coated surface. The immobilized Az-rh-gelatin on the polymer surfaces was able to capture engineered growth factors with collagen affinity, and the bound growth factors stimulated the growth of cells dose-dependently. It was also possible to immobilize Az-rh-gelatin in micropatterns (stripe, grid, and so on) using photomasks, and the cells grew according to the patterns. These results suggest that the photoreactive human gelatin, in combination with collagen-binding growth factors, will be clinically useful for surface modification of synthetic materials for cell culture systems and tissue engineering.

  20. Challenges in cardiac tissue engineering.

    PubMed

    Vunjak-Novakovic, Gordana; Tandon, Nina; Godier, Amandine; Maidhof, Robert; Marsano, Anna; Martens, Timothy P; Radisic, Milica

    2010-04-01

    Cardiac tissue engineering aims to create functional tissue constructs that can reestablish the structure and function of injured myocardium. Engineered constructs can also serve as high-fidelity models for studies of cardiac development and disease. In a general case, the biological potential of the cell-the actual "tissue engineer"-is mobilized by providing highly controllable three-dimensional environments that can mediate cell differentiation and functional assembly. For cardiac regeneration, some of the key requirements that need to be met are the selection of a human cell source, establishment of cardiac tissue matrix, electromechanical cell coupling, robust and stable contractile function, and functional vascularization. We review here the potential and challenges of cardiac tissue engineering for developing therapies that could prevent or reverse heart failure.

  1. Engineering Orthopedic Tissue Interfaces

    PubMed Central

    Yang, Peter J.

    2009-01-01

    While a wide variety of approaches to engineering orthopedic tissues have been proposed, less attention has been paid to the interfaces, the specialized areas that connect two tissues of different biochemical and mechanical properties. The interface tissue plays an important role in transitioning mechanical load between disparate tissues. Thus, the relatively new field of interfacial tissue engineering presents new challenges—to not only consider the regeneration of individual orthopedic tissues, but also to design the biochemical and cellular composition of the linking tissue. Approaches to interfacial tissue engineering may be distinguished based on if the goal is to recreate the interface itself, or generate an entire integrated tissue unit (such as an osteochondral plug). As background for future efforts in engineering orthopedic interfaces, a brief review of the biology and mechanics of each interface (cartilage–bone, ligament–bone, meniscus–bone, and muscle–tendon) is presented, followed by an overview of the state-of-the-art in engineering each tissue, including advances and challenges specific to regenerating the interfaces. PMID:19231983

  2. Optimization of human tendon tissue engineering: peracetic acid oxidation for enhanced reseeding of acellularized intrasynovial tendon.

    PubMed

    Woon, Colin Y L; Pridgen, Brian C; Kraus, Armin; Bari, Sina; Pham, Hung; Chang, James

    2011-03-01

    Tissue engineering of human flexor tendons combines tendon scaffolds with recipient cells to create complete cell-tendon constructs. Allogenic acellularized human flexor tendon has been shown to be a useful natural scaffold. However, there is difficulty repopulating acellularized tendon with recipient cells, as cell penetration is restricted by a tightly woven tendon matrix. The authors evaluated peracetic acid treatment in optimizing intratendinous cell penetration. Cadaveric human flexor tendons were harvested, acellularized, and divided into experimental groups. These groups were treated with peracetic acid in varying concentrations (2%, 5%, and 10%) and for varying time periods (4 and 20 hours) to determine the optimal treatment protocol. Experimental tendons were analyzed for differences in tendon microarchitecture. Additional specimens were reseeded by incubation in a fibroblast cell suspension at 1 × 10(6) cells/ml. This group was then analyzed for reseeding efficacy. A final group underwent biomechanical studies for strength. The optimal treatment protocol comprising peracetic acid at 5% concentration for 4 hours produced increased scaffold porosity, improving cell penetration and migration. Treated scaffolds did not show reduced collagen or glycosaminoglycan content compared with controls (p = 0.37 and p = 0.65, respectively). Treated scaffolds were cytotoxic to neither attached cells nor the surrounding cell suspension. Treated scaffolds also did not show inferior ultimate tensile stress or elastic modulus compared with controls (p = 0.26 and p = 0.28, respectively). Peracetic acid treatment of acellularized tendon scaffolds increases matrix porosity, leading to greater reseeding. It may prove to be an important step in tissue engineering of human flexor tendon using natural scaffolds.

  3. Mechanical and biochemical mapping of human auricular cartilage for reliable assessment of tissue-engineered constructs.

    PubMed

    Nimeskern, Luc; Pleumeekers, Mieke M; Pawson, Duncan J; Koevoet, Wendy L M; Lehtoviita, Iina; Soyka, Michael B; Röösli, Christof; Holzmann, David; van Osch, Gerjo J V M; Müller, Ralph; Stok, Kathryn S

    2015-07-16

    It is key for successful auricular (AUR) cartilage tissue-engineering (TE) to ensure that the engineered cartilage mimics the mechanics of the native tissue. This study provides a spatial map of the mechanical and biochemical properties of human auricular cartilage, thus establishing a benchmark for the evaluation of functional competency in AUR cartilage TE. Stress-relaxation indentation (instantaneous modulus, Ein; maximum stress, σmax; equilibrium modulus, Eeq; relaxation half-life time, t1/2; thickness, h) and biochemical parameters (content of DNA; sulfated-glycosaminoglycan, sGAG; hydroxyproline, HYP; elastin, ELN) of fresh human AUR cartilage were evaluated. Samples were categorized into age groups and according to their harvesting region in the human auricle (for AUR cartilage only). AUR cartilage displayed significantly lower Ein, σmax, Eeq, sGAG content; and significantly higher t1/2, and DNA content than NAS cartilage. Large amounts of ELN were measured in AUR cartilage (>15% ELN content per sample wet mass). No effect of gender was observed for either auricular or nasoseptal samples. For auricular samples, significant differences between age groups for h, sGAG and HYP, and significant regional variations for Ein, σmax, Eeq, t1/2, h, DNA and sGAG were measured. However, only low correlations between mechanical and biochemical parameters were seen (R<0.44). In conclusion, this study established the first comprehensive mechanical and biochemical map of human auricular cartilage. Regional variations in mechanical and biochemical properties were demonstrated in the auricle. This finding highlights the importance of focusing future research on efforts to produce cartilage grafts with spatially tunable mechanics.

  4. Characterization of a new tissue-engineered human skin equivalent with hair.

    PubMed

    Michel, M; L'Heureux, N; Pouliot, R; Xu, W; Auger, F A; Germain, L

    1999-06-01

    We designed a new tissue-engineered skin equivalent in which complete pilosebaceous units were integrated. This model was produced exclusively from human fibroblasts and keratinocytes and did not contain any synthetic material. Fibroblasts were cultured for 35 d with ascorbic acid and formed a thick fibrous sheet in the culture dish. The dermal equivalent was composed of stacked fibroblast sheets and exhibited some ultrastructural organization found in normal connective tissues. Keratinocytes seeded on this tissue formed a stratified and cornified epidermis and expressed typical markers of differentiation (keratin 10, filaggrin, and transglutaminase). After 4 wk of culture, a continuous and ultrastructurally organized basement membrane was observed and associated with the expression of laminin and collagen IV and VII. Complete pilosebaceous units were obtained by thermolysin digestion and inserted in this skin equivalent in order to assess the role of the transfollicular route in percutaneous absorption. The presence of hair follicles abolished the lag-time observed during hydrocortisone diffusion and increased significantly its rate of penetration in comparison to the control (skin equivalent with sham hair insertion). Therefore, this new hairy human skin equivalent model allowed an experimental design in which the only variable was the presence of pilosebaceous units and provided new data confirming the importance of hair follicles in percutaneous absorption.

  5. Tissue engineering of a human sized and shaped auricle using a mold.

    PubMed

    Kamil, S H; Vacanti, M P; Aminuddin, B S; Jackson, M J; Vacanti, C A; Eavey, R D

    2004-05-01

    The creation of a tissue-engineered auricle was initially successful in an immunocompromised nude mouse model. Subsequently, an immunocompetent porcine model successfully generated a helical construct. We wished to evaluate the novel technique of using a mold to create a complete, anatomically refined auricle in a large animal model. Mixtures of autogenous chondrocytes and biodegradable polymers were used inside a perforated, auricle shaped hollow gold mold. Three biodegradable polymers (calcium alginate, pluronic F-127, and polyglycolic acid) were used to retain the seeded chondrocytes inside the mold. These molds, along with a control, were implanted subcutaneously in the abdominal area of 10 animals (pigs and sheep). The constructs were removed after 8 to 20 weeks and were assessed by gross morphology and histology. All the gold implants were well tolerated by the animals. The implants using calcium alginate (n = 3) generated constructs of the exact shape and size of a normal human ear; the histology demonstrated mostly normal cartilage with some persistent alginate. The implants with pluronic F-127 (n = 3) resulted in cartilage with essentially normal histology, although leakage outside the molds and external cartilage generation was noted. Polyglycolic acid implants (n = 3) produced no useful cartilage because of an inflammatory reaction with fibrosis. The empty control mold (n = 1) demonstrated only a very small amount of fibrous tissue inside. A tissue-engineered human sized auricle of normal anatomic definition can be generated in an immunocompetent large-animal model using a mold technique. Although further refinements will be necessary, the technique appears promising for potential use in patients with microtia.

  6. Diffusion studies of nanometer polymersomes across tissue engineered human oral mucosa.

    PubMed

    Hearnden, Vanessa; Lomas, Hannah; Macneil, Sheila; Thornhill, Martin; Murdoch, Craig; Lewis, Andrew; Madsen, Jeppe; Blanazs, Adam; Armes, Steve; Battaglia, Giuseppe

    2009-07-01

    To measure the diffusion of nanometer polymersomes through tissue engineered human oral mucosa. In vitro models of full thickness tissue engineered oral mucosa (TEOM) were used to assess the penetration properties of two chemically different polymersomes comprising two of block copolymers, PMPC-PDPA and PEO-PDPA. These copolymers self-assemble into membrane-enclosed vesicular structures. Polymersomes were conjugated with fluorescent rhodamine in order to track polymersome diffusion. Imaging and quantification of the diffusion properties were assessed by confocal laser scanning microscopy (CLSM). TEOM is morphologically similar to natural oral mucosa. Using CLSM, both formulations were detectable in the TEOM within 6 h and after 48 h both penetrated up to 80 microm into the TEOM. Diffusion of PMPC-PDPA polymersomes was widespread across the epithelium with intra-epithelial uptake, while PEO-PDPA polymersomes also diffused into the epithelium. CLSM was found to be an effective and versatile method for analysing the level of diffusion of polymersomes into TEOM. The penetration and retention of PMPC-PDPA and PEO-PDPA polymersomes means they may have potential for intra-epithelial drug delivery and/or trans-epithelial delivery of therapeutic agents.

  7. Functional cardiac tissue engineering

    PubMed Central

    Liau, Brian; Zhang, Donghui; Bursac, Nenad

    2013-01-01

    Heart attack remains the leading cause of death in both men and women worldwide. Stem cell-based therapies, including the use of engineered cardiac tissues, have the potential to treat the massive cell loss and pathological remodeling resulting from heart attack. Specifically, embryonic and induced pluripotent stem cells are a promising source for generation of therapeutically relevant numbers of functional cardiomyocytes and engineering of cardiac tissues in vitro. This review will describe methodologies for successful differentiation of pluripotent stem cells towards the cardiovascular cell lineages as they pertain to the field of cardiac tissue engineering. The emphasis will be placed on comparing the functional maturation in engineered cardiac tissues and developing heart and on methods to quantify cardiac electrical and mechanical function at different spatial scales. PMID:22397609

  8. Flexor tendon tissue engineering: acellularization of human flexor tendons with preservation of biomechanical properties and biocompatibility.

    PubMed

    Pridgen, Brian C; Woon, Colin Y L; Kim, Maxwell; Thorfinn, Johan; Lindsey, Derek; Pham, Hung; Chang, James

    2011-08-01

    Acellular human tendons are a candidate scaffold for tissue engineering flexor tendons of the hand. This study compared acellularization methods and their compatibility with allogeneic human cells. Human flexor tendons were pretreated with 0.1% ethylenediaminetetracetic acid (EDTA) for 4  h followed by 24  h treatments of 1% Triton X-100, 1% tri(n-butyl)phosphate, or 0.1% or 1% sodium dodecyl sulfate (SDS) in 0.1% EDTA. Outcomes were assessed histologically by hematoxylin and eosin and SYTO green fluorescent nucleic acid stains and biochemically by a QIAGEN DNeasy kit, Sircol collagen assay, and 1,9 dimethylmethylene blue glycosaminoglycan assay. Mechanical data were collected using a Materials Testing System to pull to failure tendons acellularized with 0.1% SDS. Acellularized tendons were re-seeded in a suspension of human dermal fibroblasts. Attachment of viable cells to acellularized tendon was assessed biochemically by a cell viability assay and histologically by a live/dead stain. Data are reported as mean±standard deviation. Compared with the DNA content of fresh tendons (551±212  ng DNA/mg tendon), only SDS treatments significantly decreased DNA content (1% SDS [202.8±37.4  ng DNA/mg dry weight tendon]; 0.1% SDS [189±104  ng DNA/mg tendon]). These findings were confirmed by histology. There was no decrease in glycosaminoglycans or collagen following acellularization with SDS. There was no difference in the ultimate tensile stress (55.3±19.2 [fresh] vs. 51.5±6.9 [0.1% SDS] MPa). Re-seeded tendons demonstrated attachment of viable cells to the tendon surface using a viability assay and histology. Human flexor tendons were acellularized with 0.1% SDS in 0.1% EDTA for 24  h with preservation of mechanical properties. Preservation of collagen and glycoaminoglycans and re-seeding with human cells suggest that this scaffold is biocompatible. This will provide a promising scaffold for future human flexor tendon tissue engineering studies to

  9. Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization.

    PubMed

    Mazza, Giuseppe; Al-Akkad, Walid; Telese, Andrea; Longato, Lisa; Urbani, Luca; Robinson, Benjamin; Hall, Andrew; Kong, Kenny; Frenguelli, Luca; Marrone, Giusi; Willacy, Oliver; Shaeri, Mohsen; Burns, Alan; Malago, Massimo; Gilbertson, Janet; Rendell, Nigel; Moore, Kevin; Hughes, David; Notingher, Ioan; Jell, Gavin; Del Rio Hernandez, Armando; De Coppi, Paolo; Rombouts, Krista; Pinzani, Massimo

    2017-07-17

    The development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of human acellular liver tissue cubes (ALTCs) using normal liver tissue unsuitable for transplantation. The application of high shear stress is a key methodological determinant accelerating the process of tissue decellularization while maintaining ECM protein composition, 3D-architecture and physico-chemical properties of the native tissue. ALTCs were engineered with human parenchymal and non-parenchymal liver cell lines (HepG2 and LX2 cells, respectively), human umbilical vein endothelial cells (HUVEC), as well as primary human hepatocytes and hepatic stellate cells. Both parenchymal and non-parenchymal liver cells grown in ALTCs exhibited markedly different gene expression when compared to standard 2D cell cultures. Remarkably, HUVEC cells naturally migrated in the ECM scaffold and spontaneously repopulated the lining of decellularized vessels. The metabolic function and protein synthesis of engineered liver scaffolds with human primary hepatocytes reseeded under dynamic conditions were maintained. These results provide a solid basis for the establishment of effective protocols aimed at recreating human liver tissue in vitro.

  10. Nano-regenerative medicine towards clinical outcome of stem cell and tissue engineering in humans

    PubMed Central

    Arora, Pooja; Sindhu, Annu; Dilbaghi, Neeraj; Chaudhury, Ashok; Rajakumar, Govindasamy; Rahuman, Abdul Abdul

    2012-01-01

    Nanotechnology is a fast growing area of research that aims to create nanomaterials or nanostructures development in stem cell and tissue-based therapies. Concepts and discoveries from the fields of bio nano research provide exciting opportunities of using stem cells for regeneration of tissues and organs. The application of nanotechnology to stem-cell biology would be able to address the challenges of disease therapeutics. This review covers the potential of nanotechnology approaches towards regenerative medicine. Furthermore, it focuses on current aspects of stem- and tissue-cell engineering. The magnetic nanoparticles-based applications in stem-cell research open new frontiers in cell and tissue engineering. PMID:22260258

  11. Scaffold-assisted cartilage tissue engineering using infant chondrocytes from human hip cartilage.

    PubMed

    Kreuz, P C; Gentili, C; Samans, B; Martinelli, D; Krüger, J P; Mittelmeier, W; Endres, M; Cancedda, R; Kaps, C

    2013-12-01

    Studies about cartilage repair in the hip and infant chondrocytes are rare. The aim of our study was to evaluate the use of infant articular hip chondrocytes for tissue engineering of scaffold-assisted cartilage grafts. Hip cartilage was obtained from five human donors (age 1-10 years). Expanded chondrocytes were cultured in polyglycolic acid (PGA)-fibrin scaffolds. De- and re-differentiation of chondrocytes were assessed by histological staining and gene expression analysis of typical chondrocytic marker genes. In vivo, cartilage matrix formation was assessed by histology after subcutaneous transplantation of chondrocyte-seeded PGA-fibrin scaffolds in immunocompromised mice. The donor tissue was heterogenous showing differentiated articular cartilage and non-differentiated tissue and considerable expression of type I and II collagens. Gene expression analysis showed repression of typical chondrocyte and/or mesenchymal marker genes during cell expansion, while markers were re-induced when expanded cells were cultured in PGA-fibrin scaffolds. Cartilage formation after subcutaneous transplantation of chondrocyte loaded PGA-fibrin scaffolds in nude mice was variable, with grafts showing resorption and host cell infiltration or formation of hyaline cartilage rich in type II collagen. Addition of human platelet rich plasma (PRP) to cartilage grafts resulted robustly in formation of hyaline-like cartilage that showed type II collagen and regions with type X collagen. These results suggest that culture of expanded and/or de-differentiated infant hip cartilage cells in PGA-fibrin scaffolds initiates chondrocyte re-differentiation. The heterogenous donor tissue containing immature chondrocytes bears the risk of cartilage repair failure in vivo, which may be possibly overcome by the addition of PRP. Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  12. Challenges in Cardiac Tissue Engineering

    PubMed Central

    Tandon, Nina; Godier, Amandine; Maidhof, Robert; Marsano, Anna; Martens, Timothy P.; Radisic, Milica

    2010-01-01

    Cardiac tissue engineering aims to create functional tissue constructs that can reestablish the structure and function of injured myocardium. Engineered constructs can also serve as high-fidelity models for studies of cardiac development and disease. In a general case, the biological potential of the cell—the actual “tissue engineer”—is mobilized by providing highly controllable three-dimensional environments that can mediate cell differentiation and functional assembly. For cardiac regeneration, some of the key requirements that need to be met are the selection of a human cell source, establishment of cardiac tissue matrix, electromechanical cell coupling, robust and stable contractile function, and functional vascularization. We review here the potential and challenges of cardiac tissue engineering for developing therapies that could prevent or reverse heart failure. PMID:19698068

  13. Characterizing human pluripotent-stem-cell-derived vascular cells for tissue engineering applications.

    PubMed

    Kusuma, Sravanti; Facklam, Amanda; Gerecht, Sharon

    2015-02-15

    Tissue-engineered constructs are rendered useless without a functional vasculature owing to a lack of nutrients and oxygen. Cell-based approaches to reconstruct blood vessels can yield structures that mimic native vasculature and aid transplantation. Vascular derivatives of human induced pluripotent stem cells (hiPSCs) offer opportunities to generate patient-specific therapies and potentially provide unlimited amounts of vascular cells. To be used in engineered vascular constructs and confer therapeutic benefit, vascular derivatives must exhibit additional key properties, including extracellular matrix (ECM) production to confer structural integrity and growth factor production to facilitate integration. In this study, we examine the hypothesis that vascular cells derived from hiPSCs exhibit these critical properties to facilitate their use in engineered tissues. hiPSCs were codifferentiated toward early vascular cells (EVCs), a bicellular population of endothelial cells (ECs) and pericytes, under varying low-oxygen differentiation conditions; subsequently, ECs were isolated and passaged. We found that EVCs differentiated under low-oxygen conditions produced copious amounts of collagen IV and fibronectin as well as vascular endothelial growth factor and angiopoietin 2. EVCs differentiated under atmospheric conditions did not demonstrate such abundant ECM expression, but exhibited greater expression of angiopoietin 1. Isolated ECs could proliferate up to three passages while maintaining the EC marker vascular endothelial cadherin. Isolated ECs demonstrated an increased propensity to produce ECM compared with their EVC correlates and took on an arterial-like fate. These findings illustrate that hiPSC vascular derivates hold great potential for therapeutic use and should continue to be a preferred cell source for vascular construction.

  14. DENTAL PULP TISSUE ENGINEERING

    PubMed Central

    Demarco, FF; Conde, MCM; Cavalcanti, B; Casagrande, L; Sakai, V; Nör, JE

    2013-01-01

    Dental pulp is a highly specialized mesenchymal tissue, which have a restrict regeneration capacity due to anatomical arrangement and post-mitotic nature of odontoblastic cells. Entire pulp amputation followed by pulp-space disinfection and filling with an artificial material cause loss of a significant amount of dentin leaving as life-lasting sequelae a non-vital and weakened tooth. However, regenerative endodontics is an emerging field of modern tissue engineering that demonstrated promising results using stem cells associated with scaffolds and responsive molecules. Thereby, this article will review the most recent endeavors to regenerate pulp tissue based on tissue engineering principles and providing insightful information to readers about the different aspects enrolled in tissue engineering. Here, we speculate that the search for the ideal combination of cells, scaffolds, and morphogenic factors for dental pulp tissue engineering may be extended over future years and result in significant advances in other areas of dental and craniofacial research. The finds collected in our review showed that we are now at a stage in which engineering a complex tissue, such as the dental pulp, is no longer an unachievable and the next decade will certainly be an exciting time for dental and craniofacial research. PMID:21519641

  15. Pluripotency of Stem Cells from Human Exfoliated Deciduous Teeth for Tissue Engineering

    PubMed Central

    Rosa, Vinicius; Dubey, Nileshkumar; Islam, Intekhab; Min, Kyung-San; Nör, Jacques E.

    2016-01-01

    Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative pluripotent cells that can be retrieved from primary teeth. Although SHED are isolated from the dental pulp, their differentiation potential is not limited to odontoblasts only. In fact, SHED can differentiate into several cell types including neurons, osteoblasts, adipocytes, and endothelial cells. The high plasticity makes SHED an interesting stem cell model for research in several biomedical areas. This review will discuss key findings about the characterization and differentiation of SHED into odontoblasts, neurons, and hormone secreting cells (e.g., hepatocytes and islet-like cell aggregates). The outcomes of the studies presented here support the multipotency of SHED and their potential to be used for tissue engineering-based therapies. PMID:27313627

  16. Ex-Vivo Tissues Engineering Modeling for Reconstructive Surgery Using Human Adult Adipose Stem Cells and Polymeric Nanostructured Matrix

    PubMed Central

    Morena, Francesco; Argentati, Chiara; Calzoni, Eleonora; Cordellini, Marino; Emiliani, Carla; D’Angelo, Francesco; Martino, Sabata

    2016-01-01

    The major challenge for stem cell translation regenerative medicine is the regeneration of damaged tissues by creating biological substitutes capable of recapitulating the missing function in the recipient host. Therefore, the current paradigm of tissue engineering strategies is the combination of a selected stem cell type, based on their capability to differentiate toward committed cell lineages, and a biomaterial, that, due to own characteristics (e.g., chemical, electric, mechanical property, nano-topography, and nanostructured molecular components), could serve as active scaffold to generate a bio-hybrid tissue/organ. Thus, effort has been made on the generation of in vitro tissue engineering modeling. Here, we present an in vitro model where human adipose stem cells isolated from lipoaspirate adipose tissue and breast adipose tissue, cultured on polymeric INTEGRA® Meshed Bilayer Wound Matrix (selected based on conventional clinical applications) are evaluated for their potential application for reconstructive surgery toward bone and adipose tissue. We demonstrated that human adipose stem cells isolated from lipoaspirate and breast tissue have similar stemness properties and are suitable for tissue engineering applications. Finally, the overall results highlighted lipoaspirate adipose tissue as a good source for the generation of adult adipose stem cells.

  17. Biomaterials for Tissue Engineering

    PubMed Central

    Lee, Esther J.; Kasper, F. Kurtis; Mikos, Antonios G.

    2013-01-01

    Biomaterials serve as an integral component of tissue engineering. They are designed to provide architectural framework reminiscent of native extracellular matrix in order to encourage cell growth and eventual tissue regeneration. Bone and cartilage represent two distinct tissues with varying compositional and mechanical properties. Despite these differences, both meet at the osteochondral interface. This article presents an overview of current biomaterials employed in bone and cartilage applications, discusses some design considerations, and alludes to future prospects within this field of research. PMID:23820768

  18. Biomaterials for tissue engineering.

    PubMed

    Lee, Esther J; Kasper, F Kurtis; Mikos, Antonios G

    2014-02-01

    Biomaterials serve as an integral component of tissue engineering. They are designed to provide architectural framework reminiscent of native extracellular matrix in order to encourage cell growth and eventual tissue regeneration. Bone and cartilage represent two distinct tissues with varying compositional and mechanical properties. Despite these differences, both meet at the osteochondral interface. This article presents an overview of current biomaterials employed in bone and cartilage applications, discusses some design considerations, and alludes to future prospects within this field of research.

  19. Human flexor tendon tissue engineering: in vivo effects of stem cell reseeding.

    PubMed

    Schmitt, Taliah; Fox, Paige M; Woon, Colin Y; Farnebo, Simon J; Bronstein, Joel A; Behn, Anthony; Pham, Hung; Chang, James

    2013-10-01

    Tissue-engineered human flexor tendons may be an option to aid in reconstruction of complex upper extremity injuries with significant tendon loss. The authors hypothesize that human adipose-derived stem cells remain viable following reseeding on human tendon scaffolds in vivo and aid in graft integration. Decellularized human flexor tendons harvested from fresh-frozen cadavers and reseeded with green fluorescent protein-labeled pooled human adipose-derived stem cells were examined with bioluminescent imaging and immunohistochemistry. Reseeded repaired tendons were compared biomechanically with unseeded controls following implantation in athymic rats at 2 and 4 weeks. The ratio of collagen I to collagen III at the repair site was examined using Sirius red staining. To confirm cell migration, reseeded and unseeded tendons were placed either in contact or with a 1-mm gap for 12 days. Green fluorescent protein signal was then detected. Following reseeding, viable cells were visualized at 12 days in vitro and 4 weeks in vivo. Biomechanical testing revealed no significant difference in ultimate load to failure and 2-mm gap force. Histologic evaluation showed host cell invasion and proliferation of the repair sites. No increase in collagen III was noted in reseeded constructs. Cell migration was confirmed from reseeded constructs to unseeded tendon scaffolds with tendon contact. Human adipose-derived stem cells reseeded onto decellularized allograft scaffolds are viable over 4 weeks in vivo. The movement of host cells into the scaffold and movement of adipose-derived stem cells along and into the scaffold suggests biointegration of the allograft.

  20. Rapid tissue engineering of biomimetic human corneal limbal crypts with 3D niche architecture.

    PubMed

    Levis, Hannah J; Massie, Isobel; Dziasko, Marc A; Kaasi, Andreas; Daniels, Julie T

    2013-11-01

    Limbal epithelial stem cells are responsible for the maintenance of the human corneal epithelium and these cells reside in a specialised stem cell niche. They are located at the base of limbal crypts, in a physically protected microenvironment in close proximity to a variety of neighbouring niche cells. Design and recreation of elements of various stem cell niches have allowed researchers to simplify aspects of these complex microenvironments for further study in vitro. We have developed a method to rapidly and reproducibly create bioengineered limbal crypts (BLCs) in a collagen construct using a simple one-step method. Liquid is removed from collagen hydrogels using hydrophilic porous absorbers (HPAs) that have custom moulded micro-ridges on the base. The resulting topography on the surface of the thin collagen constructs resembles the dimensions of the stromal crypts of the human limbus. Human limbal epithelial cells seeded onto the surface of the constructs populate these BLCs and form numerous layers with a high proportion of the cells lining the crypts expressing putative stem cell marker, p63α. The HPAs are produced using a moulding process that is flexible and can be adapted depending on the requirements of the end user. Creation of defined topographical features using this process could be applicable to numerous tissue-engineering applications where varied 3-dimensional niche architectures are required.

  1. Therapeutic efficiency of tissue-engineered human corneal endothelium transplants on rabbit primary corneal endotheliopathy.

    PubMed

    Fan, Ting-jun; Zhao, Jun; Hu, Xiu-zhong; Ma, Xi-ya; Zhang, Wen-bo; Yang, Chao-zhong

    2011-06-01

    To evaluate the therapeutic efficiency of tissue-engineered human corneal endothelia (TE-HCEs) on rabbit primary corneal endotheliopathy (PCEP), TE-HCEs reconstructed with monoclonal human corneal endothelial cells (mcHCECs) and modified denuded amniotic membranes (mdAMs) were transplanted into PCEP models of New Zealand white rabbits using penetrating keratoplasty. The TE-HCEs were examined using diverse techniques including slit-lamp biomicroscopy observation and pachymeter and tonometer measurements in vivo, and fluorescent microscopy, alizarin red staining, paraffin sectioning, scanning and transmission electron microscopy observations in vitro. The corneas of transplanted eyes maintained transparency for as long as 200 d without obvious edema or immune rejection. The corneal thickness of transplanted eyes decreased gradually after transplanting, reaching almost the thickness of normal eyes after 156 d, while the TE-HCE non-transplanted eyes were turbid and showed obvious corneal edema. The polygonal corneal endothelial cells in the transplanted area originated from the TE-HCE transplant. An intact monolayer corneal endothelium had been reconstructed with the morphology, cell density and structure similar to those of normal rabbit corneal endothelium. In conclusion, the transplanted TE-HCE can reconstruct the integrality of corneal endothelium and restore corneal transparency and thickness in PCEP rabbits. The TE-HCE functions normally as an endothelial barrier and pump and promises to be an equivalent of HCE for clinical therapy of human PCEP.

  2. Application of eGFP to label human periodontal ligament stem cells in periodontal tissue engineering.

    PubMed

    Wen, Yong; Lan, Jing; Huang, Haiyun; Yu, Meijiao; Cui, Jun; Liang, Jin; Jiang, Baoqi; Xu, Xin

    2012-09-01

    To establish human periodontal ligament stem cells (hPDLSC) with high and stable expression of enhanced green fluorescent protein (eGFP) and to obtain an ideal vector expression system that suitable for gene therapy in periodontal tissue engineering. hPDLSCs were transfected with eGFP for 48h via different MOI (25, 50, 100, 200 and 400) by lentiviral vector, the transfection efficiency was evaluated by fluorescent microscopy and flow cytometry, and transfected hPDLSCs proliferation was evaluated by MTT. Pluripotent, differentiation capacity and ALP expression status were determined further. Osteoblast-associated genes expressions for osteogenesis were evaluated by quantitative-PCR. In addition, rat molar periodontal fenestration defect model was used for evaluating periodontal tissue engineering. The transfection efficiency after 48h were 44.7%, 60.9%, 71.7%, 85.8%, and 86.9% respectively. There was no significant effect of transfection (at different MOI levels of 25, 50, 100, and 200) on the proliferation of hPDLSCs (designated as eGFP-hPDLSCs) compared with hPDLSCs (P>0.05). However, proliferation of eGFP hPDLSCs at MOI 400 became slower (P<0.05). Both eGFP hPDLSCs and hPDLSCs were able to differentiate into osteocytes and adipocytes under certain conditioned media. At 7 days, expression levels of COL-1, RUNX2 in hPDLSCS were higher than those in eGFP hPDLSCs (P<0.05); expression levels of ALP and OPN in eGFP hPDLSCs were similar to those in hPDLSCs (P>0.05). Newly regenerated bone formation was observed in the defect model used. Among the transfection conditions, 48h transfection at MOI 200 is optimal for labelling hPDLSCs with eGFP in a lentiviral vector. There is no change in capability of the eGFP hPDLSCs osteogenesis. The lentiviral vector with eGFP is an appropriate expression vector system and hPDLSCs are ideal seeding cells for gene therapy in periodontal tissue engineering. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Human umbilical cord stem cell encapsulation in novel macroporous and injectable fibrin for muscle tissue engineering

    PubMed Central

    Liu, Jun; Xu, Hockin H.K.; Zhou, Hongzhi; Weir, Michael D.; Chen, Qianming; Trotman, Carroll Ann

    2012-01-01

    There has been little research on the seeding of human umbilical cord mesenchymal stem cells (hUCMSCs) in three-dimensional scaffolds for muscle tissue engineering. The objectives of this study were: (i) to seed hUCMSCs in a fibrin hydrogel containing fast-degradable microbeads (dMBs) to create macropores to enhance cell viability; and (ii) to investigate the encapsulated cell proliferation and myogenic differentiation for muscle tissue engineering. Mass fractions of 0–80% of dMBs were tested, and 35% of dMBs in fibrin was shown to avoid fibrin shrinkage while creating macropores and promoting cell viability. This construct was referred to as “dMB35”. Fibrin without dMBs was termed “dMB0”. Microbead degradation created macropores in fibrin and improved cell viability. The percentage of live cells in dMB35 reached 91% at 16 days, higher than the 81% in dMB0 (p < 0.05). Live cell density in dMB35 was 1.6-fold that of dMB0 (p < 0.05). The encapsulated hUCMSCs proliferated, increasing the cell density by 2.6 times in dMB35 from 1 to 16 days. MTT activity for dMB35 was substantially higher than that for dMB0 at 16 days (p < 0.05). hUCMSCs in dMB35 had high gene expressions of myotube markers of myosin heavy chain 1 (MYH1) and alpha-actinin 3 (ACTN3). Elongated, multinucleated cells were formed with positive staining of myogenic specific proteins including myogenin, MYH, ACTN and actin alpha 1. Moreover, a significant increase in cell fusion was detected with myogenic induction. In conclusion, hUCMSCs were encapsulated in fibrin with degradable microbeads for the first time, achieving greatly enhanced cell viability and successful myogenic differentiation with formation of multinucleated myotubes. The injectable and macroporous fibrin–dMB–hUCMSC construct may be promising for muscle tissue engineering applications. PMID:22902812

  4. Characterization of In Vitro Engineered Human Adipose Tissues: Relevant Adipokine Secretion and Impact of TNF-α

    PubMed Central

    Aubin, Kim; Safoine, Meryem; Proulx, Maryse; Audet-Casgrain, Marie-Alice; Côté, Jean-François; Têtu, Félix-André; Roy, Alphonse; Fradette, Julie

    2015-01-01

    Representative modelling of human adipose tissue functions is central to metabolic research. Tridimensional models able to recreate human adipogenesis in a physiological tissue-like context in vitro are still scarce. We describe the engineering of white adipose tissues reconstructed from their cultured adipose-derived stromal precursor cells. We hypothesize that these reconstructed tissues can recapitulate key functions of AT under basal and pro-inflammatory conditions. These tissues, featuring human adipocytes surrounded by stroma, were stable and metabolically active in long-term cultures (at least 11 weeks). Secretion of major adipokines and growth factors by the reconstructed tissues was determined and compared to media conditioned by human native fat explants. Interestingly, the secretory profiles of the reconstructed adipose tissues indicated an abundant production of leptin, PAI-1 and angiopoietin-1 proteins, while higher HGF levels were detected for the human fat explants. We next demonstrated the responsiveness of the tissues to the pro-inflammatory stimulus TNF-α, as reflected by modulation of MCP-1, NGF and HGF secretion, while VEGF and leptin protein expression did not vary. TNF-α exposure induced changes in gene expression for adipocyte metabolism-associated mRNAs such as SLC2A4, FASN and LIPE, as well as for genes implicated in NF-κB activation. Finally, this model was customized to feature adipocytes representative of progressive stages of differentiation, thereby allowing investigations using newly differentiated or more mature adipocytes. In conclusion, we produced tridimensional tissues engineered in vitro that are able to recapitulate key characteristics of subcutaneous white adipose tissue. These tissues are produced from human cells and their neo-synthesized matrix elements without exogenous or synthetic biomaterials. Therefore, they represent unique tools to investigate the effects of pharmacologically active products on human stromal cells

  5. Characterization of In Vitro Engineered Human Adipose Tissues: Relevant Adipokine Secretion and Impact of TNF-α.

    PubMed

    Aubin, Kim; Safoine, Meryem; Proulx, Maryse; Audet-Casgrain, Marie-Alice; Côté, Jean-François; Têtu, Félix-André; Roy, Alphonse; Fradette, Julie

    2015-01-01

    Representative modelling of human adipose tissue functions is central to metabolic research. Tridimensional models able to recreate human adipogenesis in a physiological tissue-like context in vitro are still scarce. We describe the engineering of white adipose tissues reconstructed from their cultured adipose-derived stromal precursor cells. We hypothesize that these reconstructed tissues can recapitulate key functions of AT under basal and pro-inflammatory conditions. These tissues, featuring human adipocytes surrounded by stroma, were stable and metabolically active in long-term cultures (at least 11 weeks). Secretion of major adipokines and growth factors by the reconstructed tissues was determined and compared to media conditioned by human native fat explants. Interestingly, the secretory profiles of the reconstructed adipose tissues indicated an abundant production of leptin, PAI-1 and angiopoietin-1 proteins, while higher HGF levels were detected for the human fat explants. We next demonstrated the responsiveness of the tissues to the pro-inflammatory stimulus TNF-α, as reflected by modulation of MCP-1, NGF and HGF secretion, while VEGF and leptin protein expression did not vary. TNF-α exposure induced changes in gene expression for adipocyte metabolism-associated mRNAs such as SLC2A4, FASN and LIPE, as well as for genes implicated in NF-κB activation. Finally, this model was customized to feature adipocytes representative of progressive stages of differentiation, thereby allowing investigations using newly differentiated or more mature adipocytes. In conclusion, we produced tridimensional tissues engineered in vitro that are able to recapitulate key characteristics of subcutaneous white adipose tissue. These tissues are produced from human cells and their neo-synthesized matrix elements without exogenous or synthetic biomaterials. Therefore, they represent unique tools to investigate the effects of pharmacologically active products on human stromal cells

  6. Human adipose-derived stem cells: definition, isolation, tissue-engineering applications.

    PubMed

    Nae, S; Bordeianu, I; Stăncioiu, A T; Antohi, N

    2013-01-01

    Recent researches have demonstrated that the most effective repair system of the body is represented by stem cells - unspecialized cells, capable of self-renewal through successive mitoses, which have also the ability to transform into different cell types through differentiation. The discovery of adult stem cells represented an important step in regenerative medicine because they no longer raises ethical or legal issues and are more accessible. Only in 2002, stem cells isolated from adipose tissue were described as multipotent stem cells. Adipose tissue stem cells benefits in tissue engineering and regenerative medicine are numerous. Development of adipose tissue engineering techniques offers a great potential in surpassing the existing limits faced by the classical approaches used in plastic and reconstructive surgery. Adipose tissue engineering clinical applications are wide and varied, including reconstructive, corrective and cosmetic procedures. Nowadays, adipose tissue engineering is a fast developing field, both in terms of fundamental researches and medical applications, addressing issues related to current clinical pathology or trauma management of soft tissue injuries in different body locations.

  7. Mechanical Stimulation Protocols of Human Derived Cells in Articular Cartilage Tissue Engineering - A Systematic Review.

    PubMed

    Khozoee, Baktash; Mafi, Pouya; Mafi, Reza; Khan, Wasim S

    2017-01-01

    Mechanical stimulation is a key factor in articular cartilage generation and maintenance. Bioreactor systems have been designed and built in order to deliver specific types of mechanical stimulation. The focus has been twofold, applying a type of preconditioning in order to stimulate cell differentiation, and to simulate in vivo conditions in order to gain further insight into how cells respond to different stimulatory patterns. Due to the complex forces at work within joints, it is difficult to simulate mechanical conditions using a bioreactor. The aim of this review is to gain a deeper understanding of the complexities of mechanical stimulation protocols by comparing those employed in bioreactors in the context of tissue engineering for articular cartilage, and to consider their effects on cultured cells. Allied and Complementary Medicine 1985 to 2016, Ovid MEDLINE[R] 1946 to 2016, and Embase 1974 to 2016 were searched using key terms. Results were subject to inclusion and exclusion criteria, key findings summarised into a table and subsequently discussed. Based on this review it is overwhelmingly clear that mechanical stimulation leads to increased chondrogenic properties in the context of bioreactor articular cartilage tissue engineering using human cells. However, given the variability and lack of controlled factors between research articles, results are difficult to compare, and a standardised method of evaluating stimulation protocols proved challenging. With improved standardisation in mechanical stimulation protocol reporting, bioreactor design and building processes, along with a better understanding of joint behaviours, we hope to perform a meta-analysis on stimulation protocols and methods. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  8. Alkylation of human hair keratin for tunable hydrogel erosion and drug delivery in tissue engineering applications.

    PubMed

    Han, Sangheon; Ham, Trevor R; Haque, Salma; Sparks, Jessica L; Saul, Justin M

    2015-09-01

    Polymeric biomaterials that provide a matrix for cell attachment and proliferation while achieving delivery of therapeutic agents are an important component of tissue engineering and regenerative medicine strategies. Keratins are a class of proteins that have received attention for numerous tissue engineering applications because, like other natural polymers, they promote favorable cell interactions and have non-toxic degradation products. Keratins can be extracted from various sources including human hair, and they are characterized by a high percentage of cysteine residues. Thiol groups on reductively extracted keratin (kerateine) form disulfide bonds, providing a more stable cross-linked hydrogel network than oxidatively extracted keratin (keratose) that cannot form disulfide crosslinks. We hypothesized that an iodoacetamide alkylation (or "capping") of cysteine thiol groups on the kerateine form of keratin could be used as a simple method to modulate the levels of disulfide crosslinking in keratin hydrogels, providing tunable rates of gel erosion and therapeutic agent release. After alkylation, the alkylated kerateines still formed hydrogels and the alkylation led to changes in the mechanical and visco-elastic properties of the materials consistent with loss of disulfide crosslinking. The alkylated kerateines did not lead to toxicity in MC3T3-E1 pre-osteoblasts. These cells adhered to keratin at levels comparable to fibronectin and greater than collagen. Alkylated kerateine gels eroded more rapidly than non-alkylated kerateine and this control over erosion led to tunable rates of delivery of rhBMP-2, rhIGF-1, and ciprofloxacin. These results demonstrate that alkylation of kerateine cysteine residues provides a cell-compatible approach to tune rates of hydrogel erosion and therapeutic agent release within the context of a naturally-derived polymeric system.

  9. Magnetic Resonance Imaging of Cardiac Strain Pattern Following Transplantation of Human Tissue Engineered Heart Muscles

    PubMed Central

    Qin, Xulei; Riegler, Johannes; Tiburcy, Malte; Zhao, Xin; Chour, Tony; Ndoye, Babacar; Nguyen, Michael; Adams, Jackson; Ameen, Mohamed; Denney, Thomas S.; Yang, Phillip C.; Nguyen, Patricia; Zimmermann, Wolfram H.; Wu, Joseph C.

    2017-01-01

    Background The use of tissue engineering approaches in combination with exogenously produced cardiomyocytes offers the potential to restore contractile function after myocardial injury. However, current techniques assessing changes in global cardiac performance following such treatments are plagued by relatively low detection ability. As the treatment is locally performed, this detection could be improved by myocardial strain imaging that measures regional contractility. Methods and Results Tissue engineered heart muscles (EHMs) were generated by casting human embryonic stem cell-derived cardiomyocytes with collagen in preformed molds. EHMs were transplanted (n=12) to cover infarct and border zones of recipient rat hearts one month after ischemia reperfusion injury. A control group (n=10) received only sham placement of sutures without EHMs. To assess the efficacy of EHMs, MRI and ultrasound-based strain imaging were performed prior to and four weeks after transplantation. In addition to strain imaging, global cardiac performance was estimated from cardiac MRI. Although no significant differences were found with global changes in left ventricular ejection fraction (EF) (Control −9.6±1.3% vs. EHM −6.2±1.9%, P=0.17), regional myocardial strain from tagged MRI was able to detect preserved systolic function in EHM-treated animals compared to control (Control 4.4±1.0% vs. EHM 1.0±0.6%, P=0.04). However, ultrasound-based strain failed to detect any significant change (Control 2.1±3.0% vs. EHM 6.3±2.9%, P=0.46). Conclusions This study highlights the feasibility of using cardiac strain from tagged MRI to assess functional changes in rat models due to localized regenerative therapies, which may not be detected by conventional measures of global systolic performance. PMID:27903535

  10. PHB/PHBHHx scaffolds and human adipose-derived stem cells for cartilage tissue engineering.

    PubMed

    Ye, Chuan; Hu, Ping; Ma, Min-Xian; Xiang, Yang; Liu, Ri-Guang; Shang, Xian-Wen

    2009-09-01

    The goal of this study was to investigate the potential of polyhydroxybutyrate (PHB)/poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) (PHB/PHBHHx) to produce neocartilage upon seeding with differentiated human adipose-derived stem cells (hASCs). hASCs were grown on a three-dimensional PHB/PHBHHx scaffold in vitro with or without chondrogenic media for 14 days. Scanning electron microscopy showed that differentiated cells produced abundant extracellular matrices with increasing culture time. No cytotoxicity was observed by the live/dead cell viability assay. GAG and total collagen content in the differentiated cells increased significantly with in vitro culture time. After 14 days of in vitro culture, the differentiated cells grown on the (PHB/PHBHHx) scaffold (differentiated cells/(PHB/PHBHHx)) were implanted into the subcutaneous layer nude mice for 12 or 24 weeks, non-differentiated cells/(PHB/PHBHHx) were implanted as the control group. The differentiated cells/(PHB/PHBHHx) implants formed cartilage-like tissue after 24 weeks of implantation, and stained positive for collagen type II, safranin O, and toluidine blue. In addition, typical cartilage lacuna was observed, and there were no remnants of PHB/PHBHHx. Collagen type II was detected by Western blot at 12 and 24 weeks of implantation. In the control group, no cartilage formation was observed. This study demonstrated that PHB/PHBHHx is a suitable material for cartilage tissue engineering.

  11. Implantable Tissue-Engineered Blood Vessels from Human Induced Pluripotent Stem Cells

    PubMed Central

    Gui, Liqiong; Dash, Biraja C.; Luo, Jiesi; Qin, Lingfeng; Zhao, Liping; Yamamoto, Kota; Hashimoto, Takuya; Wu, Hongwei; Dardik, Alan; Tellides, George; Niklason, Laura E.; Qyang, Yibing

    2016-01-01

    Derivation of functional vascular smooth muscle cells (VSMCs) from human induced pluripotent stem cells (hiPSCs) to generate tissue-engineered blood vessels (TEBVs) holds great potential in treating patients with vascular diseases. Herein, hiPSCs were differentiated into alpha-smooth muscle actin (α-SMA) and calponin-positive VSMCs, which were seeded onto polymer scaffolds in bioreactors for vascular tissue growth. A functional TEBV with abundant collagenous matrix and sound mechanics resulted, which contained cells largely positive for α-SMA and smooth muscle myosin heavy chain (SM-MHC). Moreover, when hiPSC-derived TEBV segments were implanted into nude rats as abdominal aorta interposition grafts, they remained unruptured and patent with active vascular remodeling, and showed no evidence of teratoma formation during a 2-week proof-of-principle study. Our studies represent the development of the first implantable TEBVs based on hiPSCs, and pave the way for developing autologous or allogeneic grafts for clinical use in patients with vascular disease. PMID:27336184

  12. Tissue engineering research in oral implant surgery.

    PubMed

    Ueda, M; Tohnai, I; Nakai, H

    2001-03-01

    In this article, we introduce some of the more extensively evaluated technologies using concepts of tissue engineering. We report on hard tissue engineering and soft tissue engineering and their utility for dental implant therapy. For hard tissue engineering, we evaluated human recombinant bone morphogenetic protein-2 and marrow mesenchymal stem cells using a model of sinus augmentation procedure in rabbit. We also describe distraction osteogenesis as another category for hard tissue engineering. In addition, we evaluate soft tissue management using cultured epithelial grafting for soft tissue engineering. The results of our tissue regeneration materials and methods in this study are positive. When the tissue engineering materials are used in clinics in the future, implant surgery could be the leading field.

  13. Biomechanical and cellular segmental characterization of human meniscus: building the basis for Tissue Engineering therapies.

    PubMed

    Pereira, H; Caridade, S G; Frias, A M; Silva-Correia, J; Pereira, D R; Cengiz, I F; Mano, J F; Oliveira, J M; Espregueira-Mendes, J; Reis, R L

    2014-09-01

    To overcome current limitations of Tissue Engineering (TE) strategies, deeper comprehension on meniscus biology is required. This study aims to combine biomechanical segmental analysis of fresh human meniscus tissues and its correlation with architectural and cellular characterization. Morphologically intact menisci, from 44 live donors were studied after division into three radial segments. Dynamic mechanical analysis (DMA) was performed at physiological-like conditions. Micro-computed tomography (CT) analysis of freeze-dried samples assessed micro-structure. Flow cytometry, histology and histomorphometry were used for cellular study and quantification. Anterior segments present significantly higher damping properties. Mid body fresh medial meniscus presents higher values of E' compared to lateral. Cyclic loads influence the viscoelastic behavior of menisci. By increasing the frequency leads to an increase in stiffness. Conversely, with increasing frequencies, the capacity to dissipate energy and damping properties initially decrease and then rise again. Age and gender directly correlate with higher E' and tan δ. Micro-CT analysis revealed that mean porosity was 55.5 (21.2-89.8)% and 64.7 (47.7-81.8)% for freeze-dried lateral and medial meniscus, respectively. Predominant cells are positive for CD44, CD73, CD90 and CD105, and lack CD31, CD34 and CD45 (present in smaller populations). Histomorphometry revealed that cellularity decreases from vascular zone 1 to zone 3. Anterior segments of lateral and medial meniscus have inferior cellularity as compared to mid body and posterior ones. Menisci are not uniform structures. Anterior segments have lower cellularity and higher damping. Cyclic loads influence viscoelastic characteristics. Future TE therapies should consider segmental architecture, cellularity and biomechanics of fresh tissue. Copyright © 2014. Published by Elsevier Ltd.

  14. Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels.

    PubMed

    Jung, Youngmee; Ji, HaYeun; Chen, Zaozao; Fai Chan, Hon; Atchison, Leigh; Klitzman, Bruce; Truskey, George; Leong, Kam W

    2015-10-12

    Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

  15. Human engineered heart tissue as a model system for drug testing.

    PubMed

    Eder, Alexandra; Vollert, Ingra; Hansen, Arne; Eschenhagen, Thomas

    2016-01-15

    Drug development is time- and cost-intensive and, despite extensive efforts, still hampered by the limited value of current preclinical test systems to predict side effects, including proarrhythmic and cardiotoxic effects in clinical practice. Part of the problem may be related to species-dependent differences in cardiomyocyte biology. Therefore, the event of readily available human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) has raised hopes that this human test bed could improve preclinical safety pharmacology as well as drug discovery approaches. However, hiPSC-CM are immature and exhibit peculiarities in terms of ion channel function, gene expression, structural organization and functional responses to drugs that limit their present usefulness. Current efforts are thus directed towards improving hiPSC-CM maturity and high-content readouts. Culturing hiPSC-CM as 3-dimensional engineered heart tissue (EHT) improves CM maturity and anisotropy and, in a 24-well format using silicone racks, enables automated, multiplexed high content readout of contractile function. This review summarizes the principal technology and focuses on advantages and disadvantages of this technology and its potential for preclinical drug screening.

  16. Cartilage tissue engineering.

    PubMed

    Moreira-Teixeira, Liliana S; Georgi, Nicole; Leijten, Jeroen; Wu, Ling; Karperien, Marcel

    2011-01-01

    Cartilage tissue engineering is the art aimed at repairing defects in the articular cartilage which covers the bony ends in the joints. Since its introduction in the early 1990s of the past century, cartilage tissue engineering using ACI has been used in thousands of patients to repair articular cartilage defects. This review focuses on emerging strategies to improve cartilage repair by incorporating fundamental knowledge of developmental and cell biology in the design of optimized strategies for cell delivery at the defect site and to locally stimulate cartilage repair responses. Copyright © 2011 S. Karger AG, Basel.

  17. Functional improvement and maturation of rat and human engineered heart tissue by chronic electrical stimulation.

    PubMed

    Hirt, Marc N; Boeddinghaus, Jasper; Mitchell, Alice; Schaaf, Sebastian; Börnchen, Christian; Müller, Christian; Schulz, Herbert; Hubner, Norbert; Stenzig, Justus; Stoehr, Andrea; Neuber, Christiane; Eder, Alexandra; Luther, Pradeep K; Hansen, Arne; Eschenhagen, Thomas

    2014-09-01

    Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca(2+)-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT.

  18. Enzymatic cross-linking of human recombinant elastin (HELP) as biomimetic approach in vascular tissue engineering.

    PubMed

    Bozzini, Sabrina; Giuliano, Liliana; Altomare, Lina; Petrini, Paola; Bandiera, Antonella; Conconi, Maria Teresa; Farè, Silvia; Tanzi, Maria Cristina

    2011-12-01

    The use of polymers naturally occurring in the extracellular matrix (ECM) is a promising strategy in regenerative medicine. If compared to natural ECM proteins, proteins obtained by recombinant DNA technology have intrinsic advantages including reproducible macromolecular composition, sequence and molecular mass, and overcoming the potential pathogens transmission related to polymers of animal origin. Among ECM-mimicking materials, the family of recombinant elastin-like polymers is proposed for drug delivery applications and for the repair of damaged elastic tissues. This work aims to evaluate the potentiality of a recombinant human elastin-like polypeptide (HELP) as a base material of cross-linked matrices for regenerative medicine. The cross-linking of HELP was accomplished by the insertion of cross-linking sites, glutamine and lysine, in the recombinant polymer and generating ε-(γ-glutamyl) lysine links through the enzyme transglutaminase. The cross-linking efficacy was estimated by infrared spectroscopy. Freeze-dried cross-linked matrices showed swelling ratios in deionized water (≈2500%) with good structural stability up to 24 h. Mechanical compression tests, performed at 37°C in wet conditions, in a frequency sweep mode, indicated a storage modulus of 2/3 kPa, with no significant changes when increasing number of cycles or frequency. These results demonstrate the possibility to obtain mechanically resistant hydrogels via enzymatic crosslinking of HELP. Cytotoxicity tests of cross-linked HELP were performed with human umbilical vein endothelial cells, by use of transwell filter chambers for 1-7 days, or with its extracts in the opportune culture medium for 24 h. In both cases no cytotoxic effects were observed in comparison with the control cultures. On the whole, the results suggest the potentiality of this genetically engineered HELP for regenerative medicine applications, particularly for vascular tissue regeneration.

  19. The cultivation of human multipotent mesenchymal stromal cells in clinical grade medium for bone tissue engineering.

    PubMed

    Pytlík, Robert; Stehlík, David; Soukup, Tomás; Kalbácová, Marie; Rypácek, Frantisek; Trc, Tomás; Mulinková, Katarína; Michnová, Petra; Kideryová, Linda; Zivný, Jan; Klener, Pavel; Veselá, Romana; Trnený, Marek; Klener, Pavel

    2009-07-01

    Clinical application of human multipotent mesenchymal stromal cells (hMSCs) requires their expansion to be safe and rapid. We aimed to develop an expansion protocol which would avoid xenogeneic proteins, including fetal calf serum (FCS), and which would shorten the cultivation time and avoid multiple passaging. First, we have compared research-grade alpha-MEM medium with clinical grade CellGro for Hematopoietic Cells' Medium. When FCS was used for supplementation and non-adherent cells were discarded, both media were comparable. Both media were comparable also when pooled human serum (hS) was used instead of FCS, but the numbers of hMSCs were lower when non-adherent cells were discarded. However, significantly more hMSCs were obtained both in alpha-MEM and in CellGro supplemented with hS when the non-adherent cells were left in the culture. Furthermore, addition of recombinant cytokines and other supplements (EGF, PDGF-BB, M-CSF, FGF-2, dexamethasone, insulin and ascorbic acid) to the CellGro co-culture system with hS led to 40-fold increase of hMSCs' yield after two weeks of cultivation compared to alpha-MEM with FCS. The hMSCs expanded in the described co-culture system retain their osteogenic, adipogenic and chondrogenic differentiation potential in vitro and produce bone-like mineralized tissue when propagated on 3D polylactide scaffolds in immunodeficient mice. Our protocol thus allows for very effective one-step, xenogeneic protein-free expansion of hMSCs, which can be easily transferred into good manufacturing practice (GMP) conditions for large-scale, clinical-grade production of hMSCs for purposes of tissue engineering.

  20. Engineered cartilaginous tubes for tracheal tissue replacement via self-assembly and fusion of human mesenchymal stem cell constructs

    PubMed Central

    Dikina, Anna D.; Strobel, Hannah A.; Lai, Bradley P.; Rolle, Marsha W.; Alsberg, Eben

    2015-01-01

    There is a critical need to engineer a neotrachea because currently there are no long-term treatments for tracheal stenoses affecting large portions of the airway. In this work, a modular tracheal tissue replacement strategy was developed. High-cell density, scaffold-free human mesenchymal stem cell-derived cartilaginous rings and tubes were successfully generated through employment of custom designed culture wells and a ring-to-tube assembly system. Furthermore, incorporation of transforming growth factor-β1-delivering gelatin microspheres into the engineered tissues enhanced chondrogenesis with regard to tissue size and matrix production and distribution in the ring- and tube-shaped constructs, as well as luminal rigidity of the tubes. Importantly, all engineered tissues had similar or improved biomechanical properties compared to rat tracheas, which suggests they could be transplanted in a small animal model for airway defects. The modular, bottom up approach used to grow stem cell-based cartilaginous tubes in this report is a promising platform to engineer complex organs (e.g., trachea), with control over tissue size and geometry, and has the potential to be used to generate autologous tissue implants for human clinical applications. PMID:25818451

  1. Engineered cartilaginous tubes for tracheal tissue replacement via self-assembly and fusion of human mesenchymal stem cell constructs.

    PubMed

    Dikina, Anna D; Strobel, Hannah A; Lai, Bradley P; Rolle, Marsha W; Alsberg, Eben

    2015-06-01

    There is a critical need to engineer a neotrachea because currently there are no long-term treatments for tracheal stenoses affecting large portions of the airway. In this work, a modular tracheal tissue replacement strategy was developed. High-cell density, scaffold-free human mesenchymal stem cell-derived cartilaginous rings and tubes were successfully generated through employment of custom designed culture wells and a ring-to-tube assembly system. Furthermore, incorporation of transforming growth factor-β1-delivering gelatin microspheres into the engineered tissues enhanced chondrogenesis with regard to tissue size and matrix production and distribution in the ring- and tube-shaped constructs, as well as luminal rigidity of the tubes. Importantly, all engineered tissues had similar or improved biomechanical properties compared to rat tracheas, which suggests they could be transplanted into a small animal model for airway defects. The modular, bottom up approach used to grow stem cell-based cartilaginous tubes in this report is a promising platform to engineer complex organs (e.g., trachea), with control over tissue size and geometry, and has the potential to be used to generate autologous tissue implants for human clinical applications.

  2. Engineering graded tissue interfaces.

    PubMed

    Phillips, Jennifer E; Burns, Kellie L; Le Doux, Joseph M; Guldberg, Robert E; García, Andrés J

    2008-08-26

    Interfacial zones between tissues provide specialized, transitional junctions central to normal tissue function. Regenerative medicine strategies focused on multiple cell types and/or bi/tri-layered scaffolds do not provide continuously graded interfaces, severely limiting the integration and biological performance of engineered tissue substitutes. Inspired by the bone-soft tissue interface, we describe a biomaterial-mediated gene transfer strategy for spatially regulated genetic modification and differentiation of primary dermal fibroblasts within tissue-engineered constructs. We demonstrate that zonal organization of osteoblastic and fibroblastic cellular phenotypes can be engineered by a simple, one-step seeding of fibroblasts onto scaffolds containing a spatial distribution of retrovirus encoding the osteogenic transcription factor Runx2/Cbfa1. Gradients of immobilized retrovirus, achieved via deposition of controlled poly(L-lysine) densities, resulted in spatial patterns of transcription factor expression, osteoblastic differentiation, and mineralized matrix deposition. Notably, this graded distribution of mineral deposition and mechanical properties was maintained when implanted in vivo in an ectopic site. Development of this facile and robust strategy is significant toward the regeneration of continuous interfacial zones that mimic the cellular and microstructural characteristics of native tissue.

  3. Mechanical properties of completely autologous human tissue engineered blood vessels compared to human saphenous vein and mammary artery

    PubMed Central

    Konig, Gerhardt; McAllister, Todd N; Dusserre, Nathalie; Garrido, Sergio A; Iyican, Corey; Marini, Alicia; Fiorillo, Alex; Avila, Hernan; Wystrychowski, Wojciech; Zagalski, Krzysztof; Maruszewski, Marcin; Jones, Alyce Linthurst; Cierpka, Lech; de la Fuente, Luis M; L’Heureux, Nicolas

    2009-01-01

    We have previously reported initial clinical feasibility with our small diameter tissue engineered blood vessel (TEBV). Here we present in vitro results of the mechanical properties of the TEBVs of the first 25 patients enrolled in an arterio-venous (A-V) shunt safety trial, and compare these properties with those of risk-matched human vein and artery. TEBV average burst pressures (3,490 +/− 892 mmHg, n=230) were higher than native saphenous vein (SV) (1,599 +/− 877 mmHg, n=7), and not significantly different than native internal mammary artery (IMA) (3,196 +/− 1,264 mmHg, n=16). Suture retention strength for the TEBVs (152 +/− 50 gmf) was also not significantly different than IMA (138 +/− 50 gmf). Compliance for the TEBVs prior to implantation (3.4 +/− 1.6 %/100 mmHg) was lower than IMA (11.5 +/− 3.9 %/100 mmHg). By 6 months post-implant, the TEBV compliance (8.8 +/− 4.2 %/100 mmHg, n=5) had increased to values comparable to IMA, and showed no evidence of dilation or aneurysm formation. With clinical time points beyond 21 months as an A-V shunt without intervention, the mechanical tests and subsequent lot release criteria reported here would seem appropriate minimum standards for clinical use of tissue engineered vessels. PMID:19111338

  4. Mechanical properties of completely autologous human tissue engineered blood vessels compared to human saphenous vein and mammary artery.

    PubMed

    Konig, Gerhardt; McAllister, Todd N; Dusserre, Nathalie; Garrido, Sergio A; Iyican, Corey; Marini, Alicia; Fiorillo, Alex; Avila, Hernan; Wystrychowski, Wojciech; Zagalski, Krzysztof; Maruszewski, Marcin; Jones, Alyce Linthurst; Cierpka, Lech; de la Fuente, Luis M; L'Heureux, Nicolas

    2009-03-01

    We have previously reported the initial clinical feasibility with our small diameter tissue engineered blood vessel (TEBV). Here we present in vitro results of the mechanical properties of the TEBVs of the first 25 patients enrolled in an arterio-venous (A-V) shunt safety trial, and compare these properties with those of risk-matched human vein and artery. TEBV average burst pressures (3490+/-892 mmHg, n=230) were higher than native saphenous vein (SV) (1599+/-877 mmHg, n=7), and not significantly different from native internal mammary artery (IMA) (3196+/-1264 mmHg, n=16). Suture retention strength for the TEBVs (152+/-50 gmf) was also not significantly different than IMA (138+/-50 gmf). Compliance for the TEBVs prior to implantation (3.4+/-1.6%/100 mmHg) was lower than IMA (11.5+/-3.9%/100 mmHg). By 6 months post-implant, the TEBV compliance (8.8+/-4.2%/100 mmHg, n=5) had increased to values comparable to IMA, and showed no evidence of dilation or aneurysm formation. With clinical time points beyond 21 months as an A-V shunt without intervention, the mechanical tests and subsequent lot release criteria reported here would seem appropriate minimum standards for clinical use of tissue engineered vessels.

  5. Neoproteoglycans in tissue engineering

    PubMed Central

    Weyers, Amanda; Linhardt, Robert J.

    2014-01-01

    Proteoglycans, comprised of a core protein to which glycosaminoglycan chains are covalently linked, are an important structural and functional family of macromolecules found in the extracellular matrix. Advances in our understanding of biological interactions have lead to a greater appreciation for the need to design tissue engineering scaffolds that incorporate mimetics of key extracellular matrix components. A variety of synthetic and semisynthetic molecules and polymers have been examined by tissue engineers that serve as structural, chemical and biological replacements for proteoglycans. These proteoglycan mimetics have been referred to as neoproteoglycans and serve as functional and therapeutic replacements for natural proteoglycans that are often unavailable for tissue engineering studies. Although neoproteoglycans have important limitations, such as limited signaling ability and biocompatibility, they have shown promise in replacing the natural activity of proteoglycans through cell and protein binding interactions. This review focuses on the recent in vivo and in vitro tissue engineering applications of three basic types of neoproteoglycan structures, protein–glycosaminoglycan conjugates, nano-glycosaminoglycan composites and polymer–glycosaminoglycan complexes. PMID:23399318

  6. Tissue Engineering Research

    DTIC Science & Technology

    2002-01-01

    European Molecular Biology Laboratory (EMBL) ......................................................................... 117 German Cancer Research Center...National Cancer Center Research Institute...................................................................................... 173 National Institute for...Green in the United States, has been the focus of skin tissue engineering at Nagoya University. Work at the National Cancer Center Institute in Tokyo

  7. In vivo tissue engineering of musculoskeletal tissues.

    PubMed

    McCullen, Seth D; Chow, Andre G Y; Stevens, Molly M

    2011-10-01

    Tissue engineering of musculoskeletal tissues often involves the in vitro manipulation and culture of progenitor cells, growth factors and biomaterial scaffolds. Though in vitro tissue engineering has greatly increased our understanding of cellular behavior and cell-material interactions, this methodology is often unable to recreate tissue with the hierarchical organization and vascularization found within native tissues. Accordingly, investigators have focused on alternative in vivo tissue engineering strategies, whereby the traditional triad (cells, growth factors, scaffolds) or a combination thereof are directly implanted at the damaged tissue site or within ectopic sites capable of supporting neo-tissue formation. In vivo tissue engineering may offer a preferential route for regeneration of musculoskeletal and other tissues with distinct advantages over in vitro methods based on the specific location of endogenous cultivation, recruitment of autologous cells, and patient-specific regenerated tissues.

  8. Rapid manufacturing techniques for the tissue engineering of human heart valves.

    PubMed

    Lueders, Cora; Jastram, Ben; Hetzer, Roland; Schwandt, Hartmut

    2014-10-01

    Three-dimensional (3D) printing technologies have reached a level of quality that justifies considering rapid manufacturing for medical applications. Herein, we introduce a new approach using 3D printing to simplify and improve the fabrication of human heart valve scaffolds by tissue engineering (TE). Custom-made human heart valve scaffolds are to be fabricated on a selective laser-sintering 3D printer for subsequent seeding with vascular cells from human umbilical cords. The scaffolds will be produced from resorbable polymers that must feature a number of specific properties: the structure, i.e. particle granularity and shape, and thermic properties must be feasible for the printing process. They must be suitable for the cell-seeding process and at the same time should be resorbable. They must be applicable for implementation in the human body and flexible enough to support the full functionality of the valve. The research focuses mainly on the search for a suitable scaffold material that allows the implementation of both the printing process to produce the scaffolds and the cell-seeding process, while meeting all of the above requirements. Computer tomographic data from patients were transformed into a 3D data model suitable for the 3D printer. Our current activities involve various aspects of the printing process, material research and the implementation of the cell-seeding process. Different resorbable polymeric materials have been examined and used to fabricate heart valve scaffolds by rapid manufacturing. Human vascular cells attached to the scaffold surface should migrate additionally into the inner structure of the polymeric samples. The ultimate intention of our approach is to establish a heart valve fabrication process based on 3D rapid manufacturing and TE. Based on the computer tomographic data of a patient, a custom-made scaffold for a valve will be produced on a 3D printer and populated preferably by autologous cells. The long-term goal is to support

  9. Human Mesenchymal Stem Cells Reendothelialize Porcine Heart Valve Scaffolds: Novel Perspectives in Heart Valve Tissue Engineering

    PubMed Central

    Lanuti, Paola; Serafini, Francesco; Pierdomenico, Laura; Simeone, Pasquale; Bologna, Giuseppina; Ercolino, Eva; Di Silvestre, Sara; Guarnieri, Simone; Canosa, Carlo; Impicciatore, Gianna Gabriella; Chiarini, Stella; Magnacca, Francesco; Mariggiò, Maria Addolorata; Pandolfi, Assunta; Marchisio, Marco; Di Giammarco, Gabriele; Miscia, Sebastiano

    2015-01-01

    Abstract Heart valve diseases are usually treated by surgical intervention addressed for the replacement of the damaged valve with a biosynthetic or mechanical prosthesis. Although this approach guarantees a good quality of life for patients, it is not free from drawbacks (structural deterioration, nonstructural dysfunction, and reintervention). To overcome these limitations, the heart valve tissue engineering (HVTE) is developing new strategies to synthesize novel types of valve substitutes, by identifying efficient sources of both ideal scaffolds and cells. In particular, a natural matrix, able to interact with cellular components, appears to be a suitable solution. On the other hand, the well-known Wharton's jelly mesenchymal stem cells (WJ-MSCs) plasticity, regenerative abilities, and their immunomodulatory capacities make them highly promising for HVTE applications. In the present study, we investigated the possibility to use porcine valve matrix to regenerate in vitro the valve endothelium by WJ-MSCs differentiated along the endothelial lineage, paralleled with human umbilical vein endothelial cells (HUVECs), used as positive control. Here, we were able to successfully decellularize porcine heart valves, which were then recellularized with both differentiated-WJ-MSCs and HUVECs. Data demonstrated that both cell types were able to reconstitute a cellular monolayer. Cells were able to positively interact with the natural matrix and demonstrated the surface expression of typical endothelial markers. Altogether, these data suggest that the interaction between a biological scaffold and WJ-MSCs allows the regeneration of a morphologically well-structured endothelium, opening new perspectives in the field of HVTE. PMID:26309804

  10. Mechanical loading regulates human MSC differentiation in a multi-layer hydrogel for osteochondral tissue engineering.

    PubMed

    Steinmetz, Neven J; Aisenbrey, Elizabeth A; Westbrook, Kristofer K; Qi, H Jerry; Bryant, Stephanie J

    2015-07-01

    A bioinspired multi-layer hydrogel was developed for the encapsulation of human mesenchymal stem cells (hMSCs) as a platform for osteochondral tissue engineering. The spatial presentation of biochemical cues, via incorporation of extracellular matrix analogs, and mechanical cues, via both hydrogel crosslink density and externally applied mechanical loads, were characterized in each layer. A simple sequential photopolymerization method was employed to form stable poly(ethylene glycol)-based hydrogels with a soft cartilage-like layer of chondroitin sulfate and low RGD concentrations, a stiff bone-like layer with high RGD concentrations, and an intermediate interfacial layer. Under a compressive load, the variation in hydrogel stiffness within each layer produced high strains in the soft cartilage-like layer, low strains in the stiff bone-like layer, and moderate strains in the interfacial layer. When hMSC-laden hydrogels were cultured statically in osteochondral differentiation media, the local biochemical and matrix stiffness cues were not sufficient to spatially guide hMSC differentiation after 21 days. However dynamic mechanical stimulation led to differentially high expression of collagens with collagen II in the cartilage-like layer, collagen X in the interfacial layer and collagen I in the bone-like layer and mineral deposits localized to the bone layer. Overall, these findings point to external mechanical stimulation as a potent regulator of hMSC differentiation toward osteochondral cellular phenotypes. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  11. Human Pluripotent Stem Cell Mechanobiology: Manipulating the Biophysical Microenvironment for Regenerative Medicine and Tissue Engineering Applications.

    PubMed

    Ireland, Ronald G; Simmons, Craig A

    2015-11-01

    A stem cell in its microenvironment is subjected to a myriad of soluble chemical cues and mechanical forces that act in concert to orchestrate cell fate. Intuitively, many of these soluble and biophysical factors have been the focus of intense study to successfully influence and direct cell differentiation in vitro. Human pluripotent stem cells (hPSCs) have been of considerable interest in these studies due to their great promise for regenerative medicine. Culturing and directing differentiation of hPSCs, however, is currently extremely labor-intensive and lacks the efficiency required to generate large populations of clinical-grade cells. Improved efficiency may come from efforts to understand how the cell biophysical signals can complement biochemical signals to regulate cell pluripotency and direct differentiation. In this concise review, we explore hPSC mechanobiology and how the hPSC biophysical microenvironment can be manipulated to maintain and differentiate hPSCs into functional cell types for regenerative medicine and tissue engineering applications. © 2015 AlphaMed Press.

  12. Human hypertrophic and keloid scar models: principles, limitations and future challenges from a tissue engineering perspective

    PubMed Central

    van den Broek, Lenie J; Limandjaja, Grace C; Niessen, Frank B; Gibbs, Susan

    2014-01-01

    Most cutaneous wounds heal with scar formation. Ideally, an inconspicuous normotrophic scar is formed, but an abnormal scar (hypertrophic scar or keloid) can also develop. A major challenge to scientists and physicians is to prevent adverse scar formation after severe trauma (e.g. burn injury) and understand why some individuals will form adverse scars even after relatively minor injury. Currently, many different models exist to study scar formation, ranging from simple monolayer cell culture to 3D tissue-engineered models even to humanized mouse models. Currently, these high-/medium-throughput test models avoid the main questions referring to why an adverse scar forms instead of a normotrophic scar and what causes a hypertrophic scar to form rather than a keloid scar and also, how is the genetic predisposition of the individual and the immune system involved. This information is essential if we are to identify new drug targets and develop optimal strategies in the future to prevent adverse scar formation. This viewpoint review summarizes the progress on in vitro and animal scar models, stresses the limitations in the current models and identifies the future challenges if scar-free healing is to be achieved in the future. PMID:24750541

  13. Human hypertrophic and keloid scar models: principles, limitations and future challenges from a tissue engineering perspective.

    PubMed

    van den Broek, Lenie J; Limandjaja, Grace C; Niessen, Frank B; Gibbs, Susan

    2014-06-01

    Most cutaneous wounds heal with scar formation. Ideally, an inconspicuous normotrophic scar is formed, but an abnormal scar (hypertrophic scar or keloid) can also develop. A major challenge to scientists and physicians is to prevent adverse scar formation after severe trauma (e.g. burn injury) and understand why some individuals will form adverse scars even after relatively minor injury. Currently, many different models exist to study scar formation, ranging from simple monolayer cell culture to 3D tissue-engineered models even to humanized mouse models. Currently, these high-/medium-throughput test models avoid the main questions referring to why an adverse scar forms instead of a normotrophic scar and what causes a hypertrophic scar to form rather than a keloid scar and also, how is the genetic predisposition of the individual and the immune system involved. This information is essential if we are to identify new drug targets and develop optimal strategies in the future to prevent adverse scar formation. This viewpoint review summarizes the progress on in vitro and animal scar models, stresses the limitations in the current models and identifies the future challenges if scar-free healing is to be achieved in the future. © 2014 The Authors. Experimental Dermatology. Published by John Wiley & Sons Ltd.

  14. Interaction of Tissue Engineering Substrates with Serum Proteins and Its Influence on Human Primary Endothelial Cells.

    PubMed

    Mohan, Tamilselvan; Niegelhell, Katrin; Nagaraj, Chandran; Reishofer, David; Spirk, Stefan; Olschewski, Andrea; Stana Kleinschek, Karin; Kargl, Rupert

    2017-02-13

    Polymer-based biomaterials particularly polycaprolactone (PCL) are one of the most promising substrates for tissue engineering. The surface chemistry of these materials plays a major role since it governs protein adsorption, cell adhesion, viability, degradation, and biocompatibility in the first place. This study correlates the interaction of the most abundant serum proteins (albumin, immunoglobulins, fibrinogen) with the surface properties of PCL and its influence on the morphology and metabolic activity of primary human arterial endothelial cells that are seeded on the materials. Prior to that, thin films of PCL are manufactured by spin-coating and characterized in detail. A quartz crystal microbalance with dissipation (QCM-D), a multiparameter surface plasmon resonance spectroscopy instrument (MP-SPR), wettability data, and atomic force microscopy are combined to elucidate the pH-dependent protein adsorption on the PCL substrates. Primary endothelial cells are cultured on the protein modified polymer, and conclusions are drawn on the significant impact of type and form of proteins coatings on cell morphology and metabolic activity.

  15. Automated decellularization of intact, human-sized lungs for tissue engineering.

    PubMed

    Price, Andrew P; Godin, Lindsay M; Domek, Alex; Cotter, Trevor; D'Cunha, Jonathan; Taylor, Doris A; Panoskaltsis-Mortari, Angela

    2015-01-01

    We developed an automated system that can be used to decellularize whole human-sized organs and have shown lung as an example. Lungs from 20 to 30 kg pigs were excised en bloc with the trachea and decellularized with our established protocol of deionized water, detergents, sodium chloride, and porcine pancreatic DNase. A software program was written to control a valve manifold assembly that we built for selection and timing of decellularization fluid perfusion through the airway and the vasculature. This system was interfaced with a prototypic bioreactor chamber that was connected to another program, from a commercial source, which controlled the volume and flow pressure of fluids. Lung matrix that was decellularized by the automated method was compared to a manual method previously used by us and others. Automation resulted in more consistent acellular matrix preparations as demonstrated by measuring levels of DNA, hydroxyproline (collagen), elastin, laminin, and glycosaminoglycans. It also proved highly beneficial in saving time as the decellularization procedure was reduced from days down to just 24 h. Developing a rapid, controllable, automated system for production of reproducible matrices in a closed system is a major step forward in whole-organ tissue engineering.

  16. Fast-Degradable Microbeads Encapsulating Human Umbilical Cord Stem Cells in Alginate for Muscle Tissue Engineering

    PubMed Central

    Liu, Jun; Zhou, Hongzhi; Weir, Michael D.

    2012-01-01

    Human umbilical cord mesenchymal stem cells (hUCMSCs) are inexhaustible and can be obtained without an invasive surgery. To date, there has been no report on seeding hUCMSCs in three-dimensional scaffolds for muscle tissue engineering. The objectives of this study were to (1) investigate hUCMSC seeding in a scaffold for muscle engineering and (2) develop a novel construct consisting of hUCMSC-encapsulating and fast-degradable microbeads inside a hydrogel matrix. The rationale was that the hydrogel matrix would maintain the defect volume, while the microbeads would degrade to release the cells and concomitantly create macropores in the matrix. hUCMSCs were encapsulated in alginate-fibrin microbeads, which were packed in an Arg-Gly-Asp (RGD)-modified alginate matrix (AM). This construct is referred to as hUCMSC-microbead-AM. The control consisted of the usual cell encapsulation in AM without microbeads (referred to as hUCMSC-AM). In the hUCMSC-AM construct, the hUCMSCs showed as round dots with no spreading at 1–14 days. In contrast, cells in the hUCMSC-microbead-AM construct had a healthy spreading and elongated morphology. The microbeads successfully degraded and released the cells at 8 days. Myogenic expressions for hUCMSC-microbead-AM were more than threefold those of hUCMSC-AM (p<0.05). Immunofluorescence for myogenic markers was much stronger for hUCMSC-microbead-AM than hUCMSC-AM. Muscle creatine kinase of hUCMSC-microbead-AM at 14 days was twofold that of hUCMSC-AM (p<0.05). In conclusion, hUCMSC encapsulation in novel fast-degradable microbeads inside a hydrogel matrix was investigated for muscle engineering. Compared to the usual method of seeding cells in a hydrogel matrix, hUCMSC-microbead-AM construct had greatly improved cell viability and myogenic differentiation, and hence, is promising to enhance muscle regeneration. PMID:22697426

  17. Craniofacial bone tissue engineering.

    PubMed

    Wan, Derrick C; Nacamuli, Randall P; Longaker, Michael T

    2006-04-01

    Repair and reconstruction of the craniofacial skeleton represents a significant biomedical burden, with thousands of procedures per-formed annually secondary to injuries and congenital malformations. Given the multitude of current approaches, the need for more effective strategies to repair these bone deficits is apparent. This article explores two major modalities for craniofacial bone tissue engineering: distraction osteogenesis and cellular based therapies. Current understanding of the guiding principles for each of these modalities is elaborated on along with the knowledge gained from clinical and investigative studies. By laying this foundation, future directions for craniofacial distraction and cell-based bone engineering have emerged with great promise for the advancement of clinical practice.

  18. Concise review: humanized models of tumor immunology in the 21st century: convergence of cancer research and tissue engineering.

    PubMed

    Holzapfel, Boris Michael; Wagner, Ferdinand; Thibaudeau, Laure; Levesque, Jean-Pierre; Hutmacher, Dietmar Werner

    2015-06-01

    Despite positive testing in animal studies, more than 80% of novel drug candidates fail to proof their efficacy when tested in humans. This is primarily due to the use of preclinical models that are not able to recapitulate the physiological or pathological processes in humans. Hence, one of the key challenges in the field of translational medicine is to "make the model organism mouse more human." To get answers to questions that would be prognostic of outcomes in human medicine, the mouse's genome can be altered in order to create a more permissive host that allows the engraftment of human cell systems. It has been shown in the past that these strategies can improve our understanding of tumor immunology. However, the translational benefits of these platforms have still to be proven. In the 21st century, several research groups and consortia around the world take up the challenge to improve our understanding of how to humanize the animal's genetic code, its cells and, based on tissue engineering principles, its extracellular microenvironment, its tissues, or entire organs with the ultimate goal to foster the translation of new therapeutic strategies from bench to bedside. This article provides an overview of the state of the art of humanized models of tumor immunology and highlights future developments in the field such as the application of tissue engineering and regenerative medicine strategies to further enhance humanized murine model systems.

  19. Multiphoton tomography for tissue engineering

    NASA Astrophysics Data System (ADS)

    König, Karsten

    2008-02-01

    Femtosecond laser multiphoton tomography has been employed in the field of tissue engineering to perform 3D high-resolution imaging of the extracellular matrix proteins elastin and collagen as well as of living cells without any fixation, slicing, and staining. Near infrared 80 MHz picojoule femtosecond laser pulses are able to excite the endogenous fluorophores NAD(P)H, flavoproteins, melanin, and elastin via a non-resonant two-photon excitation process. In addition, collagen can be imaged by second harmonic generation. Using a two-PMT detection system, the ratio of elastin to collagen was determined during optical sectioning. A high submicron spatial resolution and 50 picosecond temporal resolution was achieved using galvoscan mirrors and piezodriven focusing optics as well as a time-correlated single photon counting module with a fast microchannel plate detector and fast photomultipliers. Multiphoton tomography has been used to optimize the tissue engineering of heart valves and vessels in bioincubators as well as to characterize artificial skin. Stem cell characterization and manipulation are of major interest for the field of tissue engineering. Using the novel sub-20 femtosecond multiphoton nanoprocessing laser microscope FemtOgene, the differentiation of human stem cells within spheroids has been in vivo monitored with submicron resolution. In addition, the efficient targeted transfection has been demonstrated. Clinical studies on the interaction of tissue-engineered products with the natural tissue environment can be performed with in vivo multiphoton tomograph DermaInspect.

  20. Synthetic biology meets tissue engineering.

    PubMed

    Davies, Jamie A; Cachat, Elise

    2016-06-15

    Classical tissue engineering is aimed mainly at producing anatomically and physiologically realistic replacements for normal human tissues. It is done either by encouraging cellular colonization of manufactured matrices or cellular recolonization of decellularized natural extracellular matrices from donor organs, or by allowing cells to self-organize into organs as they do during fetal life. For repair of normal bodies, this will be adequate but there are reasons for making unusual, non-evolved tissues (repair of unusual bodies, interface to electromechanical prostheses, incorporating living cells into life-support machines). Synthetic biology is aimed mainly at engineering cells so that they can perform custom functions: applying synthetic biological approaches to tissue engineering may be one way of engineering custom structures. In this article, we outline the 'embryological cycle' of patterning, differentiation and morphogenesis and review progress that has been made in constructing synthetic biological systems to reproduce these processes in new ways. The state-of-the-art remains a long way from making truly synthetic tissues, but there are now at least foundations for future work. © 2016 Authors; published by Portland Press Limited.

  1. Synthetic biology meets tissue engineering

    PubMed Central

    Davies, Jamie A.; Cachat, Elise

    2016-01-01

    Classical tissue engineering is aimed mainly at producing anatomically and physiologically realistic replacements for normal human tissues. It is done either by encouraging cellular colonization of manufactured matrices or cellular recolonization of decellularized natural extracellular matrices from donor organs, or by allowing cells to self-organize into organs as they do during fetal life. For repair of normal bodies, this will be adequate but there are reasons for making unusual, non-evolved tissues (repair of unusual bodies, interface to electromechanical prostheses, incorporating living cells into life-support machines). Synthetic biology is aimed mainly at engineering cells so that they can perform custom functions: applying synthetic biological approaches to tissue engineering may be one way of engineering custom structures. In this article, we outline the ‘embryological cycle’ of patterning, differentiation and morphogenesis and review progress that has been made in constructing synthetic biological systems to reproduce these processes in new ways. The state-of-the-art remains a long way from making truly synthetic tissues, but there are now at least foundations for future work. PMID:27284030

  2. Trichostatin A enhances differentiation of human induced pluripotent stem cells to cardiogenic cells for cardiac tissue engineering.

    PubMed

    Lim, Shiang Y; Sivakumaran, Priyadharshini; Crombie, Duncan E; Dusting, Gregory J; Pébay, Alice; Dilley, Rodney J

    2013-09-01

    Human induced pluripotent stem (iPS) cells are a promising source of autologous cardiomyocytes to repair and regenerate myocardium for treatment of heart disease. In this study, we have identified a novel strategy to enhance cardiac differentiation of human iPS cells by treating embryoid bodies (EBs) with a histone deacetylase inhibitor, trichostatin A (TSA), together with activin A and bone morphogenetic protein 4 (BMP4). Over a narrow window of concentrations, TSA (1 ng/ml) directed the differentiation of human iPS cells into a cardiomyocyte lineage. TSA also exerted an additive effect with activin A (100 ng/ml) and BMP4 (20 ng/ml). The resulting cardiomyocytes expressed several cardiac-specific transcription factors and contractile proteins at both gene and protein levels. Functionally, the contractile EBs displayed calcium cycling and were responsive to the chronotropic agents isoprenaline (0.1 μM) and carbachol (1 μM). Implanting microdissected beating areas of iPS cells into tissue engineering chambers in immunocompromised rats produced engineered constructs that supported their survival, and they maintained spontaneous contraction. Human cardiomyocytes were identified as compact patches of muscle tissue incorporated within a host fibrocellular stroma and were vascularized by host neovessels. In conclusion, human iPS cell-derived cardiomyocytes can be used to engineer functional cardiac muscle tissue for studying the pathophysiology of cardiac disease, for drug discovery test beds, and potentially for generation of cardiac grafts to surgically replace damaged myocardium.

  3. Tissue engineering for lateral ridge augmentation with recombinant human bone morphogenetic protein 2 combination therapy: a case report.

    PubMed

    Mandelaris, George A; Spagnoli, Daniel B; Rosenfeld, Alan L; McKee, James; Lu, Mei

    2015-01-01

    This case report describes a tissue-engineered reconstruction with recombinant human bone morphogenetic protein 2/acellular collagen sponge (rhBMP-2/ ACS) + cancellous allograft and space maintenance via Medpor Contain mesh in the treatment of a patient requiring maxillary and mandibular horizontal ridge augmentation to enable implant placement. The patient underwent a previously unsuccessful corticocancellous bone graft at these sites. Multiple and contiguous sites in the maxilla and in the mandibular anterior, demonstrating advanced lateral ridge deficiencies, were managed using a tissue engineering approach as an alternative to autogenous bone harvesting. Four maxillary and three mandibular implants were placed 9 and 10 months, respectively, after tissue engineering reconstruction, and all were functioning successfully after 24 months of follow-up. Histomorphometric analysis of a bone core obtained at the time of the maxillary implant placement demonstrated a mean of 76.1% new vital bone formation, 22.2% marrow/cells, and 1.7% residual graft tissue. Tissue engineering for lateral ridge augmentation with combination therapy requires further research to determine predictability and limitations.

  4. Formation of in vivo tissue engineered human hyaline cartilage in the shape of a trachea with internal support.

    PubMed

    Ruszymah, Binti Haji Idrus; Chua, Kienhui; Latif, Mazlyzam Abdul; Hussein, Fuzina Nor; Saim, Aminuddin Bin

    2005-11-01

    Treatment and management of congenital as well as post-traumatic trachea stenosis remains a challenge in pediatric surgery. The aim of this study was to reconstruct a trachea with human nasal septum chondrocytes by using the combination of biodegradable hydrogel and non-biodegradable high-density polyethylene (HDP) as the internal predetermined shape scaffold. Human nasal septum cartilage was harvested as excessive tissue after elective septoplasty and digested in 0.6% collagenase II. Chondrocytes were cultured in an equal volume mix of Ham's F12 medium and Dulbecco's modified eagle medium added with 10% fetal bovine serum and basic fibroblast growth factor. After two passages, the cultured chondrocytes were trypsinized and mixed with biodegradable hydrogel Pluronic F127. The chondrocytes-hydrogel admixture was then painted over the HDP as the internal support in a predetermined trachea shape. The composite was then implanted subcutaneously in athymic mice. After 8 weeks of in vivo implantation, the tissue engineered trachea constructs were harvested. Macroscopic appearance of the tissue engineered trachea constructs demonstrated that the HDP were 80-90% covered with yellowish glistering cartilage like tissue without any sign of inflammation. The tissue engineered trachea cartilage consisted of evenly spaced lacunae embedded in basophilic matrix and stained red with Safranin-O staining denoting abundant proteoglycans production. Type II collagen gene which was expressed in native cartilage was highly expressed in this tissue engineered trachea cartilage. We have successfully reconstructed a trachea in vivo with human nasal septum chondrocytes using HDP as the internal support. This construct has the advantage of bio-inert and strength in which both are important properties in tracheal reconstruction.

  5. Stereolithography in tissue engineering.

    PubMed

    Skoog, Shelby A; Goering, Peter L; Narayan, Roger J

    2014-03-01

    Several recent research efforts have focused on use of computer-aided additive fabrication technologies, commonly referred to as additive manufacturing, rapid prototyping, solid freeform fabrication, or three-dimensional printing technologies, to create structures for tissue engineering. For example, scaffolds for tissue engineering may be processed using rapid prototyping technologies, which serve as matrices for cell ingrowth, vascularization, as well as transport of nutrients and waste. Stereolithography is a photopolymerization-based rapid prototyping technology that involves computer-driven and spatially controlled irradiation of liquid resin. This technology enables structures with precise microscale features to be prepared directly from a computer model. In this review, use of stereolithography for processing trimethylene carbonate, polycaprolactone, and poly(D,L-lactide) poly(propylene fumarate)-based materials is considered. In addition, incorporation of bioceramic fillers for fabrication of bioceramic scaffolds is reviewed. Use of stereolithography for processing of patient-specific implantable scaffolds is also discussed. In addition, use of photopolymerization-based rapid prototyping technology, known as two-photon polymerization, for production of tissue engineering scaffolds with smaller features than conventional stereolithography technology is considered.

  6. Simple Modular Bioreactors for Tissue Engineering: A System for Characterization of Oxygen Gradients, Human Mesenchymal Stem Cell Differentiation, and Prevascularization

    PubMed Central

    Lovett, Michael; Rockwood, Danielle; Baryshyan, Amanda

    2010-01-01

    Large-scale tissue engineering is limited by nutrient perfusion and mass transport limitations, especially oxygen diffusion, which restrict construct development to smaller than clinically relevant dimensions and limit the ability for in vivo integration. The goal of this work was to develop a modular approach to tissue engineering, where scaffold and tissue size, transport issues, and surgical implantation in vivo are considered from the outset. Human mesenchymal stem cells (hMSCs) were used as the model cell type, as their differentiation has been studied for several different cell lineages and often with conflicting results. Changes in the expression profiles of hMSCs differentiated under varied oxygen tensions are presented, demonstrating tissue-specific oxygen requirements for both adipogenic (20% O2) and chondrogenic (5% O2) differentiation. Oxygen and nutrient transport were enhanced by developing a bioreactor system for perfusing hMSC-seeded collagen gels using porous silk tubes, resulting in enhanced oxygen transport and cell viability within the gels. These systems are simple to use and scaled for versatility, to allow for the systematic study of relationships between cell content, oxygen, and cell function. The data may be combined with oxygen transport modeling to derive minimally sized modular units for construction of clinically relevant tissue-engineered constructs, a generic strategy that may be employed for vascularized target tissues. PMID:20528664

  7. Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering.

    PubMed

    Butler, Colin R; Hynds, Robert E; Gowers, Kate H C; Lee, Dani Do Hyang; Brown, James M; Crowley, Claire; Teixeira, Vitor H; Smith, Claire M; Urbani, Luca; Hamilton, Nicholas J; Thakrar, Ricky M; Booth, Helen L; Birchall, Martin A; De Coppi, Paolo; Giangreco, Adam; O'Callaghan, Christopher; Janes, Sam M

    2016-07-15

    Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. To define a scalable cell culture system to deliver airway epithelium to clinical grafts. Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures. 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.

  8. Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

    PubMed Central

    Gowers, Kate H. C.; Lee, Dani Do Hyang; Brown, James M.; Crowley, Claire; Teixeira, Vitor H.; Smith, Claire M.; Urbani, Luca; Hamilton, Nicholas J.; Thakrar, Ricky M.; Booth, Helen L.; Birchall, Martin A.; De Coppi, Paolo; Giangreco, Adam; O’Callaghan, Christopher

    2016-01-01

    Rationale: Stem cell–based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell–seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. Objectives: To define a scalable cell culture system to deliver airway epithelium to clinical grafts. Methods: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air–liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air–liquid interface cultures. Measurements and Main Results: 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. Conclusions: Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical

  9. [Safety evaluation of tissue engineered medical devices using normal human mesenchymal stem cells].

    PubMed

    Sawada, Rumi; Ito, Tomomi; Tsuchiya, Toshie

    2007-05-01

    Several recent studies demonstrated the potential of bioengineering using somatic stem cells in regenerative medicine. Adult human mesenchymal stem cells (hMSCs) derived from bone marrow have the pluripotency to differentiate into cells of mesodermal origin, e.g., bone, cartilage, adipose, and muscle cells; they, therefore, have many potential clinical applications. On the other hand, stem cells possess a self-renewal capability similar to cancer cells. For safety evaluation of tissue engineered medical devices using normal hMSCs, in this study, we investigated the expression levels of several genes that affect cell proliferation in hMSCs during in vitro culture. We focused on the relationship between the hMSC proliferation and their transforming growth factor beta (TGFbeta) signaling during in vitro culture. The proliferation rate of hMSCs gradually decreased and cellular senescence was observed for about 3 months. The mRNA expressions of TGFbeta1, TGFbeta2, and TGFbeta receptor type I (TGFbetaRI) in hMSCs increased with the length of cell culture. The mRNA expressions of Smad3 increased, but those of c-myc and nucleostemin decreased with the length hMSCs were in in vitro culture. In addition, the expression profiles of the genes which regulate cellular proliferation in hMSCs were significantly different from those of cancer cells. In conclusion, hMSCs derived from bone marrow seldom underwent spontaneous transformation during 1-2 months in vitro culture for use in clinical applications. In hMSCs as well as in epithelial cells, growth might be controlled by the TGFbeta family signaling.

  10. Value of human amniotic epithelial cells in tissue engineering for cornea.

    PubMed

    Fatimah, Simat Siti; Ng, Sook Luan; Chua, Kien Hui; Hayati, Abdul Rahman; Tan, Ay Eeng; Tan, Geok Chin

    2010-11-01

    Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco's modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.

  11. Raman fiberoptic probe for monitoring human tissue engineered oral mucosa constructs

    NASA Astrophysics Data System (ADS)

    Khmaladze, Alexander; Kuo, Shiuhyang; Okagbare, Paul; Marcelo, Cynthia L.; Feinberg, Stephen E.; Morris, Michael D.

    2013-02-01

    In oral and maxillofacial surgery, there is a need for tissue engineered constructs for dental implants, reconstructions due to trauma, oral cancer or congenital defects. A non-invasive quality monitoring of the fabrication of tissue engineered constructs during their production and implantation is a required component of any successful tissue engineering technique. We demonstrate the design and application of a Raman spectroscopic probe for rapid and noninvasive monitoring of Ex Vivo Produced Oral Mucosa Equivalent constructs (EVPOMEs). We conducted in vivo studies to identify Raman spectroscopic failure indicators for EVPOMEs (already developed in vitro), and found that Raman spectra of EVPOMEs exposed to thermal stress showed correlation of the band height ratio of CH2 deformation to phenylalanine ring breathing modes, providing a Raman metric to distinguish between viable and nonviable constructs. This is the first step towards the ultimate goal to design a stand-alone system, which will be usable in a clinical setting, as the data processing and analysis will be performed with minimal user intervention, based on already established and tested Raman spectroscopic indicators for EVPOMEs.

  12. Human oral mucosa tissue-engineered constructs monitored by Raman fiber-optic probe.

    PubMed

    Khmaladze, Alexander; Kuo, Shiuhyang; Kim, Roderick Y; Matthews, Robert V; Marcelo, Cynthia L; Feinberg, Stephen E; Morris, Michael D

    2015-01-01

    In maxillofacial and oral surgery, there is a need for the development of tissue-engineered constructs. They are used for reconstructions due to trauma, dental implants, congenital defects, or oral cancer. A noninvasive monitoring of the fabrication of tissue-engineered constructs at the production and implantation stages done in real time is extremely important for predicting the success of tissue-engineered grafts. We demonstrated a Raman spectroscopic probe system, its design and application, for real-time ex vivo produced oral mucosa equivalent (EVPOME) constructs noninvasive monitoring. We performed in vivo studies to find Raman spectroscopic indicators for postimplanted EVPOME failure and determined that Raman spectra of EVPOMEs preexposed to thermal stress during manufacturing procedures displayed correlation of the band height ratio of CH2 deformation to phenylalanine ring breathing modes, giving a Raman metric to distinguish between healthy and compromised postimplanted constructs. This study is the step toward our ultimate goal to develop a stand-alone system, to be used in a clinical setting, where the data collection and analysis are conducted on the basis of these spectroscopic indicators with minimal user intervention.

  13. "The state of the heart": Recent advances in engineering human cardiac tissue from pluripotent stem cells.

    PubMed

    Sirabella, Dario; Cimetta, Elisa; Vunjak-Novakovic, Gordana

    2015-08-01

    The pressing need for effective cell therapy for the heart has led to the investigation of suitable cell sources for tissue replacement. In recent years, human pluripotent stem cell research expanded tremendously, in particular since the derivation of human-induced pluripotent stem cells. In parallel, bioengineering technologies have led to novel approaches for in vitro cell culture. The combination of these two fields holds potential for in vitro generation of high-fidelity heart tissue, both for basic research and for therapeutic applications. However, this new multidisciplinary science is still at an early stage. Many questions need to be answered and improvements need to be made before clinical applications become a reality. Here we discuss the current status of human stem cell differentiation into cardiomyocytes and the combined use of bioengineering approaches for cardiac tissue formation and maturation in developmental studies, disease modeling, drug testing, and regenerative medicine.

  14. Concise Review: Human Dermis as an Autologous Source of Stem Cells for Tissue Engineering and Regenerative Medicine

    PubMed Central

    Vapniarsky, Natalia; Arzi, Boaz; Hu, Jerry C.; Nolta, Jan A.

    2015-01-01

    The exciting potential for regenerating organs from autologous stem cells is on the near horizon, and adult dermis stem cells (DSCs) are particularly appealing because of the ease and relative minimal invasiveness of skin collection. A substantial number of reports have described DSCs and their potential for regenerating tissues from mesenchymal, ectodermal, and endodermal lineages; however, the exact niches of these stem cells in various skin types and their antigenic surface makeup are not yet clearly defined. The multilineage potential of DSCs appears to be similar, despite great variability in isolation and in vitro propagation methods. Despite this great potential, only limited amounts of tissues and clinical applications for organ regeneration have been developed from DSCs. This review summarizes the literature on DSCs regarding their niches and the specific markers they express. The concept of the niches and the differentiation capacity of cells residing in them along particular lineages is discussed. Furthermore, the advantages and disadvantages of widely used methods to demonstrate lineage differentiation are considered. In addition, safety considerations and the most recent advancements in the field of tissue engineering and regeneration using DSCs are discussed. This review concludes with thoughts on how to prospectively approach engineering of tissues and organ regeneration using DSCs. Our expectation is that implementation of the major points highlighted in this review will lead to major advancements in the fields of regenerative medicine and tissue engineering. Significance Autologous dermis-derived stem cells are generating great excitement and efforts in the field of regenerative medicine and tissue engineering. The substantial impact of this review lies in its critical coverage of the available literature and in providing insight regarding niches, characteristics, and isolation methods of stem cells derived from the human dermis. Furthermore, it

  15. Concise Review: Human Dermis as an Autologous Source of Stem Cells for Tissue Engineering and Regenerative Medicine.

    PubMed

    Vapniarsky, Natalia; Arzi, Boaz; Hu, Jerry C; Nolta, Jan A; Athanasiou, Kyriacos A

    2015-10-01

    The exciting potential for regenerating organs from autologous stem cells is on the near horizon, and adult dermis stem cells (DSCs) are particularly appealing because of the ease and relative minimal invasiveness of skin collection. A substantial number of reports have described DSCs and their potential for regenerating tissues from mesenchymal, ectodermal, and endodermal lineages; however, the exact niches of these stem cells in various skin types and their antigenic surface makeup are not yet clearly defined. The multilineage potential of DSCs appears to be similar, despite great variability in isolation and in vitro propagation methods. Despite this great potential, only limited amounts of tissues and clinical applications for organ regeneration have been developed from DSCs. This review summarizes the literature on DSCs regarding their niches and the specific markers they express. The concept of the niches and the differentiation capacity of cells residing in them along particular lineages is discussed. Furthermore, the advantages and disadvantages of widely used methods to demonstrate lineage differentiation are considered. In addition, safety considerations and the most recent advancements in the field of tissue engineering and regeneration using DSCs are discussed. This review concludes with thoughts on how to prospectively approach engineering of tissues and organ regeneration using DSCs. Our expectation is that implementation of the major points highlighted in this review will lead to major advancements in the fields of regenerative medicine and tissue engineering. Autologous dermis-derived stem cells are generating great excitement and efforts in the field of regenerative medicine and tissue engineering. The substantial impact of this review lies in its critical coverage of the available literature and in providing insight regarding niches, characteristics, and isolation methods of stem cells derived from the human dermis. Furthermore, it provides

  16. Skin tissue engineering.

    PubMed

    Mansbridge, Jonathan

    2008-01-01

    The major applications of tissue-engineered skin substitutes are in promoting the healing of acute and chronic wounds. Several approaches have been taken by commercial companies to develop products to address these conditions. Skin substitutes include both acellular and cellular devices. While acellular skin substitutes act as a template for dermal formation, this discussion mainly covers cellular devices. In addressing therapeutic applications in tissue engineering generally, a valuable precursor is an understanding of the mechanism of the underlying pathology. While this is straightforward in many cases, it has not been available for wound healing. Investigation of the mode of action of the tissue-engineered skin substitutes has led to considerable insight into the mechanism of formation, maintenance and treatment of chronic wounds. Four aspects mediating healing are considered here for their mechanism of action: (i) colonization of the wound bed by live fibroblasts in the implant, (ii) the secretion of growth factors, (iii) provision of a suitable substrate for cell migration, particularly keratinocytes and immune cells, and (iv) modification of the immune system by secretion of neutrophil recruiting chemokines. An early event in acute wound healing is an influx of neutrophils that destroy planktonic bacteria. However, if the bacteria are able to form biofilm, they become resistant to neutrophil action and prevent reepithelialization. In this situation the wound becomes chronic. In chronic wounds, fibroblasts show a senescence-like phenotype with decreased secretion of neutrophil chemoattractants that make it more likely that biofilms become established. Treatment of the chronic wounds involves debridement to eliminate biofilm, and the use of antimicrobials. A role of skin substitutes is to provide non-senescent fibroblasts that attract and activate neutrophils to prevent biofilm re-establishment. The emphasis of the conclusion is the importance of preventing

  17. Tissue engineering strategies applied in the regeneration of the human intervertebral disk.

    PubMed

    Silva-Correia, Joana; Correia, Sandra I; Oliveira, Joaquim M; Reis, Rui L

    2013-12-01

    Low back pain (LBP) is one of the most common painful conditions that lead to work absenteeism, medical visits, and hospitalization. The majority of cases showing signs of LBP are due to age-related degenerative changes in the intervertebral disk (IVD), which are, in fact, associated with multiple spine pathologies. Traditional and more conservative procedures/clinical approaches only treat the symptoms of disease and not the underlying pathology, thus limiting their long-term efficiency. In the last few years, research and development of new approaches aiming to substitute the nucleus pulposus and annulus fibrosus tissue and stimulate its regeneration has been conducted. Regeneration of the damaged IVD using tissue engineering strategies appears particularly promising in pre-clinical studies. Meanwhile, surgical techniques must be adapted to this new approach in order to be as minimally invasive as possible, reducing recovering time and side effects associated to traditional surgeries. In this review, the current knowledge on IVD, its associated pathologies and current surgical procedures are summarized. Furthermore, it also provides a succinct and up-to-date overview on regenerative medicine research, especially on the newest tissue engineering strategies for IVD regeneration.

  18. The chorioallantoic membrane (CAM) assay for the study of human bone regeneration: a refinement animal model for tissue engineering

    PubMed Central

    Moreno-Jiménez, Inés; Hulsart-Billstrom, Gry; Lanham, Stuart A.; Janeczek, Agnieszka A.; Kontouli, Nasia; Kanczler, Janos M.; Evans, Nicholas D.; Oreffo, Richard OC

    2016-01-01

    Biomaterial development for tissue engineering applications is rapidly increasing but necessitates efficacy and safety testing prior to clinical application. Current in vitro and in vivo models hold a number of limitations, including expense, lack of correlation between animal models and human outcomes and the need to perform invasive procedures on animals; hence requiring new predictive screening methods. In the present study we tested the hypothesis that the chick embryo chorioallantoic membrane (CAM) can be used as a bioreactor to culture and study the regeneration of human living bone. We extracted bone cylinders from human femoral heads, simulated an injury using a drill-hole defect, and implanted the bone on CAM or in vitro control-culture. Micro-computed tomography (μCT) was used to quantify the magnitude and location of bone volume changes followed by histological analyses to assess bone repair. CAM blood vessels were observed to infiltrate the human bone cylinder and maintain human cell viability. Histological evaluation revealed extensive extracellular matrix deposition in proximity to endochondral condensations (Sox9+) on the CAM-implanted bone cylinders, correlating with a significant increase in bone volume by μCT analysis (p < 0.01). This human-avian system offers a simple refinement model for animal research and a step towards a humanized in vivo model for tissue engineering. PMID:27577960

  19. The chorioallantoic membrane (CAM) assay for the study of human bone regeneration: a refinement animal model for tissue engineering

    NASA Astrophysics Data System (ADS)

    Moreno-Jiménez, Inés; Hulsart-Billstrom, Gry; Lanham, Stuart A.; Janeczek, Agnieszka A.; Kontouli, Nasia; Kanczler, Janos M.; Evans, Nicholas D.; Oreffo, Richard Oc

    2016-08-01

    Biomaterial development for tissue engineering applications is rapidly increasing but necessitates efficacy and safety testing prior to clinical application. Current in vitro and in vivo models hold a number of limitations, including expense, lack of correlation between animal models and human outcomes and the need to perform invasive procedures on animals; hence requiring new predictive screening methods. In the present study we tested the hypothesis that the chick embryo chorioallantoic membrane (CAM) can be used as a bioreactor to culture and study the regeneration of human living bone. We extracted bone cylinders from human femoral heads, simulated an injury using a drill-hole defect, and implanted the bone on CAM or in vitro control-culture. Micro-computed tomography (μCT) was used to quantify the magnitude and location of bone volume changes followed by histological analyses to assess bone repair. CAM blood vessels were observed to infiltrate the human bone cylinder and maintain human cell viability. Histological evaluation revealed extensive extracellular matrix deposition in proximity to endochondral condensations (Sox9+) on the CAM-implanted bone cylinders, correlating with a significant increase in bone volume by μCT analysis (p < 0.01). This human-avian system offers a simple refinement model for animal research and a step towards a humanized in vivo model for tissue engineering.

  20. The chorioallantoic membrane (CAM) assay for the study of human bone regeneration: a refinement animal model for tissue engineering.

    PubMed

    Moreno-Jiménez, Inés; Hulsart-Billstrom, Gry; Lanham, Stuart A; Janeczek, Agnieszka A; Kontouli, Nasia; Kanczler, Janos M; Evans, Nicholas D; Oreffo, Richard Oc

    2016-08-31

    Biomaterial development for tissue engineering applications is rapidly increasing but necessitates efficacy and safety testing prior to clinical application. Current in vitro and in vivo models hold a number of limitations, including expense, lack of correlation between animal models and human outcomes and the need to perform invasive procedures on animals; hence requiring new predictive screening methods. In the present study we tested the hypothesis that the chick embryo chorioallantoic membrane (CAM) can be used as a bioreactor to culture and study the regeneration of human living bone. We extracted bone cylinders from human femoral heads, simulated an injury using a drill-hole defect, and implanted the bone on CAM or in vitro control-culture. Micro-computed tomography (μCT) was used to quantify the magnitude and location of bone volume changes followed by histological analyses to assess bone repair. CAM blood vessels were observed to infiltrate the human bone cylinder and maintain human cell viability. Histological evaluation revealed extensive extracellular matrix deposition in proximity to endochondral condensations (Sox9+) on the CAM-implanted bone cylinders, correlating with a significant increase in bone volume by μCT analysis (p < 0.01). This human-avian system offers a simple refinement model for animal research and a step towards a humanized in vivo model for tissue engineering.

  1. [Tissue engineering in reconstructive urology].

    PubMed

    Engel, O; Soave, A; Rink, M; Dahlem, R; Hellwinkel, O; Chun, F K; Fisch, M

    2015-05-01

    The term tissue engineering incorporates various techniques for the production of replacement tissues and organs. In urology tissue engineering offers many promising possibilities for the reconstruction of the urinary tract. Currently, buccal mucosa and urothelial cells are most commonly used for tissue engineering of the urinary tract. Various materials have been tested for their suitability as tissue scaffolds. The ideal scaffold, however, has not yet been found. In addition to material sciences and cell culture methods, surgical techniques play an important role in reconstructive urology for the successful implantation of tissue engineered transplants.

  2. Multipotent mesenchymal stem cells from human subacromial bursa: potential for cell based tendon tissue engineering.

    PubMed

    Song, Na; Armstrong, April D; Li, Feng; Ouyang, Hongsheng; Niyibizi, Christopher

    2014-01-01

    Rotator cuff injuries are a common clinical problem either as a result of overuse or aging. Biological approaches to tendon repair that involve use of scaffolding materials or cell-based approaches are currently being investigated. The cell-based approaches are focused on applying multipotent mesenchymal stem cells (MSCs) mostly harvested from bone marrow. In the present study, we focused on characterizing cells harvested from tissues associated with rotator cuff tendons based on an assumption that these cells would be more appropriate for tendon repair. We isolated MSCs from bursa tissue associated with rotator cuff tendons and characterized them for multilineage differentiation in vitro and in vivo. Human bursa was obtained from patients undergoing rotator cuff surgery and cells within were isolated using collagenase and dispase digestion. The cells isolated from the tissues were characterized for osteoblastic, adipogenic, chondrogenic, and tenogenic differentiation in vitro and in vivo. The results showed that the cells isolated from bursa tissue exhibited MSCs characteristics as evidenced by the expression of putative cell surface markers attributed to MSCs. The cells exhibited high proliferative capacity and differentiated toward cells of mesenchymal lineages with high efficiency. Bursa-derived cells expressed markers of tenocytes when treated with bone morphogenetic protein-12 (BMP-12) and assumed aligned morphology in culture. Bursa cells pretreated with BMP-12 and seeded in ceramic scaffolds formed extensive bone, as well as tendon-like tissue in vivo. Bone formation was demonstrated by histological analysis and immunofluorescence for DMP-1 in tissue sections made from the scaffolds seeded with the cells. Tendon-like tissue formed in vivo consisted of parallel collagen fibres typical of tendon tissues. Bursa-derived cells also formed a fibrocartilagenous tissue in the ceramic scaffolds. Taken together, the results demonstrate a new source of MSCs with a

  3. Modular Tissue Engineering: Engineering Biological Tissues from the Bottom Up.

    PubMed

    Nichol, Jason W; Khademhosseini, Ali

    2009-01-01

    Tissue engineering creates biological tissues that aim to improve the function of diseased or damaged tissues. To enhance the function of engineered tissues there is a need to generate structures that mimic the intricate architecture and complexity of native organs and tissues. With the desire to create more complex tissues with features such as developed and functional microvasculature, cell binding motifs and tissue specific morphology, tissue engineering techniques are beginning to focus on building modular microtissues with repeated functional units. The emerging field known as modular tissue engineering focuses on fabricating tissue building blocks with specific microarchitectural features and using these modular units to engineer biological tissues from the bottom up. In this review we will examine the promise and shortcomings of "bottom-up" approaches to creating engineered biological tissues. Specifically, we will survey the current techniques for controlling cell aggregation, proliferation and extracellular matrix deposition, as well as approaches to generating shape-controlled tissue modules. We will then highlight techniques utilized to create macroscale engineered biological tissues from modular microscale units.

  4. Informing future cartilage repair strategies: a comparative study of three different human cell types for cartilage tissue engineering.

    PubMed

    Saha, Sushmita; Kirkham, Jennifer; Wood, David; Curran, Stephen; Yang, Xuebin B

    2013-06-01

    A major clinical need exists for cartilage repair and regeneration. Despite many different strategies having been pursued, the identification of an optimised cell type and of pre-treatment conditions remains a challenge. This study compares the cartilage-like tissue generated by human bone marrow stromal cells (HBMSCs) and human neonatal and adult chondrocytes cultured on three-dimensional (3D) scaffolds under various conditions in vitro and in vivo with the aim of informing future cartilage repair strategies based upon tissue-engineering approaches. After 3 weeks in vitro culture, all three cell types showed cartilage-like tissue formation on 3D poly (lactide-co-glycolide) acid scaffolds only when cultured in chondrogenic medium. After 6 weeks of chondro-induction, neonatal chondrocyte constructs revealed the most cartilage-like tissue formation with a prominent superficial zone-like layer, a middle zone-like structure and the thinnest fibrous capsule. HBMSC constructs had the thickest fibrous capsule formation. Under basal culture conditions, neonatal articular chondrocytes failed to form any tissue, whereas HBMSCs and adult chondrocytes showed thick fibrous capsule formation at 6 weeks. After in vivo implantation, all groups generated more compact tissues compared with in vitro constructs. Pre-culturing in chondrogenic media for 1 week before implantation reduced fibrous tissue formation in all cell constructs at week 3. After 6 weeks, only the adult chondrocyte group pre-cultured in chondrogenic media was able to maintain a more chondrogenic/less fibrocartilaginous phenotype. Thus, pre-culture under chondrogenic conditions is required to maintain a long-term chondrogenic phenotype, with adult chondrocytes being a more promising cell source than HBMSCs for articular cartilage tissue engineering.

  5. Tissue engineering of blood vessels.

    PubMed

    Baguneid, M S; Seifalian, A M; Salacinski, H J; Murray, D; Hamilton, G; Walker, M G

    2006-03-01

    Tissue engineering techniques have been employed successfully in the management of wounds, burns and cartilage repair. Current prosthetic alternatives to autologous vascular bypass grafts remain poor in terms of patency and infection risk. Growing biological blood vessels has been proposed as an alternative. This review is based on a literature search using Medline, PubMed, ISIS and CAS of original articles and reviews, and unpublished material and abstracts. Complete incorporation into host tissues and the maintenance of a viable and self-renewing endothelial layer are the fundamental goals to be achieved when developing a tissue-engineered blood vessel. Sourcing of cells and modulating their interaction with extracellular matrix and supporting scaffold have been the focus of intense research. Although the use of tissue-engineered blood vessels in humans is so far limited, advances in our knowledge of stem cell precursors and the development of new biomaterials should enable this technology to reach routine clinical practice within a decade. Copyright (c) 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

  6. Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types.

    PubMed

    Proulx, Stéphanie; d'Arc Uwamaliya, Jeanne; Carrier, Patrick; Deschambeault, Alexandre; Audet, Caroline; Giasson, Claude J; Guérin, Sylvain L; Auger, François A; Germain, Lucie

    2010-10-29

    The purpose of this study was to produce and characterize human tissue-engineered corneas reconstructed using all three corneal cell types (epithelial, stromal, and endothelial cells) by the self-assembly approach. Fibroblasts cultured in medium containing serum and ascorbic acid secreted their own extracellular matrix and formed sheets that were superposed to reconstruct a stromal tissue. Endothelial and epithelial cells were seeded on each side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology and transmission electron microscopy (TEM). Immunofluorescence labeling of epithelial keratins, basement membrane components, Na+/K+-ATPase α1, and collagen type I was also performed. Epithelial and endothelial cells adhered to the reconstructed stroma. After 10 days at the air-liquid interface, the corneal epithelial cells stratified (4 to 5 cell layers) and differentiated into well defined basal and wing cells that also expressed Na+/K+-ATPase α1 protein, keratin 3/12, and basic keratins. Basal epithelial cells from the reconstructed epithelium formed many hemidesmosomes and secreted a well defined basement membrane rich in laminin V and collagen VII. Endothelial cells formed a monolayer of tightly-packed cells and also expressed the function related protein Na+/K+-ATPase α1. This study demonstrates the feasibility of producing a complete tissue-engineered human cornea, similar to native corneas, using untransformed fibroblasts, epithelial and endothelial cells, without the need for exogenous biomaterial.

  7. Decellularized Wharton’s Jelly from human umbilical cord as a novel 3D scaffolding material for tissue engineering applications

    PubMed Central

    Jadalannagari, Sushma; Converse, Gabriel; McFall, Christopher; Buse, Eric; Filla, Michael; Villar, Maria T.; Artigues, Antonio; Mellot, Adam J.; Wang, Jinxi; Detamore, Michael S.; Hopkins, Richard A.; Aljitawi, Omar S.

    2017-01-01

    In tissue engineering, an ideal scaffold attracts and supports cells thus providing them with the necessary mechanical support and architecture as they reconstruct new tissue in vitro and in vivo. This manuscript details a novel matrix derived from decellularized Wharton’s jelly (WJ) obtained from human umbilical cord for use as a scaffold for tissue engineering application. This decellularized Wharton’s jelly matrix (DWJM) contained 0.66 ± 0.12 μg/mg sulfated glycosaminoglycans (GAGs), and was abundant in hyaluronic acid, and completely devoid of cells. Mass spectroscopy revealed the presence of collagen types II, VI and XII, fibronectin-I, and lumican I. When seeded onto DWJM, WJ mesenchymal stem cells (WJMSCs), successfully attached to, and penetrated the porous matrix resulting in a slower rate of cell proliferation. Gene expression analysis of WJ and bone marrow (BM) MSCs cultured on DWJM demonstrated decreased expression of proliferation genes with no clear pattern of differentiation. When this matrix was implanted into a murine calvarial defect model with, green fluorescent protein (GFP) labeled osteocytes, the osteocytes were observed to migrate into the matrix as early as 24 hours. They were also identified in the matrix up to 14 days after transplantation. Together with these findings, we conclude that DWJM can be used as a 3D porous, bioactive and biocompatible scaffold for tissue engineering and regenerative medicine applications. PMID:28222169

  8. Effective combination of hydrostatic pressure and aligned nanofibrous scaffolds on human bladder smooth muscle cells: implication for bladder tissue engineering.

    PubMed

    Ahvaz, Hana Hanaee; Soleimani, Masoud; Mobasheri, Hamid; Bakhshandeh, Behnaz; Shakhssalim, Naser; Soudi, Sara; Hafizi, Maryam; Vasei, Mohammad; Dodel, Masumeh

    2012-09-01

    Bladder tissue engineering has been the focus of many studies due to its highly therapeutic potential. In this regard many aspects such as biochemical and biomechanical factors need to be studied extensively. Mechanical stimulations such as hydrostatic pressure and topology of the matrices are critical features which affect the normal functions of cells involved in bladder regeneration. In this study, hydrostatic pressure (10 cm H(2)O) and stretch forces were exerted on human bladder smooth muscle cells (hBSMCs) seeded on aligned nanofibrous polycaprolactone/PLLA scaffolds, and the alterations in gene and protein expressions were studied. The gene transcription patterns for collagen type I, III, IV, elastin, α-SMA, calponin and caldesmon were monitored on days 3 and 5 quantitatively. Changes in the expressions of α-SMA, desmin, collagen type I and III were quantified by Enzyme-linked immuno-sorbent assay. The scaffolds were characterized using scanning electron microscope, contact angle measurement and tensile testing. The positive effect of mechanical forces on the functional improvement of the engineered tissue was supported by translational down-regulation of α-SMA and VWF, up-regulation of desmin and improvement of collagen type III:I ratio. Altogether, our study reveals that proper hydrostatic pressure in combination with appropriate surface stimulation on hBSMCs causes a tissue-specific phenotype that needs to be considered in bladder tissue engineering.

  9. Decellularized Wharton's Jelly from human umbilical cord as a novel 3D scaffolding material for tissue engineering applications.

    PubMed

    Jadalannagari, Sushma; Converse, Gabriel; McFall, Christopher; Buse, Eric; Filla, Michael; Villar, Maria T; Artigues, Antonio; Mellot, Adam J; Wang, Jinxi; Detamore, Michael S; Hopkins, Richard A; Aljitawi, Omar S

    2017-01-01

    In tissue engineering, an ideal scaffold attracts and supports cells thus providing them with the necessary mechanical support and architecture as they reconstruct new tissue in vitro and in vivo. This manuscript details a novel matrix derived from decellularized Wharton's jelly (WJ) obtained from human umbilical cord for use as a scaffold for tissue engineering application. This decellularized Wharton's jelly matrix (DWJM) contained 0.66 ± 0.12 μg/mg sulfated glycosaminoglycans (GAGs), and was abundant in hyaluronic acid, and completely devoid of cells. Mass spectroscopy revealed the presence of collagen types II, VI and XII, fibronectin-I, and lumican I. When seeded onto DWJM, WJ mesenchymal stem cells (WJMSCs), successfully attached to, and penetrated the porous matrix resulting in a slower rate of cell proliferation. Gene expression analysis of WJ and bone marrow (BM) MSCs cultured on DWJM demonstrated decreased expression of proliferation genes with no clear pattern of differentiation. When this matrix was implanted into a murine calvarial defect model with, green fluorescent protein (GFP) labeled osteocytes, the osteocytes were observed to migrate into the matrix as early as 24 hours. They were also identified in the matrix up to 14 days after transplantation. Together with these findings, we conclude that DWJM can be used as a 3D porous, bioactive and biocompatible scaffold for tissue engineering and regenerative medicine applications.

  10. Engineering functionally graded tissue engineering scaffolds.

    PubMed

    Leong, K F; Chua, C K; Sudarmadji, N; Yeong, W Y

    2008-04-01

    Tissue Engineering (TE) aims to create biological substitutes to repair or replace failing organs or tissues due to trauma or ageing. One of the more promising approaches in TE is to grow cells on biodegradable scaffolds, which act as temporary supports for the cells to attach, proliferate and differentiate; after which the scaffold will degrade, leaving behind a healthy regenerated tissue. Tissues in nature, including human tissues, exhibit gradients across a spatial volume, in which each identifiable layer has specific functions to perform so that the whole tissue/organ can behave normally. Such a gradient is termed a functional gradient. A good TE scaffold should mimic such a gradient, which fulfils the biological and mechanical requirements of the target tissue. Thus, the design and fabrication process of such scaffolds become more complex and the introduction of computer-aided tools will lend themselves well to ease these challenges. This paper reviews the needs and characterization of these functional gradients and the computer-aided systems used to ease the complexity of the scaffold design stage. These include the fabrication techniques capable of building functionally graded scaffolds (FGS) using both conventional and rapid prototyping (RP) techniques. They are able to fabricate both continuous and discrete types of FGS. The challenge in fabricating continuous FGS using RP techniques lies in the development of suitable computer aided systems to facilitate continuous FGS design. What have been missing are the appropriate models that relate the scaffold gradient, e.g. pore size, porosity or material gradient, to the biological and mechanical requirements for the regeneration of the target tissue. The establishment of these relationships will provide the foundation to develop better computer-aided systems to help design a suitable customized FGS.

  11. Fascia tissue engineering with human adipose-derived stem cells in a murine model: Implications for pelvic floor reconstruction.

    PubMed

    Hung, Man-Jung; Wen, Mei-Chin; Huang, Ying-Ting; Chen, Gin-Den; Chou, Min-Min; Yang, Vivian Cheng

    2014-10-01

    Mesh-augmented vaginal surgery for treatment of pelvic organ prolapse (POP) does not meet patients' needs. This study aims to test the hypothesis that fascia tissue engineering using adipose-derived stem cells (ADSCs) might be a potential therapeutic strategy for reconstructing the pelvic floor. Human ADSCs were isolated, differentiated, and characterized in vitro. Both ADSCs and fibroblastic-differentiated ADSCs were used to fabricate tissue-engineered fascia equivalents, which were then transplanted under the back skin of experimental nude mice. ADSCs prepared in our laboratory were characterized as a group of mesenchymal stem cells. In vitro fibroblastic differentiation of ADSCs showed significantly increased gene expression of cellular collagen type I and elastin (p < 0.05) concomitantly with morphological changes. By contrast, ADSCs cultured in control medium did not demonstrate these changes. Both of the engrafted fascia equivalents could be traced up to 12 weeks after transplantation in the subsequent animal study. Furthermore, the histological outcomes differed with a thin (111.0 ± 19.8 μm) lamellar connective tissue or a thick (414.3 ± 114.9 μm) adhesive fibrous tissue formation between the transplantation of ADSCs and fibroblastic-differentiated ADSCs, respectively. Nonetheless, the implantation of a scaffold without cell seeding (the control group) resulted in a thin (102.0 ± 17.1 μm) fibrotic band and tissue contracture. Our results suggest the ADSC-seeded implant is better than the implant alone in enhancing tissue regeneration after transplantation. ADSCs with or without fibroblastic differentiation might have a potential but different role in fascia tissue engineering to repair POP in the future. Copyright © 2013. Published by Elsevier B.V.

  12. Tissue-engineered constructs of human oral mucosa examined by Raman spectroscopy.

    PubMed

    Khmaladze, Alexander; Ganguly, Arindam; Kuo, Shiuhyang; Raghavan, Mekhala; Kainkaryam, Raghu; Cole, Jacqueline H; Izumi, Kenji; Marcelo, Cynthia L; Feinberg, Stephen E; Morris, Michael D

    2013-04-01

    A noninvasive quality monitoring of tissue-engineered constructs is a required component of any successful tissue-engineering technique. During a 2-week production period, ex vivo produced oral mucosa-equivalent constructs (EVPOMEs) may encounter adverse culturing conditions that might compromise their quality and render them ineffective. We demonstrate the application of near-infrared Raman spectroscopy to in vitro monitoring of EVPOMEs during their manufacturing process, with the ultimate goal of applying this technology in situ to monitor the grafted EVPOMEs. We identify Raman spectroscopic failure indicators for less-than optimal EVPOMEs that are stressed by higher temperature and exposure to higher than normal concentration of calcium ions. Raman spectra of EVPOMEs exposed to thermal and calcium stress showed correlation of the band height ratio of CH(2) deformation to phenylalanine ring breathing modes, providing a Raman metric to distinguish between viable and nonviable constructs. We compared these results to histology and glucose consumption measurements, demonstrating that Raman spectroscopy is more sensitive and specific to changes in proteins' secondary structure not visible by H&E histology. We also exposed the EVPOMEs to rapamycin, a cell growth inhibitor and cell proliferation capacity preserver, and distinguished between EVPOMEs pretreated with 2 nM rapamycin and controls, using the ratio of the Amide III envelope to the phenylalanine band as an indicator.

  13. Tissue-Engineered Constructs of Human Oral Mucosa Examined by Raman Spectroscopy

    PubMed Central

    Khmaladze, Alexander; Ganguly, Arindam; Kuo, Shiuhyang; Raghavan, Mekhala; Kainkaryam, Raghu; Cole, Jacqueline H.; Izumi, Kenji; Marcelo, Cynthia L.; Feinberg, Stephen E.

    2013-01-01

    A noninvasive quality monitoring of tissue-engineered constructs is a required component of any successful tissue-engineering technique. During a 2-week production period, ex vivo produced oral mucosa-equivalent constructs (EVPOMEs) may encounter adverse culturing conditions that might compromise their quality and render them ineffective. We demonstrate the application of near-infrared Raman spectroscopy to in vitro monitoring of EVPOMEs during their manufacturing process, with the ultimate goal of applying this technology in situ to monitor the grafted EVPOMEs. We identify Raman spectroscopic failure indicators for less-than optimal EVPOMEs that are stressed by higher temperature and exposure to higher than normal concentration of calcium ions. Raman spectra of EVPOMEs exposed to thermal and calcium stress showed correlation of the band height ratio of CH2 deformation to phenylalanine ring breathing modes, providing a Raman metric to distinguish between viable and nonviable constructs. We compared these results to histology and glucose consumption measurements, demonstrating that Raman spectroscopy is more sensitive and specific to changes in proteins' secondary structure not visible by H&E histology. We also exposed the EVPOMEs to rapamycin, a cell growth inhibitor and cell proliferation capacity preserver, and distinguished between EVPOMEs pretreated with 2 nM rapamycin and controls, using the ratio of the Amide III envelope to the phenylalanine band as an indicator. PMID:22992065

  14. Engineering of implantable liver tissues.

    PubMed

    Sakai, Yasuyuki; Nishikawa, M; Evenou, F; Hamon, M; Huang, H; Montagne, K P; Kojima, N; Fujii, T; Niino, T

    2012-01-01

    In this chapter, from the engineering point of view, we introduce the results from our group and related research on three typical configurations of engineered liver tissues; cell sheet-based tissues, sheet-like macroporous scaffold-based tissues, and tissues based on special scaffolds that comprise a flow channel network. The former two do not necessitate in vitro prevascularization and are thus promising in actual human clinical trials for liver diseases that can be recovered by relatively smaller tissue mass. The third approach can implant a much larger mass but is still not yet feasible. In all cases, oxygen supply is the key engineering factor. For the first configuration, direct oxygen supply using an oxygen-permeable polydimethylsiloxane membrane enables various liver cells to exhibit distinct behaviors, complete double layers of mature hepatocytes and fibroblasts, spontaneous thick tissue formation of hepatocarcinoma cells and fetal hepatocytes. Actual oxygen concentration at the cell level can be strictly controlled in this culture system. Using this property, we found that initially low then subsequently high oxygen concentrations were favorable to growth and maturation of fetal cells. For the second configuration, combination of poly-L: -lactic acid 3D scaffolds and appropriate growth factor cocktails provides a suitable microenvironment for the maturation of cells in vitro but the cell growth is limited to a certain distance from the inner surfaces of the macropores. However, implantation to the mesentery leaves of animals allows the cells again to proliferate and pack the remaining spaces of the macroporous structure, suggesting the high feasibility of 3D culture of hepatocyte progenitors for liver tissue-based therapies. For the third configuration, we proposed a design criterion concerning the dimensions of flow channels based on oxygen diffusion and consumption around the channel. Due to the current limitation in the resolution of 3D

  15. Scaffold's surface geometry significantly affects human stem cell bone tissue engineering.

    PubMed

    Graziano, Antonio; d'Aquino, Riccardo; Cusella-De Angelis, Maria Gabriella; De Francesco, Francesco; Giordano, Antonio; Laino, Gregorio; Piattelli, Adriano; Traini, Tonino; De Rosa, Alfredo; Papaccio, Gianpaolo

    2008-01-01

    In this study, we have observed dental pulp stem cells (SBP-DPSCs) performances on different scaffolds, such as PLGA 85:15, hydroxyapatite chips (HA) and titanium. Stem cells were challenged with each engineered surface, either in plane cultures or in a rotating apparatus, for a month. Gingival fibroblasts were used as controls. Results showed that stem cells exerted a different response, depending on the different type of textured surface: in fact, microconcavities significantly affected SBP-DPSC differentiation into osteoblasts, both temporally and quantitatively, with respect to the other textured surfaces. Actually, stem cells challenged with concave surfaces differentiated quicker and showed nuclear polarity, an index of secretion, cellular activity and matrix formation. Moreover, bone-specific proteins were significantly expressed and the obtained bone tissue was of significant thickness. Thus, cells cultured on the concave textured surface had better cell-scaffold interactions and were induced to secrete factors that, due to their autocrine effects, quickly lead to osteodifferentiation, bone tissue formation, and vascularization. The worst cell performance was obtained using convex surfaces, due to the scarce cell proliferation on to the scaffold and the poor matrix secretion. In conclusion, this study stresses that for a suitable and successful bone tissue reconstruction the surface texture is of paramount importance. (c) 2007 Wiley-Liss, Inc.

  16. On the decellularization of fresh or frozen human umbilical arteries: implications for small-diameter tissue engineered vascular grafts.

    PubMed

    Tuan-Mu, Ho-Yi; Yu, Chen-Hsiang; Hu, Jin-Jia

    2014-06-01

    Most tissues, including those to be decellularized for tissue engineering applications, are frozen for long term preservation. Such conventional cryopreservation has been shown to alter the structure and mechanical properties of tissues. Little is known, however, how freezing affects decellularization of tissues. The purpose of this study was two-fold: to examine the effects of freezing on decellularization of human umbilical arteries (HUAs), which represent a potential scaffolding material for small-diameter tissue-engineered vascular grafts, and to examine how decellularization affects the mechanical properties of frozen HUAs. Among many decellularization methods, hypotonic sodium dodecyl sulfate solution was selected as the decellularizing agent and tested on fresh HUAs to optimize decellularization conditions. The efficiency of decellularization was evaluated by DNA assay and histology every 12 up to 48 h. The optimized decellularization protocol was then performed on frozen HUAs. The stiffness, burst pressure, and suture retention strength of fresh HUAs and frozen HUAs before and after decellularization were also examined. It appeared that freezing decreased the efficiency of decellularization, which may be attributed to the condensed extracellular matrix caused by freezing. While the stiffness of fresh HUAs did not change significantly after decellularization, decellularization reduced the compliance of frozen HUAs. Interestingly, the stiffness of decellularized frozen HUAs was similar to that of decellularized fresh HUAs. Although little difference in stiffness was observed, we suggest avoiding freezing if more efficient and complete decellularization is desired.

  17. Tissue engineering and ENT surgery.

    PubMed

    Patel, Nimesh N; Butler, Peter E M; Buttery, Lee; Polak, Julia M; Tolley, Neil S

    2002-03-01

    Tissue engineering is the development of biological substitutes for the repair and regeneration of damaged tissues. We explain the principles of this emerging field of biotechology. The present and potential applications of tissue engineering technologies in ENT surgery are then reviewed.

  18. Tissue-engineered skin substitutes.

    PubMed

    Mansbridge, Jonathan

    2002-01-01

    The last two years have seen new tissue-engineered skin substitutes come onto the market and begin to resolve the various roles to which each is best suited. It is becoming evident that some of the very expensive cell-based products have cost-benefit advantage despite their high price and are valuable within the restricted applications for which they are intended. The use of skin substitutes for testing purposes has extended from epidermal keratinocytes to other integumentary epithelia and into preparations containing multiple cell types in which reactions resulting from paracrine interactions can be examined. Challenges remain in the application of gene therapy techniques to skin substitutes, both the control of transgene expression and in the selection of suitable genes to transfect. A coming challenge is the production of tissue-engineered products without the use of animal products other than human cells. A challenge that may be diminishing is the importance of acute rejection of allogeneic tissue-engineered skin substitutes.

  19. Human collagen-based multilayer scaffolds for tendon-to-bone interface tissue engineering.

    PubMed

    Kim, Beob Soo; Kim, Eun Ji; Choi, Ji Suk; Jeong, Ji Hoon; Jo, Chris Hyunchul; Cho, Yong Woo

    2014-11-01

    The natural tendon-to-bone region has a gradient in structure and composition, which is translated into a spatial variation of chemical, physical, and biological properties. This unique transitional tissue between bone and tendon is not normally recreated during natural bone-to-tendon healing. In this study, we have developed a human collagen-based multilayer scaffold mimicking the tendon-to-bone region. The scaffold consists of four different layers with the following composition gradient: (a) a tendon layer composed of collagen; (b) an uncalcified fibrocartilage layer composed of collagen and chondroitin sulfate; (c) a calcified fibrocartilage layer composed of collagen and less apatite; (d) a bone layer composed of collagen and apatite. The chemical, physical, and mechanical properties of the scaffold were characterized by a scanning electron microscope, porosimeter, universal tensile machine, Fourier transform infrared spectrometer, energy dispersive X-ray analysis apparatus, and thermogravimetric analysis apparatus. The multilayer scaffold provided a gradual transition of the physical, chemical, and mechanical environment and supported the adhesion and proliferation of human fibroblasts, chondrocytes, and osteoblasts toward each corresponding matrix. Overall, our results suggest the feasibility of a human collagen-based multilayer scaffold for regeneration of hard-to-soft interface tissues. © 2014 Wiley Periodicals, Inc.

  20. [Generation of a substitute for human oral mucosa and verification of its viability by tissue-engineering].

    PubMed

    Marañés Gálvez, C; Liceras Liceras, E; Alaminos, M; Fernández Valadés, R; Ruiz Montes, A M; Garzón, I; Sánchez-Quevedo, M C; Campos, A

    2011-01-01

    Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues. This way, tissue ingineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we have developed a new model for artificial oral mucosa generated by tissue engineering using a fibrin-agarosa scaffold. For that purpose, we have generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal mucosa oral using enzymatic treatments. Then, we have determined the viability of cultured cells by electron probe quantitative X-ray microanalysis, and we have demonstrated that most of the cells in the primary cultures were alive and hd high K/Na ratios. Once cell viability was determined, we used cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarosa extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and de oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.

  1. Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilage.

    PubMed

    Sakai, Shinsuke; Mishima, Hajime; Ishii, Tomoo; Akaogi, Hiroshi; Yoshioka, Tomokazu; Ohyabu, Yoshimi; Chang, Fei; Ochiai, Naoyuki; Uemura, Toshimasa

    2009-04-01

    The method of constructing cartilage tissue from bone marrow-derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow-derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per microg DNA of the tissues were 10.01 +/- 3.49 microg/microg DNA in the case of the RWV bioreactor and 6.27 +/- 3.41 microg/microg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three-dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow-derived cells.

  2. Cardiac tissue engineering: state of the art.

    PubMed

    Hirt, Marc N; Hansen, Arne; Eschenhagen, Thomas

    2014-01-17

    The engineering of 3-dimensional (3D) heart muscles has undergone exciting progress for the past decade. Profound advances in human stem cell biology and technology, tissue engineering and material sciences, as well as prevascularization and in vitro assay technologies make the first clinical application of engineered cardiac tissues a realistic option and predict that cardiac tissue engineering techniques will find widespread use in the preclinical research and drug development in the near future. Tasks that need to be solved for this purpose include standardization of human myocyte production protocols, establishment of simple methods for the in vitro vascularization of 3D constructs and better maturation of myocytes, and, finally, thorough definition of the predictive value of these methods for preclinical safety pharmacology. The present article gives an overview of the present state of the art, bottlenecks, and perspectives of cardiac tissue engineering for cardiac repair and in vitro testing.

  3. Direct Hydrogel Encapsulation of Pluripotent Stem Cells Enables Ontomimetic Differentiation and Growth of Engineered Human Heart Tissues

    PubMed Central

    Kerscher, Petra; Turnbull, Irene C; Hodge, Alexander J; Kim, Joonyul; Seliktar, Dror; Easley, Christopher J; Costa, Kevin D; Lipke, Elizabeth A

    2016-01-01

    Human engineered heart tissues have potential to revolutionize cardiac development research, drug-testing, and treatment of heart disease; however, implementation is limited by the need to use pre-differentiated cardiomyocytes (CMs). Here we show that by providing a 3D poly(ethylene glycol)-fibrinogen hydrogel microenvironment, we can directly differentiate human pluripotent stem cells (hPSCs) into contracting heart tissues. Our straight-forward, ontomimetic approach, imitating the process of development, requires only a single cell-handling step, provides reproducible results for a range of tested geometries and size scales, and overcomes inherent limitations in cell maintenance and maturation, while achieving high yields of CMs with developmentally appropriate temporal changes in gene expression. Here we demonstrate that hPSCs encapsulated within this biomimetic 3D hydrogel microenvironment develop into functional cardiac tissues composed of self-aligned CMs with evidence of ultrastructural maturation, mimicking heart development, and enabling investigation of disease mechanisms and screening of compounds on developing human heart tissue. PMID:26826618

  4. Direct hydrogel encapsulation of pluripotent stem cells enables ontomimetic differentiation and growth of engineered human heart tissues.

    PubMed

    Kerscher, Petra; Turnbull, Irene C; Hodge, Alexander J; Kim, Joonyul; Seliktar, Dror; Easley, Christopher J; Costa, Kevin D; Lipke, Elizabeth A

    2016-03-01

    Human engineered heart tissues have potential to revolutionize cardiac development research, drug-testing, and treatment of heart disease; however, implementation is limited by the need to use pre-differentiated cardiomyocytes (CMs). Here we show that by providing a 3D poly(ethylene glycol)-fibrinogen hydrogel microenvironment, we can directly differentiate human pluripotent stem cells (hPSCs) into contracting heart tissues. Our straight-forward, ontomimetic approach, imitating the process of development, requires only a single cell-handling step, provides reproducible results for a range of tested geometries and size scales, and overcomes inherent limitations in cell maintenance and maturation, while achieving high yields of CMs with developmentally appropriate temporal changes in gene expression. We demonstrate that hPSCs encapsulated within this biomimetic 3D hydrogel microenvironment develop into functional cardiac tissues composed of self-aligned CMs with evidence of ultrastructural maturation, mimicking heart development, and enabling investigation of disease mechanisms and screening of compounds on developing human heart tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Vascularization strategies for tissue engineers.

    PubMed

    Dew, Lindsey; MacNeil, Sheila; Chong, Chuh Khiun

    2015-01-01

    All tissue-engineered substitutes (with the exception of cornea and cartilage) require a vascular network to provide the nutrient and oxygen supply needed for their survival in vivo. Unfortunately the process of vascular ingrowth into an engineered tissue can take weeks to occur naturally and during this time the tissues become starved of essential nutrients, leading to tissue death. This review initially gives a brief overview of the processes and factors involved in the formation of new vasculature. It then summarizes the different approaches that are being applied or developed to overcome the issue of slow neovascularization in a range of tissue-engineered substitutes. Some potential future strategies are then discussed.

  6. [URETERAL TISSUE ENGINEERING: CHALLENGES AND PROSPECTS].

    PubMed

    Glybochko, P V; Alaev, Ju G; Vinarov, A Z; Butnaru, D V; Titov, A S; Bibikova, E E; Sevostjanova, S I

    2015-01-01

    A broad range of pathologic conditions of the ureter (strictures, obliterations, fistulas, and so on) requiring reconstructive plastic surgery is a challenging urological problem. A variety of approaches to solve the problem indicates the need of searching for new opportunities. A new direction in reconstructive surgery of the ureter is the tissue engineering. Tissue engineering involves the usage of matrices and cells. The matrices can be used both with cultured cells, and without them. This review represents the results of preclinical studies on feasibility of tissue engineering using as a matrix both natural and synthetic materials for different ureter impairments. Presently, there are no data on the use of tissue-engineering for the ureter reconstruction in clinical trials (i.e. involving human subjects). The results of studies presented in the review inspire certain optimism, but ureteral tissue-engineering is a difficult task requiring a balanced approach and well-thought-out design of preclinical studies.

  7. Label-free multiphoton fluorescence imaging monitors metabolism in living primary human cells used for tissue engineering

    NASA Astrophysics Data System (ADS)

    Chen, Leng-Chun; Lloyd, William R.; Kuo, Shiuhyang; Marcelo, Cynthia L.; Feinberg, Stephen E.; Mycek, Mary-Ann

    2012-03-01

    Fluorescence redox imaging was employed to monitor the metabolic activity of primary human oral keratinocytes prior to the development of tissue-engineered constructs. Keratinocytes with controlled culture conditions were treated with varying levels of chemical stimuli, resulting in differing cellular morphology, growth rate, and metabolic activity. Fluorescence images of keratinocytes were noninvasively acquired from endogenous intracellular metabolic fluorophores NAD(P)H and FAD. A redox ratio quantitatively analyzed each pair of images, showing that fluorescence redox imaging may be a novel technique to characterize live cell viability

  8. High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties.

    PubMed

    Cigan, Alexander D; Roach, Brendan L; Nims, Robert J; Tan, Andrea R; Albro, Michael B; Stoker, Aaron M; Cook, James L; Vunjak-Novakovic, Gordana; Hung, Clark T; Ateshian, Gerard A

    2016-06-14

    Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring ∅3-4mm×2.3mm, with cell seeding densities ranging from 15 to 90×10(6)cells/mL. Subsets of constructs were subjected to transient or sustained TGF-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young's moduli of up to ~250kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950kPa (0.01Hz), and contained 5.7% wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5%/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-β treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes.

  9. Heart Regeneration with Engineered Myocardial Tissue

    PubMed Central

    Bajpai, Vivek K.; Andreadis, Stelios T.; Murry, Charles E.

    2014-01-01

    Heart disease is the leading cause of morbidity and mortality worldwide, and regenerative therapies that replace damaged myocardium could benefit millions of patients annually. The many cell types in the heart, including cardiomyocytes, endothelial cells, vascular smooth muscle cells, pericytes, and cardiac fibroblasts, communicate via intercellular signaling and modulate each other’s function. Although much progress has been made in generating cells of the cardiovascular lineage from human pluripotent stem cells, a major challenge now is creating the tissue architecture to integrate a microvascular circulation and afferent arterioles into such an engineered tissue. Recent advances in cardiac and vascular tissue engineering will move us closer to the goal of generating functionally mature tissue. Using the biology of the myocardium as the foundation for designing engineered tissue and addressing the challenges to implantation and integration, we can bridge the gap from bench to bedside for a clinically tractable engineered cardiac tissue. PMID:24819474

  10. Three-Dimensional Human Cardiac Tissue Engineered by Centrifugation of Stacked Cell Sheets and Cross-Sectional Observation of Its Synchronous Beatings by Optical Coherence Tomography

    PubMed Central

    Hasegawa, Akiyuki; Matsuura, Katsuhisa; Kobayashi, Mari; Iwana, Shin-ichi; Kabetani, Yasuhiro

    2017-01-01

    Three-dimensional (3D) tissues are engineered by stacking cell sheets, and these tissues have been applied in clinical regenerative therapies. The optimal fabrication technique of 3D human tissues and the real-time observation system for these tissues are important in tissue engineering, regenerative medicine, cardiac physiology, and the safety testing of candidate chemicals. In this study, for aiming the clinical application, 3D human cardiac tissues were rapidly fabricated by human induced pluripotent stem (iPS) cell-derived cardiac cell sheets with centrifugation, and the structures and beatings in the cardiac tissues were observed cross-sectionally and noninvasively by two optical coherence tomography (OCT) systems. The fabrication time was reduced to approximately one-quarter by centrifugation. The cross-sectional observation showed that multilayered cardiac cell sheets adhered tightly just after centrifugation. Additionally, the cross-sectional transmissions of beatings within multilayered human cardiac tissues were clearly detected by OCT. The observation showed the synchronous beatings of the thicker 3D human cardiac tissues, which were fabricated rapidly by cell sheet technology and centrifugation. The rapid tissue-fabrication technique and OCT technology will show a powerful potential in cardiac tissue engineering, regenerative medicine, and drug discovery research. PMID:28326324

  11. Mesenchymal Stem Cells for Osteochondral Tissue Engineering

    PubMed Central

    Ng, Johnathan; Bernhard, Jonathan; Vunjak-Novakovic, Gordana

    2017-01-01

    Summary Mesenchymal stem cells (MSC) are of major interest to regenerative medicine, because of the ease of harvesting from a variety of sources (including bone marrow and fat aspirates) and ability to form a range of mesenchymal tissues, in vitro and in vivo. We focus here on the use of MSCs for engineering of cartilage, bone, and complex osteochondral tissue constructs, using protocols that replicate some aspects of the natural mesodermal development. For engineering of human bone, we discuss some of the current advances, and highlight the use of perfusion bioreactors for supporting anatomically exact human bone grafts. For engineering of human cartilage, we discuss limitations of current approaches, and highlight engineering of stratified, mechanically functional human cartilage interfaced with bone by mesenchymal condensation of MSCs. Taken together, the current advances enable engineering physiologically relevant bone, cartilage and osteochondral composites, and physiologically relevant studies of osteochondral development and disease. PMID:27236665

  12. Tissue engineering for neurodegenerative diseases using human amniotic membrane and umbilical cord.

    PubMed

    Sanluis-Verdes, Anahí; Sanluis-Verdes, Namibia; Manso-Revilla, María Jesús; Castro-Castro, Antonio Manuel; Pombo-Otero, Jorge; Fraga-Mariño, María; Sanchez-Ibañez, Jacinto; Doménech, Nieves; Rendal-Vázquez, María Esther

    2017-03-01

    Regenerative medicine, based on the use of stem cells, scaffolds and growth factors, has the potential to be a good approach for restoring damaged tissues of the central nervous system. This study investigated the use of human amniotic mesenchymal stem cells (hAMSC), human amniotic epithelial stem cells (hAESC), and human Wharton's jelly mesenchymal stem cells (hWJMSC) derived from human umbilical cord as a source of stem cells, and the potential of the human amniotic membrane (HAM) as a scaffold and/or source of growth factors to promote nerve regeneration. The hAMSC and hAESC obtained from HAM and the hWJMSC from umbilical cords were cultured in induction medium to obtain neural-like cells. The morphological differentiation of hAMSC, hAESC and hWJMSC into neural-like cells was evident after 4-5 days, when they acquired an elongated and multipolar shape, and at 21 days, when they expressed neural and glial markers. On other way, the HAM was completely decellularized without affecting the components of the basement membrane or the matrix. Subsequently, hAMSC, hAESC and hWJMSC differentiated into neural-like cells were seeded onto the decellularized HAM, maintaining their morphology. Finally, conditioned media from the HAM allowed proliferation of hAMSC, hAESC and hWJMSC differentiated to neural-like cells. Both HAM and umbilical cord are biomaterials with great potential for use in regenerative medicine for the treatment of neurodegenerative diseases.

  13. Biomaterials for tissue engineering: summary

    NASA Technical Reports Server (NTRS)

    Christenson, L.; Mikos, A. G.; Gibbons, D. F.; Picciolo, G. L.; McIntire, L. V. (Principal Investigator)

    1997-01-01

    This article summarizes presentations and discussion at the workshop "Enabling Biomaterial Technology for Tissue Engineering," which was held during the Fifth World Biomaterials Congress in May 1996. Presentations covered the areas of material substrate architecture, barrier effects, and cellular response, including analysis of biomaterials challenges involved in producing specific tissue-engineered products.

  14. Biomaterials for tissue engineering: summary.

    PubMed

    Christenson, L; Mikos, A G; Gibbons, D F; Picciolo, G L

    1997-01-01

    This article summarizes presentations and discussion at the workshop "Enabling Biomaterial Technology for Tissue Engineering," which was held during the Fifth World Biomaterials Congress in May 1996. Presentations covered the areas of material substrate architecture, barrier effects, and cellular response, including analysis of biomaterials challenges involved in producing specific tissue-engineered products.

  15. Biomaterials for tissue engineering: summary

    NASA Technical Reports Server (NTRS)

    Christenson, L.; Mikos, A. G.; Gibbons, D. F.; Picciolo, G. L.; McIntire, L. V. (Principal Investigator)

    1997-01-01

    This article summarizes presentations and discussion at the workshop "Enabling Biomaterial Technology for Tissue Engineering," which was held during the Fifth World Biomaterials Congress in May 1996. Presentations covered the areas of material substrate architecture, barrier effects, and cellular response, including analysis of biomaterials challenges involved in producing specific tissue-engineered products.

  16. Engineering vascular tissue with functional smooth muscle cells derived from human iPS cells and nanofibrous scaffolds.

    PubMed

    Wang, Yongyu; Hu, Jiang; Jiao, Jiao; Liu, Zhongning; Zhou, Zhou; Zhao, Chao; Chang, Lung-Ji; Chen, Y Eugene; Ma, Peter X; Yang, Bo

    2014-10-01

    Tissue-engineered blood vessels (TEBVs) are promising in the replacement of diseased vascular tissues. However, it remains a great challenge to obtain a sufficient number of functional smooth muscle cells (SMCs) in a clinical setting to construct patient-specific TEBVs. In addition, it is critical to develop a scaffold to accommodate these cells and retain their functional phenotype for the regeneration of TEBVs. In this study, human induced pluripotent stem cells (iPSCs) were established from primary human aortic fibroblasts, and characterized with the pluripotency markers expression and cells' capabilities to differentiate into all three germ layer cells. A highly efficient method was then developed to induce these human iPSCs into proliferative SMCs. After multiple times of expansion, the expanded SMCs retained the potential to be induced into the functional contractile phenotype of mature SMCs, which was characterized by the contractile response to carbachol treatment, up-regulation of specific collagen genes under transforming growth factor β1 treatment, and up-regulation of specific matrix metalloproteinase genes under cytokine stimulation. We also developed an advanced macroporous and nanofibrous (NF) poly(l-lactic acid) (PLLA) scaffold with suitable pore size and interpore connectivity to seed these human iPSC-derived SMCs and maintain their differentiated phenotype. Subcutaneous implantation of the SMC-scaffold construct in nude mice demonstrated vascular tissue formation, with robust collagenous matrix deposition inside the scaffold and the maintenance of differentiated SMC phenotype. Taken together, this study established an exciting approach towards the construction of patient-specific TEBVs. We established patient-specific human iPSCs, derived proliferative SMCs for expansion, turned on their mature contractile SMC phenotype, and developed an advanced scaffold for these cells to regenerate vascular tissue in vivo.

  17. Genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using Gata6.

    PubMed

    Guye, Patrick; Ebrahimkhani, Mohammad R; Kipniss, Nathan; Velazquez, Jeremy J; Schoenfeld, Eldi; Kiani, Samira; Griffith, Linda G; Weiss, Ron

    2016-01-06

    Human induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells, there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for generating and then co-differentiating hiPSC-derived progenitors. With a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression, we initiate rapid emergence of all three germ layers as a complex function of GATA6 expression levels and tissue context. Within 2 weeks we obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype, including haematopoietic and stromal cells as well as a neuronal niche. Collectively, our approach demonstrates derivation of complex tissues from hiPSCs using a single autologous hiPSCs as source and generates a range of stromal cells that co-develop with parenchymal cells to form tissues.

  18. Should human chondrocytes fly? The impact of electromagnetic irradiation on chondrocyte viability and implications for their use in tissue engineering.

    PubMed

    Koehler, C; Niederbichler, A D; Scholz, T; Bode, B; Roos, J; Jung, F J; Hoerstrup, S P; Hellermann, J P; Wedler, V

    2006-12-01

    A significant logistic factor as to the successful clinical application of the autologous tissue engineering concept is efficient transportation: the donor cells need to be delivered to tissue processing facilities which in most cases requires air transportation. This study was designed to evaluate how human chondrocytes react to X-ray exposure. Primary cell cultures were established, cultured, incubated and exposed to different doses and time periods of radiation. Subsequently, quantitative cell proliferation assays were done and qualitative evaluation of cellular protein production were performed. Our results show that after irradiation of chondrocytes with different doses, no significant differences in terms of cellular viability occurred compared with the control group. These results were obtained when chondrocytes were exposed to luggage transillumination doses as well as exposure to clinically used radiation doses. Any damage affecting cell growth or quality was not observed in our study. However, information about damage of cellular DNA remains incomplete.

  19. Human acellular cartilage matrix powders as a biological scaffold for cartilage tissue engineering with synovium-derived mesenchymal stem cells.

    PubMed

    Chang, Chih-Hung; Chen, Chia-Chun; Liao, Cheng-Hao; Lin, Feng-Huei; Hsu, Yuan-Ming; Fang, Hsu-Wei

    2014-07-01

    In our previous study, we found that cartilage fragments from osteoarthritic knee promoted chondrogenesis of mesenchymal stem cells. In this study, we further transformed the cartilage tissues into acellular cartilage matrix (ACM) and explored the feasibility of using ACM as a biological scaffold. Nonworn parts of cartilage tissues were obtained during total knee arthroplasty (TKA) surgery and were successfully fabricated into ACM powders. The ACM powders and human synovium-derived mesenchymal stem cells (SMSCs) were mixed into collagen gel for in vitro culture. Histological results showed a synergistic effect of ACM powders and chondrogenic growth factors in the formation of engineered cartilage. The findings of real-time polymerase chain reaction (PCR) suggested that ACM powders had the potential of promoting type II collagen gene expression in the growth factors-absent environment. Moreover, with growth factors induction, the ACM powders could reduce the hypertrophy in chondrogenesis of SMSCs. In summary, ACM powders could serve as a functional scaffold that benefited the chondrogenesis of SMSCs for cartilage tissue engineering.

  20. Tissue engineering of reproductive tissues and organs.

    PubMed

    Atala, Anthony

    2012-07-01

    Regenerative medicine and tissue engineering technology may soon offer new hope for patients with serious injuries and end-stage reproductive organ failure. Scientists are now applying the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that can restore and maintain normal function in diseased and injured reproductive tissues. In addition, the stem cell field is advancing, and new discoveries in this field will lead to new therapeutic strategies. For example, newly discovered types of stem cells have been retrieved from uterine tissues such as amniotic fluid and placental stem cells. The process of therapeutic cloning and the creation of induced pluripotent cells provide still other potential sources of stem cells for cell-based tissue engineering applications. Although stem cells are still in the research phase, some therapies arising from tissue engineering endeavors that make use of autologous adult cells have already entered the clinic. This article discusses these tissue engineering strategies for various organs in the male and female reproductive tract.

  1. In-depth evaluation of commercially available human vascular smooth muscle cells phenotype: Implications for vascular tissue engineering.

    PubMed

    Timraz, Sara B H; Farhat, Ilyas A H; Alhussein, Ghada; Christoforou, Nicolas; Teo, Jeremy C M

    2016-05-01

    In vitro research on vascular tissue engineering has extensively used isolated primary human or animal smooth muscle cells (SMC). Research programs that lack such facilities tend towards commercially available primary cells sources. Here, we aim to evaluate the capacity of commercially available human SMC to maintain their contractile phenotype, and determine if dedifferentiation towards the synthetic phenotype occurs in response to conventional cell culture and passaging without any external biochemical or mechanical stimuli. Lower passage SMC adopted a contractile phenotype marked by a relatively slower proliferation rate, higher expression of proteins of the contractile apparatus and smoothelin, elongated morphology, and reduced deposition of collagen types I and III. As the passage number increased, migratory capacity was enhanced, average cell speed, total distance and net distance travelled increased up to passage 8. Through the various assays, corroborative evidence pinpoints SMC at passage 7 as the transition point between the contractile and synthetic phenotypes, while passage 8 distinctly and consistently exhibited characteristics of synthetic phenotype. This knowledge is particularly useful in selecting SMC of appropriate passage number for the target vascular tissue engineering application, for example, a homeostatic vascular graft for blood vessel replacement versus recreating atherosclerotic blood vessel model in vitro. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Evaluation of decellularized human umbilical vein (HUV) for vascular tissue engineering - comparison with endothelium-denuded HUV.

    PubMed

    Mangold, Silvia; Schrammel, Siegfried; Huber, Georgine; Niemeyer, Markus; Schmid, Christof; Stangassinger, Manfred; Hoenicka, Markus

    2015-01-01

    Human umbilical vessels have been recognized as a valuable and widely available resource for vascular tissue engineering. Whereas endothelium-denuded human umbilical veins (HUVs) have been successfully seeded with a patient-derived neoendothelium, decellularized vessels may have additional advantages, due to their lower antigenicity. The present study investigated the effects of three different decellularization procedures on the histological, mechanical and seeding properties of HUVs. Vessels were decellularized by detergent treatment (Triton X-100, sodium deoxycholate, IGEPAL-CA630), osmotic lysis (3 m NaCl, distilled water) and peroxyacetic acid treatment. In all cases, nuclease treatments were required to remove residual nucleic acids. Decellularization resulted in a partial loss of fibronectin and laminin staining in the subendothelial layer and affected the appearance of elastic fibres. In addition to removing residual nucleic acids, nuclease treatment weakened all stainings and substantially altered surface properties, as seen in scanning electron micrographs, indicating additional non-specific effects. Detergent treatment and osmotic lysis caused failure stresses to decrease significantly. Although conditioned medium prepared from decellularized HUV did not severely affect endothelial cell growth, cells seeded on decellularized HUV did not remain viable. This may be attributed to the partial removal of essential extracellular matrix components as well as to changes of surface properties. Therefore, decellularized HUVs appear to require additional modifications in order to support successful cell seeding. Replacing the vessels' endothelium may thus be a superior alternative to decellularization when creating tissue-engineered blood vessels with non-immunogenic luminal interfaces.

  3. Myocardial Tissue Engineering for Regenerative Applications.

    PubMed

    Fujita, Buntaro; Zimmermann, Wolfram-Hubertus

    2017-09-01

    This review provides an overview of the current state of tissue-engineered heart repair with a special focus on the anticipated modes of action of tissue-engineered therapy candidates and particular implications as to transplant immunology. Myocardial tissue engineering technologies have made tremendous advances in recent years. Numerous different strategies are under investigation and have reached different stages on their way to clinical translation. Studies in animal models demonstrated that heart repair requires either remuscularization by delivery of bona fide cardiomyocytes or paracrine support for the activation of endogenous repair mechanisms. Tissue engineering approaches result in enhanced cardiomyocyte retention and sustained remuscularization, but may also be explored for targeted paracrine or mechanical support. Some of the more advanced tissue engineering approaches are already tested clinically; others are at late stages of pre-clinical development. Process optimization towards cGMP compatibility and clinical scalability of contractile engineered human myocardium is an essential step towards clinical translation. Long-term allograft retention can be achieved under immune suppression. HLA matching may be an option to enhance graft retention and reduce the need for comprehensive immune suppression. Tissue-engineered heart repair is entering the clinical stage of the translational pipeline. Like in any effective therapy, side effects must be anticipated and carefully controlled. Allograft implantation under immune suppression is the most likely clinical scenario. Strategies to overcome transplant rejection are evolving and may further boost the clinical acceptance of tissue-engineered heart repair.

  4. [Preliminary study on tissue-engineered cartilage with human dermal fibroblasts co-cultured with porcine chondrocytes in vitro].

    PubMed

    Liu, Xia; Zhou, Guang-dong; Liu, Wei; Cao, Yi-lin

    2009-11-01

    To explore the feasibility of constructing tissue-engineered cartilage with human dermal fibroblasts (HDFs) in vitro. Porcine articular chondrocytes and HDFs were isolated and in vitro expanded respectively. Then they were mixed at the ratio of 1:1 (chondrocytes: fibroblasts) . The mixed cells were seeded onto polyglycolic acid (PGA) scaffold at the ultimate concentration of 5.0 x 10(7)/ml as co-culture group. Chondrocytes and HDFs at the same ultimate concentration were seeded respectively onto the scaffold as chondrocyte group ( positive control group) and fibroblast group ( negative control group). The specimens were collected after in vitro culture for 8 weeks. Gross observation, histology and immunohistochemistry were used to evaluate the results. In chondrocyte group, the cell-scaffold constructs could maintain the original size and shape during in vitro culture. The new formed cartilage-like tissue had typical histological structure and extracellular matrix staining similar to normal cartilage. In co-culture group the constructs shrunk slightly at 8 weeks, cartilage-like tissue formed and GAG could be detected for strong expression by Safranin O staining. Furthermore, using the specific identification, a few HDFs derived cells were found to form lacuna structure at the peripheral area of cartilage-like tissue. In fibroblast group, the constructs deformed and shrunk gradually without mature cartilage lacuna in histology. The 3D-co-culture system can effectively induce the differentiation of HDFs to chondrocytes. The tissue-engineered cartilage can be constructed in vitro with the 3D-co-culture system.

  5. Karyotyping of human chondrocytes in scaffold-assisted cartilage tissue engineering.

    PubMed

    Trimborn, Marc; Endres, Michaela; Bommer, Christiane; Janke, Una; Krüger, Jan-Philipp; Morawietz, Lars; Kreuz, Peter C; Kaps, Christian

    2012-04-01

    Scaffold-assisted autologous chondrocyte implantation (ACI) is an effective clinical procedure for cartilage repair. The aim of our study was to evaluate the chromosomal stability of human chondrocytes subjected to typical cell culture procedures needed for regenerative approaches in polymer-scaffold-assisted cartilage repair. Chondrocytes derived from post mortem donors and from donors scheduled for ACI were expanded, cryopreserved and re-arranged in polyglycolic acid (PGA)-fibrin scaffolds for tissue culture. Chondrocyte redifferentiation was analyzed by electron microscopy, histology and gene expression analysis. Karyotyping was performed using GTG banding and fluorescence in situ hybridization on a single cell basis. Chondrocytes showed de- and redifferentiation accompanied by the formation of extracellular matrix and induction of typical chondrocyte marker genes like type II collagen in PGA-fibrin scaffolds. Post mortem chondrocytes showed up to 1.7% structural and high numbers of numerical (up to 26.7%) chromosomal aberrations, while chondrocytes from living donors scheduled for ACI showed up to 1.8% structural and up to 1.3% numerical alterations. Cytogenetically, cell culture procedures and PGA-fibrin scaffolds did not significantly alter chromosomal integrity of the chondrocyte genome. Human chondrocytes derived from living donors subjected to regenerative medicine cell culture procedures like cell expansion, cryopreservation and culture in resorbable polymer-based scaffolds show normal chromosomal integrity and normal karyotypes. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  6. An engineered human conjunctival-like tissue to study ocular surface inflammatory diseases.

    PubMed

    García-Posadas, Laura; Soriano-Romaní, Laura; López-García, Antonio; Diebold, Yolanda

    2017-01-01

    The aim of this study was to develop a three-dimensional model of the human conjunctiva that can be used to perform physiology and pathophysiology experiments. Fibrin-based matrices (derived from human plasma or plasma cryoprecipitate) were used as scaffolds, and primary cells were obtained from conjunctival tissue. Conjunctival constructs were analyzed by immunofluorescent staining and scanning electron microscopy and cell proliferation was measured with alamarBlue® assay. After characterizing the constructs, four different experimental conditions were analyzed in cryoprecipitate matrices: controls, air-lifted cultures (to increase cell stratification), partially desiccated cultures (to mimic dry eye disease), and IL-13-treated cultures (to mimic allergy). Constructs were stained with hematoxylin/eosin to observe changes in morphology. High molecular weight glycoconjugates were identified by HPA staining. MUC5AC and IL-6 secretion was evaluated by ELISA. The fibrin-based matrices supported conjunctival cell growth. Epithelial cells grew on the surface of the scaffolds and underwent stratification that increased over time. These cells had microvilli, which suggests cell polarization and functionality. Fibroblasts were integrated in the scaffold and showed elongated shape. Compared to controls, air-lifted construct had increased epithelial stratification and upregulated MUC5AC secretion. Increased MUC5AC secretion also occurred in partially desiccated and IL-13-treated cultures. The inflammatory status of cells was evaluated by IL-6 levels which were increased in air-lifted and partially desiccated cultures, but not in IL-13-treated ones. In conclusion, we have developed a new three-dimensional model of human conjunctiva that can be used to study ocular surface inflammatory diseases.

  7. An engineered human conjunctival-like tissue to study ocular surface inflammatory diseases

    PubMed Central

    García-Posadas, Laura; Soriano-Romaní, Laura; López-García, Antonio; Diebold, Yolanda

    2017-01-01

    The aim of this study was to develop a three-dimensional model of the human conjunctiva that can be used to perform physiology and pathophysiology experiments. Fibrin-based matrices (derived from human plasma or plasma cryoprecipitate) were used as scaffolds, and primary cells were obtained from conjunctival tissue. Conjunctival constructs were analyzed by immunofluorescent staining and scanning electron microscopy and cell proliferation was measured with alamarBlue® assay. After characterizing the constructs, four different experimental conditions were analyzed in cryoprecipitate matrices: controls, air-lifted cultures (to increase cell stratification), partially desiccated cultures (to mimic dry eye disease), and IL-13-treated cultures (to mimic allergy). Constructs were stained with hematoxylin/eosin to observe changes in morphology. High molecular weight glycoconjugates were identified by HPA staining. MUC5AC and IL-6 secretion was evaluated by ELISA. The fibrin-based matrices supported conjunctival cell growth. Epithelial cells grew on the surface of the scaffolds and underwent stratification that increased over time. These cells had microvilli, which suggests cell polarization and functionality. Fibroblasts were integrated in the scaffold and showed elongated shape. Compared to controls, air-lifted construct had increased epithelial stratification and upregulated MUC5AC secretion. Increased MUC5AC secretion also occurred in partially desiccated and IL-13-treated cultures. The inflammatory status of cells was evaluated by IL-6 levels which were increased in air-lifted and partially desiccated cultures, but not in IL-13-treated ones. In conclusion, we have developed a new three-dimensional model of human conjunctiva that can be used to study ocular surface inflammatory diseases. PMID:28248962

  8. The cell-engineered construct of cartilage on the basis of biopolymer hydrogel matrix and human adipose tissue-derived mesenchymal stromal cells (in vitro study).

    PubMed

    Surguchenko, Valentina A; Ponomareva, Anna S; Kirsanova, Ljudmila A; Skaleckij, Nikolaj N; Sevastianov, Viktor I

    2015-02-01

    The study results of in vitro formation of tissue-engineered cartilage construct on the basis of cell-engineered construct composed of biopolymer hydrogel matrix and human adipose tissue-derived mesenchymal stromal cells (hADSCs) are presented. It was revealed that hADSCs in biopolymer hydrogel matrix Sphero®GEL under chondrogenic conditions generate three-dimensional structures and produce cartilaginous extracellular matrix components: collagen type II and glycosaminoglycans.

  9. Tissue engineering of a human 3D in vitro tumor test system.

    PubMed

    Moll, Corinna; Reboredo, Jenny; Schwarz, Thomas; Appelt, Antje; Schürlein, Sebastian; Walles, Heike; Nietzer, Sarah

    2013-08-06

    Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system. Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns). Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow. In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

  10. Microfluidic hydrogels for tissue engineering.

    PubMed

    Huang, Guo You; Zhou, Li Hong; Zhang, Qian Cheng; Chen, Yong Mei; Sun, Wei; Xu, Feng; Lu, Tian Jian

    2011-03-01

    With advanced properties similar to the native extracellular matrix, hydrogels have found widespread applications in tissue engineering. Hydrogel-based cellular constructs have been successfully developed to engineer different tissues such as skin, cartilage and bladder. Whilst significant advances have been made, it is still challenging to fabricate large and complex functional tissues due mainly to the limited diffusion capability of hydrogels. The integration of microfluidic networks and hydrogels can greatly enhance mass transport in hydrogels and spatiotemporally control the chemical microenvironment of cells, mimicking the function of native microvessels. In this review, we present and discuss recent advances in the fabrication of microfluidic hydrogels from the viewpoint of tissue engineering. Further development of new hydrogels and microengineering technologies will have a great impact on tissue engineering.

  11. Tissue engineering for periodontal regeneration.

    PubMed

    Kao, Richard T; Conte, Greg; Nishimine, Dee; Dault, Scott

    2005-03-01

    As a result of periodontal regeneration research, a series of clinical techniques have emerged that permit tissue engineering to be performed for more efficient regeneration and repair of periodontal defects and improved implant site development. Historically, periodontal regeneration research has focused on a quest for "magic filler" material. This search has led to the development of techniques utilizing autologous bone and bone marrow, allografts, xenografts, and various man-made bone substitutes. Though these techniques have had limited success, the desire for a more effective regenerative approach has resulted in the development of tissue engineering techniques. Tissue engineering is a relatively new field of reconstructive biology which utilizes mechanical, cellular, or biologic mediators to facilitate reconstruction/regeneration of a particular tissue. In periodontology, the concept of tissue engineering had its beginnings with guided tissue regeneration, a mechanical approach utilizing nonresorbable membranes to obtain regeneration in defects. In dental implantology, guided bone regeneration membranes +/- mechanical support are used for bone augmentation of proposed implant placement sites. With the availability of partially purified protein mixture from developing teeth and growth factors from recombinant technology, a new era of tissue engineering whereby biologic mediators can be used for periodontal regeneration. The advantage of recombinant growth factors is this tissue engineering device is consistent in its regenerative capacity, and variations in regenerative response are due to individual healing response and/or poor surgical techniques. In this article, the authors review how tissue engineering has advanced and discuss its impact on the clinical management of both periodontal and osseous defects in preparation for implant placement. An understanding of these new tissue engineering techniques is essential for comprehending today's ever

  12. Tissue Engineering of Corneal Endothelium

    PubMed Central

    Mimura, Tatsuya; Yokoo, Seiichi; Yamagami, Satoru

    2012-01-01

    Human corneal endothelial cells (HCECs) do not replicate after wounding. Therefore, corneal endothelial deficiency can result in irreversible corneal edema. Descemet stripping automated endothelial keratoplasty (DSAEK) allows selective replacement of the diseased corneal endothelium. However, DSAEK requires a donor cornea and the worldwide shortage of corneas limits its application. This review presents current knowledge on the tissue engineering of corneal endothelium using cultured HCECs. We also provide our recent work on tissue engineering for DSAEK grafts using cultured HCECs. We reconstructed DSAEK grafts by seeding cultured DiI-labelled HCECs on collagen sheets. Then HCEC sheets were transplanted onto the posterior stroma after descemetorhexis in the DSAEK group. Severe stromal edema was detected in the control group, but not in the DSAEK group throughout the observation period. Fluorescein microscopy one month after surgery showed numerous DiI-labelled cells on the posterior corneal surface in the DSAEK group. Frozen sections showed a monolayer of DiI-labelled cells on Descemet’s membrane. These findings indicate that cultured adult HCECs, transplanted with DSAEK surgery, maintain corneal transparency after transplantation and suggest the feasibility of performing DSAEK with HCECs to treat endothelial dysfunction. PMID:24955745

  13. [Tissue engineering of parathyroid gland].

    PubMed

    Iovino, F; Armano, G; Auriemma, P P; Sergio, R; De Sena, G; Capuozzo, V; Rosso, F; Marino, G; Papale, F; Grimaldi, A; Barbarisi, A

    2010-01-01

    The postoperative hypoparathyroidism is a not rare complication after total thyroidectomy and/or total parathyroidectomy. Attempts to transplant parathyroid tissue began in 1975 with the work of Wells, but still today results are disappointing. However, with the development of tissue engineering techniques, some experimental approaches to build artificial parathyroid are been made. Bioengineered device, actively secreting PTH, for transplant in patients with iatrogenic hypoparathyroidism is unavailable. Parathyroid cells were obtained from three chronic uremic patients in hemodialysis, operated for secondary hyperparathyroidism. Cell cultures in RPMI medium were subsequently seeded on collagen scaffold (three-dimensional matrix with slow biodegradation). Collagen is the major component of the extracellular matrix and thus is a good substrate for cell adhesion and growth. Culture media, with a low calcium concentration, were optimised to physiologically stimulate parathyroid hormone secretion. Cell cultures were morphologically observed in optical and electron (ESEM) microscopy and metabolically assayed by MTT method until the tenth week. Besides, concentration of parathyroid hormone in the culture medium has been measured for several weeks. After 24 hours of culture in RPMI, cells extracted from human parathyroid glands were nearly all adherent and organised in clusters to resemble the glandular organization. The cellular population consisted predominantly of parathyroid cells (90-95%). On collagen scaffolds, cells maintains an epithelial-like morphology also after 10 weeks, colonizing the scaffold surface and keeping a good proliferative rate with a discrete production of parathyroid hormone. The use of parathyroid cells extracted from patients with secondary hyperparathyroidism was certainly an appropriate choice that enabled us to achieve these results, that albeit partial bode well for the experimental in vivo animal model. The bioengineered scaffolds when

  14. Biomimetic Materials for Tissue Engineering

    PubMed Central

    Ma, Peter X

    2008-01-01

    Tissue engineering and regenerative medicine is an exciting research area that aims at regenerative alternatives to harvested tissues for transplantation. Biomaterials play a pivotal role as scaffolds to provide three-dimensional templates and synthetic extracellular-matrix environments for tissue regeneration. It is often beneficial for the scaffolds to mimic certain advantageous characteristics of the natural extracellular matrix, or developmental or would healing programs. This article reviews current biomimetic materials approaches in tissue engineering. These include synthesis to achieve certain compositions or properties similar to those of the extracellular matrix, novel processing technologies to achieve structural features mimicking the extracellular matrix on various levels, approaches to emulate cell-extracellular matrix interactions, and biologic delivery strategies to recapitulate a signaling cascade or developmental/would-healing program. The article also provides examples of enhanced cellular/tissue functions and regenerative outcomes, demonstrating the excitement and significance of the biomimetic materials for tissue engineering and regeneration. PMID:18045729

  15. Biomimetic materials for tissue engineering.

    PubMed

    Ma, Peter X

    2008-01-14

    Tissue engineering and regenerative medicine is an exciting research area that aims at regenerative alternatives to harvested tissues for transplantation. Biomaterials play a pivotal role as scaffolds to provide three-dimensional templates and synthetic extracellular matrix environments for tissue regeneration. It is often beneficial for the scaffolds to mimic certain advantageous characteristics of the natural extracellular matrix, or developmental or wound healing programs. This article reviews current biomimetic materials approaches in tissue engineering. These include synthesis to achieve certain compositions or properties similar to those of the extracellular matrix, novel processing technologies to achieve structural features mimicking the extracellular matrix on various levels, approaches to emulate cell-extracellular matrix interactions, and biologic delivery strategies to recapitulate a signaling cascade or developmental/wound healing program. The article also provides examples of enhanced cellular/tissue functions and regenerative outcomes, demonstrating the excitement and significance of the biomimetic materials for tissue engineering and regeneration.

  16. Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering.

    PubMed

    Rnjak-Kovacina, Jelena; Wise, Steven G; Li, Zhe; Maitz, Peter K M; Young, Cara J; Wang, Yiwei; Weiss, Anthony S

    2011-10-01

    We obtained low and high porosity synthetic human elastin scaffolds by adapting low (1 mL/h) and high (3 mL/h) flow rates respectively during electrospinning. Physical, mechanical and biological properties of these scaffolds were screened to identify the best candidates for the bioengineering of dermal tissue. SHE scaffolds that were electrospun at the higher flow rate presented increased fiber diameter and greater average pore size and over doubling of overall scaffold porosity. Both types of scaffold displayed Young's moduli comparable to that of native elastin, but the high porosity scaffolds possessed higher tensile strength. Low and high porosity scaffolds supported early attachment, spreading and proliferation of primary dermal fibroblasts, but only high porosity scaffolds supported active cell migration and infiltration into the scaffold. High porosity SHE scaffolds promoted cell persistence and scaffold remodeling in vitro with only moderate scaffold contraction. The scaffolds persisted for at least 6 weeks in a mouse subcutaneous implantation study with fibroblasts on the exterior and infiltrating, evidence of scaffold remodeling including de novo collagen synthesis and early stage angiogenesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Murine and Human Tissue-Engineered Esophagus Form from Sufficient Stem/Progenitor Cells and Do Not Require Microdesigned Biomaterials

    PubMed Central

    Spurrier, Ryan Gregory; Speer, Allison L.; Hou, Xiaogang; El-Nachef, Wael N.

    2015-01-01

    Purpose: Tissue-engineered esophagus (TEE) may serve as a therapeutic replacement for absent foregut. Most prior esophagus studies have favored microdesigned biomaterials and yielded epithelial growth alone. None have generated human TEE with mesenchymal components. We hypothesized that sufficient progenitor cells might only require basic support for successful generation of murine and human TEE. Materials and Methods: Esophageal organoid units (EOUs) were isolated from murine or human esophagi and implanted on a polyglycolic acid/poly-l-lactic acid collagen-coated scaffold in adult allogeneic or immune-deficient mice. Alternatively, EOU were cultured for 10 days in vitro prior to implantation. Results: TEE recapitulated all key components of native esophagus with an epithelium and subjacent muscularis. Differentiated suprabasal and proliferative basal layers of esophageal epithelium, muscle, and nerve were identified. Lineage tracing demonstrated that multiple EOU could contribute to the epithelium and mesenchyme of a single TEE. Cultured murine EOU grew as an expanding sphere of proliferative basal cells on a neuromuscular network that demonstrated spontaneous peristalsis in culture. Subsequently, cultured EOU generated TEE. Conclusions: TEE forms after transplantation of mouse and human organ-specific stem/progenitor cells in vivo on a relatively simple biodegradable scaffold. This is a first step toward future human therapies. PMID:25298083

  18. Tissue engineering: A live disc

    NASA Astrophysics Data System (ADS)

    Hukins, David W. L.

    2005-12-01

    A material-cell hybrid device that mimics the anatomic shape of the intervertebral disc has been made and successfully implanted into mice to show that tissue engineering may, in the future, benefit sufferers from back pain.

  19. Polymeric Nanofibers in Tissue Engineering

    PubMed Central

    Dahlin, Rebecca L.; Kasper, F. Kurtis

    2011-01-01

    Polymeric nanofibers can be produced using methods such as electrospinning, phase separation, and self-assembly, and the fiber composition, diameter, alignment, degradation, and mechanical properties can be tailored to the intended application. Nanofibers possess unique advantages for tissue engineering. The small diameter closely matches that of extracellular matrix fibers, and the relatively large surface area is beneficial for cell attachment and bioactive factor loading. This review will update the reader on the aspects of nanofiber fabrication and characterization important to tissue engineering, including control of porous structure, cell infiltration, and fiber degradation. Bioactive factor loading will be discussed with specific relevance to tissue engineering. Finally, applications of polymeric nanofibers in the fields of bone, cartilage, ligament and tendon, cardiovascular, and neural tissue engineering will be reviewed. PMID:21699434

  20. Tissue engineering in the vasculature.

    PubMed

    Naito, Yuji; Rocco, Kevin; Kurobe, Hirotsugu; Maxfield, Mark; Breuer, Christopher; Shinoka, Toshiharu

    2014-01-01

    Tissue engineering holds great promise to address complications and limitations encountered with the use of traditional prosthetic materials, such as thrombogenicity, infection, and future degeneration which represent the major morbidity and mortality after device implant surgery. The general concept of tissue engineering consists of three main components: a scaffold material, a cell type for seeding the scaffold, and biochemical, physio-chemical signaling and remodeling process. This remodeling process is guided by cell signals derived from both seeded cells and host inflammatory cells that infiltrate the scaffold and deposit extracellular matrix, forming the neotissue. Vascular tissue engineering is at the forefront in the translation of this technology to clinical practice, as tissue engineered vascular grafts (TEVGs) have now been successfully implanted in children with congenital heart disease. In this report, we review the history, advances, and state of the art in TEVGs. Copyright © 2013 Wiley Periodicals, Inc.

  1. Polymeric nanofibers in tissue engineering.

    PubMed

    Dahlin, Rebecca L; Kasper, F Kurtis; Mikos, Antonios G

    2011-10-01

    Polymeric nanofibers can be produced using methods such as electrospinning, phase separation, and self-assembly, and the fiber composition, diameter, alignment, degradation, and mechanical properties can be tailored to the intended application. Nanofibers possess unique advantages for tissue engineering. The small diameter closely matches that of extracellular matrix fibers, and the relatively large surface area is beneficial for cell attachment and bioactive factor loading. This review will update the reader on the aspects of nanofiber fabrication and characterization important to tissue engineering, including control of porous structure, cell infiltration, and fiber degradation. Bioactive factor loading will be discussed with specific relevance to tissue engineering. Finally, applications of polymeric nanofibers in the fields of bone, cartilage, ligament and tendon, cardiovascular, and neural tissue engineering will be reviewed.

  2. Mechanobioreactors for Cartilage Tissue Engineering.

    PubMed

    Weber, Joanna F; Perez, Roman; Waldman, Stephen D

    2015-01-01

    Mechanical stimulation is an effective method to increase extracellular matrix synthesis and to improve the mechanical properties of tissue-engineered cartilage constructs. In this chapter, we describe valuable methods of imposing direct mechanical stimuli (compression or shear) to tissue-engineered cartilage constructs as well as some common analytical methods used to quantify the effects of mechanical stimuli after short-term or long-term loading.

  3. Angiogenesis and Tissue Engineering Research

    DTIC Science & Technology

    2010-08-01

    11, 305, 2002. 5. Shin’oka, T., Matsumura, G., Hibino, N., Naito, Y., Watanabe, M., Konuma, T., Sakamoto, T., Nagatsu, M., and Kurosawa , H. Midterm...Ikada, Y., Kurosawa , H., and Shin’oka, T. Successful application of tissue engineered vas- cular autografts: clinical experience. Biomaterials 24...2303, 2003. 35. Matsumura, G., Ishihara, Y., Miyagawa-Tomita, S., Ikada, Y., Matsuda, S., Kurosawa , H., and Shin’oka, T. Evaluation of tissue-engineered

  4. Liposomes in tissue engineering and regenerative medicine

    PubMed Central

    Monteiro, Nelson; Martins, Albino; Reis, Rui L.; Neves, Nuno M.

    2014-01-01

    Liposomes are vesicular structures made of lipids that are formed in aqueous solutions. Structurally, they resemble the lipid membrane of living cells. Therefore, they have been widely investigated, since the 1960s, as models to study the cell membrane, and as carriers for protection and/or delivery of bioactive agents. They have been used in different areas of research including vaccines, imaging, applications in cosmetics and tissue engineering. Tissue engineering is defined as a strategy for promoting the regeneration of tissues for the human body. This strategy may involve the coordinated application of defined cell types with structured biomaterial scaffolds to produce living structures. To create a new tissue, based on this strategy, a controlled stimulation of cultured cells is needed, through a systematic combination of bioactive agents and mechanical signals. In this review, we highlight the potential role of liposomes as a platform for the sustained and local delivery of bioactive agents for tissue engineering and regenerative medicine approaches. PMID:25401172

  5. Release of Tensile Strain on Engineered Human Tendon Tissue Disturbs Cell Adhesions, Changes Matrix Architecture, and Induces an Inflammatory Phenotype

    PubMed Central

    Bayer, Monika L.; Schjerling, Peter; Herchenhan, Andreas; Zeltz, Cedric; Heinemeier, Katja M.; Christensen, Lise; Krogsgaard, Michael; Gullberg, Donald; Kjaer, Michael

    2014-01-01

    Mechanical loading of tendon cells results in an upregulation of mechanotransduction signaling pathways, cell-matrix adhesion and collagen synthesis, but whether unloading removes these responses is unclear. We investigated the response to tension release, with regard to matrix proteins, pro-inflammatory mediators and tendon phenotypic specific molecules, in an in vitro model where tendon-like tissue was engineered from human tendon cells. Tissue sampling was performed 1, 2, 4 and 6 days after surgical de-tensioning of the tendon construct. When tensile stimulus was removed, integrin type collagen receptors showed a contrasting response with a clear drop in integrin subunit α11 mRNA and protein expression, and an increase in α2 integrin mRNA and protein levels. Further, specific markers for tendon cell differentiation declined and normal tendon architecture was disturbed, whereas pro-inflammatory molecules were upregulated. Stimulation with the cytokine TGF-β1 had distinct effects on some tendon-related genes in both tensioned and de-tensioned tissue. These findings indicate an important role of mechanical loading for cellular and matrix responses in tendon, including that loss of tension leads to a decrease in phenotypical markers for tendon, while expression of pro-inflammatory mediators is induced. PMID:24465881

  6. Detergent decellularization of heart valves for tissue engineering: toxicological effects of residual detergents on human endothelial cells.

    PubMed

    Cebotari, Serghei; Tudorache, Igor; Jaekel, Thomas; Hilfiker, Andres; Dorfman, Suzanne; Ternes, Waldemar; Haverich, Axel; Lichtenberg, Artur

    2010-03-01

    Detergents are powerful agents for tissue decellularization. Despite this, the high toxicity of detergent residua can be a major limitation. This study evaluated the efficacy of detergent removal from decellularized pulmonary valves (PVs) and the consequences of repopulation with human endothelial cells (HECs). Porcine PVs were treated with 1% sodium deoxycholate (SDC), group A; 1% sodium dodecyl sulfate (SDS), group B; and a mixture of 0.5% SDC/0.5% SDS, group C (n = 5 each). After each of 10 succeeding wash cycles (WCs), samples of the washing solution (WS) were analyzed by solid phase extraction and high performance liquid chromatography for the presence of detergents. Metabolic activity of HEC was also assessed in the WS samples (cytotoxicity and MTS assays). Decellularized and washed PVs were reseeded with HEC. Histological analysis demonstrated efficient tissue decellularization in all groups. Detergents' concentration in all WSs decreased exponentially and was below 50 mg/L after 6, 8, and 4 WCs in groups A, B, and C, respectively. This concentration resulted in no significant toxic influence on cell cultures, and scaffolds could be efficiently reseeded with HEC. In conclusion, intensive washing of detergent decellularized valvular scaffolds lowers the residual contamination below a hazardous threshold and allows their successful repopulation with HEC for tissue engineering purposes.

  7. Commercial considerations in tissue engineering

    PubMed Central

    Mansbridge, Jonathan

    2006-01-01

    Tissue engineering is a field with immense promise. Using the example of an early tissue-engineered skin implant, Dermagraft, factors involved in the successful commercial development of devices of this type are explored. Tissue engineering has to strike a balance between tissue culture, which is a resource-intensive activity, and business considerations that are concerned with minimizing cost and maximizing customer convenience. Bioreactor design takes place in a highly regulated environment, so factors to be incorporated into the concept include not only tissue culture considerations but also matters related to asepsis, scaleup, automation and ease of use by the final customer. Dermagraft is an allogeneic tissue. Stasis preservation, in this case cryopreservation, is essential in allogeneic tissue engineering, allowing sterility testing, inventory control and, in the case of Dermagraft, a cellular stress that may be important for hormesis following implantation. Although the use of allogeneic cells provides advantages in manufacturing under suitable conditions, it raises the spectre of immunological rejection. Such rejection has not been experienced with Dermagraft. Possible reasons for this and the vision of further application of allogeneic tissues are important considerations in future tissue-engineered cellular devices. This review illustrates approaches that indicate some of the criteria that may provide a basis for further developments. Marketing is a further requirement for success, which entails understanding of the mechanism of action of the procedure, and is illustrated for Dermagraft. The success of a tissue-engineered product is dependent on many interacting operations, some discussed here, each of which must be performed simultaneously and well. PMID:17005024

  8. Projection Stereolithographic Fabrication of Human Adipose Stem Cell-Incorporated Biodegradable Scaffolds for Cartilage Tissue Engineering.

    PubMed

    Sun, Aaron X; Lin, Hang; Beck, Angela M; Kilroy, Evan J; Tuan, Rocky S

    2015-01-01

    The poor self-healing ability of cartilage necessitates the development of methods for cartilage regeneration. Scaffold construction with live stem cell incorporation and subsequent differentiation presents a promising route. Projection stereolithography (PSL) offers high resolution and processing speed as well as the ability to fabricate scaffolds that precisely fit the anatomy of cartilage defects using medical imaging as the design template. We report here the use of a visible-light-based PSL (VL-PSL) system to encapsulate human adipose-derived stem cells (hASCs) into a biodegradable polymer [poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid (PDLLA-PEG)]/hyaluronic acid (HA) matrix to produce live cell constructs with customized architectures. After fabrication, hASCs showed high viability (84%) and were uniformly distributed throughout the constructs, which possessed high mechanical properties with a compressive modulus of 780 kPa. The hASC-seeded constructs were then cultured in control or TGF-β3-containing chondrogenic medium for up to 28 days. In chondrogenic medium-treated group (TGF-β3 group), hASCs maintained 77% viability and expressed chondrogenic genes Sox9, collagen type II, and aggrecan at 11, 232, and 2.29 × 10(5) fold increases, respectively compared to levels at day 0 in non-chondrogenic medium. The TGF-β3 group also produced a collagen type II and glycosaminoglycan-rich extracellular matrix, detected by immunohistochemistry, Alcian blue staining, and Safranin O staining suggesting robust chondrogenesis within the scaffold. Without chondroinductive addition (Control group), cell viability decreased with time (65% at 28 days) and showed poor cartilage matrix deposition. After 28 days, mechanical strength of the TGF-β3 group remained high at 240 kPa. Thus, the PSL and PDLLA-PEG/HA-based fabrication method using adult stem cells is a promising approach in producing mechanically competent engineered cartilage for joint

  9. Projection Stereolithographic Fabrication of Human Adipose Stem Cell-Incorporated Biodegradable Scaffolds for Cartilage Tissue Engineering

    PubMed Central

    Sun, Aaron X.; Lin, Hang; Beck, Angela M.; Kilroy, Evan J.; Tuan, Rocky S.

    2015-01-01

    The poor self-healing ability of cartilage necessitates the development of methods for cartilage regeneration. Scaffold construction with live stem cell incorporation and subsequent differentiation presents a promising route. Projection stereolithography (PSL) offers high resolution and processing speed as well as the ability to fabricate scaffolds that precisely fit the anatomy of cartilage defects using medical imaging as the design template. We report here the use of a visible-light-based PSL (VL-PSL) system to encapsulate human adipose-derived stem cells (hASCs) into a biodegradable polymer [poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid (PDLLA-PEG)]/hyaluronic acid (HA) matrix to produce live cell constructs with customized architectures. After fabrication, hASCs showed high viability (84%) and were uniformly distributed throughout the constructs, which possessed high mechanical properties with a compressive modulus of 780 kPa. The hASC-seeded constructs were then cultured in control or TGF-β3-containing chondrogenic medium for up to 28 days. In chondrogenic medium-treated group (TGF-β3 group), hASCs maintained 77% viability and expressed chondrogenic genes Sox9, collagen type II, and aggrecan at 11, 232, and 2.29 × 105 fold increases, respectively compared to levels at day 0 in non-chondrogenic medium. The TGF-β3 group also produced a collagen type II and glycosaminoglycan-rich extracellular matrix, detected by immunohistochemistry, Alcian blue staining, and Safranin O staining suggesting robust chondrogenesis within the scaffold. Without chondroinductive addition (Control group), cell viability decreased with time (65% at 28 days) and showed poor cartilage matrix deposition. After 28 days, mechanical strength of the TGF-β3 group remained high at 240 kPa. Thus, the PSL and PDLLA-PEG/HA-based fabrication method using adult stem cells is a promising approach in producing mechanically competent engineered cartilage for joint

  10. Hyperbranched poly(NIPAM) polymers modified with antibiotics for the reduction of bacterial burden in infected human tissue engineered skin.

    PubMed

    Shepherd, Joanna; Sarker, Prodip; Rimmer, Stephen; Swanson, Linda; MacNeil, Sheila; Douglas, Ian

    2011-01-01

    The escalating global incidence of bacterial infection, particularly in chronic wounds, is a problem that requires significant improvements to existing therapies. We have developed hyperbranched poly(NIPAM) polymers functionalized with the antibiotics Vancomycin and Polymyxin-B that are sensitive to the presence of bacteria in solution. Binding of bacteria to the polymers causes a conformational change, resulting in collapse of the polymers and the formation of insoluble polymer/bacteria complexes. We have applied these novel polymers to our tissue engineered human skin model of a burn wound infected with Pseudomonas aeruginosa and Staphylococcus aureus. When the polymers were removed from the infected skin, either in a polymer gel solution or in the form of hydrogel membranes, they removed bound bacteria, thus reducing the bacterial load in the infected skin model. These bacteria-binding polymers have many potential uses, including coatings for wound dressings.

  11. Bone tissue engineering via human induced pluripotent, umbilical cord and bone marrow mesenchymal stem cells in rat cranium.

    PubMed

    Wang, Ping; Liu, Xian; Zhao, Liang; Weir, Michael D; Sun, Jirun; Chen, Wenchuan; Man, Yi; Xu, Hockin H K

    2015-05-01

    Human induced pluripotent stem cells (hiPSCs) are an exciting cell source with great potential for tissue engineering. Human bone marrow mesenchymal stem cells (hBMSCs) have been used in clinics but are limited by several disadvantages, hence alternative sources of MSCs such as umbilical cord MSCs (hUCMSCs) are being investigated. However, there has been no report comparing hiPSCs, hUCMSCs and hBMSCs for bone regeneration. The objectives of this pilot study were to investigate hiPSCs, hUCMSCs and hBMSCs for bone tissue engineering, and compare their bone regeneration via seeding on biofunctionalized macroporous calcium phosphate cement (CPC) in rat cranial defects. For all three types of cells, approximately 90% of the cells remained alive on CPC scaffolds. Osteogenic genes were up-regulated, and mineral synthesis by cells increased with time in vitro for all three types of cells. The new bone area fractions at 12weeks (mean±sd; n=6) were (30.4±5.8)%, (27.4±9.7)% and (22.6±4.7)% in hiPSC-MSC-CPC, hUCMSC-CPC and hBMSC-CPC respectively, compared to (11.0±6.3)% for control (p<0.05). No significant differences were detected among the three types of stem cells (p>0.1). New blood vessel density was higher in cell-seeded groups than control (p<0.05). De novo bone formation and participation by implanted cells was confirmed via immunohistochemical staining. In conclusion, (1) hiPSCs, hUCMSCs and hBMSCs greatly enhanced bone regeneration, more than doubling the new bone amount of cell-free CPC control; (2) hiPSC-MSCs and hUCMSCs represented viable alternatives to hBMSCs; (3) biofunctionalized macroporous CPC-stem cell constructs had a robust capacity for bone regeneration.

  12. Extracellular matrix expression of human tenocytes in three-dimensional air-liquid and PLGA cultures compared with tendon tissue: implications for tendon tissue engineering.

    PubMed

    Stoll, Christiane; John, Thilo; Endres, Michaela; Rosen, Christian; Kaps, Christian; Kohl, Benjamin; Sittinger, Michael; Ertel, Wolfgang; Schulze-Tanzil, Gundula

    2010-09-01

    Tenocyte transplantation may prove to be an approach to support healing of tendon defects. Cell-cell and cell-matrix contacts within three-dimensional (3D) cultures may prevent tenocyte dedifferentiation observed in monolayer (2D) culture. The present study compares both neotissue formation and tenocyte extracellular matrix (ECM) expression in 2D and 3D cultures directly with that of native tendon, in order to determine optimal conditions for tendon tissue engineering. Primary human tenocytes were embedded in poly[lactic-co-glycolic-acid] (PLGA)-scaffolds and high-density cultures. Neotissue formation was examined by hematoxyline-eosine (H&E) and immunofluorescence staining. Gene expression of ECM proteins and vascular endothelial growth factor (VEGF) was compared at days 0 (2D), 14, and 28 in 3D cultures and tendon. Histomorphology of 3D culture showed tendon-like tissue as tenocyte cell nuclei became more elongated and ECM accumulated. Type I collagen gene expression was higher in 2D culture than in tendon and decreased in 4-week-old 3D cultures, whereas type III collagen was only elevated in high-density culture compared with tendon. Decorin and COMP were reduced in 2D and increased in 3D culture almost to ex vivo level. These results suggest that the 3D high-density or biodegradable scaffolds cultures encourage the differentiation of expanded monolayer tenocytes in vitro to tendon-like tissue. (c) 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  13. Phase II Clinical Trial of Intraoral Grafting of Human Tissue-Engineered Oral Mucosa

    DTIC Science & Technology

    2016-10-01

    graft contracture and Wound Healing Index; and ancillary outcome measures of tissue perfusion measured graft color and laser Doppler flowmetry, and...infection, fluid loss, and foreign material contamination and relapse secondary to wound contracture. Oral mucosa is in limited supply for use in...reconstructive procedures in the oral cavity. This is especially prevalent after large avulsed soft tissue wounds involving the mouth and lips seen in

  14. Scaffolds in Tendon Tissue Engineering

    PubMed Central

    Longo, Umile Giuseppe; Lamberti, Alfredo; Petrillo, Stefano; Maffulli, Nicola; Denaro, Vincenzo

    2012-01-01

    Tissue engineering techniques using novel scaffold materials offer potential alternatives for managing tendon disorders. Tissue engineering strategies to improve tendon repair healing include the use of scaffolds, growth factors, cell seeding, or a combination of these approaches. Scaffolds have been the most common strategy investigated to date. Available scaffolds for tendon repair include both biological scaffolds, obtained from mammalian tissues, and synthetic scaffolds, manufactured from chemical compounds. Preliminary studies support the idea that scaffolds can provide an alternative for tendon augmentation with an enormous therapeutic potential. However, available data are lacking to allow definitive conclusion on the use of scaffolds for tendon augmentation. We review the current basic science and clinical understanding in the field of scaffolds and tissue engineering for tendon repair. PMID:22190961

  15. Nanomaterials, Inflammation and Tissue Engineering

    PubMed Central

    Padmanabhan, Jagannath

    2014-01-01

    Nanomaterials exhibit unique properties that are absent in the bulk material because decreasing material size leads to an exponential increase in surface area, surface area to volume ratio, and effective stiffness, resulting in altered physiochemical properties. Diverse categories of nanomaterials such as nanoparticles, nanoporous scaffolds, nanopatterned surfaces, nanofibers and carbon nanotubes can be generated using advanced fabrication and processing techniques. These materials are being increasingly incorporated in tissue engineering scaffolds to facilitate the development of biomimetic substitutes to replace damaged tissues and organs. Long term success of nanomaterials in tissue engineering is contingent upon the inflammatory responses they elicit in vivo. This review seeks to summarize the recent developments in our understanding of biochemical and biophysical attributes of nanomaterials and the inflammatory responses they elicit, with a focus on strategies for nanomaterial design in tissue engineering applications. PMID:25421333

  16. Harvesting the potential of the human umbilical cord: isolation and characterisation of four cell types for tissue engineering applications.

    PubMed

    Hayward, Cindy J; Fradette, Julie; Galbraith, Todd; Rémy, Murielle; Guignard, Rina; Gauvin, Robert; Germain, Lucie; Auger, François A

    2013-01-01

    The human umbilical cord (UC) has attracted interest as a source of cells for many research applications. UC solid tissues contain four cell types: epithelial, stromal, smooth muscle and endothelial cells. We have developed a unique protocol for the sequential extraction of all four cell types from a single UC, allowing tissue reconstruction using multiple cell types from the same source. By combining perfusion, immersion and explant techniques, all four cell types have been successfully expanded in monolayer cultures. We have also characterised epithelial and Wharton's jelly cells (WJC) by immunolabelling of specific proteins. Epithelial cell yields averaged at 2.3 × 10(5) cells per centimetre UC, and the cells expressed an unusual combination of keratins typical of simple, mucous and stratified epithelia. Stromal cells in the Wharton's jelly expressed desmin, α-smooth muscle actin, elastin, keratins (K12, K16, K18 and K19), vimentin and collagens. Expression patterns in cultured cells resembled those found in situ except for basement membrane components and type III collagen. These stromal cells featured a sustained proliferation rate up to passage 12 after thawing. The mesenchymal stem cell (MSC) character of the WJC was confirmed by their expression of typical MSC surface markers and by adipogenic and osteogenic differentiation assays. To emphasise and demonstrate their potential for regenerative medicine, UC cell types were successfully used to produce human tissue-engineered constructs. Both bilayered stromal/epithelial and vascular substitutes were produced, establishing the versatility and importance of these cells for research and therapeutic applications. Copyright © 2012 S. Karger AG, Basel.

  17. Trends in Tissue Engineering for Blood Vessels

    PubMed Central

    Nemeno-Guanzon, Judee Grace; Lee, Soojung; Berg, Johan Robert; Jo, Yong Hwa; Yeo, Jee Eun; Nam, Bo Mi; Koh, Yong-Gon; Lee, Jeong Ik

    2012-01-01

    Over the years, cardiovascular diseases continue to increase and affect not only human health but also the economic stability worldwide. The advancement in tissue engineering is contributing a lot in dealing with this immediate need of alleviating human health. Blood vessel diseases are considered as major cardiovascular health problems. Although blood vessel transplantation is the most convenient treatment, it has been delimited due to scarcity of donors and the patient's conditions. However, tissue-engineered blood vessels are promising alternatives as mode of treatment for blood vessel defects. The purpose of this paper is to show the importance of the advancement on biofabrication technology for treatment of soft tissue defects particularly for vascular tissues. This will also provide an overview and update on the current status of tissue reconstruction especially from autologous stem cells, scaffolds, and scaffold-free cellular transplantable constructs. The discussion of this paper will be focused on the historical view of cardiovascular tissue engineering and stem cell biology. The representative studies featured in this paper are limited within the last decade in order to trace the trend and evolution of techniques for blood vessel tissue engineering. PMID:23251085

  18. Trends in tissue engineering for blood vessels.

    PubMed

    Nemeno-Guanzon, Judee Grace; Lee, Soojung; Berg, Johan Robert; Jo, Yong Hwa; Yeo, Jee Eun; Nam, Bo Mi; Koh, Yong-Gon; Lee, Jeong Ik

    2012-01-01

    Over the years, cardiovascular diseases continue to increase and affect not only human health but also the economic stability worldwide. The advancement in tissue engineering is contributing a lot in dealing with this immediate need of alleviating human health. Blood vessel diseases are considered as major cardiovascular health problems. Although blood vessel transplantation is the most convenient treatment, it has been delimited due to scarcity of donors and the patient's conditions. However, tissue-engineered blood vessels are promising alternatives as mode of treatment for blood vessel defects. The purpose of this paper is to show the importance of the advancement on biofabrication technology for treatment of soft tissue defects particularly for vascular tissues. This will also provide an overview and update on the current status of tissue reconstruction especially from autologous stem cells, scaffolds, and scaffold-free cellular transplantable constructs. The discussion of this paper will be focused on the historical view of cardiovascular tissue engineering and stem cell biology. The representative studies featured in this paper are limited within the last decade in order to trace the trend and evolution of techniques for blood vessel tissue engineering.

  19. Effect of different growth factors on human osteoblasts activities: a possible application in bone regeneration for tissue engineering.

    PubMed

    Bosetti, Michela; Boccafoschi, Francesca; Leigheb, Massimiliano; Cannas, Mario F

    2007-12-01

    Cultured human primary osteoblasts reproduce the phenotypic differentiation and maturation of cells in vivo. We have investigated the influence of three isoforms of transforming growth factor beta (TGF-beta1, TGF-beta2 and TGF-beta3), three fibroblast growth factors (FGF-2, FGF-4 and FGF-6) and the active metabolite of Vitamin D [1,25-(OH)(2)D3] on proliferation, alkaline phosphatase activity and mineralization of human osteoblasts during a period of 24 days of culture. TGF-beta isoforms and three FGFs examined have been proved to be inducers of osteoblasts proliferation (higher extent for TGF-beta and FGF-2) and inhibitors of alkaline phosphatase activity and osteoblasts mineralization. Combination of these growth factors with the active form of Vitamin D induced osteodifferentiation. In fact Vitamin D showed an additive effect on alkaline phosphatase activity and calcium content, induced by FGF-2 and TGF-beta in human osteoblast. These results highlight the potential of proliferating cytokines' combination with mineralizing agents for in vitro bone growth induction in bone tissue engineering.

  20. Tooth Tissue Engineering: The Importance of Blood Products as a Supplement in Tissue Culture Medium for Human Pulp Dental Stem Cells.

    PubMed

    Pisciolaro, Ricardo Luiz; Duailibi, Monica Talarico; Novo, Neil Ferreira; Juliano, Yara; Pallos, Debora; Yelick, Pamela Crotty; Vacanti, Joseph Phillip; Ferreira, Lydia Masako; Duailibi, Silvio Eduardo

    2015-11-01

    One of the goals in using cells for tissue engineering (TE) and cell therapy consists of optimizing the medium for cell culture. The present study compares three different blood product supplements for improved cell proliferation and protection against DNA damage in cultured human dental pulp stem cells for tooth TE applications. Human cells from dental pulp were first characterized as adult stem cells (ectomesenchymal mixed origin) by flow cytometry. Next, four different cell culture conditions were tested: I, supplement-free; II, supplemented with fetal bovine serum; III, allogeneic human serum; and IV, autologous human serum. Cultured cells were then characterized for cell proliferation, mineralized nodule formation, and colony-forming units (CFU) capability. After 28 days in culture, the comet assay was performed to assess possible damage in cellular DNA. Our results revealed that Protocol IV achieved higher cell proliferation than Protocol I (p = 0.0112). Protocols II and III resulted in higher cell proliferation than Protocol I, but no statistical differences were found relative to Protocol IV. The comet assay revealed less cell damage in cells cultured using Protocol IV as compared to Protocols II and III. The damage percentage observed on Protocol II was significantly higher than all other protocols. CFUs capability was highest using Protocol IV (p = 0.0018) and III, respectively, and the highest degree of mineralization was observed using Protocol IV as compared to Protocols II and III. Protocol IV resulted in significantly improved cell proliferation, and no cell damage was observed. These results demonstrate that human blood product supplements can be used as feasible supplements for culturing adult human dental stem cells.

  1. Large scale expansion of human umbilical cord cells in a rotating bed system bioreactor for cardiovascular tissue engineering applications.

    PubMed

    Reichardt, Anne; Polchow, Bianca; Shakibaei, Mehdi; Henrich, Wolfgang; Hetzer, Roland; Lueders, Cora

    2013-01-01

    Widespread use of human umbilical cord cells for cardiovascular tissue engineering requires production of large numbers of well-characterized cells under controlled conditions. In current research projects, the expansion of cells to be used to create a tissue construct is usually performed in static cell culture systems which are, however, often not satisfactory due to limitations in nutrient and oxygen supply. To overcome these limitations dynamic cell expansion in bioreactor systems under controllable conditions could be an important tool providing continuous perfusion for the generation of large numbers of viable pre-conditioned cells in a short time period. For this purpose cells derived from human umbilical cord arteries were expanded in a rotating bed system bioreactor for up to 9 days. For a comparative study, cells were cultivated under static conditions in standard culture devices. Our results demonstrated that the microenvironment in the perfusion bioreactor was more favorable than that of the standard cell culture flasks. Data suggested that cells in the bioreactor expanded 39 fold (38.7 ± 6.1 fold) in comparison to statically cultured cells (31.8 ± 3.0 fold). Large-scale production of cells in the bioreactor resulted in more than 3 x 10(8) cells from a single umbilical cord fragment within 9 days. Furthermore cell doubling time was lower in the bioreactor system and production of extracellular matrix components was higher. With this study, we present an appropriate method to expand human umbilical cord artery derived cells with high cellular proliferation rates in a well-defined bioreactor system under GMP conditions.

  2. Bone tissue engineering in osteoporosis.

    PubMed

    Jakob, Franz; Ebert, Regina; Ignatius, Anita; Matsushita, Takashi; Watanabe, Yoshinobu; Groll, Juergen; Walles, Heike

    2013-06-01

    Osteoporosis is a polygenetic, environmentally modifiable disease, which precipitates into fragility fractures of vertebrae, hip and radius and also confers a high risk of fractures in accidents and trauma. Aging and the genetic molecular background of osteoporosis cause delayed healing and impair regeneration. The worldwide burden of disease is huge and steadily increasing while the average life expectancy is also on the rise. The clinical need for bone regeneration applications, systemic or in situ guided bone regeneration and bone tissue engineering, will increase and become a challenge for health care systems. Apart from in situ guided tissue regeneration classical ex vivo tissue engineering of bone has not yet reached the level of routine clinical application although a wealth of scaffolds and growth factors has been developed. Engineering of complex bone constructs in vitro requires scaffolds, growth and differentiation factors, precursor cells for angiogenesis and osteogenesis and suitable bioreactors in various combinations. The development of applications for ex vivo tissue engineering of bone faces technical challenges concerning rapid vascularization for the survival of constructs in vivo. Recent new ideas and developments in the fields of bone biology, materials science and bioreactor technology will enable us to develop standard operating procedures for ex vivo tissue engineering of bone in the near future. Once prototyped such applications will rapidly be tailored for compromised conditions like vitamin D and sex hormone deficiencies, cellular deficits and high production of regeneration inhibitors, as they are prevalent in osteoporosis and in higher age.

  3. Three-Dimensional Engineered High Fidelity Normal Human Lung Tissue-Like Assemblies (TLA) as Targets for Human Respiratory Virus Infections

    NASA Technical Reports Server (NTRS)

    Goodwin, T. J.; Deatly, A. M.; Suderman, M. T.; Lin, Y.-H.; Chen, W.; Gupta, C. K.; Randolph, V. B.; Udem, S. A.

    2003-01-01

    Unlike traditional two-dimensional (2D) cell cultures, three-dimensional (3D) tissue-like assemblies (TLA) (Goodwin et aI, 1992, 1993, 2000 and Nickerson et aI. , 2001,2002) offer high organ fidelity with the potential to emulate the infective dynamics of viruses and bacteria in vivo. Thus, utilizing NASA micro gravity Rotating Wall Vessel (RWV) technology, in vitro human broncho-epithelial (HBE) TLAs were engineered to mimic in vivo tissue for study of human respiratory viruses. These 3D HBE TLAs were propagated from a human broncho-tracheal cell line with a mesenchymal component (HBTC) as the foundation matrix and either an adult human broncho-epithelial cell (BEAS-2B) or human neonatal epithelial cell (16HBE140-) as the overlying element. Resulting TLAs share several characteristic features with in vivo human respiratory epithelium including tight junctions, desmosomes and cilia (SEM, TEM). The presence of epithelium and specific lung epithelium markers furthers the contention that these HBE cells differentiate into TLAs paralleling in vivo tissues. A time course of infection of these 3D HBE TLAs with human respiratory syncytial virus (hRSV) wild type A2 strain, indicates that virus replication and virus budding are supported and manifested by increasing virus titer and detection of membrane-bound F and G glycoproteins. Infected 3D HBE TLAs remain intact for up to 12 days compared to infected 2D cultures that are destroyed in 2-3 days. Infected cells show an increased vacuolation and cellular destruction (by transmission electron microscopy) by day 9; whereas, uninfected cells remain robust and morphologically intact. Therefore, the 3D HBE TLAs mimic aspects of human respiratory epithelium providing a unique opportunity to analyze, for the first time, simulated in vivo viral infection independent of host immune response.

  4. Optimization of human tendon tissue engineering: synergistic effects of growth factors for use in tendon scaffold repopulation.

    PubMed

    Raghavan, Shyam S; Woon, Colin Y L; Kraus, Armin; Megerle, Kai; Pham, Hung; Chang, James

    2012-02-01

    Tissue-engineered flexor tendon grafts may allow reconstruction of severe tendon losses. One critical factor is the optimization of cell proliferation and reseeding. Use of growth factors--basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-1, and platelet-derived growth factor (PDGF)-BB--may improve culture conditions for human fibroblasts, tenocytes, and adipose-derived stem cells and increase repopulation of a tendon scaffold. All cell types were plated at a density of 10,000 cells per well and cultured in F12 media supplemented with varying concentrations of bFGF, IGF-1, and PDGF-BB. After 72 hours, cell proliferation was determined using the CellTiter assay. Human flexor tendon segments were acellularized and reseeded in a cell suspension of 5 × 10(5) cells/ml. After 5 days, tendon repopulation was determined using the MTS assay and histology. Statistical significance was determined with analysis of variance and a t test. For all cell types, there was enhanced proliferation with growth factors. Among single growth factors, PDGF-BB at 50 ng/ml was the most efficient stimulator of proliferation. With multiple growth factors, the optimal concentration was determined to be 5 ng/ml bFGF, 50 ng/ml IGF-1, and 50 ng/ml PDGF-BB (increase when compared with control: fibroblasts, 2.92-fold; tenocytes, 2.3-fold; and adipose-derived stem cells, 2.4-fold; p < 0.05). Tendons reseeded with this optimal combination of growth factors showed improved reseeding compared with the control group (fibroblasts, 2.01-fold; tenocytes, 1.78-fold; and adipose-derived stem cells, 1.76-fold; p < 0.05). bFGF, IGF-1, and PDGF-BB can be used to improve cellular proliferation and repopulation of an acellularized scaffold. The use of growth factors may be an important step in the tissue engineering of human flexor tendons.

  5. Image-guided tissue engineering

    PubMed Central

    Ballyns, Jeffrey J; Bonassar, Lawrence J

    2009-01-01

    Replication of anatomic shape is a significant challenge in developing implants for regenerative medicine. This has lead to significant interest in using medical imaging techniques such as magnetic resonance imaging and computed tomography to design tissue engineered constructs. Implementation of medical imaging and computer aided design in combination with technologies for rapid prototyping of living implants enables the generation of highly reproducible constructs with spatial resolution up to 25 μm. In this paper, we review the medical imaging modalities available and a paradigm for choosing a particular imaging technique. We also present fabrication techniques and methodologies for producing cellular engineered constructs. Finally, we comment on future challenges involved with image guided tissue engineering and efforts to generate engineered constructs ready for implantation. PMID:19583811

  6. Non-invasive Imaging and Tracking of Engineered Human Muscle Precursor Cells for Skeletal Muscle Tissue Engineering Using Positron Emission Tomography

    PubMed Central

    Haralampieva, Deana; Betzel, Thomas; Dinulovic, Ivana; Salemi, Souzan; Stoelting, Meline; Kraemer, Stefanie; Schibli, Roger; Sulser, Tullio; Handschin, Christoph; Eberli, Daniel; Ametamey, Simon M.

    2016-01-01

    Transplantation of human muscle precursor cells (hMPCs) is envisioned for the treatment of various muscle diseases. However, a feasible non-invasive tool to monitor cell survival, migration and integration into the host tissue is still missing. Methods In this study, we designed an adenoviral delivery system to genetically modify hMPCs to express a signaling-deficient form of a human dopamine D2 receptor (hD2R). The gene expression levels of the receptor were evaluated by Reverse Transcriptase Polymerase Chain Reaction (RTPCR) and infection efficiency was visualized by fluorescent microscopy. Viability, proliferation and differentiation capacity of the transduced cells were confirmed and their sustained myogenic phenotype was shown by flow cytometry analysis and fluorescent microscopy. 18F-Fallypride and 18F-FMISO, two well-established PET radioligands, were successfully synthesized and evaluated for their potential to image engineered hMPCs in a mouse model. Furthermore, biodistribution studies and autoradiography were also performed to determine the extent of signal specificity. Results To address the feasibility of the presented approach for tracking of hMPCs in an in vivo model, we first evaluated the safety of the adenoviral gene-delivery, which showed no detrimental effects on the primary human cells. Specific binding of 18F-Fallypride to hD2R_hMPCs was demonstrated in vitro, as well as in vivo, by performing autoradiography, biodistribution and PET experiments, respectively. Furthermore, 18F-FMISO uptake was evaluated at different time-points after cell inoculation in vivo, showing high signal only at the early stages. Finally, histological assessment of the harvested tissues confirmed the sustained survival of the transplanted cells at different time-points with formation of muscle tissue at the site of injection. Conclusion We here propose a signaling-deficient human D2R as a potent reporter for in vivo hMPCs PET tracking by 18F-Fallypride. This approach

  7. Bone tissue engineering by using a combination of polymer/Bioglass composites with human adipose-derived stem cells.

    PubMed

    Lu, Wei; Ji, Kun; Kirkham, Jennifer; Yan, Yu; Boccaccini, Aldo R; Kellett, Margaret; Jin, Yan; Yang, Xuebin B

    2014-04-01

    Translational research in bone tissue engineering is essential for "bench to bedside" patient benefit. However, the ideal combination of stem cells and biomaterial scaffolds for bone repair/regeneration is still unclear. The aim of this study is to investigate the osteogenic capacity of a combination of poly(DL-lactic acid) (PDLLA) porous foams containing 5 wt% and 40 wt% of Bioglass particles with human adipose-derived stem cells (ADSCs) in vitro and in vivo. Live/dead fluorescent markers, confocal microscopy and scanning electron microscopy showed that PDLLA/Bioglass porous scaffolds supported ADSC attachment, growth and osteogenic differentiation, as confirmed by enhanced alkaline phosphatase (ALP) activity. Higher Bioglass content of the PDLLA foams increased ALP activity compared with the PDLLA only group. Extracellular matrix deposition after 8 weeks in the in vitro cultures was evident by Alcian blue/Sirius red staining. In vivo bone formation was assessed by using scaffold/ADSC constructs in diffusion chambers transplanted intraperitoneally into nude mice and recovered after 8 weeks. Histological and immunohistochemical assays indicated significant new bone formation in the 40 wt% and 5 wt% Bioglass constructs compared with the PDLLA only group. Thus, the combination of a well-developed biodegradable bioactive porous PDLLA/Bioglass composite scaffold with a high-potential stem cell source (human ADSCs) could be a promising approach for bone regeneration in a clinical setting.

  8. Tissue engineering on matrix: future of autologous tissue replacement.

    PubMed

    Weber, Benedikt; Emmert, Maximilian Y; Schoenauer, Roman; Brokopp, Chad; Baumgartner, Laura; Hoerstrup, Simon P

    2011-05-01

    Tissue engineering aims at the creation of living neo-tissues identical or close to their native human counterparts. As basis of this approach, temporary biodegradable supporter matrices are fabricated in the shape of a desired construct, which promote tissue strength and provide functionality until sufficient neo-tissue is formed. Besides fully synthetic polymer-based scaffolds, decellularized biological tissue of xenogenic or homogenic origin can be used. In a second step, these scaffolds are seeded with autologous cells attaching to the scaffold microstructure. In order to promote neo-tissue formation and maturation, the seeded scaffolds are exposed to different forms of stimulation. In cardiovascular tissue engineering, this "conditioning" can be achieved via culture media and biomimetic in vitro exposure, e.g., using flow bioreactors. This aims at adequate cellular differentiation, proliferation, and extracellular matrix production to form a living tissue called the construct. These living autologous constructs, such as heart valves or vascular grafts, are created in vitro, comprising a viable interstitium with repair and remodeling capabilities already prior to implantation. In situ further in vivo remodeling is intended to recapitulate physiological vascular architecture and function. The remodeling mechanisms were shown to be dominated by monocytic infiltration and chemotactic host-cell attraction leading into a multifaceted inflammatory process and neo-tissue formation. Key molecules of these processes can be integrated into the scaffold matrix to direct cell and tissue fate in vivo.

  9. Silk fibroin scaffolds for urologic tissue engineering

    PubMed Central

    Sack, Bryan S.; Mauney, Joshua R.; Estrada, Carlos R.

    2016-01-01

    Urologic tissue engineering efforts have been largely focused on bladder and urethral defect repair. The current surgical gold standard for treatment of poorly compliant pathological bladders and severe urethral stricture disease is enterocystoplasty and onlay urethroplasty with autologous tissue, respectively. The complications associated with autologous tissue use and harvesting have led to efforts to develop tissue-engineered alternatives. Natural and synthetic materials have been used with varying degrees of success, but none has proved consistently reliable for urologic tissue defect repair in humans. Silk fibroin (SF) scaffolds have been tested in bladder and urethral repair because of their favorable biomechanical properties including structural strength, elasticity, biodegradability and biocompatibility. SF scaffolds have been used in multiple animal models, and have demonstrated robust regeneration of smooth muscle and urothelium. The pre-clinical data involving SF scaffolds in urologic defect repair are encouraging and suggest that they hold potential for future clinical use. PMID:26801192

  10. Cell sheet-based cardiac tissue engineering.

    PubMed

    Matsuura, Katsuhisa; Masuda, Shinako; Shimizu, Tatsuya

    2014-01-01

    Tissue engineering is indispensable for the advancement of regenerative medicine and the development of tissue models. Cell sheet-based method is one the promising strategies for cardiac tissue engineering. To date, cell sheet transplantation using wide variety of cells has been performed for the treatment of various heart diseases. These cell sheet transplantations have shown to ameliorate cardiac dysfunction and improve symptoms of heart failure. Recent progress of the technologies on the layering of cardiac cell sheets accompanied with vascularization and the large scale cultivation system of embryonic stem cell and induced pluripotent stem cell is about to turn the fabrication of thickened human cardiac tissue for transplant and tissue models into reality. Copyright © 2013 Wiley Periodicals, Inc.

  11. Vascularization Strategies for Tissue Engineering

    PubMed Central

    Lovett, Michael; Lee, Kyongbum; Edwards, Aurelie

    2009-01-01

    Tissue engineering is currently limited by the inability to adequately vascularize tissues in vitro or in vivo. Issues of nutrient perfusion and mass transport limitations, especially oxygen diffusion, restrict construct development to smaller than clinically relevant dimensions and limit the ability for in vivo integration. There is much interest in the field as researchers have undertaken a variety of approaches to vascularization, including material functionalization, scaffold design, microfabrication, bioreactor development, endothelial cell seeding, modular assembly, and in vivo systems. Efforts to model and measure oxygen diffusion and consumption within these engineered tissues have sought to quantitatively assess and improve these design strategies. This review assesses the current state of the field by outlining the prevailing approaches taken toward producing vascularized tissues and highlighting their strengths and weaknesses. PMID:19496677

  12. Incorporation of exudates of human platelet-rich fibrin gel in biodegradable fibrin scaffolds for tissue engineering of cartilage.

    PubMed

    Chien, Chi-Sheng; Ho, Hsiu-O; Liang, Yu-Chih; Ko, Pai-Hung; Sheu, Ming-Thau; Chen, Chien-Ho

    2012-05-01

    The goal of this study was to assess the incorporation of exudates of human platelet-rich fibrin (hPRF) that is abundant in platelet cytokines and growth factors into biodegradable fibrin (FB) scaffolds as a regeneration matrix for promoting chondrocyte proliferation and re-differentiation. hPRF was obtained from human blood by centrifugation without an anticoagulant, and the exudate of hPRF was collected and mixed with bovine fibrinogen, and then thrombin was added to form the FB scaffold. Proliferation and differentiation of human primary chondrocytes and a human chondrosarcoma cell line, the SW-1353, embedded in the three-dimensional (3D) scaffolds and on the two-dimensional (2D) surface of the FB scaffolds so produced were evaluated in comparison with an agarose (AG) scaffold serving as the control. Results demonstrated that the amounts of these cytokines and growth factors in hPRF exudates were higher than those in the blood-derived products except for TGF-β1. Chondrocytes and SW1353 cells on the 2D and 3D FB scaffolds with the addition of the exudates of PRF exhibited more-available proliferation and differentiation than cells on 2D and 3D FB and AG scaffolds. It was concluded that FB scaffolds can provide an appropriate environment for chondrocyte proliferation and re-differentiation, and it could be improved by adding exudates of hPRF. These 3D scaffolds have great promise for cartilage tissue engineering. Copyright © 2012 Wiley Periodicals, Inc.

  13. Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model.

    PubMed

    Green, Nicola H; Corfe, Bernard M; Bury, Jonathan P; MacNeil, Sheila

    2015-05-18

    The incidence of both esophageal adenocarcinoma and its precursor, Barrett's Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa. A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa. This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett's Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

  14. Engineering of human tracheal tissue with collagen-enforced poly-lactic-glycolic acid non-woven mesh: a preliminary study in nude mice.

    PubMed

    Wu, Wei; Feng, Xue; Mao, Tianqiu; Feng, Xinghua; Ouyang, Hong-Wei; Zhao, Guifang; Chen, Fulin

    2007-06-01

    The purpose of the current study is to fabricate tissue engineered trachea with poly-lactic-glycolic acid (PLGA) non-woven mesh enforced by collagen type I. PLGA fibres coated with collagen solution were put together and fabricated into the shape of a human trachea, after drying and cross-linking treatment, a non-woven mesh with "C" shape formed. Chondrocytes from sheep nasal septum cartilage were expanded in vitro and seeded into PLGA/collagen non-woven mesh in the density of 5.0 x 10(7)mL(-1). After 5 days of in vitro incubation, six Cell-PLGA/collagen composites were implanted subcutaneously into the back of 6 nude mice to prefabricate a tissue engineering trachea. Eight weeks later, the cartilage formation was observed by gross inspection and histological examination. Cartilage-like tissue in the shape of the initial PLGA/collagen scaffold had been regenerated successfully without obvious inflammatory response. The tissue engineered trachea cartilage consisted of evenly spaced lacunae embedded in matrix stained red with safranin-O staining. The amount of GAGs in tissue engineered trachea cartilage reached 71.42% of normal value in native cartilage. This study demonstrated that collagen-enforced PLGA non-woven mesh facilitated the adhesion and proliferation of chondrocytes, it also owned adequate mechanical strength to serve as an ideal scaffold for trachea tissue engineering without internal support.

  15. Cell communication and tissue engineering.

    PubMed

    Rossello, Ricardo A; H, David

    2010-01-01

    Gap junction intercellular communication (GJIC) is ubiquitous in the majority of cells and is indispensable for proper development and function of most tissues. The loss of gap junction mediated cell to cell communication leads to compromised development in many tissues and organs, and also facilitates tumorigenesis and autonomous cell behavior in cancerous cells. Because cells embedded in an extracellular matrix constantly interact through gap junctions to coordinate normal tissue functions and homeostasis, our group hypothesized that increasing cell to cell communication, via genetically engineering cells to overexpress gap junction proteins, could improve cell signaling and increase differentiation in interior regions of engineered tissue equivalents. In a recent paper,1 we presented a platform to regenerate full 3D equivalents of engineered tissue, providing a strategy to overcome a barrier in regenerative medicine. These findings suggest that both targeted delivery and cell-based strategies can be used as treatments to enhance communication in 3D living tissue.2 In this addendum, we address the effects of extracellular calcium (Ca(2+) (e)) on intracellular calcium (Ca(2+) (i)), GJIC and osteogenic differentiation under conditions in which bone marrow stromal cells (BMSCs) also exhibit higher cell-to-cell communication. As a key secondary messenger in many biological processes, the levels of Ca(2+) (e) and Ca(2+) (i) play a role in cell differentiation and may be a tunable signal in tissue regeneration. Higher cell-to-cell communication was achieved by both genetically engineering cells to overexpress connexin 43 (Cx43) and by a high density cell seeding technique, denoted micromass seeding (MM). The results presented in this addendum show that the intensity and duration of a second messenger, like calcium, can be augmented in a platform that enables higher cell-to-cell communication. The ability to modulate calcium signaling, combined with our previous

  16. Composite tissue engineering on polycaprolactone nanofiber scaffolds.

    PubMed

    Reed, Courtney R; Han, Li; Andrady, Anthony; Caballero, Montserrat; Jack, Megan C; Collins, James B; Saba, Salim C; Loboa, Elizabeth G; Cairns, Bruce A; van Aalst, John A

    2009-05-01

    Tissue engineering has largely focused on single tissue-type reconstruction (such as bone); however, the basic unit of healing in any clinically relevant scenario is a compound tissue type (such as bone, periosteum, and skin). Nanofibers are submicron fibrils that mimic the extracellular matrix, promoting cellular adhesion, proliferation, and migration. Stem cell manipulation on nanofiber scaffolds holds significant promise for future tissue engineering. This work represents our initial efforts to create the building blocks for composite tissue reflecting the basic unit of healing. Polycaprolactone (PCL) nanofibers were electrospun using standard techniques. Human foreskin fibroblasts, murine keratinocytes, and periosteal cells (4-mm punch biopsy) harvested from children undergoing palate repair were grown in appropriate media on PCL nanofibers. Human fat-derived mesenchymal stem cells were osteoinduced on PCL nanofibers. Cell growth was assessed with fluorescent viability staining; cocultured cells were differentiated using antibodies to fibroblast- and keratinocyte-specific surface markers. Osteoinduction was assessed with Alizarin red S. PCL nanofiber scaffolds supported robust growth of fibroblasts, keratinocytes, and periosteal cells. Cocultured periosteal cells (with fibroblasts) and keratinocytes showed improved longevity of the keratinocytes, though growth of these cell types was randomly distributed throughout the scaffold. Robust osteoinduction was noted on PCL nanofibers. Composite tissue engineering using PCL nanofiber scaffolds is possible, though the major obstacles to the trilaminar construct are maintaining an appropriate interface between the tissue types and neovascularization of the composite structure.

  17. Chitosan as a Modifying Component of Artificial Scaffold for Human Skin Tissue Engineering.

    PubMed

    Romanova, O A; Grigor'ev, T E; Goncharov, M E; Rudyak, S G; Solov'yova, E V; Krasheninnikov, S T; Saprykin, V P; Sytina, E V; Chvalun, S N; Pal'tsev, M A; Panteleev, A A

    2015-08-01

    We compared the structure and mechanical properties of scaffolds based on pure collagen, pure chitosan, and a mixture of these polymers. The role of the composition and structure of scaffolds in the maintenance of cell functions (proliferation, differentiation, and migration) was demonstrated in two experimental models: homogeneous tissue analogues (scaffold populated by fibroblasts) and complex skin equivalents (fibroblasts and keratinocytes). In contrast to collagen scaffolds, pure chitosan inhibited the growth of fibroblasts that did not form contacts with chitosan fibers, but formed specific cellular conglomerates, spheroids, and lose their ability to synthesize natural extracellular matrix. However, the use of chitosan as an additive stimulated proliferative activity of fibroblasts on collagen, which can be associated with improvement of mechanical properties of the collagen scaffolds. The effectiveness of chitosan as an additional cross-linking agent also manifested in its ability to improve significantly the resistance of collagen scaffolds to fibroblast contraction in comparison with glutaraldehyde treatment. Polymer scaffolds (without cells) accelerated complete healing of skin wounds in vivo irrespective of their composition healing, pure chitosan sponge being most effective. We concluded that the use of chitosan as the scaffold for skin equivalents populated with skin cells is impractical, whereas it can be an effective modifier of polymer scaffolds.

  18. Bioactive glass in tissue engineering

    PubMed Central

    Rahaman, Mohamed N.; Day, Delbert E.; Bal, B. Sonny; Fu, Qiang; Jung, Steven B.; Bonewald, Lynda F.; Tomsia, Antoni P.

    2011-01-01

    This review focuses on recent advances in the development and use of bioactive glass for tissue engineering applications. Despite its inherent brittleness, bioactive glass has several appealing characteristics as a scaffold material for bone tissue engineering. New bioactive glasses based on borate and borosilicate compositions have shown the ability to enhance new bone formation when compared to silicate bioactive glass. Borate-based bioactive glasses also have controllable degradation rates, so the degradation of the bioactive glass implant can be more closely matched to the rate of new bone formation. Bioactive glasses can be doped with trace quantities of elements such as Cu, Zn and Sr, which are known to be beneficial for healthy bone growth. In addition to the new bioactive glasses, recent advances in biomaterials processing have resulted in the creation of scaffold architectures with a range of mechanical properties suitable for the substitution of loaded as well as non-loaded bone. While bioactive glass has been extensively investigated for bone repair, there has been relatively little research on the application of bioactive glass to the repair of soft tissues. However, recent work has shown the ability of bioactive glass to promote angiogenesis, which is critical to numerous applications in tissue regeneration, such as neovascularization for bone regeneration and the healing of soft tissue wounds. Bioactive glass has also been shown to enhance neocartilage formation during in vitro culture of chondrocyte-seeded hydrogels, and to serve as a subchondral substrate for tissue-engineered osteochondral constructs. Methods used to manipulate the structure and performance of bioactive glass in these tissue engineering applications are analyzed. PMID:21421084

  19. Effects of gel concentration, human fibronectin, and cation supplement on the tissue-engineered cartilage.

    PubMed

    Kuo, Yung-Chih; Ku, I-Nan

    2007-01-01

    Cultivation of bovine knee chondrocytes (BKCs) in various cationic additives was studied using chitosan-gelatin scaffolds, whose surfaces were modified by human fibronectin (HFN). Here, the genipin-crosslinked scaffolds were fabricated by the freezing/lyophilization method with various concentrations of the precursory gels. The experimental results indicated that a lower freezing temperature led to higher moisture content, porosity, and specific surface area of a scaffold. The higher the precursor concentration, the larger the moisture content of a scaffold. A fast biodegradation of scaffold matrix was generated by a high porosity with BKCs. A higher concentration of HFN coated on scaffold surfaces yielded a faster rate of BKC attachment from the culture medium. The amounts of BKCs, glycosaminoglycans, and collagen over 28-day cultivation increased with the scaffold porosity, the coating concentration of HFN, the seeding density of BKCs, and the calcium concentration in medium.

  20. Polymer concepts in tissue engineering.

    PubMed

    Peter, S J; Miller, M J; Yasko, A W; Yaszemski, M J; Mikos, A G

    1998-01-01

    Traumatic injuries, cancer treatment, and congenital abnormalities are often associated with abnormal bone shape or segmental bone loss. Restoration of normal structure and function in these cases requires replacement of the missing bone that may be accomplished by surgical transfer of natural tissue from an uninjured location elsewhere in the body. However, this procedure is limited by availability, adequate blood supply, and secondary deformities at the donor site. One strategy to overcome these problems is to develop living tissue substitutes based on synthetic biodegradable polymers. Three methods of bone regeneration using biodegradable polymers are being studied in our laboratory: tissue induction, cell transplantation, and fabrication of vascularized bone flaps. Injectable polymers are used for filling skeletal defects and guiding bone tissue growth. Their main advantage is minimizing the surgical intervention or the severity of the surgery. Polymer-cell constructs also hold great promise in the field of tissue engineering. They provide a scaffold on which cells grow and organize themselves. As the cells begin to secrete their own extracellular matrix, the polymer degrades and is eventually eliminated from the body, resulting in completely natural tissue replacement. Bone flaps can be fabricated ectopically into precise shapes and sizes. With an attached vascular supply, these flaps can be transferred into areas deficient in vascularity. This article discusses polymer concepts regarding bone tissue engineering and reviews recent advances of our laboratory on guided bone regeneration using biodegradable polymer scaffolds.

  1. Engineered human vaccines

    SciTech Connect

    Sandhu, J.S. . Div. of Immunology and Neurobiology)

    1994-01-01

    The limitations of human vaccines in use at present and the design requirements for a new generation of human vaccines are discussed. The progress in engineering of human vaccines for bacteria, viruses, parasites, and cancer is reviewed, and the data from human studies with the engineered vaccines are discussed, especially for cancer and AIDS vaccines. The final section of the review deals with the possible future developments in the field of engineered human vaccines and the requirement for effective new human adjuvants.

  2. Keratin-chitosan membranes as scaffold for tissue engineering of human cornea.

    PubMed

    Vázquez, Natalia; Chacón, Manuel; Meana, Álvaro; Menéndez-Menéndez, Yolanda; Ferrero-Gutierrez, Amaia; Cereijo-Martín, David; Naveiras, Miguel; Merayo-Lloves, Jesús

    2015-07-01

    To study the attachment and growth of human corneal cells on keratin-chitosan membranes. The end goal is to develop a bioengineered cornea based on this material. Keratin-chitosan membranes were prepared as previously described by Tanabe et al., 2002. Briefly, 7.15 mg/cm2 of keratin dialysate was mixed with 10 wt% chitosan solution and 20 wt% glycerol. The solution was cast into a silicone mold and dried at 50ºC for 36 hours. Eyes were attained from a local eye bank after penetrant-keratoplastic surgery. Human epithelial, stromal and endothelial cells were obtained of the limbal, stromal and endothelial regions. Cells were cultured on keratin-chitosan membranes, as well as on plastic dishes as controls. When cultured cells reached confluence, they were fixed, incubated with primary antibodies (E-cadherin, cytokeratin high molecular weight (CK), vimentin and Na+/K+ ATPase) and visualized by indirect immunocytochemistry. Epithelial, stromal and endothelial cells were able to attach and grow on keratin-chitosan membranes. All the cells maintained their morphology and cellular markers, both in the membrane and on the culture plate. Epithelial cells stained positively for CK and E-cadherin. A positive vimentin stain was observed in all stromal cells, while endothelial cells were positive for vimentin and Na+/K+ ATPase, but negative for E-cadherin. Keratin-chitosan membranes have been shown to be a good scaffold for culturing epithelial, stromal and endothelial corneal cells; therefore, future applications of keratin-chitosan membranes may be developed for reconstruction of the cornea.

  3. Fetal tissue engineering: diaphragmatic replacement.

    PubMed

    Fauza, D O; Marler, J J; Koka, R; Forse, R A; Mayer, J E; Vacanti, J P

    2001-01-01

    Prosthetic repair of congenital diaphragmatic hernia has been associated with high complication rates. This study was aimed at applying fetal tissue engineering to diaphragmatic replacement. Fetal lambs underwent harvest of skeletal muscle specimens. Once expanded in vitro, fetal myoblasts were suspended in a collagen hydrogel submitted to controlled radial tension. The construct was then placed in a bioreactor. After birth, all animals underwent creation of 2 diaphragmatic defects. One defect was repaired with the autologous-engineered construct placed in between 2 acellular supporting membranes and the other with an identical construct but without any cells. Each animal was its own control (graft, n = 10). Animals were killed at different time-points postimplantation for histologic examination. Statistical analysis was by analysis of variance (ANOVA). Fetal myoblasts expanded up to twice as fast as neonatal cells. Hydrogel-based radial tension enhanced construct architecture by eliciting cell organization within the scaffold. No eventration was present in 4 of 5 engineered constructs but in 0 of 5 acellular grafts (P<.05). At harvest, engineered constructs were thick and histologically resembled normal skeletal muscle, whereas acellular grafts were thin, floppy, and showed low cell density with increased fibrosis. Unlike acellular grafts, engineered cellular diaphragmatic constructs are anatomically and histologically similar to normal muscle. Fetal tissue engineering may be a viable alternative for diaphragmatic replacement.

  4. Tendon Tissue Engineering: Mechanism and Effects of Human Tenocyte Coculture With Adipose-Derived Stem Cells.

    PubMed

    Long, Chao; Wang, Zhen; Legrand, Anais; Chattopadhyay, Arhana; Chang, James; Fox, Paige M

    2017-09-06

    Adipose-derived stem cells (ASCs) are a potential candidate for cell-based therapy targeting tendon injury; however, their therapeutic benefit relies on their ability to interact with native tenocytes. This study examines the mechanism and effects of coculturing human tenocytes and ASCs. Tenocytes (T) were directly cocultured with either ASCs (A) or fibroblasts (F) (negative control) in the following ratios: 50% T/50% A or F; 25% T/75% A or F; and 75% T/25% A or F. Cells were indirectly cocultured using a transwell insert that allowed for exchange of soluble factors only. Proliferation and collagen I production were measured and compared with monoculture controls. Synergy was quantified using the interaction index (II), which normalizes measured values by the expected values assuming no interaction (no synergy when II = 1). The ability of ASCs to elicit tenocyte migration was examined in vitro using a transwell migration assay and ex vivo using decellularized human flexor tendon explants. Compared with monoculture controls, II of proliferation was greater than 1 for all tenocyte and ASC direct coculture ratios, but not for tenocyte and fibroblast direct coculture ratios or for tenocyte and ASC indirect coculture. The ASCs elicited greater tenocyte migration in vitro and ex vivo. The II of collagen I production was greater than 1 for direct coculture groups with 25% T/75% A and 75% T/25% A. Direct coculture of ASCs and tenocytes demonstrated synergistic proliferation and collagen I production, and ASCs elicited tenocyte migration in vitro and ex vivo. These interactions play a key role in tendon healing and were absent when ASCs were replaced with fibroblasts, supporting the use of ASCs for cell-based therapy targeting tendon injuries. When ASCs are delivered for cell-based therapy, they directly interact with native tenocytes to increase cell proliferation, collagen I production, and tenocyte migration, which may enhance tendon healing. Copyright © 2017

  5. PHBV and predifferentiated human adipose-derived stem cells for cartilage tissue engineering.

    PubMed

    Liu, Jiong; Zhao, Bin; Zhang, Yunqiang; Lin, Yunfeng; Hu, Ping; Ye, Chuan

    2010-08-01

    This study was conducted to investigate whether in vitro chondrogenic differentiated human adipose-derived stem cells (hASCs) can maintain the chondrogenic phenotype in (3-hydroxybutrate-co-3-hydroxyvalerate) (PHBV) scaffolds and whether differentiated hASCs/PHBV construct can produce neocartilage in a heterotopic animal model. hASCs were cultured with or without chondrogenic media in vitro and then seeded on PHBV foams. Differentiated cell/PHBV constructs were subcutaneously implanted in nude mice for 8 or 16 weeks; nondifferentiated cell/PHBV constructs were implanted in the control group. The results in the control group showed no cartilage formation and the disappearance of the scaffold at 8 weeks. Conversely, all differentiated hASCs/PHBV implants kept their original shape throughout 16 weeks. These implants at 16 weeks had stronger chondrocytes-specific histochemical staining than those at 8 weeks, with GAG, total collagen, and compressive moduli increased with implantation time. Cartilage lacunae were observed in all retrieved implants at 16 weeks. The chondrocytes-specific genes were detected by RT-PCR at 16 weeks. The remnants of PHBV were observed in the implants throughout 16 weeks. This study demonstrates that chondrogenic predifferentiated hASCs have the ability to maintain a chondrogenic phenotype in PHBV and that cell/PHBV constructs can produce neocartilage in a heterotopic site, but the degradation rates of PHBV in different environments needs more investigation.

  6. Insulin-like growth factor I enhances collagen synthesis in engineered human tendon tissue.

    PubMed

    Herchenhan, Andreas; Bayer, Monika L; Eliasson, Pernilla; Magnusson, S Peter; Kjaer, Michael

    2015-02-01

    Isolated human tendon cells form 3D tendon constructs that demonstrate collagen fibrillogenesis and feature structural similarities to tendon when cultured under tensile load. The exact role of circulating growth factors for collagen formation in tendon is sparsely examined. We investigated the influence of insulin-like growth factor I (IGF-I) on tendon construct formation in 3D cell culture. Tendon constructs were grown in 0.5 or 10% FBS with or without IGF-I (250 mg/ml) supplementation. Collagen content (fluorometric), mRNA levels (PCR) and fibril diameter (transmission electron microscopy) were determined at 7, 10, 14, 21 and 28 days. IGF-I revealed a stimulating effect on fibril diameter (up to day 21), mRNA for collagen (to day 28), tenomodulin (to day 28) and scleraxis (at days 10 and 14), and on overall collagen content. 10% FBS diminished the development of fibril diameter (day 14), collagen content (at days 21 and 28) and mRNA expression for collagen, tenomodulin and scleraxis. IGF-I supplementation promotes early onset of tensile load induced collagen formation and tendon structural arrangement, whereas the FBS concentration routinely used in cultures diminishes collagen expression, collagen content and fibril formation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Analysis of blood and lymph vascularization patterns in tissue-engineered human dermo-epidermal skin analogs of different pigmentation.

    PubMed

    Klar, Agnieszka S; Böttcher-Haberzeth, Sophie; Biedermann, Thomas; Schiestl, Clemens; Reichmann, Ernst; Meuli, Martin

    2014-02-01

    Bioengineered dermo-epidermal skin analogs containing melanocytes represent a promising approach to cover large skin defects including restoration of the patient's own skin color. So far, little is known about the development of blood and lymphatic vessels in pigmented skin analogs after transplantation. In this experimental study, we analyzed the advancement and differences of host blood and lymphatic vessel ingrowth into light- and dark-pigmented human tissue-engineered skin analogs in a rat model. Keratinocytes, melanocytes, and fibroblasts from light- and dark-pigmented skin biopsies were isolated, cultured, and expanded. For each donor, melanocytes and keratinocytes were seeded in ratios of 1:1, 1:5, and 1:10 onto fibroblast-containing collagen gels. The skin analogs were subsequently transplanted onto full-thickness wounds of immuno-incompetent rats and quantitatively analyzed for vascular and lymphatic vessel density after 8 and 15 weeks. The skin analogs revealed a significant difference in vascularization patterns between light- and dark-pigmented constructs after 8 weeks, with a higher amount of blood vessels in light compared to dark skin. In contrast, no obvious difference could be detected within the light- and dark-pigmented group when varying melanocyte/keratinocyte ratios were used. However, after 15 weeks, the aforementioned difference in blood vessel density between light and dark constructs could no longer be detected. Regarding lymphatic vessels, light and dark analogs showed similar vessel density after 8 and 15 weeks, while there were generally less lymphatic than blood vessels. These data suggest that, at least during early skin maturation, keratinocytes, melanocytes, and fibroblasts from different skin color types used to construct pigmented dermo-epidermal skin analogs have distinct influences on the host tissue after transplantation. We speculate that different VEGF expression patterns might be involved in this disparate revascularization

  8. Polyphosphazene functionalized polyester fiber matrices for tendon tissue engineering: in vitro evaluation with human mesenchymal stem cells.

    PubMed

    Peach, M Sean; James, Roshan; Toti, Udaya S; Deng, Meng; Morozowich, Nicole L; Allcock, Harry R; Laurencin, Cato T; Kumbar, Sangamesh G

    2012-08-01

    Poly[(ethyl alanato)(1)(p-methyl phenoxy)(1)] phosphazene (PNEA-mPh) was used to modify the surface of electrospun poly(ε-caprolactone) (PCL) nanofiber matrices having an average fiber diameter of 3000 ± 1700 nm for the purpose of tendon tissue engineering and augmentation. This study reports the effect of polyphosphazene surface functionalization on human mesenchymal stem cell (hMSC) adhesion, cell-construct infiltration, proliferation and tendon differentiation, as well as long term cellular construct mechanical properties. PCL fiber matrices functionalized with PNEA-mPh acquired a rougher surface morphology and led to enhanced cell adhesion as well as superior cell-construct infiltration when compared to smooth PCL fiber matrices. Long-term in vitro hMSC cultures on both fiber matrices were able to produce clinically relevant moduli. Both fibrous constructs expressed scleraxis, an early tendon differentiation marker, and a bimodal peak in expression of the late tendon differentiation marker tenomodulin, a pattern that was not observed in PCL thin film controls. Functionalized matrices achieved a more prominent tenogenic differentiation, possessing greater tenomodulin expression and superior phenotypic maturity according to the ratio of collagen I to collagen III expression. These findings indicate that PNEA-mPh functionalization is an efficient method for improving cell interactions with electrospun PCL matrices for the purpose of tendon repair.

  9. Impact of Cell Composition and Geometry on Human Induced Pluripotent Stem Cells-Derived Engineered Cardiac Tissue

    PubMed Central

    Nakane, Takeichiro; Masumoto, Hidetoshi; Tinney, Joseph P.; Yuan, Fangping; Kowalski, William J.; Ye, Fei; LeBlanc, Amanda J.; Sakata, Ryuzo; Yamashita, Jun K.; Keller, Bradley B.

    2017-01-01

    The current study describes a scalable, porous large-format engineered cardiac tissue (LF-ECT) composed of human induced pluripotent stem cells (hiPSCs) derived multiple lineage cardiac cells with varied 3D geometries and cell densities developed towards the goal of scale-up for large animal pre-clinical studies. We explored multiple 15 × 15 mm ECT geometries using molds with rectangular internal staggered posts (mesh, ME), without posts (plain sheet, PS), or long parallel posts (multiple linear bundles, ML) and a gel matrix containing hiPSC-derived cardiomyocytes, endothelial, and vascular mural cells matured in vitro for 14 days. ME-ECTs displayed the lowest dead cell ratio (p < 0.001) and matured into 0.5 mm diameter myofiber bundles with greater 3D cell alignment and higher active stress than PS-ECTs. Increased initial ECT cell number beyond 6 M per construct resulted in reduced cell survival and lower active stress. The 6M-ME-ECTs implanted onto 1 week post-infarct immune tolerant rat hearts engrafted, displayed evidence for host vascular coupling, and recovered myocardial structure and function with reduced scar area. We generated a larger (30 × 30 mm) ME-ECT to confirm scalability. Thus, large-format ECTs generated from hiPSC-derived cardiac cells may be feasible for large animal preclinical cardiac regeneration paradigms. PMID:28368043

  10. Bioreactor Technology in Cardiovascular Tissue Engineering

    NASA Astrophysics Data System (ADS)

    Mertsching, H.; Hansmann, J.

    Cardiovascular tissue engineering is a fast evolving field of biomedical science and technology to manufacture viable blood vessels, heart valves, myocar-dial substitutes and vascularised complex tissues. In consideration of the specific role of the haemodynamics of human circulation, bioreactors are a fundamental of this field. The development of perfusion bioreactor technology is a consequence of successes in extracorporeal circulation techniques, to provide an in vitro environment mimicking in vivo conditions. The bioreactor system should enable an automatic hydrodynamic regime control. Furthermore, the systematic studies regarding the cellular responses to various mechanical and biochemical cues guarantee the viability, bio-monitoring, testing, storage and transportation of the growing tissue.

  11. Prevascularization of self-organizing engineered heart tissue by human umbilical vein endothelial cells abrogates contractile performance.

    PubMed

    Sondergaard, Claus Svane; Witt, Russell; Mathews, Grant; Najibi, Skender; Le, Lisa; Clift, Tracy; Si, Ming-Sing

    2012-12-01

    Establishing vascularization is a critical obstacle to the generation of engineered heart tissue (EHT) of substantial thickness. Addition of endothelial cells to the formative stages of EHT has been demonstrated to result in prevascularization, or the formation of capillary-like structures. The detailed study of the effects of prevascularization on EHT contractile function is lacking. Here, we evaluated the functional impact of prevascularization by human umbilical vein endothelial cells (HUVECs) in self-organizing EHT. EHT fibers were generated by the self-organization of neonatal rat cardiac cells on a fibrin hydrogel scaffold with or without HUVECs. Contractile function was measured and force-length relationship and rate of force production were assessed. Immunofluorescent studies were used to evaluate arrangement and distribution of HUVECs within the EHT fibers. RT-PCR was used to assess the transcript levels of hypoxia inducible factor-1a (Hif-1α). EHT with HUVECs manifested tubule-like structures at the periphery during fiber formation. After fiber formation, HUVECs were heterogeneously located throughout the EHT fiber and human CD31+ tubule-like structures were identified. The expression level of Hif-1α did not change with the addition of HUVECs. However, maximal force and rate of force generation were not improved in HUVECs containing EHT as compared to control EHT fibers. The addition of HUVECs may result in sparse microvascularization of EHT. However, this perceived benefit is overshadowed by a significant decrease in contractile function and highlights the need for perfused vascularization strategies in order to generate EHT that approaches clinically relevant dimensions.

  12. Enhanced Differentiation of Human Embryonic Stem Cells on Extracellular Matrix-Containing Osteomimetic Scaffolds for Bone Tissue Engineering

    PubMed Central

    Rutledge, Katy; Cheng, Qingsu; Pryzhkova, Marina; Harris, Greg M.

    2014-01-01

    Current methods of treating critical size bone defects include autografts and allografts, however, both present major limitations including donor-site morbidity, risk of disease transmission, and immune rejection. Tissue engineering provides a promising alternative to circumvent these shortcomings through the use of autologous cells, three-dimensional scaffolds, and growth factors. We investigated the development of a scaffold with native bone extracellular matrix (ECM) components for directing the osteogenic differentiation of human embryonic stem cells (hESCs). Toward this goal, a microsphere-sintering technique was used to fabricate poly(lactic-co-glycolic acid) (PLGA) scaffolds with optimum mechanical and structural properties. Human osteoblasts (hOBs) were seeded on these scaffolds to deposit bone ECM for 14 days. This was followed by a decellularization step leaving the mineralized matrix intact. Characterization of the decellularized PLGA scaffolds confirmed the deposition of calcium, collagen II, and alkaline phosphatase by osteoblasts. hESCs were seeded on the osteomimetic substrates in the presence of osteogenic growth medium, and osteogenicity was determined according to calcium content, osteocalcin expression, and bone marker gene regulation. Cell proliferation studies showed a constant increase in number for hESCs seeded on both PLGA and ECM-coated PLGA scaffolds. Calcium deposition by hESCs was significantly higher on the osteomimetic scaffolds compared with the control groups. Consistently, immunofluorescence staining demonstrated an increased expression of osteocalcin in hESCs seeded on ECM-coated osteomimetic PLGA scaffolds. Gene expression analysis of RUNX2 and osteocalcin further confirmed osteogenic differentiation of hESCs at the highest expression level on osteomimetic PLGA. These results together demonstrate the potential of PLGA scaffolds with native bone ECM components to direct osteogenic differentiation of hESCs and induce bone formation

  13. Force generation of different human cardiac valve interstitial cells: relevance to individual valve function and tissue engineering.

    PubMed

    Smith, Sally; Taylor, Patricia M; Chester, Adrian H; Allen, Sean P; Dreger, Sally A; Eastwood, Mark; Yacoub, Magdi H

    2007-07-01

    Cardiac valves perform highly sophisticated functions that depend upon the specific characteristics of the component interstitial cells (ICs). The ability of valve ICs to contribute to these functions may be related to the generation of different types of tension within the valve structure. The study aim was to characterize cellular morphology and the forces generated by valve ICs and to compare this with morphology and forces generated by other cell types. Cultured human valve ICs, pericardial fibroblasts and vascular smooth muscle cells were seeded in 3-D collagen gels and placed in a device that accurately measures the forces generated. Cell morphology was determined in seeded gels fixed in glutaraldehyde, stained with toluidine blue and visualized using a high-definition stereo light microscope. Valve ICs generated an average peak force of 30.9 +/- 10.4 dynes over a 24-h period which, unlike other cell types tested, increased as cell density decreased (R = 0.67, p <0.0001). The temporal pattern of force generation in mitral valve cells was significantly faster than in aortic or tricuspid cells (p <0.05). Microscopic examination revealed the formation of cellular processes establishing a cell/cell and cell/matrix network. When externally induced changes in matrix tension occurred, the valve ICs unlike the other cell types - did not respond to restore the previous level of tension. Human cardiac valve ICs produce a specific pattern of force generation that may be related to the individual function of each heart valve. The specialized function of these cells may serve as a guide for the choice of candidate cells for tissue engineering heart valves.

  14. Enhanced differentiation of human embryonic stem cells on extracellular matrix-containing osteomimetic scaffolds for bone tissue engineering.

    PubMed

    Rutledge, Katy; Cheng, Qingsu; Pryzhkova, Marina; Harris, Greg M; Jabbarzadeh, Ehsan

    2014-11-01

    Current methods of treating critical size bone defects include autografts and allografts, however, both present major limitations including donor-site morbidity, risk of disease transmission, and immune rejection. Tissue engineering provides a promising alternative to circumvent these shortcomings through the use of autologous cells, three-dimensional scaffolds, and growth factors. We investigated the development of a scaffold with native bone extracellular matrix (ECM) components for directing the osteogenic differentiation of human embryonic stem cells (hESCs). Toward this goal, a microsphere-sintering technique was used to fabricate poly(lactic-co-glycolic acid) (PLGA) scaffolds with optimum mechanical and structural properties. Human osteoblasts (hOBs) were seeded on these scaffolds to deposit bone ECM for 14 days. This was followed by a decellularization step leaving the mineralized matrix intact. Characterization of the decellularized PLGA scaffolds confirmed the deposition of calcium, collagen II, and alkaline phosphatase by osteoblasts. hESCs were seeded on the osteomimetic substrates in the presence of osteogenic growth medium, and osteogenicity was determined according to calcium content, osteocalcin expression, and bone marker gene regulation. Cell proliferation studies showed a constant increase in number for hESCs seeded on both PLGA and ECM-coated PLGA scaffolds. Calcium deposition by hESCs was significantly higher on the osteomimetic scaffolds compared with the control groups. Consistently, immunofluorescence staining demonstrated an increased expression of osteocalcin in hESCs seeded on ECM-coated osteomimetic PLGA scaffolds. Gene expression analysis of RUNX2 and osteocalcin further confirmed osteogenic differentiation of hESCs at the highest expression level on osteomimetic PLGA. These results together demonstrate the potential of PLGA scaffolds with native bone ECM components to direct osteogenic differentiation of hESCs and induce bone formation.

  15. Use of Clotted Human Plasma and Aprotinin in Skin Tissue Engineering: A Novel Approach to Engineering Composite Skin on a Porous Scaffold.

    PubMed

    Paul, Michelle; Kaur, Pritinder; Herson, Marisa; Cheshire, Perdita; Cleland, Heather; Akbarzadeh, Shiva

    2015-10-01

    Tissue-engineered composite skin is a promising therapy for the treatment of chronic and acute wounds, including burns. Providing the wound bed with a dermal scaffold populated by autologous dermal and epidermal cellular components can further entice host cell infiltration and vascularization to achieve permanent wound closure in a single stage. However, the high porosity and the lack of a supportive basement membrane in most commercially available dermal scaffolds hinders organized keratinocyte proliferation and stratification in vitro and may delay re-epithelization in vivo. The objective of this study was to develop a method to enable the in vitro production of a human skin equivalent (HSE) that included a porous scaffold and dermal and epidermal cells expanded ex vivo, with the potential to be used for definitive treatment of skin defects in a single procedure. A collagen-glycosaminoglycan dermal scaffold (Integra(®)) was populated with adult fibroblasts. A near-normal skin architecture was achieved by the addition of coagulated human plasma to the fibroblast-populated scaffold before seeding cultured keratinocytes. This resulted in reducing scaffold pore size and improving contact surfaces. Skin architecture and basement membrane formation was further improved by the addition of aprotinin (a serine protease inhibitor) to the culture media to inhibit premature clot digestion. Histological assessment of the novel HSE revealed expression of keratin 14 and keratin 10 similar to native skin, with a multilayered neoepidermis morphologically comparable to human skin. Furthermore, deposition of collagen IV and laminin-511 were detected by immunofluorescence, indicating the formation of a continuous basement membrane at the dermal-epidermal junction. The proposed method was efficient in producing an in vitro near native HSE using the chosen off-the-shelf porous scaffold (Integra). The same principles and promising outcomes should be applicable to other biodegradable

  16. Tissue engineering in urothelium regeneration.

    PubMed

    Vaegler, Martin; Maurer, Sabine; Toomey, Patricia; Amend, Bastian; Sievert, Karl-Dietrich

    2015-03-01

    The development of therapeutic treatments to regenerate urothelium, manufacture tissue equivalents or neourethras for in-vivo application is a significant challenge in the field of tissue engineering. Many studies have focused on urethral defects that, in most cases, inadequately address current therapies. This article reviews the primary tissue engineering strategies aimed at the clinical requirements for urothelium regeneration while concentrating on promising investigations in the use of grafts, cellular preparations, as well as seeded or unseeded natural and synthetic materials. Despite significant progress being made in the development of scaffolds and matrices, buccal mucosa transplants have not been replaced. Recently, graft tissues appear to have an advantage over the use of matrices. These therapies depend on cell isolation and propagation in vitro that require, not only substantial laboratory resources, but also subsequent surgical implant procedures. The choice of the correct cell source is crucial when determining an in-vivo application because of the risks of tissue changes and abnormalities that may result in donor site morbidity. Addressing an appropriately-designed animal model and relevant regulatory issues is of fundamental importance for the principal investigators when a therapy using cellular components has been developed for clinical use.

  17. [DEVELOPMENT OF CELL SHEET ENGINEERING TECHNOLOGY IN ENGINEERING VASCULARIZED TISSUE].

    PubMed

    Chen, Jia; Ma, Dongyang; Ren, Liling

    2015-03-01

    To review the development of cell sheet engineering technology in engineering vascularized tissue. The literature about cell sheet engineering technology and engineering vascularized tissue was reviewed, analyzed, and summarized. Although there are many methods to engineer vascularized tissue, cell sheet engineering technology provides a promising potential to develop a vascularized tissue. Recently, cell sheet engineering technology has become a hot topic in engineering vascularized tissue. Co-culturing endothelial cells on a cell sheet, endothelial cells are able to form three-dimensional prevascularized networks and microvascular cavities in the cell sheet, which facilitate the formation of functional vascular networks in the transplanted tissue. Cell sheet engineering technology is a promising strategy to engineer vascularized tissue, which is still being studied to explore more potential.

  18. Developmental biology and tissue engineering.

    PubMed

    Marga, Francoise; Neagu, Adrian; Kosztin, Ioan; Forgacs, Gabor

    2007-12-01

    Morphogenesis implies the controlled spatial organization of cells that gives rise to tissues and organs in early embryonic development. While morphogenesis is under strict genetic control, the formation of specialized biological structures of specific shape hinges on physical processes. Tissue engineering (TE) aims at reproducing morphogenesis in the laboratory, i.e., in vitro, to fabricate replacement organs for regenerative medicine. The classical approach to generate tissues/organs is by seeding and expanding cells in appropriately shaped biocompatible scaffolds, in the hope that the maturation process will result in the desired structure. To accomplish this goal more naturally and efficiently, we set up and implemented a novel TE method that is based on principles of developmental biology and employs bioprinting, the automated delivery of cellular composites into a three-dimensional (3D) biocompatible environment. The novel technology relies on the concept of tissue liquidity according to which multicellular aggregates composed of adhesive and motile cells behave in analogy with liquids: in particular, they fuse. We emphasize the major role played by tissue fusion in the embryo and explain how the parameters (surface tension, viscosity) that govern tissue fusion can be used both experimentally and theoretically to control and simulate the self-assembly of cellular spheroids into 3D living structures. The experimentally observed postprinting shape evolution of tube- and sheet-like constructs is presented. Computer simulations, based on a liquid model, support the idea that tissue liquidity may provide a mechanism for in vitro organ building.

  19. The Use of Total Human Bone Marrow Fraction in a Direct Three-Dimensional Expansion Approach for Bone Tissue Engineering Applications: Focus on Angiogenesis and Osteogenesis

    PubMed Central

    Oliveira, Hugo; Catros, Sylvain; Siadous, Robin; Derkaoui, Sidi-Mohammed; Bareille, Reine; Letourneur, Didier; Amédée, Joëlle

    2015-01-01

    Current approaches in bone tissue engineering have shown limited success, mostly owing to insufficient vascularization of the construct. A common approach consists of co-culture of endothelial cells and osteoblastic cells. This strategy uses cells from different sources and differentiation states, thus increasing the complexity upstream of a clinical application. The source of reparative cells is paramount for the success of bone tissue engineering applications. In this context, stem cells obtained from human bone marrow hold much promise. Here, we analyzed the potential of human whole bone marrow cells directly expanded in a three-dimensional (3D) polymer matrix and focused on the further characterization of this heterogeneous population and on their ability to promote angiogenesis and osteogenesis, both in vitro and in vivo, in a subcutaneous model. Cellular aggregates were formed within 24 h and over the 12-day culture period expressed endothelial and bone-specific markers and a specific junctional protein. Ectopic implantation of the tissue-engineered constructs revealed osteoid tissue and vessel formation both at the periphery and within the implant. This work sheds light on the potential clinical use of human whole bone marrow for bone regeneration strategies, focusing on a simplified approach to develop a direct 3D culture without two-dimensional isolation or expansion. PMID:25333855

  20. Biomaterials for liver tissue engineering.

    PubMed

    Jain, Era; Damania, Apeksha; Kumar, Ashok

    2014-04-01

    Liver extracellular matrix (ECM) composition, topography and biomechanical properties influence cell-matrix interactions. The ECM presents guiding cues for hepatocyte phenotype maintenance, differentiation and proliferation both in vitro and in vivo. Current understanding of such cell-guiding cues along with advancement of techniques for scaffold fabrication has led to evolution of matrices for liver tissue culture from simple porous scaffolds to more complex 3D matrices with microarchitecture similar to in vivo. Natural and synthetic polymeric biomaterials fabricated in different topographies and porous matrices have been used for hepatocyte culture. Heterotypic and homotypic cell interactions are necessary for developing an adult liver as well as an artificial liver. A high oxygen demand of hepatocytes as well as graded oxygen distribution in liver is another challenging attribute of the normal liver architecture that further adds to the complexity of engineered substrate design. A balanced interplay of cell-matrix interactions along with cell-cell interactions and adequate supply of oxygen and nutrient determines the success of an engineered substrate for liver cells. Techniques devised to incorporate these features of hepatic function and mimic liver architecture range from maintaining liver cells in mm-sized tailor-made scaffolds to a more bottoms up approach that starts from building the microscopic subunit of the whole tissue. In this review, we discuss briefly various biomaterials used for liver tissue engineering with respect to design parameters such as scaffold composition and chemistry, biomechanical properties, topography, cell-cell interactions and oxygenation.

  1. Formation of tissue engineered composite construct of cartilage and skin using high density polyethylene as inner scaffold in the shape of human helix.

    PubMed

    Ruszymah, B H I; Chua, K H; Mazlyzam, A L; Aminuddin, B S

    2011-06-01

    Formation of external ear via tissue engineering has created interest amongst surgeons as an alternative for ear reconstruction in congenital microtia. To reconstruct a composite human construct of cartilage and skin in the shape of human ear helix in athymic mice. Six human nasal cartilages were used and digested with Collagenase II. Chondrocytes were passaged in 175 cm(2) culture flasks at a density of 10,000 cells/cm(2). Frozen human plasma was then mixed with human chondrocytes. Six human skin samples were cut into small pieces trypsinized and resuspended. The keratinocytes were plated in six-well plate culture dishes at a density of 2×105 cells per well. Dermis tissues were digested and the fibroblast cells resuspended in six-well plate at the density of 10,000 cells per well. Fibrin-fibroblast layer and fibrin-keratinocytes were formed by mixing with human plasma to create 6 bilayered human skin equivalent (BSE) constructs. The admixture of fibrin chondrocytes layers was wrapped around high density polyethylene (HDP), and implanted at the dorsum of the athymic mice. The construct was left for 4 weeks and after maturation the mice skin above the implanted construct was removed and replaced by BSE for another 4 weeks. Haematoxylin and Eosin showed that the construct consists of fine arrangement and organized tissue structure starting with HDP followed by cartilage, dermis and epidermis. Safranin-O staining was positive for proteoglycan matrix production. Monoclonal mouse antihuman cytokeratin, 34βE12 staining displayed positive result for human keratin protein. The study has shown the possibility to reconstruct ear helix with HDP and tissue engineered human cartilage and skin. This is another step to form a human ear and hopefully will be an alternative in reconstructive ear surgery. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  2. Biomaterials in myocardial tissue engineering

    PubMed Central

    Reis, Lewis A.; Chiu, Loraine L. Y.; Feric, Nicole; Fu, Lara; Radisic, Milica

    2016-01-01

    Cardiovascular disease is the leading cause of death in the developed world, and as such there is a pressing need for treatment options. Cardiac tissue engineering emerged from the need to develop alternate sources and methods of replacing tissue damaged by cardiovascular diseases, as the ultimate treatment option for many who suffer from end-stage heart failure is a heart transplant. In this review we focus on biomaterial approaches to augment injured or impaired myocardium with specific emphasis on: the design criteria for these biomaterials; the types of scaffolds—composed of natural or synthetic biomaterials, or decellularized extracellular matrix—that have been used to develop cardiac patches and tissue models; methods to vascularize scaffolds and engineered tissue, and finally injectable biomaterials (hydrogels)designed for endogenous repair, exogenous repair or as bulking agents to maintain ventricular geometry post-infarct. The challenges facing the field and obstacles that must be overcome to develop truly clinically viable cardiac therapies are also discussed. PMID:25066525

  3. Biomaterials in myocardial tissue engineering.

    PubMed

    Reis, Lewis A; Chiu, Loraine L Y; Feric, Nicole; Fu, Lara; Radisic, Milica

    2016-01-01

    Cardiovascular disease is the leading cause of death in the developed world, and as such there is a pressing need for treatment options. Cardiac tissue engineering emerged from the need to develop alternative sources and methods of replacing tissue damaged by cardiovascular diseases, as the ultimate treatment option for many who suffer from end-stage heart failure is a heart transplant. In this review we focus on biomaterial approaches to augmenting injured or impaired myocardium, with specific emphasis on: the design criteria for these biomaterials; the types of scaffolds - composed of natural or synthetic biomaterials or decellularized extracellular matrix - that have been used to develop cardiac patches and tissue models; methods to vascularize scaffolds and engineered tissue; and finally, injectable biomaterials (hydrogels) designed for endogenous repair, exogenous repair or as bulking agents to maintain ventricular geometry post-infarct. The challenges facing the field and obstacles that must be overcome to develop truly clinically viable cardiac therapies are also discussed. Copyright © 2014 John Wiley & Sons, Ltd.

  4. Attenuation of skeletal muscle wasting with recombinant human growth hormone secreted from a tissue-engineered bioartificial muscle

    NASA Technical Reports Server (NTRS)

    Vandenburgh, H.; Del Tatto, M.; Shansky, J.; Goldstein, L.; Russell, K.; Genes, N.; Chromiak, J.; Yamada, S.

    1998-01-01

    Skeletal muscle wasting is a significant problem in elderly and debilitated patients. Growth hormone (GH) is an anabolic growth factor for skeletal muscle but is difficult to deliver in a therapeutic manner by injection owing to its in vivo instability. A novel method is presented for the sustained secretion of recombinant human GH (rhGH) from genetically modified skeletal muscle implants, which reduces host muscle wasting. Proliferating murine C2C12 skeletal myoblasts stably transduced with the rhGH gene were tissue engineered in vitro into bioartificial muscles (C2-BAMs) containing organized postmitotic myofibers secreting 3-5 microg of rhGH/day in vitro. When implanted subcutaneously into syngeneic mice, C2-BAMs delivered a sustained physiologic dose of 2.5 to 11.3 ng of rhGH per milliliter of serum. rhGH synthesized and secreted by the myofibers was in the 22-kDa monomeric form and was biologically active, based on downregulation of a GH-sensitive protein synthesized in the liver. Skeletal muscle disuse atrophy was induced in mice by hindlimb unloading, causing the fast plantaris and slow soleus muscles to atrophy by 21 to 35% ( < 0.02). This atrophy was significantly attenuated 41 to 55% (p < 0.02) in animals that received C2-BAM implants, but not in animals receiving daily injections of purified rhGH (1 mg/kg/day). These data support the concept that delivery of rhGH from BAMs may be efficacious in treating muscle-wasting disorders.

  5. Collagen-coated nano-electrospun PCL seeded with human endometrial stem cells for skin tissue engineering applications.

    PubMed

    Sharif, Shiva; Ai, Jafar; Azami, Mahmoud; Verdi, Javad; Atlasi, Mohammad Ali; Shirian, Sadegh; Samadikuchaksaraei, Ali

    2017-08-09

    Human endometrial stem cells (hEnSCs) are known as an attractive source of stem cells for regenerative medicine. hEnSCs are easily isolated and are capable of repairing uterine through their strong ability of creating new capillaries. In this study, a three-dimensional (3D) nanofibrous polycaprolactone (PCL)/collagen scaffold was fabricated and characterized in order to be applied as a new approach for skin reconstruction. Furthermore, the behavior of hEnSCs on this scaffold was investigated. First, a PCL 3D scaffold was constructed using electrospinning technique. Plasma treated and PCL was grafted by collagen. The constructs were characterized for mechanical and structural properties. Cell attachment, proliferation, viability, and differentiation of hEnSCs were assessed after being seeded on PCL and PCL/collagen scaffolds using scanning electron microscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and real-time polymerase chain reaction tests. The results showed higher wettability for the PCL/collagen scaffold with desirable mechanical and structural characteristics compared to PCL and collagen alone. The attachment and proliferation rates of hEnSCs on the PCL/collagen scaffold were higher compared to those on the bare PCL. Hence, hEnSCs are newly discovered stem cell source for skin tissue engineering in vitro, particularly when developed on PCL/collagen nanofiber scaffolds. Therefore, application of hEnSCs for skin regeneration is a novel therapeutic approach for temporary skin substitute. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017. © 2017 Wiley Periodicals, Inc.

  6. Human Adipose Stem Cells Differentiated on Braided Polylactide Scaffolds Is a Potential Approach for Tendon Tissue Engineering.

    PubMed

    Vuornos, Kaisa; Björninen, Miina; Talvitie, Elina; Paakinaho, Kaarlo; Kellomäki, Minna; Huhtala, Heini; Miettinen, Susanna; Seppänen-Kaijansinkko, Riitta; Haimi, Suvi

    2016-03-01

    Growing number of musculoskeletal defects increases the demand for engineered tendon. Our aim was to find an efficient strategy to produce tendon-like matrix in vitro. To allow efficient differentiation of human adipose stem cells (hASCs) toward tendon tissue, we tested different medium compositions, biomaterials, and scaffold structures in preliminary tests. This is the first study to report that medium supplementation with 50 ng/mL of growth and differentiation factor-5 (GDF-5) and 280 μM l-ascorbic acid are essential for tenogenic differentiation of hASCs. Tenogenic medium (TM) was shown to significantly enhance tendon-like matrix production of hASCs compared to other tested media groups. Cell adhesion, proliferation, and tenogenic differentiation of hASCs were supported on braided poly(l/d)lactide (PLA) 96l/4d copolymer filament scaffolds in TM condition compared to foamed poly(l-lactide-co-ɛ-caprolactone) (PLCL) 70L/30CL scaffolds. A uniform cell layer formed on braided PLA 96/4 scaffolds when hASCs were cultured in TM compared to maintenance medium (MM) condition after 14 days of culture. Furthermore, total collagen content and gene expression of tenogenic marker genes were significantly higher in TM condition after 2 weeks of culture. The elastic modulus of PLA 96/4 scaffold was more similar to the elastic modulus reported for native Achilles tendon. Our study showed that the optimized TM is needed for efficient and rapid in vitro tenogenic extracellular matrix production of hASCs. PLA 96/4 scaffolds together with TM significantly stimulated hASCs, thus demonstrating the potential clinical relevance of this novel and emerging approach to tendon injury treatments in the future.

  7. Osteogenic potential of adipogenic predifferentiated human bone-marrow-derived multipotent stromal cells for bone tissue engineering.

    PubMed

    Moya, Adrien; Larochette, Nathanaël; Bourguignon, Marianne; El-Hafci, Hanane; Potier, Esther; Petite, Hervé; Logeart-Avramoglou, Delphine

    2017-09-06

    In the present study, we evaluated the benefits of an adipogenic predifferentiation, the pathway most closely related to osteoblastogenesis, on the pro-osteogenic potential of human adult multipotent bone marrow stromal cells (hBMSCs), both in vitro and in vivo. Adipogenic differentiation of hBMSCs for 14 days resulted in a heterogeneous cell population from which the most adipogenic-committed cells were eliminated by their lack of re-adhesion ability. Our results provided evidence that the select adherent adipogenic differentiated hBMSCs (sAD+ cells) express a gene profile characteristic of both adipogenic and osteogenic lineages. In vitro, when cultured in osteogenic medium, sAD+ differentiated along the osteogenic lineage faster than undifferentiated hBMSCs. In vivo, in an ectopic mouse model, sAD+ exhibited a significantly higher bone formation capability compared to undifferentiated hBMSCs. We sought, then, to investigate the underlying mechanisms responsible for such beneficial effects of adipogenic pre-differentiation on bone formation and found that this outcome was not linked to a better cell survival post-implantation. The secretome of sAD+ was both pro-angiogenic and chemo-attractant but its potential did not supersede the one of undifferentiated hBMSCs. However, using co-culture systems, we observed that the sAD+ paracrine factors were pro-osteogenic on undifferentiated hBMSCs. In conclusion, adipogenic priming endows hBMSCs with high osteogenic potential as well as pro-osteogenic paracrine-mediated activity. This preconditioning appears as a promising strategy for bone tissue engineering technology in order to improve the hBMSC osteogenic potency in vivo. This article is protected by copyright. All rights reserved.

  8. An in vitro expansion score for tissue-engineering applications with human bone marrow-derived mesenchymal stem cells.

    PubMed

    Bertolo, Alessandro; Mehr, Marco; Janner-Jametti, Tiziana; Graumann, Ursula; Aebli, Niklaus; Baur, Martin; Ferguson, Stephen J; Stoyanov, Jivko V

    2016-02-01

    Human bone marrow-derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue-engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony-forming unit (CFU) assay, population doubling time (PDT), senescence-associated β-galactosidase (SA-β-Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA-β-Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late-passage cells than in early-passage cells. Early-passage cells underwent efficient osteogenic differentiation, with mid-passage cells performing best in chondrogenic differentiation. Late-passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number.

  9. Attenuation of skeletal muscle wasting with recombinant human growth hormone secreted from a tissue-engineered bioartificial muscle

    NASA Technical Reports Server (NTRS)

    Vandenburgh, H.; Del Tatto, M.; Shansky, J.; Goldstein, L.; Russell, K.; Genes, N.; Chromiak, J.; Yamada, S.

    1998-01-01

    Skeletal muscle wasting is a significant problem in elderly and debilitated patients. Growth hormone (GH) is an anabolic growth factor for skeletal muscle but is difficult to deliver in a therapeutic manner by injection owing to its in vivo instability. A novel method is presented for the sustained secretion of recombinant human GH (rhGH) from genetically modified skeletal muscle implants, which reduces host muscle wasting. Proliferating murine C2C12 skeletal myoblasts stably transduced with the rhGH gene were tissue engineered in vitro into bioartificial muscles (C2-BAMs) containing organized postmitotic myofibers secreting 3-5 microg of rhGH/day in vitro. When implanted subcutaneously into syngeneic mice, C2-BAMs delivered a sustained physiologic dose of 2.5 to 11.3 ng of rhGH per milliliter of serum. rhGH synthesized and secreted by the myofibers was in the 22-kDa monomeric form and was biologically active, based on downregulation of a GH-sensitive protein synthesized in the liver. Skeletal muscle disuse atrophy was induced in mice by hindlimb unloading, causing the fast plantaris and slow soleus muscles to atrophy by 21 to 35% ( < 0.02). This atrophy was significantly attenuated 41 to 55% (p < 0.02) in animals that received C2-BAM implants, but not in animals receiving daily injections of purified rhGH (1 mg/kg/day). These data support the concept that delivery of rhGH from BAMs may be efficacious in treating muscle-wasting disorders.

  10. Polymer-ceramic spiral structured scaffolds for bone tissue engineering: effect of hydroxyapatite composition on human fetal osteoblasts.

    PubMed

    Zhang, Xiaojun; Chang, Wei; Lee, Paul; Wang, Yuhao; Yang, Min; Li, Jun; Kumbar, Sangamesh G; Yu, Xiaojun

    2014-01-01

    For successful bone tissue engineering, a scaffold needs to be osteoconductive, porous, and biodegradable, thus able to support attachment and proliferation of bone cells and guide bone formation. Recently, hydroxyapatites (HA), a major inorganic component of natural bone, and biodegrade polymers have drawn much attention as bone scaffolds. The present study was designed to investigate whether the bone regenerative properties of nano-HA/polycaprolactone (PCL) spiral scaffolds are augmented in an HA dose dependent manner, thereby establishing a suitable composition as a bone formation material. Nano-HA/PCL spiral scaffolds were prepared with different weight ratios of HA and PCL, while porosity was introduced by a modified salt leaching technique. Human fetal osteoblasts (hFOBs) were cultured on the nano-HA/PCL spiral scaffolds up to 14 days. Cellular responses in terms of cell adhesion, viability, proliferation, differentiation, and the expression of bone-related genes were investigated. These scaffolds supported hFOBs adhesion, viability and proliferation. Cell proliferation trend was quite similar on polymer-ceramic and neat polymer spiral scaffolds on days 1, 7, and 14. However, the significantly increased amount of alkaline phosphatase (ALP) activity and mineralized matrix synthesis was evident on the nano-HA/PCL spiral scaffolds. The HA composition in the scaffolds showed a significant effect on ALP and mineralization. Bone phenotypic markers such as bone sialoprotein (BSP), osteonectin (ON), osteocalcin (OC), and type I collagen (Col-1) were semi-quantitatively estimated by reverse transcriptase polymerase chain reaction analysis. All of these results suggested the osteoconductive characteristics of HA/PCL nanocomposite and cell maturation were HA dose dependent. For instance, HA∶PCL = 1∶4 group showed significantly higher ALP mineralization and elevated levels of BSP, ON, OC and Col-I expression as compared other lower or higher ceramic ratios

  11. Polymer-Ceramic Spiral Structured Scaffolds for Bone Tissue Engineering: Effect of Hydroxyapatite Composition on Human Fetal Osteoblasts

    PubMed Central

    Zhang, Xiaojun; Chang, Wei; Lee, Paul; Wang, Yuhao; Yang, Min; Li, Jun; Kumbar, Sangamesh G.; Yu, Xiaojun

    2014-01-01

    For successful bone tissue engineering, a scaffold needs to be osteoconductive, porous, and biodegradable, thus able to support attachment and proliferation of bone cells and guide bone formation. Recently, hydroxyapatites (HA), a major inorganic component of natural bone, and biodegrade polymers have drawn much attention as bone scaffolds. The present study was designed to investigate whether the bone regenerative properties of nano-HA/polycaprolactone (PCL) spiral scaffolds are augmented in an HA dose dependent manner, thereby establishing a suitable composition as a bone formation material. Nano-HA/PCL spiral scaffolds were prepared with different weight ratios of HA and PCL, while porosity was introduced by a modified salt leaching technique. Human fetal osteoblasts (hFOBs) were cultured on the nano-HA/PCL spiral scaffolds up to 14 days. Cellular responses in terms of cell adhesion, viability, proliferation, differentiation, and the expression of bone-related genes were investigated. These scaffolds supported hFOBs adhesion, viability and proliferation. Cell proliferation trend was quite similar on polymer-ceramic and neat polymer spiral scaffolds on days 1, 7, and 14. However, the significantly increased amount of alkaline phosphatase (ALP) activity and mineralized matrix synthesis was evident on the nano-HA/PCL spiral scaffolds. The HA composition in the scaffolds showed a significant effect on ALP and mineralization. Bone phenotypic markers such as bone sialoprotein (BSP), osteonectin (ON), osteocalcin (OC), and type I collagen (Col-1) were semi-quantitatively estimated by reverse transcriptase polymerase chain reaction analysis. All of these results suggested the osteoconductive characteristics of HA/PCL nanocomposite and cell maturation were HA dose dependent. For instance, HA∶PCL = 1∶4 group showed significantly higher ALP mineralization and elevated levels of BSP, ON, OC and Col-I expression as compared other lower or higher ceramic ratios

  12. Videofetoscopically assisted fetal tissue engineering: bladder augmentation.

    PubMed

    Fauza, D O; Fishman, S J; Mehegan, K; Atala, A

    1998-01-01

    were more compliant (P < .05) and had greater capacity pressures greater than 20 mm Hg (P < .05) than those closed primarily. Histological analysis of the engineered tissue showed a multilayered urothelial lining on the luminal side and overlying layers of smooth muscle cells surrounded by connective tissue. Videofetoscopically assisted fetal bladder engineering may be a viable alternative for prompt bladder reconstruction at birth. The architecture of autologous engineered fetal bladder tissue resembles that of native bladder. This concept may prove useful for the treatment of certain human neonatal conditions such as bladder and cloacal exstrophies.

  13. Tissue engineering of blood vessel

    PubMed Central

    Zhang, Wen Jie; Liu, Wei; Cui, Lei; Cao, Yilin

    2007-01-01

    Abstract Vascular grafts are in large demand for coronary and peripheral bypass surgeries. Although synthetic grafts have been developed, replacement of vessels with purely synthetic polymeric conduits often leads to the failure of such graft, especially in the grafts less than 6 mm in diameter or in the areas of low blood flow, mainly due to the early formation of thrombosis. Moreover, the commonly used materials lack growth potential, and long-term results have revealed several material-related failures, such as stenosis, thromboembolization, calcium deposition and infection. Tissue engineering has become a promising approach for generating a bio-compatible vessel graft with growth potential. Since the first success of constructing blood vessels with collagen and cultured vascular cells by Weinberg and Bell, there has been considerable progress in the area of vessel engineering. To date, tissue- engineered blood vessels (TEBVs) could be successfully constructed in vitro, and be used to repair the vascular defects in animal models. This review describes the major progress in the field, including the seeding cell sources, the biodegradable scaffolds, the construction technologies, as well as the encouraging achievements in clinical applications. The remaining challenges are also discussed. PMID:17979876

  14. Kidney diseases and tissue engineering.

    PubMed

    Moon, Kyung Hyun; Ko, In Kap; Yoo, James J; Atala, Anthony

    2016-04-15

    Kidney disease is a worldwide public health problem. Renal failure follows several disease stages including acute and chronic kidney symptoms. Acute kidney injury (AKI) may lead to chronic kidney disease (CKD), which can progress to end-stage renal disease (ESRD) with a mortality rate. Current treatment options are limited to dialysis and kidney transplantation; however, problems such as donor organ shortage, graft failure and numerous complications remain a concern. To address this issue, cell-based approaches using tissue engineering (TE) and regenerative medicine (RM) may provide attractive approaches to replace the damaged kidney cells with functional renal specific cells, leading to restoration of normal kidney functions. While development of renal tissue engineering is in a steady state due to the complex composition and highly regulated functionality of the kidney, cell therapy using stem cells and primary kidney cells has demonstrated promising therapeutic outcomes in terms of restoration of renal functions in AKI and CKD. In this review, basic components needed for successful renal kidney engineering are discussed, and recent TE and RM approaches to treatment of specific kidney diseases will be presented.

  15. Different ratios of bone marrow mesenchymal stem cells and chondrocytes used in tissue-engineered cartilage and its application for human ear-shaped substitutes in vitro.

    PubMed

    Kang, Ning; Liu, Xia; Yan, Li; Wang, Qian; Cao, Yilin; Xiao, Ran

    2013-01-01

    The application of chondrocyte-based cartilage tissue engineering is limited because of the lack of autologous cartilage sources and chondrocyte dedifferentiation after in vitro expansion. Coculture of bone marrow mesenchymal stem cells (BMSCs) and chondrocytes has been a promising strategy for cartilage engineering as chondrocytes can provide a chondrogenic environment for BMSCs. However, there are no systematic comparison studies for engineered cartilage constructed using different mixing ratios of BMSCs and chondrocytes, and the most effective mixing ratio with the lowest number of chondrocytes is unknown. Here, we set a gradient of mixing ratios of BMSCs to chondrocytes for an in vitro coculture system and compared the shape retention and quality of the engineered cartilage using macroscopic and histological assays, glycosaminoglycan content assessment and immunohistochemical staining of type II collagen, biomechanical evaluation and hypertrophy-related gene expression analysis. The results showed that at least 30% chondrocytes were required to generate cartilage tissue with satisfactory shape and quality. Therefore, we preliminarily assessed the feasibility of engineering a human ear-shaped substitute using a coculture system with a 7:3 ratio of BMSCs to chondrocytes. After 8 weeks of in vitro culture, the precise architecture of the human ear-shaped construct was well maintained with the typical cartilaginous composition confirmed by histological assays.

  16. Cardiac Conduction through Engineered Tissue

    PubMed Central

    Choi, Yeong-Hoon; Stamm, Christof; Hammer, Peter E.; Kwaku, Kevin F.; Marler, Jennifer J.; Friehs, Ingeborg; Jones, Mara; Rader, Christine M.; Roy, Nathalie; Eddy, Mau-Thek; Triedman, John K.; Walsh, Edward P.; McGowan, Francis X.; del Nido, Pedro J.; Cowan, Douglas B.

    2006-01-01

    In children, interruption of cardiac atrioventricular (AV) electrical conduction can result from congenital defects, surgical interventions, and maternal autoimmune diseases during pregnancy. Complete AV conduction block is typically treated by implanting an electronic pacemaker device, although long-term pacing therapy in pediatric patients has significant complications. As a first step toward developing a substitute treatment, we implanted engineered tissue constructs in rat hearts to create an alternative AV conduction pathway. We found that skeletal muscle-derived cells in the constructs exhibited sustained electrical coupling through persistent expression and function of gap junction proteins. Using fluorescence in situ hybridization and polymerase chain reaction analyses, myogenic cells in the constructs were shown to survive in the AV groove of implanted hearts for the duration of the animal’s natural life. Perfusion of hearts with fluorescently labeled lectin demonstrated that implanted tissues became vascularized and immunostaining verified the presence of proteins important in electromechanical integration of myogenic cells with surrounding recipient rat cardiomyocytes. Finally, using optical mapping and electrophysiological analyses, we provide evidence of permanent AV conduction through the implant in one-third of recipient animals. Our experiments provide a proof-of-principle that engineered tissue constructs can function as an electrical conduit and, ultimately, may offer a substitute treatment to conventional pacing therapy. PMID:16816362

  17. Dynamic changes in high and low mammographic density human breast tissues maintained in murine tissue engineering chambers during various murine peripartum states and over time.

    PubMed

    Chew, G L; Huang, D; Huo, C W; Blick, T; Hill, P; Cawson, J; Frazer, H; Southey, M D; Hopper, J L; Henderson, M A; Haviv, I; Thompson, E W

    2013-07-01

    Mammographic density (MD) is a strong heritable risk factor for breast cancer, and may decrease with increasing parity. However, the biomolecular basis for MD-associated breast cancer remains unclear, and systemic hormonal effects on MD-associated risk is poorly understood. This study assessed the effect of murine peripartum states on high and low MD tissue maintained in a xenograft model of human MD. Method High and low MD human breast tissues were precisely sampled under radiographic guidance from prophylactic mastectomy specimens of women. The high and low MD tissues were maintained in separate vascularised biochambers in nulliparous or pregnant SCID mice for 4 weeks, or mice undergoing postpartum involution or lactation for three additional weeks. High and low MD biochamber material was harvested for histologic and radiographic comparisons during various murine peripartum states. High and low MD biochamber tissues in nulliparous mice were harvested at different timepoints for histologic and radiographic comparisons. Results High MD biochamber tissues had decreased stromal (p = 0.0027), increased adipose (p = 0.0003) and a trend to increased glandular tissue areas (p = 0.076) after murine postpartum involution. Stromal areas decreased (p = 0.042), while glandular (p = 0.001) and adipose areas (p = 0.009) increased in high MD biochamber tissues during lactation. A difference in radiographic density was observed in high (p = 0.0021) or low MD biochamber tissues (p = 0.004) between nulliparous, pregnant and involution groups. No differences in tissue composition were observed in high or low MD biochamber tissues maintained for different durations, although radiographic density increased over time. Conclusion High MD biochamber tissues had measurable histologic changes after postpartum involution or lactation. Alterations in radiographic density occurred in biochamber tissues between different peripartum states and over time. These findings

  18. Biomaterials for vascular tissue engineering.

    PubMed

    Ravi, Swathi; Chaikof, Elliot L

    2010-01-01

    Cardiovascular disease is the leading cause of mortality in the USA. The limited availability of healthy autologous vessels for bypass grafting procedures has led to the fabrication of prosthetic vascular conduits. While synthetic polymers have been extensively studied as substitutes in vascular engineering, they fall short of meeting the biological challenges at the blood-material interface. Various tissue engineering strategies have emerged to address these flaws and increase long-term patency of vascular grafts. Vascular cell seeding of scaffolds and the design of bioactive polymers for in situ arterial regeneration have yielded promising results. This article describes the advances made in biomaterials design to generate suitable materials that not only match the mechanical properties of native vasculature, but also promote cell growth, facilitate extracellular matrix production and inhibit thrombogenicity.

  19. Biomaterials for vascular tissue engineering

    PubMed Central

    Ravi, Swathi; Chaikof, Elliot L

    2010-01-01

    Cardiovascular disease is the leading cause of mortality in the USA. The limited availability of healthy autologous vessels for bypass grafting procedures has led to the fabrication of prosthetic vascular conduits. While synthetic polymers have been extensively studied as substitutes in vascular engineering, they fall short of meeting the biological challenges at the blood–material interface. Various tissue engineering strategies have emerged to address these flaws and increase long-term patency of vascular grafts. Vascular cell seeding of scaffolds and the design of bioactive polymers for in situ arterial regeneration have yielded promising results. This article describes the advances made in biomaterials design to generate suitable materials that not only match the mechanical properties of native vasculature, but also promote cell growth, facilitate extracellular matrix production and inhibit thrombogenicity. PMID:20017698

  20. ASTM lights the way for tissue engineered medical products standards: jump start for combination medical products that restore biological function of human tissues.

    PubMed

    Picciolo, G L; Stocum, D L

    2001-01-01

    Everybody hopes for better health and restoration of impaired bodily function, and now that hope is illuminated by the promise of powerful biological tools that make human cells grow and replace human tissue. ASTM Committee F04 on Medical and Surgical Materials and Devices is taking the lead by defining some of those tools as standards that can be used for the development, production, testing, and regulatory approval of medical products.

  1. Microbioreactors for Cartilage Tissue Engineering.

    PubMed

    Chang, Yu-Han; Wu, Min-Hsien

    2015-01-01

    In tissue engineering research, cell-based assays are widely utilized to fundamentally explore cellular responses to extracellular conditions. Nevertheless, the simplified cell culture models available at present have several inherent shortcomings and limitations. To tackle the issues, a wide variety of microbioreactors for cell culture have been actively proposed, especially during the past decade. Among these, micro-scale cell culture devices based on microfluidic biochip technology have particularly attracted considerable attention. In this chapter, we not only discuss the advantageous features of using micro-scale cell culture devices for cell-based assays, but also describe their fabrication, experimental setup, and application.

  2. Stem cell-based meniscus tissue engineering.

    PubMed

    Mandal, Biman B; Park, Sang-Hyug; Gil, Eun Seok; Kaplan, David L

    2011-11-01

    Knee meniscus, a fibrocartilaginous tissue, is characterized by heterogeneity in extracellular matrix (ECM) and biomechanical properties, and critical for orthopedic stability, load transmission, shock absorption, and stress distribution within the knee joint. Most damage to the meniscus cannot be effectively healed by the body due to its partial avascular nature; thus, damage caused by injury or age impairs normal knee function, predisposing patients to osteoarthritis. Meniscus tissue engineering offers a possible solution to this problem by generating replacement tissue that may be implanted into the defect site to mimic the function of natural meniscal tissue. To address this need, a multiporous, multilamellar meniscus was formed using silk protein scaffolds and stem cells. The silk scaffolds were seeded with human bone marrow stem cells and differentiated over time in chondrogenic culture in the presence of transforming growth factor-beta 3 to generate meniscus-like tissue in vitro. High cellularity along with abundant ECM leading to enhanced biomechanics similar to native tissue was found. Higher levels of collagen type I and II, sulfated glycosaminoglycans along with enhanced collagen 1-α1, aggrecan, and SOX9 gene expression further confirmed differentiation and matured cell phenotype. The results of this study are a step forward toward biomechanically competent meniscus engineering, reconstituting both form and function of the native meniscus.

  3. Multilayered silk scaffolds for meniscus tissue engineering.

    PubMed

    Mandal, Biman B; Park, Sang-Hyug; Gil, Eun S; Kaplan, David L

    2011-01-01

    Removal of injured/damaged meniscus, a vital fibrocartilaginous load-bearing tissue, impairs normal knee function and predisposes patients to osteoarthritis. Meniscus tissue engineering solution is one option to improve outcomes and relieve pain. In an attempt to fabricate knee meniscus grafts three layered wedge shaped silk meniscal scaffold system was engineered to mimic native meniscus architecture. The scaffolds were seeded with human fibroblasts (outside) and chondrocytes (inside) in a spatial separated mode similar to native tissue, in order to generate meniscus-like tissue in vitro. In chondrogenic culture in the presence of TGF-b3, cell-seeded constructs increased in cellularity and extracellular matrix (ECM) content. Histology and Immunohistochemistry confirmed maintenance of chondrocytic phenotype with higher levels of sulfated glycosaminoglycans (sGAG) and collagen types I and II. Improved scaffold mechanical properties along with ECM alignment with time in culture suggest this multiporous silk construct as a useful micro-patterned template for directed tissue growth with respect to form and function of meniscus-like tissue.

  4. Multilayered silk scaffolds for meniscus tissue engineering

    PubMed Central

    Mandal, Biman B.; Park, Sang-Hyug; Gil, Eun Seok

    2010-01-01

    Removal of injured/damaged meniscus, a vital fibrocartilaginous load-bearing tissue, impairs normal knee function and predisposes patients to osteoarthritis. Meniscus tissue engineering solution is one option to improve outcomes and relieve pain. In an attempt to fabricate knee meniscus grafts three layered wedge shaped silk meniscal scaffold system was engineered to mimic native meniscus architecture. The scaffolds were seeded with human fibroblasts (outside) and chondrocytes (inside) in a spatial separated mode similar to native tissue, in order to generate meniscus-like tissue in vitro. In chondrogenic culture in the presence of TGF-b3, cell seeded constructs increased in cellularity and extracellular matrix (ECM) content. Histology and Immunohistochemistry confirmed maintenance of chondrocytic phenotype with higher levels of sulphated glycosaminoglycans (sGAG) and collagen types I and II. Improved scaffold mechanical properties along with ECM alignment with time in culture suggest this multiporous silk construct as a useful micro-patterned template for directed tissue growth with respect to form and function of meniscus-like tissue. PMID:20926132

  5. Biomechanics and mechanobiology in functional tissue engineering.

    PubMed

    Guilak, Farshid; Butler, David L; Goldstein, Steven A; Baaijens, Frank P T

    2014-06-27

    The field of tissue engineering continues to expand and mature, and several products are now in clinical use, with numerous other preclinical and clinical studies underway. However, specific challenges still remain in the repair or regeneration of tissues that serve a predominantly biomechanical function. Furthermore, it is now clear that mechanobiological interactions between cells and scaffolds can critically influence cell behavior, even in tissues and organs that do not serve an overt biomechanical role. Over the past decade, the field of "functional tissue engineering" has grown as a subfield of tissue engineering to address the challenges and questions on the role of biomechanics and mechanobiology in tissue engineering. Originally posed as a set of principles and guidelines for engineering of load-bearing tissues, functional tissue engineering has grown to encompass several related areas that have proven to have important implications for tissue repair and regeneration. These topics include measurement and modeling of the in vivo biomechanical environment; quantitative analysis of the mechanical properties of native tissues, scaffolds, and repair tissues; development of rationale criteria for the design and assessment of engineered tissues; investigation of the effects biomechanical factors on native and repair tissues, in vivo and in vitro; and development and application of computational models of tissue growth and remodeling. Here we further expand this paradigm and provide examples of the numerous advances in the field over the past decade. Consideration of these principles in the design process will hopefully improve the safety, efficacy, and overall success of engineered tissue replacements.

  6. Strategies for Whole Lung Tissue Engineering

    PubMed Central

    Calle, Elizabeth A.; Ghaedi, Mahboobe; Sundaram, Sumati; Sivarapatna, Amogh; Tseng, Michelle K.

    2014-01-01

    Recent work has demonstrated the feasibility of using decellularized lung extracellular matrix scaffolds to support the engineering of functional lung tissue in vitro. Rendered acellular through the use of detergents and other reagents, the scaffolds are mounted in organ-specific bioreactors where cells in the scaffold are provided with nutrients and appropriate mechanical stimuli such as ventilation and perfusion. Though initial studies are encouraging, a great deal remains to be done to advance the field and transition from rodent lungs to whole human tissue engineered lungs. To do so, a variety of hurdles must be overcome. In particular, a reliable source of human-sized scaffolds, as well as a method of terminal sterilization of scaffolds, must be identified. Continued research in lung cell and developmental biology will hopefully help identify the number and types of cells that will be required to regenerate functional lung tissue. Finally, bioreactor designs must be improved in order to provide more precise ventilation stimuli and vascular perfusion in order to avoid injury to or death of the cells cultivated within the scaffold. Ultimately, the success of efforts to engineer a functional lung in vitro will critically depend on the ability to create a fully endothelialized vascular network that provides sufficient barrier function and alveolar-capillary surface area to exchange gas at rates compatible with healthy lung function. PMID:24691527

  7. Surgical retrieval, isolation and in vitro expansion of human anterior cruciate ligament-derived cells for tissue engineering applications.

    PubMed

    Gupta, Ashim; Sharif, Kevin; Walters, Megan; Woods, Mia D; Potty, Anish; Main, Benjamin J; El-Amin, Saadiq F

    2014-04-30

    Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities. The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 ºC or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is

  8. Multiphasic Scaffolds for Periodontal Tissue Engineering

    PubMed Central

    Ivanovski, S.; Vaquette, C.; Gronthos, S.; Hutmacher, D.W.; Bartold, P.M.

    2014-01-01

    For a successful clinical outcome, periodontal regeneration requires the coordinated response of multiple soft and hard tissues (periodontal ligament, gingiva, cementum, and bone) during the wound-healing process. Tissue-engineered constructs for regeneration of the periodontium must be of a complex 3-dimensional shape and adequate size and demonstrate biomechanical stability over time. A critical requirement is the ability to promote the formation of functional periodontal attachment between regenerated alveolar bone, and newly formed cementum on the root surface. This review outlines the current advances in multiphasic scaffold fabrication and how these scaffolds can be combined with cell- and growth factor–based approaches to form tissue-engineered constructs capable of recapitulating the complex temporal and spatial wound-healing events that will lead to predictable periodontal regeneration. This can be achieved through a variety of approaches, with promising strategies characterized by the use of scaffolds that can deliver and stabilize cells capable of cementogenesis onto the root surface, provide biomechanical cues that encourage perpendicular alignment of periodontal fibers to the root surface, and provide osteogenic cues and appropriate space to facilitate bone regeneration. Progress on the development of multiphasic constructs for periodontal tissue engineering is in the early stages of development, and these constructs need to be tested in large animal models and, ultimately, human clinical trials. PMID:25139362

  9. Multiphasic scaffolds for periodontal tissue engineering.

    PubMed

    Ivanovski, S; Vaquette, C; Gronthos, S; Hutmacher, D W; Bartold, P M

    2014-12-01

    For a successful clinical outcome, periodontal regeneration requires the coordinated response of multiple soft and hard tissues (periodontal ligament, gingiva, cementum, and bone) during the wound-healing process. Tissue-engineered constructs for regeneration of the periodontium must be of a complex 3-dimensional shape and adequate size and demonstrate biomechanical stability over time. A critical requirement is the ability to promote the formation of functional periodontal attachment between regenerated alveolar bone, and newly formed cementum on the root surface. This review outlines the current advances in multiphasic scaffold fabrication and how these scaffolds can be combined with cell- and growth factor-based approaches to form tissue-engineered constructs capable of recapitulating the complex temporal and spatial wound-healing events that will lead to predictable periodontal regeneration. This can be achieved through a variety of approaches, with promising strategies characterized by the use of scaffolds that can deliver and stabilize cells capable of cementogenesis onto the root surface, provide biomechanical cues that encourage perpendicular alignment of periodontal fibers to the root surface, and provide osteogenic cues and appropriate space to facilitate bone regeneration. Progress on the development of multiphasic constructs for periodontal tissue engineering is in the early stages of development, and these constructs need to be tested in large animal models and, ultimately, human clinical trials.

  10. Heart valve and arterial tissue engineering.

    PubMed

    Sarraf, C E; Harris, A B; McCulloch, A D; Eastwood, M

    2003-10-01

    In the industrialized world, cardiovascular disease alone is responsible for almost half of all deaths. Many of the conditions can be treated successfully with surgery, often using transplantation techniques; however, autologous vessels or human-donated organs are in short supply. Tissue engineering aims to create specific, matching grafts by growing cells on appropriate matrices, but there are many steps between the research laboratory and the operating theatre. Neo-tissues must be effective, durable, non-thrombogenic and non-immunogenic. Scaffolds should be bio-compatible, porous (to allow cell/cell communication) and amenable to surgery. In the early days of cardiovascular tissue engineering, autologous or allogenic cells were grown on inert matrices, but patency and thrombogenicity of grafts were disappointing. The current ethos is toward appropriate cell types grown in (most often) a polymeric matrix that degrades at a rate compatible with the cells' production of their own extracellular matrical proteins, thus gradually replacing the graft with a living counterpart. The geometry is crucial. Computer models have been made of valves, and these are used as three-dimensional patterns for mass-production of implant scaffolds. Vessel walls have integral connective tissue architecture, and application of physiological level mechanical forces conditions bio-engineered components to align in precise orientation. This article reviews the concepts involved and successes achieved to date.

  11. Human endothelial colony-forming cells expanded with an improved protocol are a useful endothelial cell source for scaffold-based tissue engineering.

    PubMed

    Denecke, Bernd; Horsch, Liska D; Radtke, Stefan; Fischer, Johannes C; Horn, Peter A; Giebel, Bernd

    2015-11-01

    One of the major challenges in tissue engineering is to supply larger three-dimensional (3D) bioengineered tissue transplants with sufficient amounts of nutrients and oxygen and to allow metabolite removal. Consequently, artificial vascularization strategies of such transplants are desired. One strategy focuses on endothelial cells capable of initiating new vessel formation, which are settled on scaffolds commonly used in tissue engineering. A bottleneck in this strategy is to obtain sufficient amounts of endothelial cells, as they can be harvested only in small quantities directly from human tissues. Thus, protocols are required to expand appropriate cells in sufficient amounts without interfering with their capability to settle on scaffold materials and to initiate vessel formation. Here, we analysed whether umbilical cord blood (CB)-derived endothelial colony-forming cells (ECFCs) fulfil these requirements. In a first set of experiments, we showed that marginally expanded ECFCs settle and survive on different scaffold biomaterials. Next, we improved ECFC culture conditions and developed a protocol for ECFC expansion compatible with 'Good Manufacturing Practice' (GMP) standards. We replaced animal sera with human platelet lysates and used a novel type of tissue-culture ware. ECFCs cultured under the new conditions revealed significantly lower apoptosis and increased proliferation rates. Simultaneously, their viability was increased. Since extensively expanded ECFCs could still settle on scaffold biomaterials and were able to form tubular structures in Matrigel assays, we conclude that these ex vivo-expanded ECFCs are a novel, very potent cell source for scaffold-based tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

  12. Nanotechnology in bone tissue engineering.

    PubMed

    Walmsley, Graham G; McArdle, Adrian; Tevlin, Ruth; Momeni, Arash; Atashroo, David; Hu, Michael S; Feroze, Abdullah H; Wong, Victor W; Lorenz, Peter H; Longaker, Michael T; Wan, Derrick C

    2015-07-01

    Nanotechnology represents a major frontier with potential to significantly advance the field of bone tissue engineering. Current limitations in regenerative strategies include impaired cellular proliferation and differentiation, insufficient mechanical strength of scaffolds, and inadequate production of extrinsic factors necessary for efficient osteogenesis. Here we review several major areas of research in nanotechnology with potential implications in bone regeneration: 1) nanoparticle-based methods for delivery of bioactive molecules, growth factors, and genetic material, 2) nanoparticle-mediated cell labeling and targeting, and 3) nano-based scaffold construction and modification to enhance physicochemical interactions, biocompatibility, mechanical stability, and cellular attachment/survival. As these technologies continue to evolve, ultimate translation to the clinical environment may allow for improved therapeutic outcomes in patients with large bone deficits and osteodegenerative diseases. Traditionally, the reconstruction of bony defects has relied on the use of bone grafts. With advances in nanotechnology, there has been significant development of synthetic biomaterials. In this article, the authors provided a comprehensive review on current research in nanoparticle-based therapies for bone tissue engineering, which should be useful reading for clinicians as well as researchers in this field. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Cardiac tissue engineering using perfusion bioreactor systems

    PubMed Central

    Radisic, Milica; Marsano, Anna; Maidhof, Robert; Wang, Yadong; Vunjak-Novakovic, Gordana

    2009-01-01

    This protocol describes tissue engineering of synchronously contractile cardiac constructs by culturing cardiac cell populations on porous scaffolds (in some cases with an array of channels) and bioreactors with perfusion of culture medium (in some cases supplemented with an oxygen carrier). The overall approach is ‘biomimetic’ in nature as it tends to provide in vivo-like oxygen supply to cultured cells and thereby overcome inherent limitations of diffusional transport in conventional culture systems. In order to mimic the capillary network, cells are cultured on channeled elastomer scaffolds that are perfused with culture medium that can contain oxygen carriers. The overall protocol takes 2–4 weeks, including assembly of the perfusion systems, preparation of scaffolds, cell seeding and cultivation, and on-line and end-point assessment methods. This model is well suited for a wide range of cardiac tissue engineering applications, including the use of human stem cells, and high-fidelity models for biological research. PMID:18388955

  14. Human and Murine Tissue-Engineered Colon Exhibit Diverse Neuronal Subtypes and Can Be Populated by Enteric Nervous System Progenitor Cells When Donor Colon Is Aganglionic.

    PubMed

    Wieck, Minna M; El-Nachef, Wael N; Hou, Xiaogang; Spurrier, Ryan G; Holoyda, Kathleen A; Schall, Kathy A; Mojica, Salvador Garcia; Collins, Malie K; Trecartin, Andrew; Cheng, Zhi; Frykman, Philip K; Grikscheit, Tracy C

    2016-01-01

    Tissue-engineered colon (TEC) might potentially replace absent or injured large intestine, but the enteric nervous system (ENS), a key component, has not been investigated. In various enteric neuropathic diseases in which the TEC is derived from aganglionic donor colon, the resulting construct might also be aganglionic, limiting tissue engineering applications in conditions such as Hirschsprung disease (HD). We hypothesized that TEC might contain a diverse population of enteric neuronal subtypes, and that aganglionic TEC can be populated by neurons and glia when supplemented with ENS progenitor cells in the form of neurospheres. Human and murine organoid units (OU) and multicellular clusters containing epithelium and mesenchyme were isolated from both mouse and human donor tissues, including from normally innervated and aganglionic colon. The OU were seeded onto a biodegradable scaffold and implanted within a host mouse, resulting in the growth of TEC. Aganglionic murine and human OU were supplemented with cultured neurospheres to populate the absent ENS not provided by the OU to rescue the HD phenotype. TEC demonstrated abundant smooth muscle and clusters of neurons and glia beneath the epithelium and deeper within the mesenchyme. Motor and afferent neuronal subtypes were identified in TEC. Aganglionic OU formed TEC with absent neural elements, but neurons and glia were abundant when aganglionic OU were supplemented with ENS progenitor cells. Murine and human TEC contain key components of the ENS that were not previously identified, including glia, neurons, and fundamental neuronal subtypes. TEC derived from aganglionic colon can be populated with neurons and glia when supplemented with neurospheres. Combining tissue engineering and cellular replacement therapies represents a new strategy for treating enteric neuropathies, particularly HD.

  15. Human and Murine Tissue-Engineered Colon Exhibit Diverse Neuronal Subtypes and Can Be Populated by Enteric Nervous System Progenitor Cells When Donor Colon Is Aganglionic

    PubMed Central

    Wieck, Minna M.; El-Nachef, Wael N.; Hou, Xiaogang; Spurrier, Ryan G.; Holoyda, Kathleen A.; Schall, Kathy A.; Mojica, Salvador Garcia; Collins, Malie K.; Trecartin, Andrew; Cheng, Zhi; Frykman, Philip K.

    2016-01-01

    Purpose: Tissue-engineered colon (TEC) might potentially replace absent or injured large intestine, but the enteric nervous system (ENS), a key component, has not been investigated. In various enteric neuropathic diseases in which the TEC is derived from aganglionic donor colon, the resulting construct might also be aganglionic, limiting tissue engineering applications in conditions such as Hirschsprung disease (HD). We hypothesized that TEC might contain a diverse population of enteric neuronal subtypes, and that aganglionic TEC can be populated by neurons and glia when supplemented with ENS progenitor cells in the form of neurospheres. Materials and Methods: Human and murine organoid units (OU) and multicellular clusters containing epithelium and mesenchyme were isolated from both mouse and human donor tissues, including from normally innervated and aganglionic colon. The OU were seeded onto a biodegradable scaffold and implanted within a host mouse, resulting in the growth of TEC. Aganglionic murine and human OU were supplemented with cultured neurospheres to populate the absent ENS not provided by the OU to rescue the HD phenotype. Results: TEC demonstrated abundant smooth muscle and clusters of neurons and glia beneath the epithelium and deeper within the mesenchyme. Motor and afferent neuronal subtypes were identified in TEC. Aganglionic OU formed TEC with absent neural elements, but neurons and glia were abundant when aganglionic OU were supplemented with ENS progenitor cells. Conclusion: Murine and human TEC contain key components of the ENS that were not previously identified, including glia, neurons, and fundamental neuronal subtypes. TEC derived from aganglionic colon can be populated with neurons and glia when supplemented with neurospheres. Combining tissue engineering and cellular replacement therapies represents a new strategy for treating enteric neuropathies, particularly HD. PMID:26414777

  16. Nanotechnological strategies for engineering complex tissues

    PubMed Central

    Dvir, Tal; Timko, Brian P.; Kohane, Daniel S.; Langer, Robert

    2014-01-01

    Tissue engineering aims at developing functional substitutes for damaged tissues and organs. Before transplantation, cells are generally seeded on biomaterial scaffolds that recapitulate the extracellular matrix and provide cells with information that is important for tissue development. Here we review the nanocomposite nature of the extracellular matrix, describe the design considerations for different tissues and discuss the impact of nanostructures on the properties of scaffolds and their uses in monitoring the behaviour of engineered tissues. We also examine the different nanodevices used to trigger certain processes for tissue development, and offer our view on the principal challenges and prospects of applying nanotechnology in tissue engineering. PMID:21151110

  17. Nanotechnological strategies for engineering complex tissues

    NASA Astrophysics Data System (ADS)

    Dvir, Tal; Timko, Brian P.; Kohane, Daniel S.; Langer, Robert

    2011-01-01

    Tissue engineering aims at developing functional substitutes for damaged tissues and organs. Before transplantation, cells are generally seeded on biomaterial scaffolds that recapitulate the extracellular matrix and provide cells with information that is important for tissue development. Here we review the nanocomposite nature of the extracellular matrix, describe the design considerations for different tissues and discuss the impact of nanostructures on the properties of scaffolds and their uses in monitoring the behaviour of engineered tissues. We also examine the different nanodevices used to trigger certain processes for tissue development, and offer our view on the principal challenges and prospects of applying nanotechnology in tissue engineering.

  18. Enhancing repair of full-thickness excisional wounds in a murine model: Impact of tissue-engineered biological dressings featuring human differentiated adipocytes.

    PubMed

    Morissette Martin, Pascal; Maux, Amandine; Laterreur, Véronique; Mayrand, Dominique; L Gagné, Valérie; Moulin, Véronique J; Fradette, Julie

    2015-08-01

    Promotion of skin repair for acute or chronic wounds through the use of tissue-engineered products is an active field of research. This study evaluates the effects mediated by tissue-engineered biological dressings containing human in vitro-differentiated adipocytes and adipose-derived stromal cells (ASCs). Re-epithelialization, granulation tissue formation and neovascularization of full-thickness cutaneous wounds were specifically assessed using a murine model featuring a fluorescent epidermis. In comparison with wounds that did not receive an adipocyte-containing biological dressing, treated wounds displayed a slight but significantly faster wound closure based on macroscopic observations over 18 days. Non-invasive imaging of GFP-expressing keratinocytes determined that the kinetics of re-epithelialization were similar for both groups. Treated wounds featured thicker granulation tissues (1.7-fold, P < 0.0001) enriched in collagens (1.3-fold, P < 0.0104). In addition, wound cryosections labeled for detection of CD31-expressing cells indicated a 2.2-fold (P < 0.0002) increased neovascularization for the treated wounds at the time of terminal biopsy. This is in accordance with the secretion of pro-angiogenic factors detected in media conditioned by the dressings. Taken together, these results establish that a new type of engineered substitutes featuring a mixture of adipocytes and ASCs can promote cutaneous healing when applied as temporary dressings, suggesting their potential relevance for chronic wound management studies. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  19. Fetal tissue engineering from amniotic fluid.

    PubMed

    Kaviani, Amir; Guleserian, Kristine; Perry, Tjörvi E; Jennings, Russell W; Ziegler, Moritz M; Fauza, Dario O

    2003-04-01

    We have recently shown, in an animal model, that amniotic fluid can be a source of cells for fetal tissue engineering. This study was aimed at determining whether fetal tissue constructs could also be engineered from cells normally found in human amniotic fluid. Cells obtained from the amniotic fluid of pregnant women at 15 to 19 weeks of gestation (n=6) were cultured in Dulbecco's Modified Eagle's medium (Sigma Chemical, St Louis, MO) containing 20% fetal bovine serum and 5 ng/mL basic fibroblast growth factor in a 95% humidified, 5% CO(2) chamber at 37 degrees C. A subpopulation of morphologically distinct cells was then mechanically isolated from the rest and selectively expanded. The lineage of this subpopulation of amniocytes was determined by immunofluorescent staining with antibodies against standard intermediate filaments and surface antigens. Cell proliferation rates were determined by oxidation assay. After cell expansion, colonies of amniocytes were statically and dynamically seeded onto both unwoven, 1-mm-thick polyglycolic acid polymer scaffold and acellular human dermis for 72 hours. The resulting constructs were analyzed by scanning electron microscopy. Amniocytes stained positively for smooth muscle actin, vimentin, cytokeratin 18, and fibroblast surface protein, and negatively for desmin, cluster of differentiation 31, and von Willebrand's factor (Dako, Carpenteria, CA). These findings are consistent with a mesenchymal, fibroblast-myofibroblast cell lineage. Mesenchymal amniocytes could be rapidly expanded in culture, based on results of the proliferation assay. Scanning electron microscopy of amniocyte constructs revealed dense, confluent layers of cells surrounding the polymer matrices and firm cell adhesion to both PGA and Alloderm (Lifecell Corp, Branchburg, NJ) scaffolds. No evidence of cell death was observed. Subpopulations of fetal mesenchymal cells can be consistently isolated from human amniotic fluid and rapidly expanded in vitro. Human

  20. Biomechanics and mechanobiology in functional tissue engineering

    PubMed Central

    Guilak, Farshid; Butler, David L.; Goldstein, Steven A.; Baaijens, Frank P.T.

    2014-01-01

    The field of tissue engineering continues to expand and mature, and several products are now in clinical use, with numerous other preclinical and clinical studies underway. However, specific challenges still remain in the repair or regeneration of tissues that serve a predominantly biomechanical function. Furthermore, it is now clear that mechanobiological interactions between cells and scaffolds can critically influence cell behavior, even in tissues and organs that do not serve an overt biomechanical role. Over the past decade, the field of “functional tissue engineering” has grown as a subfield of tissue engineering to address the challenges and questions on the role of biomechanics and mechanobiology in tissue engineering. Originally posed as a set of principles and guidelines for engineering of load-bearing tissues, functional tissue engineering has grown to encompass several related areas that have proven to have important implications for tissue repair and regeneration. These topics include measurement and modeling of the in vivo biomechanical environment; quantitative analysis of the mechanical properties of native tissues, scaffolds, and repair tissues; development of rationale criteria for the design and assessment of engineered tissues; investigation of the effects biomechanical factors on native and repair tissues, in vivo and in vitro; and development and application of computational models of tissue growth and remodeling. Here we further expand this paradigm and provide examples of the numerous advances in the field over the past decade. Consideration of these principles in the design process will hopefully improve the safety, efficacy, and overall success of engineered tissue replacements. PMID:24818797

  1. Videofetoscopically assisted fetal tissue engineering: skin replacement.

    PubMed

    Fauza, D O; Fishman, S J; Mehegan, K; Atala, A

    1998-02-01

    fibroblasts multiplied significantly faster in vitro (approximately fivefold) than expected. Fetal keratinocytes multiplied at expected postnatal rates. The engineered grafts induced faster epithelization of the wound (partial at 1 week and complete between 2 and 3 weeks postoperatively) than did the acellular ones (partial at 3 weeks and complete between 3 and 4 weeks postoperatively). Analysis of skin architecture showed a higher level of epidermal organization and less dermal scarring in the wounds that received the engineered, cell-implanted polymer scaffold. (1) Videofetoscopically assisted fetal tissue engineering is a viable method for obtaining expanded autologous tissue for prompt surgical reconstruction at birth. (2) Fetal skin can be expanded and engineered in vitro at faster rates than expected postnatally, with current tissue engineering techniques. (3) Engineered autologous fetal skin induces a faster and more organized healing of neonatal skin defects than that observed with second intention. This concept may prove useful for the treatment of certain human neonatal conditions such as giant neoplasias, ectopia cordis, and other body wall defects.

  2. Effects of Tamoxifen and oestrogen on histology and radiographic density in high and low mammographic density human breast tissues maintained in murine tissue engineering chambers.

    PubMed

    Chew, G L; Huo, C W; Huang, D; Blick, T; Hill, P; Cawson, J; Frazer, H; Southey, M C; Hopper, J L; Britt, K; Henderson, M A; Haviv, I; Thompson, E W

    2014-11-01

    Mammographic density (MD) is a strong risk factor for breast cancer. It is altered by exogenous endocrine treatments, including hormone replacement therapy and Tamoxifen. Such agents also modify breast cancer (BC) risk. However, the biomolecular basis of how systemic endocrine therapy modifies MD and MD-associated BC risk is poorly understood. This study aims to determine whether our xenograft biochamber model can be used to study the effectiveness of therapies aimed at modulating MD, by examine the effects of Tamoxifen and oestrogen on histologic and radiographic changes in high and low MD tissues maintained within the biochamber model. High and low MD human tissues were precisely sampled under radiographic guidance from prophylactic mastectomy fresh specimens of high-risk women, then inserted into separate vascularized murine biochambers. The murine hosts were concurrently implanted with Tamoxifen, oestrogen or placebo pellets, and the high and low MD biochamber tissues maintained in the murine host environment for 3 months, before the high and low MD biochamber tissues were harvested for histologic and radiographic analyses. The radiographic density of high MD tissue maintained in murine biochambers was decreased in Tamoxifen-treated mice compared to oestrogen-treated mice (p = 0.02). Tamoxifen treatment of high MD tissue in SCID mice led to a decrease in stromal (p = 0.009), and an increase in adipose (p = 0.023) percent areas, compared to placebo-treated mice. No histologic or radiographic differences were observed in low MD biochamber tissue with any treatment. High MD biochamber tissues maintained in mice implanted with Tamoxifen, oestrogen or placebo pellets had dynamic and measurable histologic compositional and radiographic changes. This further validates the dynamic nature of the MD xenograft model, and suggests the biochamber model may be useful for assessing the underlying molecular pathways of Tamoxifen-reduced MD, and in testing of other

  3. 3D bioprinting for engineering complex tissues.

    PubMed

    Mandrycky, Christian; Wang, Zongjie; Kim, Keekyoung; Kim, Deok-Ho

    2016-01-01

    Bioprinting is a 3D fabrication technology used to precisely dispense cell-laden biomaterials for the construction of complex 3D functional living tissues or artificial organs. While still in its early stages, bioprinting strategies have demonstrated their potential use in regenerative medicine to generate a variety of transplantable tissues, including skin, cartilage, and bone. However, current bioprinting approaches still have technical challenges in terms of high-resolution cell deposition, controlled cell distributions, vascularization, and innervation within complex 3D tissues. While no one-size-fits-all approach to bioprinting has emerged, it remains an on-demand, versatile fabrication technique that may address the growing organ shortage as well as provide a high-throughput method for cell patterning at the micrometer scale for broad biomedical engineering applications. In this review, we introduce the basic principles, materials, integration strategies and applications of bioprinting. We also discuss the recent developments, current challenges and future prospects of 3D bioprinting for engineering complex tissues. Combined with recent advances in human pluripotent stem cell technologies, 3D-bioprinted tissue models could serve as an enabling platform for high-throughput predictive drug screening and more effective regenerative therapies. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Decellularized musculofascial extracellular matrix for tissue engineering

    PubMed Central

    Wang, Lina; Johnson, Joshua A; Chang, David W.; Zhang, Qixu

    2016-01-01

    Ideal scaffolds that represent native extracellular matrix (ECM) properties of musculofascial tissues have great importance in musculofascial tissue engineering. However, detailed characterization of musculofascial tissues’ ECM (particularly, of fascia) from large animals is still lacking. In this study, we developed a decellularization protocol for processing pig composite musculofascial tissues. Decellularized muscle (D-muscle) and decellularized fascia (D-fascia), which are two important components of decellularized musculofascial extracellular matrix (DMM), were comprehensively characterized. D-muscle and D-fascia retained intact three-dimensional architecture, strong mechanical properties, and bioactivity of compositions such as collagen, laminin, glycosaminoglycan, and vascular endothelial growth factor. D-muscle and D-fascia provided a compatible niche for human adipose-derived stem cell integration and proliferation. Heterotopic and orthotopic implantation of D-muscle and D-fascia in a rodent model further proved their biocompatibility and myogenic properties during the remodeling process. The differing characteristics of D-muscle from D-fascia (e.g., D-muscle’s strong pro-angiogenic and pro-myogenic properties vs. D-fascia’s strong mechanical properties) indicate different clinical application opportunities of D-muscle vs. D-fascia scaffolds. DMM comprising muscle and fascia ECM as a whole unit can thus provide not only a clinically translatable platform for musculofascial tissue repair and regeneration but also a useful standard for scaffold design in musculofascial tissue engineering. PMID:23347834

  5. 3D Bioprinting for Engineering Complex Tissues

    PubMed Central

    Mandrycky, Christian; Wang, Zongjie; Kim, Keekyoung; Kim, Deok-Ho

    2016-01-01

    Bioprinting is a 3D fabrication technology used to precisely dispense cell-laden biomaterials for the construction of complex 3D functional living tissues or artificial organs. While still in its early stages, bioprinting strategies have demonstrated their potential use in regenerative medicine to generate a variety of transplantable tissues, including skin, cartilage, and bone. However, current bioprinting approaches still have technical challenges in terms of high-resolution cell deposition, controlled cell distributions, vascularization, and innervation within complex 3D tissues. While no one-size-fits-all approach to bioprinting has emerged, it remains an on-demand, versatile fabrication technique that may address the growing organ shortage as well as provide a high-throughput method for cell patterning at the micrometer scale for broad biomedical engineering applications. In this review, we introduce the basic principles, materials, integration strategies and applications of bioprinting. We also discuss the recent developments, current challenges and future prospects of 3D bioprinting for engineering complex tissues. Combined with recent advances in human pluripotent stem cell technologies, 3D-bioprinted tissue models could serve as an enabling platform for high-throughput predictive drug screening and more effective regenerative therapies. PMID:26724184

  6. Interface tissue engineering: next phase in musculoskeletal tissue repair.

    PubMed

    Sahoo, Sambit; Teh, Thomas Kh; He, Pengfei; Toh, Siew Lok; Goh, James Ch

    2011-05-01

    Increasing incidence of musculoskeletal injuries coupled with limitations in the current treatment options have necessitated tissue engineering and regenerative medicine- based approaches. Moving forward from engineering isolated musculoskeletal tissues, research strategies are now being increasingly focused on repairing and regenerating the interfaces between dissimilar musculoskeletal tissues with the aim to achieve seamless integration of engineered musculoskeletal tissues. This article reviews the state-of-the-art in the tissue engineering of musculoskeletal tissue interfaces with a focus on Singapore's contribution in this emerging field. Various biomimetic scaffold and cellbased strategies, the use of growth factors, gene therapy and mechanical loading, as well as animal models for functional validation of the tissue engineering strategies are discussed.

  7. Cell sheet engineering for heart tissue repair.

    PubMed

    Masuda, Shinako; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

    2008-01-14

    Recently, myocardial tissue engineering has emerged as one of the most promising therapies for patients suffering from severe heart failure. Nevertheless, conventional methods in tissue engineering involving the seeding of cells into biodegradable scaffolds have intrinsic shortcomings, such as inflammatory reactions and fibrous tissue formation caused by scaffold degradation. On the other hand, we have developed cell sheet engineering as scaffoldless tissue engineering, and applied it for myocardial tissue engineering. Using temperature-responsive culture surfaces, cells can be harvested as intact sheets and cell-dense thick tissues are constructed by layering these cell sheets. Myocardial cell sheets non-invasively harvested from temperature-responsive culture surfaces are successfully layered, resulting in electrically communicative 3-dimensional (3-D) cardiac constructs. Transplantation of cell sheets onto damaged hearts improved heart function in several animal models. In this review, we summarize the development of myocardial tissue engineering using cell sheets harvested from temperature-responsive culture surfaces and discuss about future views.

  8. Construction of engineering adipose-like tissue in vivo utilizing human insulin gene-modified umbilical cord mesenchymal stromal cells with silk fibroin 3D scaffolds.

    PubMed

    Li, Shi-Long; Liu, Yi; Hui, Ling

    2015-12-01

    We evaluated the use of a combination of human insulin gene-modified umbilical cord mesenchymal stromal cells (hUMSCs) with silk fibroin 3D scaffolds for adipose tissue engineering. In this study hUMSCs were isolated and cultured. HUMSCs infected with Ade-insulin-EGFP were seeded in fibroin 3D scaffolds with uniform 50-60 µm pore size. Silk fibroin scaffolds with untransfected hUMSCs were used as control. They were cultured for 4 days in adipogenic medium and transplanted under the dorsal skins of female Wistar rats after the hUMSCs had been labelled with chloromethylbenzamido-1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (CM-Dil). Macroscopical impression, fluorescence observation, histology and SEM were used for assessment after transplantation at 8 and 12 weeks. Macroscopically, newly formed adipose tissue was observed in the experimental group and control group after 8 and 12 weeks. Fluorescence observation supported that the formed adipose tissue originated from seeded hUMSCs rather than from possible infiltrating perivascular tissue. Oil red O staining of newly formed tissue showed that there was substantially more tissue regeneration in the experimental group than in the control group. SEM showed that experimental group cells had more fat-like cells, whose volume was larger than that of the control group, and degradation of the silk fibroin scaffold was greater under SEM observation. This study provides significant evidence that hUMSCs transfected by adenovirus vector have good compatibility with silk fibroin scaffold, and adenoviral transfection of the human insulin gene can be used for the construction of tissue-engineered adipose.

  9. Perivascular cells and tissue engineering: Current applications and untapped potential.

    PubMed

    Avolio, Elisa; Alvino, Valeria V; Ghorbel, Mohamed T; Campagnolo, Paola

    2017-03-01

    The recent development of tissue engineering provides exciting new perspectives for the replacement of failing organs and the repair of damaged tissues. Perivascular cells, including vascular smooth muscle cells, pericytes and other tissue specific populations residing around blood vessels, have been isolated from many organs and are known to participate to the in situ repair process and angiogenesis. Their potential has been harnessed for cell therapy of numerous pathologies; however, in this Review we will discuss the potential of perivascular cells in the development of tissue engineering solutions for healthcare. We will examine their application in the engineering of vascular grafts, cardiac patches and bone substitutes as well as other tissue engineering applications and we will focus on their extensive use in the vascularization of engineered constructs. Additionally, we will discuss the emerging potential of human pericytes for the development of efficient, vascularized and non-immunogenic engineered constructs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  10. [Vaginal reconstruction with tissue engineering technology].

    PubMed

    Zhang, Mingle; Li, Yachai; Huang, Xianghua

    2011-07-01

    To summarize the research and development of vaginal reconstruction with tissue engineering technology. The recent literature concerning vaginal reconstruction with tissue engineering technology at home and abroad was extensively reviewed and the research and development were summarized. Tissue engineering provides an ideal material as the inner tissue in vaginoplasty. The reconstructed tissue closely resembles native vaginal tissue in the cellular organization and physical properties. The clinical use of the tissue engineered vagina in vaginoplasty can not be harmful to an organism, and the neovagina has sufficient length and depth. However, the long-term follow-up is needed. Vaginal reconstruction with tissue engineering technology may have good application prospects, but further research is required.

  11. Advancing nasal reconstructive surgery: the application of tissue engineering technology.

    PubMed

    Oseni, Adelola; Crowley, Claire; Lowdell, Mark; Birchall, Martin; Butler, Peter E; Seifalian, Alexander M

    2012-11-01

    Cartilage tissue engineering is a rapidly progressing area of regenerative medicine with advances in cell biology and scaffold engineering constantly being investigated. Many groups are now capable of making neocartilage constructs with some level of morphological, biochemical, and histological likeness to native human cartilage tissues. The application of this useful technology in articular cartilage repair is well described in the literature; however, few studies have evaluated its application in head and neck reconstruction. Although there are many studies on auricular cartilage tissue engineering, there are few studies regarding cartilage tissue engineering for complex nasal reconstruction. This study therefore highlighted the challenges involved with nasal reconstruction, with special focus on nasal cartilage tissue, and examined how advancements made in cartilage tissue engineering research could be applied to improve the clinical outcomes of total nasal reconstructive surgery. Copyright © 2011 John Wiley & Sons, Ltd.

  12. Silk film biomaterials for cornea tissue engineering.

    PubMed

    Lawrence, Brian D; Marchant, Jeffrey K; Pindrus, Mariya A; Omenetto, Fiorenzo G; Kaplan, David L

    2009-03-01

    Biomaterials for corneal tissue engineering must demonstrate several critical features for potential utility in vivo, including transparency, mechanical integrity, biocompatibility and slow biodegradation. Silk film biomaterials were designed and characterized to meet these functional requirements. Silk protein films were used in a biomimetic approach to replicate corneal stromal tissue architecture. The films were 2 microm thick to emulate corneal collagen lamellae dimensions, and were surface patterned to guide cell alignment. To enhance trans-lamellar diffusion of nutrients and to promote cell-cell interaction, pores with 0.5-5.0 microm diameters were introduced into the silk films. Human and rabbit corneal fibroblast proliferation, alignment and corneal extracellular matrix expression on these films in both 2D and 3D cultures were demonstrated. The mechanical properties, optical clarity and surface patterned features of these films, combined with their ability to support corneal cell functions suggest that this new biomaterial system offers important potential benefits for corneal tissue regeneration.

  13. Scleraxis-overexpressed human embryonic stem cell-derived mesenchymal stem cells for tendon tissue engineering with knitted silk-collagen scaffold.

    PubMed

    Chen, Xiao; Yin, Zi; Chen, Jia-Lin; Liu, Huan-Huan; Shen, Wei-Liang; Fang, Zhi; Zhu, Ting; Ji, Junfeng; Ouyang, Hong-Wei; Zou, Xiao-Hui

    2014-06-01

    Despite our previous study that demonstrates that human embryonic stem cells (hESCs) can be used as seed cells for tendon tissue engineering after stepwise induction, suboptimal tendon regeneration implies that a new strategy needs to be developed for tendon repair. We investigated whether overexpression of the tendon-specific transcription factor scleraxis (SCX) in hESC-derived mesenchymal stem cells (hESC-MSCs) together with knitted silk-collagen sponge scaffold could promote tendon regeneration. hESCs were initially differentiated into MSCs and then engineered with scleraxis (SCX+hESC-MSCs). Engineered tendons were constructed with SCX+hESC-MSCs and a knitted silk-collagen sponge scaffold and then mechanical stress was applied. SCX elevated tendon gene expression in hESC-MSCs and concomitantly attenuated their adipogenic and chondrogenic potential. Mechanical stress further augmented the expression of tendon-specific genes in SCX+hESC-MSC-engineered tendon. Moreover, in vivo mechanical stimulation promoted the alignment of cells and increased the diameter of collagen fibers after ectopic transplantation. In the in vivo tendon repair model, the SCX+hESC-MSC-engineered tendon enhanced the regeneration process as shown by histological scores and superior mechanical performance compared with control cells, especially at early stages. Our study offers new evidence concerning the roles of SCX in tendon differentiation and regeneration. We demonstrated a novel strategy of combining hESCs, genetic engineering, and tissue-engineering principles for tendon regeneration, which are important for the future application of hESCs and silk scaffolds for tendon repair.

  14. Valve Tissue Engineering with Living Absorbable Threads.

    PubMed

    Liberski, Albert Ryszard; Raynaud, Christophe Michel; Ayad, Nadia; Wojciechowska, Dorota; Sathappan, Abbirami

    2017-05-01

    Tissue engineering (TE) depends on the population of scaffolds with appropriate cells, arranged in a specific physiological direction using a variety of techniques. Here, a novel technique of creating "living threads" is described based on thin (poly(ε-caprolactone) fibers of different diameters (23-243 μm). The fibers readily attract human mesenchymal stem cells (MSCs), which are firmly adhered. These versatile fibers can be used to produce dimensional shapes identical in shape to the cup-like structure of a normal human valve, while preserving the specific orientation of both the cells and the fibers. The MSCs on leaflets and the cells cultured in flask shown similar epitopes expression when analyzed by fluorescence activated cell sorting. Together, these characteristics have important functional implications as living absorbable fibers can be a valuable resource in TE of living tissues, including heart valves. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Tissue engineering of cultured skin substitutes.

    PubMed

    Horch, Raymund E; Kopp, Jürgen; Kneser, Ulrich; Beier, Justus; Bach, Alexander D

    2005-01-01

    Skin replacement has been a challenging task for surgeons ever since the introduction of skin grafts by Reverdin in 1871. Recently, skin grafting has evolved from the initial autograft and allograft preparations to biosynthetic and tissue-engineered living skin replacements. This has been fostered by the dramatically improved survival rates of major burns where the availability of autologous normal skin for grafting has become one of the limiting factors. The ideal properties of a temporary and a permanent skin substitute have been well defined. Tissue-engineered skin replacements: cultured autologous keratinocyte grafts, cultured allogeneic keratinocyte grafts, autologous/allogeneic composites, acellular biological matrices, and cellular matrices including such biological substances as fibrin sealant and various types of collagen, hyaluronic acid etc. have opened new horizons to deal with such massive skin loss. In extensive burns it has been shown that skin substitution with cultured grafts can be a life-saving measure where few alternatives exist. Future research will aim to create skin substitutes with cultured epidermis that under appropriate circumstances may provide a wound cover that could be just as durable and esthetically acceptable as conventional split-thickness skin grafts. Genetic manipulation may in addition enhance the performance of such cultured skin substitutes. If cell science, molecular biology, genetic engineering, material science and clinical expertise join their efforts to develop optimized cell culture techniques and synthetic or biological matrices then further technical advances might well lead to the production of almost skin like new tissue-engineered human skin products resembling natural human skin.

  16. Tissue engineering skeletal muscle for orthopaedic applications

    NASA Technical Reports Server (NTRS)

    Payumo, Francis C.; Kim, Hyun D.; Sherling, Michael A.; Smith, Lee P.; Powell, Courtney; Wang, Xiao; Keeping, Hugh S.; Valentini, Robert F.; Vandenburgh, Herman H.

    2002-01-01

    With current technology, tissue-engineered skeletal muscle analogues (bioartificial muscles) generate too little active force to be clinically useful in orthopaedic applications. They have been engineered genetically with numerous transgenes (growth hormone, insulinlike growth factor-1, erythropoietin, vascular endothelial growth factor), and have been shown to deliver these therapeutic proteins either locally or systemically for months in vivo. Bone morphogenetic proteins belonging to the transforming growth factor-beta superfamily are osteoinductive molecules that drive the differentiation pathway of mesenchymal cells toward the chondroblastic or osteoblastic lineage, and stimulate bone formation in vivo. To determine whether skeletal muscle cells endogenously expressing bone morphogenetic proteins might serve as a vehicle for systemic bone morphogenetic protein delivery in vivo, proliferating skeletal myoblasts (C2C12) were transduced with a replication defective retrovirus containing the gene for recombinant human bone morphogenetic protein-6 (C2BMP-6). The C2BMP-6 cells constitutively expressed recombinant human bone morphogenetic protein-6 and synthesized bioactive recombinant human bone morphogenetic protein-6, based on increased alkaline phosphatase activity in coincubated mesenchymal cells. C2BMP-6 cells did not secrete soluble, bioactive recombinant human bone morphogenetic protein-6, but retained the bioactivity in the cell layer. Therefore, genetically-engineered skeletal muscle cells might serve as a platform for long-term delivery of osteoinductive bone morphogenetic proteins locally.

  17. Multiscale tissue engineering for liver reconstruction.

    PubMed

    Sudo, Ryo

    2014-01-01

    The liver is a target of in vitro tissue engineering despite its capability to regenerate in vivo. The construction of liver tissues in vitro remains challenging. In this review, conventional 3D cultures of hepatocytes are first discussed. Recent advances in the 3D culturing of liver cells are then summarized in the context of in vitro liver tissue reconstruction at the micro- and macroscales. The application of microfluidics technology to liver tissue engineering has been introduced as a bottom-up approach performed at the microscale, whereas whole-organ bioengineering technology was introduced as a top-down approach performed at the macroscale. Mesoscale approaches are also discussed in considering the integration of micro- and macroscale approaches. Multiple parallel multiscale liver tissue engineering studies are ongoing; however, no tissue-engineered liver that is appropriate for clinical use has yet been realized. The integration of multiscale tissue engineering studies is essential for further understanding of liver reconstruction strategies.

  18. Imaging Strategies for Tissue Engineering Applications

    PubMed Central

    Nam, Seung Yun; Ricles, Laura M.; Suggs, Laura J.

    2015-01-01

    Tissue engineering has evolved with multifaceted research being conducted using advanced technologies, and it is progressing toward clinical applications. As tissue engineering technology significantly advances, it proceeds toward increasing sophistication, including nanoscale strategies for material construction and synergetic methods for combining with cells, growth factors, or other macromolecules. Therefore, to assess advanced tissue-engineered constructs, tissue engineers need versatile imaging methods capable of monitoring not only morphological but also functional and molecular information. However, there is no single imaging modality that is suitable for all tissue-engineered constructs. Each imaging method has its own range of applications and provides information based on the specific properties of the imaging technique. Therefore, according to the requirements of the tissue engineering studies, the most appropriate tool should be selected among a variety of imaging modalities. The goal of this review article is to describe available biomedical imaging methods to assess tissue engineering applications and to provide tissue engineers with criteria and insights for determining the best imaging strategies. Commonly used biomedical imaging modalities, including X-ray and computed tomography, positron emission tomography and single photon emission computed tomography, magnetic resonance imaging, ultrasound imaging, optical imaging, and emerging techniques and multimodal imaging, will be discussed, focusing on the latest trends of their applications in recent tissue engineering studies. PMID:25012069

  19. Imaging strategies for tissue engineering applications.

    PubMed

    Nam, Seung Yun; Ricles, Laura M; Suggs, Laura J; Emelianov, Stanislav Y

    2015-02-01

    Tissue engineering has evolved with multifaceted research being conducted using advanced technologies, and it is progressing toward clinical applications. As tissue engineering technology significantly advances, it proceeds toward increasing sophistication, including nanoscale strategies for material construction and synergetic methods for combining with cells, growth factors, or other macromolecules. Therefore, to assess advanced tissue-engineered constructs, tissue engineers need versatile imaging methods capable of monitoring not only morphological but also functional and molecular information. However, there is no single imaging modality that is suitable for all tissue-engineered constructs. Each imaging method has its own range of applications and provides information based on the specific properties of the imaging technique. Therefore, according to the requirements of the tissue engineering studies, the most appropriate tool should be selected among a variety of imaging modalities. The goal of this review article is to describe available biomedical imaging methods to assess tissue engineering applications and to provide tissue engineers with criteria and insights for determining the best imaging strategies. Commonly used biomedical imaging modalities, including X-ray and computed tomography, positron emission tomography and single photon emission computed tomography, magnetic resonance imaging, ultrasound imaging, optical imaging, and emerging techniques and multimodal imaging, will be discussed, focusing on the latest trends of their applications in recent tissue engineering studies.

  20. Fabrication of novel high surface area mushroom gilled fibers and their effects on human adipose derived stem cells under pulsatile fluid flow for tissue engineering applications.

    PubMed

    Tuin, Stephen A; Pourdeyhimi, Behnam; Loboa, Elizabeth G

    2016-05-01

    The fabrication and characterization of novel high surface area hollow gilled fiber tissue engineering scaffolds via industrially relevant, scalable, repeatable, high speed, and economical nonwoven carding technology is described. Scaffolds were validated as tissue engineering scaffolds using human adipose derived stem cells (hASC) exposed to pulsatile fluid flow (PFF). The effects of fiber morphology on the proliferation and viability of hASC, as well as effects of varied magnitudes of shear stress applied via PFF on the expression of the early osteogenic gene marker runt related transcription factor 2 (RUNX2) were evaluated. Gilled fiber scaffolds led to a significant increase in proliferation of hASC after seven days in static culture, and exhibited fewer dead cells compared to pure PLA round fiber controls. Further, hASC-seeded scaffolds exposed to 3 and 6dyn/cm(2) resulted in significantly increased mRNA expression of RUNX2 after one hour of PFF in the absence of soluble osteogenic induction factors. This is the first study to describe a method for the fabrication of high surface area gilled fibers and scaffolds. The scalable manufacturing process and potential fabrication across multiple nonwoven and woven platforms makes them promising candidates for a variety of applications that require high surface area fibrous materials. We report here for the first time the successful fabrication of novel high surface area gilled fiber scaffolds for tissue engineering applications. Gilled fibers led to a significant increase in proliferation of human adipose derived stem cells after one week in culture, and a greater number of viable cells compared to round fiber controls. Further, in the absence of osteogenic induction factors, gilled fibers led to significantly increased mRNA expression of an early marker for osteogenesis after exposure to pulsatile fluid flow. This is the first study to describe gilled fiber fabrication and their potential for tissue engineering

  1. Soft tissue engineering in craniomaxillofacial surgery

    PubMed Central

    Kim, Roderick Y; Fasi, Anthony C; Feinberg, Stephen E

    2014-01-01

    Craniofacial soft tissue reconstruction may be required following trauma, tumor resection, and to repair congenital deformities. Recent advances in the field of tissue engineering have significantly widened the reconstructive armamentarium of the surgeon. The successful identification and combination of tissue engineering, scaffold, progenitor cells, and physiologic signaling molecules has enabled the surgeon to design, recreate the missing tissue in its near natural form. This has resolved the issues like graft rejection, wound dehiscence, or poor vascularity. Successfully reconstructed tissue through soft tissue engineering protocols would help surgeon to restore the form and function of the lost tissue in its originality. This manuscript intends to provide a glimpse of the basic principle of tissue engineering, contemporary, and future direction of this field as applied to craniofacial surgery. PMID:24987591

  2. Nasal chondrocyte-based engineered autologous cartilage tissue for repair of articular cartilage defects: an observational first-in-human trial.

    PubMed

    Mumme, Marcus; Barbero, Andrea; Miot, Sylvie; Wixmerten, Anke; Feliciano, Sandra; Wolf, Francine; Asnaghi, Adelaide M; Baumhoer, Daniel; Bieri, Oliver; Kretzschmar, Martin; Pagenstert, Geert; Haug, Martin; Schaefer, Dirk J; Martin, Ivan; Jakob, Marcel

    2016-10-22

    Articular cartilage injuries have poor repair capacity, leading to progressive joint damage, and cannot be restored predictably by either conventional treatments or advanced therapies based on implantation of articular chondrocytes. Compared with articular chondrocytes, chondrocytes derived from the nasal septum have superior and more reproducible capacity to generate hyaline-like cartilage tissues, with the plasticity to adapt to a joint environment. We aimed to assess whether engineered autologous nasal chondrocyte-based cartilage grafts allow safe and functional restoration of knee cartilage defects. In a first-in-human trial, ten patients with symptomatic, post-traumatic, full-thickness cartilage lesions (2-6 cm(2)) on the femoral condyle or trochlea were treated at University Hospital Basel in Switzerland. Chondrocytes isolated from a 6 mm nasal septum biopsy specimen were expanded and cultured onto collagen membranes to engineer cartilage grafts (30 × 40 × 2 mm). The engineered tissues were implanted into the femoral defects via mini-arthrotomy and assessed up to 24 months after surgery. Primary outcomes were feasibility and safety of the procedure. Secondary outcomes included self-assessed clinical scores and MRI-based estimation of morphological and compositional quality of the repair tissue. This study is registered with ClinicalTrials.gov, number NCT01605201. The study is ongoing, with an approved extension to 25 patients. For every patient, it was feasible to manufacture cartilaginous grafts with nasal chondrocytes embedded in an extracellular matrix rich in glycosaminoglycan and type II collagen. Engineered tissues were stable through handling with forceps and could be secured in the injured joints. No adverse reactions were recorded and self-assessed clinical scores for pain, knee function, and quality of life were improved significantly from before surgery to 24 months after surgery. Radiological assessments indicated variable degrees of

  3. Nanomaterials for Cardiac Myocyte Tissue Engineering.

    PubMed

    Amezcua, Rodolfo; Shirolkar, Ajay; Fraze, Carolyn; Stout, David A

    2016-07-19

    Since their synthesizing introduction to the research community, nanomaterials have infiltrated almost every corner of science and engineering. Over the last decade, one such field has begun to look at using nanomaterials for beneficial applications in tissue engineering, specifically, cardiac tissue engineering. During a myocardial infarction, part of the cardiac muscle, or myocardium, is deprived of blood. Therefore, the lack of oxygen destroys cardiomyocytes, leaving dead tissue and possibly resulting in the development of arrhythmia, ventricular remodeling, and eventual heart failure. Scarred cardiac muscle results in heart failure for millions of heart attack survivors worldwide. Modern cardiac tissue engineering research has developed nanomaterial applications to combat heart failure, preserve normal heart tissue, and grow healthy myocardium around the infarcted area. This review will discuss the recent progress of nanomaterials for cardiovascular tissue engineering applications through three main nanomaterial approaches: scaffold designs, patches, and injectable materials.

  4. Nanomaterials for Cardiac Myocyte Tissue Engineering

    PubMed Central

    Amezcua, Rodolfo; Shirolkar, Ajay; Fraze, Carolyn; Stout, David A.

    2016-01-01

    Since their synthesizing introduction to the research community, nanomaterials have infiltrated almost every corner of science and engineering. Over the last decade, one such field has begun to look at using nanomaterials for beneficial applications in tissue engineering, specifically, cardiac tissue engineering. During a myocardial infarction, part of the cardiac muscle, or myocardium, is deprived of blood. Therefore, the lack of oxygen destroys cardiomyocytes, leaving dead tissue and possibly resulting in the development of arrhythmia, ventricular remodeling, and eventual heart failure. Scarred cardiac muscle results in heart failure for millions of heart attack survivors worldwide. Modern cardiac tissue engineering research has developed nanomaterial applications to combat heart failure, preserve normal heart tissue, and grow healthy myocardium around the infarcted area. This review will discuss the recent progress of nanomaterials for cardiovascular tissue engineering applications through three main nanomaterial approaches: scaffold designs, patches, and injectable materials. PMID:28335261

  5. Tissue engineering: current state of clinical application.

    PubMed

    Fauza, Dario O

    2003-06-01

    Despite several, mostly isolated successes, few controlled, prospective trials have yet validated clinical tissue engineering applications. Although this may, at least in part, be explained by the very young age of this field, tissue engineering involves the need for an elaborate and expensive infrastructure, not to mention qualified personnel. This translates into an inherent difficulty in establishing multicenter trials. Moreover, companies mostly devoted to tissue engineering have yet to prove themselves economically viable. On the other hand, although very few engineered tissues have been approved by the US Food and Drug Administration (FDA), more than 70 companies have recently been developing new products. Many challenges are yet to be overcome before "off-the-shelf" tissues can be offered commercially. Nevertheless, given the scientific promise, potential social impact, and young age of the field, many believe that it should be only a matter of time until tissue engineering reaches the mainstream of surgical practice.

  6. Meniscus tissue engineering using a novel combination of electrospun scaffolds and human meniscus cells embedded within an extracellular matrix hydrogel.

    PubMed

    Baek, Jihye; Chen, Xian; Sovani, Sujata; Jin, Sungho; Grogan, Shawn P; D'Lima, Darryl D

    2015-04-01

    Meniscus injury and degeneration have been linked to the development of secondary osteoarthritis (OA). Therapies that successfully repair or replace the meniscus are, therefore, likely to prevent or delay OA progression. We investigated the novel approach of building layers of aligned polylactic acid (PLA) electrospun (ES) scaffolds with human meniscus cells embedded in extracellular matrix (ECM) hydrogel to lead to formation of neotissues that resemble meniscus-like tissue. PLA ES scaffolds with randomly oriented or aligned fibers were seeded with human meniscus cells derived from vascular or avascular regions. Cell viability, cell morphology, and gene expression profiles were monitored via confocal microscopy, scanning electron microscopy (SEM), and real-time polymerase chain reaction (PCR), respectively. Seeded scaffolds were used to produce multilayered constructs and were examined via histology and immunohistochemistry. Morphology and mechanical properties of PLA scaffolds (with and without cells) were influenced by fiber direction of the scaffolds. Both PLA scaffolds supported meniscus tissue formation with increased COL1A1, SOX9, and COMP, yet no difference in gene expression was found between random and aligned PLA scaffolds. Overall, ES materials, which possess mechanical strength of meniscus and can support neotissue formation, show potential for use in cell-based meniscus regeneration strategies.

  7. Meniscus Tissue Engineering Using a Novel Combination of Electrospun Scaffolds and Human Meniscus Cells Embedded within an Extracellular Matrix Hydrogel

    PubMed Central

    Baek, Jihye; Chen, Xian; Sovani, Sujata; Jin, Sungho; Grogan, Shawn P; D’Lima, Darryl D

    2015-01-01

    Meniscus injury and degeneration have been linked to the development of secondary osteoarthritis (OA). Therapies that successfully repair or replace the meniscus are therefore likely to prevent or delay OA progression. We investigated the novel approach of building layers of aligned polylactic acid (PLA) electrospun (ES) scaffolds with human meniscus cells embedded in extracellular matrix (ECM) hydrogel to lead to formation of neotissues that resemble meniscus-like tissue. PLA ES scaffolds with randomly oriented or aligned fibers were seeded with human meniscus cells derived from vascular or avascular regions. Cell viability, cell morphology, and gene expression profiles were monitored via confocal microscopy, scanning electron microscopy (SEM), and real-time PCR, respectively. Seeded scaffolds were used to produce multilayered constructs and were examined via histology and immunohistochemistry. Morphology and mechanical properties of PLA scaffolds (with and without cells) were influenced by fiber direction of the scaffolds. Both PLA scaffolds supported meniscus tissue formation with increased COL1A1, SOX9, COMP, yet no difference in gene expression was found between random and aligned PLA scaffolds. Overall, ES materials, which possess mechanical strength of meniscus and can support neotissue formation, show potential for use in cell-based meniscus regeneration strategies. PMID:25640671

  8. Disparate companions: tissue engineering meets cancer research.

    PubMed

    Tilkorn, Daniel J; Lokmic, Zerina; Chaffer, Christine L; Mitchell, Geraldine M; Morrison, Wayne A; Thompson, Erik W

    2010-01-01

    Recreating an environment that supports and promotes fundamental homeostatic mechanisms is a significant challenge in tissue engineering. Optimizing cell survival, proliferation, differentiation, apoptosis and angiogenesis, and providing suitable stromal support and signalling cues are keys to successfully generating clinically useful tissues. Interestingly, those components are often subverted in the cancer setting, where aberrant angiogenesis, cellular proliferation, cell signalling and resistance to apoptosis drive malignant growth. In contrast to tissue engineering, identifying and inhibiting those pathways is a major challenge in cancer research. The recent discovery of adult tissue-specific stem cells has had a major impact on both tissue engineering and cancer research. The unique properties of these cells and their role in tissue and organ repair and regeneration hold great potential for engineering tissue-specific constructs. The emerging body of evidence implicating stem cells and progenitor cells as the source of oncogenic transformation prompts caution when using these cells for tissue-engineering purposes. While tissue engineering and cancer research may be considered as opposed fields of research with regard to their proclaimed goals, the compelling overlap in fundamental pathways underlying these processes suggests that cross-disciplinary research will benefit both fields. In this review article, tissue engineering and cancer research are brought together and explored with regard to discoveries that may be of mutual benefit.

  9. Initial evaluation of the use of USPIO cell labeling and noninvasive MR monitoring of human tissue-engineered vascular grafts in vivo.

    PubMed

    Nelson, G N; Roh, J D; Mirensky, T L; Wang, Y; Yi, T; Tellides, G; Pober, J S; Shkarin, P; Shapiro, E M; Saltzman, W M; Papademetris, X; Fahmy, T M; Breuer, C K

    2008-11-01

    This pilot study examines noninvasive MR monitoring of tissue-engineered vascular grafts (TEVGs) in vivo using cells labeled with iron oxide nanoparticles. Human aortic smooth muscle cells (hASMCs) were labeled with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. The labeled hASMCs, along with human aortic endothelial cells, were incorporated into eight TEVGs and were then surgically implanted as aortic interposition grafts in a C.B-17 SCID/bg mouse host. USPIO-labeled hASMCs persisted in the grafts throughout a 3 wk observation period and allowed noninvasive MR imaging of the human TEVGs for real-time, serial monitoring of hASMC retention. This study demonstrates the feasibility of applying noninvasive imaging techniques for evaluation of in vivo TEVG performance.

  10. Keratoconus: Tissue Engineering and Biomaterials

    PubMed Central

    Karamichos, Dimitrios; Hjortdal, Jesper

    2014-01-01

    Keratoconus (KC) is a bilateral, asymmetric, corneal disorder that is characterized by progressive thinning, steepening, and potential scarring. The prevalence of KC is stated to be 1 in 2000 persons worldwide; however, numbers vary depending on size of the study and regions. KC appears more often in South Asian, Eastern Mediterranean, and North African populations. The cause remains unknown, although a variety of factors have been considered. Genetics, cellular, and mechanical changes have all been reported; however, most of these studies have proven inconclusive. Clearly, the major problem here, like with any other ocular disease, is quality of life and the threat of vision loss. While most KC cases progress until the third or fourth decade, it varies between individuals. Patients may experience periods of several months with significant changes followed by months or years of no change, followed by another period of rapid changes. Despite the major advancements, it is still uncertain how to treat KC at early stages and prevent vision impairment. There are currently limited tissue engineering techniques and/or “smart” biomaterials that can help arrest the progression of KC. This review will focus on current treatments and how biomaterials may hold promise for the future. PMID:25215423

  11. [Electrospinning technology in tissue engineering scaffolds].

    PubMed

    Li, Haoyi; Liu, Yong; He, Xuetao; Ding, Yumei; Yan, Hua; Xie, Pengcheng; Yang, Weimin

    2012-01-01

    Tissue engineering technology provides a new method to repair ill tissue and worn-out organs. In tissue engineering, scaffolds play an important role in supporting cell growth, inducing tissue regeneration, controlling tissue structure and releasing active factor. In the last decade, electrospinning technology developed rapidly and opened vast application fields for scaffolds. In this review, we summarized the technological conditions of electrospinning for scaffolds, the study of electrospun fiber scaffolds applied in tissue cell cultivation, and some new directions of electrospinning technology for scaffolds. We also addressed development directions of electrospinning research for scaffolds.

  12. [Chondrocyte mecanobiology. Application in cartilage tissue engineering].

    PubMed

    Stoltz, Jean François; Netter, Patrick; Huselstein, Céline; de Isla, Natalia; Wei Yang, Jing; Muller, Sylvaine

    2005-11-01

    Cartilage is a hydrated connective tissue that withstands and distributes mechanical forces within joints. Chondrocytes utilize mechanical signals to maintain cartilaginous tissue homeostasis. They regulate their metabolic activity through complex biological and biophysical interactions with the extracellular matrix (ECM). Some mechanotransduction mechanisms are known, while many others no doubt remain to be discovered. Various aspects of chondrocyte mechanobiology have been applied to tissue engineering, with the creation of replacement tissue in vitro from bioresorbable or non-bioresorbable scaffolds and harvested cells. The tissues are maintained in a near-physiologic mechanical and biochemical environment. This paper is an overview of both chondrocyte mechanobiology and cartilage tissue engineering

  13. Bone Tissue Engineering with Premineralized Silk Scaffolds

    PubMed Central

    Kim, Hyeon Joo; Kim, Ung-Jin; Kim, Hyun Suk; Li, Chunmei; Wada, Masahisa; Leisk, Gary G.; Kaplan, David L.

    2009-01-01

    Silks fibroin biomaterials are being explored as novel protein-based systems for cell and tissue culture. In the present study, biomimetic growth of calcium phosphate on porous silk fibroin polymeric scaffolds was explored to generate organic/inorganic composites as scaffolds for bone tissue engineering. Aqueous-derived silk fibroin scaffolds were prepared with the addition of polyaspartic acid during processing, followed by the controlled deposition of calcium phosphate by exposure to CaCl2 and Na2HPO4. These mineralized protein-composite scaffolds were subsequently seeded with human bone marrow stem cells (hMSC) and cultured in vitro for 6 weeks under osteogenic conditions with or without BMP-2. The extent of osteoconductivity was assessed by cell numbers, alkaline phosphatase and calcium deposition, along with immunohistochemistry for bone related outcomes. The results suggest increased osteoconductive outcomes with an increase in initial content of apatite and BMP-2 in the silk fibroin porous scaffolds. The premineralization of these highly porous silk fibroin protein scaffolds provided enhanced outcomes for the bone tissue engineering. PMID:18387349

  14. A multilayer tissue engineered meniscus substitute.

    PubMed

    Halili, Albana Ndreu; Hasirci, Nesrin; Hasirci, Vasif

    2014-04-01

    Various methods have been tried to treat the main meniscus problem, meniscal tears, for which we believe tissue engineering could be a viable solution. In this study, a three dimensional, collagen-based meniscus substitute was prepared by tissue engineering using human fibrochondrocytes and a collagen based-scaffold. This construct was made with 3 different collagen-based foams interspaced with two electrospun nano/microfibrous mats. The top layer was made of collagen type I-chondroitin sulfate-hyaluronic acid (Coll-CS-HA), and the middle and the bottom layers were made of only collagen type I with different porosities and thus with different mechanical properties. The mats of aligned fibers were a blend of collagen type I and poly(L-lactic acid-co-glycolic acid) (PLGA). After seeding with human fibrochondrocytes, cell attachment, proliferation, and production of extracellular matrix and glucoseaminoglycan were studied. Cell seeding had a positive effect on the compressive properties of foams and the 3D construct. The 3D construct with all its 5 layers had better mechanical properties than the individual foams.

  15. Biomimetic strategies for engineering composite tissues.

    PubMed

    Lee, Nancy; Robinson, Jennifer; Lu, Helen

    2016-08-01

    The formation of multiple tissue types and their integration into composite tissue units presents a frontier challenge in regenerative engineering. Tissue-tissue synchrony is crucial in providing structural support for internal organs and enabling daily activities. This review highlights the state-of-the-art in composite tissue scaffold design, and explores how biomimicry can be strategically applied to avoid over-engineering the scaffold. Given the complexity of biological tissues, determining the most relevant parameters for recapitulating native structure-function relationships through strategic biomimicry will reduce the burden for clinical translation. It is anticipated that these exciting efforts in composite tissue engineering will enable integrative and functional repair of common soft tissue injuries and lay the foundation for total joint or limb regeneration.

  16. 3D Printing for Tissue Engineering

    PubMed Central

    Jia, Jia; Yao, Hai; Mei, Ying

    2016-01-01

    Tissue engineering aims to fabricate functional tissue for applications in regenerative medicine and drug testing. More recently, 3D printing has shown great promise in tissue fabrication with a structural control from micro- to macro-scale by using a layer-by-layer approach. Whether through scaffold-based or scaffold-free approaches, the standard for 3D printed tissue engineering constructs is to provide a biomimetic structural environment that facilitates tissue formation and promotes host tissue integration (e.g., cellular infiltration, vascularization, and active remodeling). This review will cover several approaches that have advanced the field of 3D printing through novel fabrication methods of tissue engineering constructs. It will also discuss the applications of synthetic and natural materials for 3D printing facilitated tissue fabrication. PMID:26869728

  17. 3D Printing for Tissue Engineering.

    PubMed

    Richards, Dylan Jack; Tan, Yu; Jia, Jia; Yao, Hai; Mei, Ying

    2013-10-01

    Tissue engineering aims to fabricate functional tissue for applications in regenerative medicine and drug testing. More recently, 3D printing has shown great promise in tissue fabrication with a structural control from micro- to macro-scale by using a layer-by-layer approach. Whether through scaffold-based or scaffold-free approaches, the standard for 3D printed tissue engineering constructs is to provide a biomimetic structural environment that facilitates tissue formation and promotes host tissue integration (e.g., cellular infiltration, vascularization, and active remodeling). This review will cover several approaches that have advanced the field of 3D printing through novel fabrication methods of tissue engineering constructs. It will also discuss the applications of synthetic and natural materials for 3D printing facilitated tissue fabrication.

  18. Novel chitin scaffolds derived from marine sponge Ianthella basta for tissue engineering approaches based on human mesenchymal stromal cells: Biocompatibility and cryopreservation.

    PubMed

    Mutsenko, Vitalii V; Gryshkov, Oleksandr; Lauterboeck, Lothar; Rogulska, Olena; Tarusin, Dmitriy N; Bazhenov, Vasilii V; Schütz, Kathleen; Brüggemeier, Sophie; Gossla, Elke; Akkineni, Ashwini R; Meißner, Heike; Lode, Anja; Meschke, Stephan; Fromont, Jane; Stelling, Allison L; Tabachnik, Konstantin R; Gelinsky, Michael; Nikulin, Sergey; Rodin, Sergey; Tonevitsky, Alexander G; Petrenko, Alexander Y; Glasmacher, Birgit; Schupp, Peter J; Ehrlich, Hermann

    2017-11-01

    The extraordinary biocompatibility and mechanical properties of chitinous scaffolds from marine sponges endows these structures with unique properties that render them ideal for diverse biomedical applications. In the present work, a technological route to produce "ready-to-use" tissue-engineered products based on poriferan chitin is comprehensively investigated for the first time. Three key stages included isolation of scaffolds from the marine demosponge Ianthella basta, confirmation of their biocompatibility with human mesenchymal stromal cells, and cryopreservation of the tissue-like structures grown within these scaffolds using a slow cooling protocol. Biocompatibility of the macroporous, flat chitin scaffolds has been confirmed by cell attachment, high cell viability and the ability to differentiate into the adipogenic lineage. The viability of cells cryopreserved on chitin scaffolds was reduced by about 30% as compared to cells cryopreserved in suspension. However, the surviving cells were able to retain their differentiation potential; and this is demonstrated for the adipogenic lineage. The results suggest that chitin from the marine demosponge I. basta is a promising, highly biocompatible biomaterial for stem cell-based tissue-engineering applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Tissue-Engineering for the Study of Cardiac Biomechanics

    PubMed Central

    Ma, Stephen P.; Vunjak-Novakovic, Gordana

    2016-01-01

    The notion that both adaptive and maladaptive cardiac remodeling occurs in response to mechanical loading has informed recent progress in cardiac tissue engineering. Today, human cardiac tissues engineered in vitro offer complementary knowledge to that currently provided by animal models, with profound implications to personalized medicine. We review here recent advances in the understanding of the roles of mechanical signals in normal and pathological cardiac function, and their application in clinical translation of tissue engineering strategies to regenerative medicine and in vitro study of disease. PMID:26720588

  20. Spinal Cord Repair with Engineered Nervous Tissue

    DTIC Science & Technology

    2012-10-01

    success of bridging a lateral hemisection in the rat spinal cord with engineered (“stretch-grown”) living nervous tissue constructs 2 . For the current...AD_________________ Award Number: W81XWH-10-1-0941 TITLE: Spinal Cord Repair with Engineered...SUBTITLE Spinal Cord Repair with Engineered Nervous Tissue 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-10-1-0941 5c. PROGRAM ELEMENT NUMBER 6

  1. Biodegradable polymeric fiber structures in tissue engineering.

    PubMed

    Tuzlakoglu, Kadriye; Reis, Rui L

    2009-03-01

    Tissue engineering offers a promising new approach to create biological alternatives to repair or restore function of damaged or diseased tissues. To obtain three-dimensional tissue constructs, stem or progenitor cells must be combined with a highly porous three-dimensional scaffold, but many of the structures purposed for tissue engineering cannot meet all the criteria required by an adequate scaffold because of lack of mechanical strength and interconnectivity, as well as poor surface characteristics. Fiber-based structures represent a wide range of morphological and geometric possibilities that can be tailored for each specific tissue-engineering application. The present article overviews the research data on tissue-engineering therapies based on the use of biodegradable fiber architectures as a scaffold.

  2. Engineered neural tissue for peripheral nerve repair.

    PubMed

    Georgiou, Melanie; Bunting, Stephen C J; Davies, Heather A; Loughlin, Alison J; Golding, Jonathan P; Phillips, James B

    2013-10-01

    A new combination of tissue engineering techniques provides a simple and effective method for building aligned cellular biomaterials. Self-alignment of Schwann cells within a tethered type-1 collagen matrix, followed by removal of interstitial fluid produces a stable tissue-like biomaterial that recreates the aligned cellular and extracellular matrix architecture associated with nerve grafts. Sheets of this engineered neural tissue supported and directed neuronal growth in a co-culture model, and initial in vivo tests showed that a device containing rods of rolled-up sheets could support neuronal growth during rat sciatic nerve repair (5 mm gap). Further testing of this device for repair of a critical-sized 15 mm gap showed that, at 8 weeks, engineered neural tissue had supported robust neuronal regeneration across the gap. This is, therefore, a useful new approach for generating anisotropic engineered tissues, and it can be used with Schwann cells to fabricate artificial neural tissue for peripheral nerve repair.

  3. The interaction between a combined knitted silk scaffold and microporous silk sponge with human mesenchymal stem cells for ligament tissue engineering.

    PubMed

    Liu, Haifeng; Fan, Hongbin; Wang, Yue; Toh, Siew Lok; Goh, James C H

    2008-02-01

    Cell seeding on knitted scaffolds often require a gel system, which was found to be practically unsuitable for anterior cruciate ligament (ACL) reconstruction as the cell-gel composite often gets dislodged from the scaffold in the in vivo dynamic situations. In order to solve this problem, we fabricated this combined silk scaffold with weblike microporous silk sponges formed in the openings of a knitted silk scaffold and subsequently combined with adult human bone marrow-derived mesenchymal stem cells (hMSCs) for in vitro ligament tissue engineering. Human MSCs adhered and grew well on the combined silk scaffolds. Moreover, in comparison with the knitted silk scaffolds seeded with hMSCs in fibroin gel the cellular function was more actively exhibited on the combined silk scaffolds, as evident by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for ligament-related gene markers (e.g., type I, III collagen and tenascin-C), immunohistochemical and western blot evaluations of ligament-related extracellular matrix (ECM) components. While the knitted structure holds the microporous silk sponges together and provides the structural strength of the combined silk scaffold, the microporous structure of the silk sponges mimic the ECM which consequently promotes cell proliferation, function, and differentiation. This feature overcomes the limitation of knitted scaffold for ligament tissue engineering application.

  4. Ligament tissue engineering using synthetic biodegradable fiber scaffolds.

    PubMed

    Lin, V S; Lee, M C; O'Neal, S; McKean, J; Sung, K L

    1999-10-01

    Tissue engineering offers the possibility of replacing damaged human ligaments with engineered ligament tissues. Hence, we attempted to culture in vitro ligament tissues by seeding human anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells onto synthetic biodegradable polymer fiber scaffolds. The ACL and MCL cells readily attached to the scaffold fibers. These cells and their secreted matrix soon surrounded the scaffold fibers and bridged the gaps in between. Beginning at 2 weeks, portions of the scaffolds were completely filled with tissue matrix. By 5 weeks, the scaffolds became single bundles of tissue. Thus the cell/fiber system appears to be a viable system for culturing ligament tissues. Additionally, cell proliferation under mechanical and biochemical stimuli was studied for up to 4 days. Whereas mechanical stimulus and transforming growth factor enhanced proliferation, inflammatory agents (lipopolysaccharide and complement C5a) had a negative effect. This work can thus contribute to a sound strategy for culturing replacement ligament tissues in vitro.

  5. Vascularized Bone Tissue Engineering: Approaches for Potential Improvement

    PubMed Central

    Nguyen, Lonnissa H.; Annabi, Nasim; Nikkhah, Mehdi; Bae, Hojae; Binan, Loïc; Park, Sangwon; Kang, Yunqing

    2012-01-01

    Significant advances have been made in bone tissue engineering (TE) in the past decade. However, classical bone TE strategies have been hampered mainly due to the lack of vascularization within the engineered bone constructs, resulting in poor implant survival and integration. In an effort toward clinical success of engineered constructs, new TE concepts have arisen to develop bone substitutes that potentially mimic native bone tissue structure and function. Large tissue replacements have failed in the past due to the slow penetration of the host vasculature, leading to necrosis at the central region of the engineered tissues. For this reason, multiple microscale strategies have been developed to induce and incorporate vascular networks within engineered bone constructs before implantation in order to achieve successful integration with the host tissue. Previous attempts to engineer vascularized bone tissue only focused on the effect of a single component among the three main components of TE (scaffold, cells, or signaling cues) and have only achieved limited success. However, with efforts to improve the engineered bone tissue substitutes, bone TE approaches have become more complex by combining multiple strategies simultaneously. The driving force behind combining various TE strategies is to produce bone replacements that more closely recapitulate human physiology. Here, we review and discuss the limitations of current bone TE approaches and possible strategies to improve vascularization in bone tissue substitutes. PMID:22765012

  6. Strategies for organ level tissue engineering

    PubMed Central

    Rustad, Kristine C; Sorkin, Michael; Levi, Benjamin; Longaker, Michael T

    2010-01-01

    The field of tissue engineering has made considerable strides since it was first described in the late 1980s. The advent and subsequent boom in stem cell biology, emergence of novel technologies for biomaterial development and further understanding of developmental biology have contributed to this accelerated progress. However, continued efforts to translate tissue-engineering strategies into clinical therapies have been hampered by the problems associated with scaling up laboratory methods to produce large, complex tissues. The significant challenges faced by tissue engineers include the production of an intact vasculature within a tissue-engineered construct and recapitulation of the size and complexity of a whole organ. Here we review the basic components necessary for bioengineering organs—biomaterials, cells and bioactive molecules—and discuss various approaches for augmenting these principles to achieve organ level tissue engineering. Ultimately, the successful translation of tissue-engineered constructs into everyday clinical practice will depend upon the ability of the tissue engineer to “scale up” every aspect of the research and development process. PMID:21197216

  7. Nanofibers and their applications in tissue engineering

    PubMed Central

    Vasita, Rajesh; Katti, Dhirendra S

    2006-01-01

    Developing scaffolds that mimic the architecture of tissue at the nanoscale is one of the major challenges in the field of tissue engineering. The development of nanofibers has greatly enhanced the scope for fabricating scaffolds that can potentially meet this challenge. Currently, there are three techniques available for the synthesis of nanofibers: electrospinning, self-assembly, and phase separation. Of these techniques, electrospinning is the most widely studied technique and has also demonstrated the most promising results in terms of tissue engineering applications. The availability of a wide range of natural and synthetic biomaterials has broadened the scope for development of nanofibrous scaffolds, especially using the electrospinning technique. The three dimensional synthetic biodegradable scaffolds designed using nanofibers serve as an excellent framework for cell adhesion, proliferation, and differentiation. Therefore, nanofibers, irrespective of their method of synthesis, have been used as scaffolds for musculoskeletal tissue engineering (including bone, cartilage, ligament, and skeletal muscle), skin tissue engineering, vascular tissue engineering, neural tissue engineering, and as carriers for the controlled delivery of drugs, proteins, and DNA. This review summarizes the currently available techniques for nanofiber synthesis and discusses the use of nanofibers in tissue engineering and drug delivery applications. PMID:17722259

  8. Amelogenin in Enamel Tissue Engineering.

    PubMed

    Uskoković, Vuk

    2015-01-01

    In this chapter the basic premises, the recent findings and the future challenges in the use of amelogenin for enamel tissue engineering are being discoursed on. Results emerging from the experiments performed to assess the fundamental physicochemical mechanisms of the interaction of amelogenin, the main protein of the enamel matrix, and the growing crystals of apatite, are mentioned, alongside a moderately comprehensive literature review of the subject at hand. The clinical importance of understanding this protein/mineral interaction at the nanoscale are highlighted as well as the potential for tooth enamel to act as an excellent model system for studying some of the essential aspects of biomineralization processes in general. The dominant paradigm stating that amelogenin directs the uniaxial growth of apatite crystals in enamel by slowing down the growth of (hk0) faces on which it adheres is being questioned based on the results demonstrating the ability of amelogenin to promote the nucleation and crystal growth of apatite under constant titration conditions designed to mimic those present in the developing enamel matrix. The role of numerous minor components of the enamel matrix is being highlighted as essential and impossible to compensate for by utilizing its more abundant ingredients only. It is concluded that the three major aspects of amelogenesis outlined hereby--(1) the assembly of amelogenin and other enamel matrix proteins, (2) the proteolytic activity, and (3) crystallization--need to be in precise synergy with each other in order for the grounds for the proper imitation of amelogenesis in the lab to be created.

  9. Amelogenin in Enamel Tissue Engineering

    PubMed Central

    2016-01-01

    In this chapter the basic premises, the recent findings and the future challenges in the use of amelogenin for enamel tissue engineering are being discoursed on. Results emerging from the experiments performed to assess the fundamental physicochemical mechanisms of the interaction of amelogenin, the main protein of the enamel matrix, and the growing crystals of apatite, are mentioned, alongside a moderately comprehensive literature review of the subject at hand. The clinical importance of understanding this protein/mineral interaction at the nanoscale are highlighted as well as the potential for tooth enamel to act as an excellent model system for studying some of the essential aspects of biomineralization processes in general. The dominant paradigm stating that amelogenin directs the uniaxial growth of apatite crystals in enamel by slowing down the growth of (hk0) faces on which it adheres is being questioned based on the results demonstrating the ability of amelogenin to promote the nucleation and crystal growth of apatite under constant titration conditions designed to mimic those present in the developing enamel matrix. The role of numerous minor components of the enamel matrix is being highlighted as essential and impossible to compensate for by utilizing its more abundant ingredients only. It is concluded that the three major aspects of amelogenesis outlined hereby – (1) the assembly of amelogenin and other enamel matrix proteins, (2) the proteolytic activity, and (3) crystallization – need to be in precise synergy with each other in order for the grounds for the proper imitation of amelogenesis in the lab to be created. PMID:26545753

  10. Tumor Engineering: The Other Face of Tissue Engineering

    SciTech Connect

    Ghajar, Cyrus M; Bissell, Mina J

    2010-03-09

    training grounds have largely consisted of small rodents, despite marked differences between human and mouse physiology, or plastic dishes, even though just like our tissues and organs most tumors exist within three-dimensional proteinacious milieus. One could argue that this is comparable to training for a desert war in the arctic. In this special issue of tissue engineering, Fischbach-Teschl and colleagues build a strong case for engineering complex cultures analogous to normal organs to tractably model aspects of the human tumor microenvironment that simply cannot be reproduced with traditional two-dimensional cell culture techniques and that cannot be studied in a controlled fashion in vivo. This idea has gained considerable traction of late as concepts presented and convincingly shown years ago have only now begun to be appreciated. Perhaps, then, it is time to organize those who wish to build complex tumor models to study cancer biology under a common umbrella. Accordingly, we propose that tumor engineering be defined as the construction of complex culture models that recapitulate aspects of the in vivo tumor microenvironment to study the dynamics of tumor development, progression, and therapy on multiple scales. Inherent in this definition is the collaboration that must occur between physical and life scientists to guide the design of patterning techniques, materials, and imaging modalities for the study of cancer from the subcellular to tissue level in physiologically relevant contexts. To date, the most successful tissue engineering approaches have employed methods that recapitulate the composition, architecture, and/or chemical presentation of native tissue. For instance, induction of blood vessel growth for therapeutic purposes has been achieved with sequential release of vascular endothelial growth factor (VEGF) and platelet derived growth factor to induce and stabilize blood vessels. This approach imitates that which occurs during physiological angiogenesis as

  11. Culturing primary human osteoblasts on electrospun poly(lactic-co-glycolic acid) and poly(lactic-co-glycolic acid)/nanohydroxyapatite scaffolds for bone tissue engineering.

    PubMed

    Li, Mengmeng; Liu, Wenwen; Sun, Jiashu; Xianyu, Yunlei; Wang, Jidong; Zhang, Wei; Zheng, Wenfu; Huang, Deyong; Di, Shiyu; Long, Yun-Ze; Jiang, Xingyu

    2013-07-10

    In this work, we fabricated polymeric fibrous scaffolds for bone tissue engineering using primary human osteoblasts (HOB) as the model cell. By employing one simple approach, electrospinning, we produced poly(lactic-co-glycolic acid) (PLGA) scaffolds with different topographies including microspheres, beaded fibers, and uniform fibers, as well as the PLGA/nanohydroxyapatite (nano-HA) composite scaffold. The bone-bonding ability of electrospun scaffolds was investigated by using simulated body fluid (SBF) solution, and the nano-HA in PLGA/nano-HA composite scaffold can significantly enhance the formation of the bonelike apatites. Furthermore, we carried out in vitro experiments to test the performance of electrospun scaffolds by utilizing both mouse preosteoblast cell line (MC 3T3 E1) and HOB. Results including cell viability, alkaline phosphatase (ALP) activity, and osteocalcin concentration demonstrated that the PLGA/nano-HA fibers can promote the proliferation of HOB efficiently, indicating that it is a promising scaffold for human bone repair.

  12. Engineered Muscle Actuators: Cells and Tissues

    DTIC Science & Technology

    2007-01-10

    platform. Outcomes by milestone: (1) Develop integrated tissue culture bioreactor systems: completed all but bulk perfusion (2) Develop appropriate...tissue culture perfusion bioreactors (B) Second generation cm-scale hybrid swimming robotic platform & control methodologies (C) Guidance of engineered...integrated tissue culture perfusion bioreactors 1. Employ rapid manufacturing techniques for bioreactors 1. accelerate system development 2. increase number

  13. Growth factor delivery to re-engineer periodontal tissues.

    PubMed

    Anusaksathien, Orasa; Giannobile, William V

    2002-06-01

    Repair of tooth-supporting structures destroyed by the chronic inflammatory disease periodontitis is a major goal of oral therapy. The field of tissue engineering combines materials science and biology to repair tissues and organs. Periodontal tissue engineering has been achieved with limited success by the utilization of guiding tissue (cell occlusive) membranes and bone grafting techniques. Over the past decade investigators have begun to utilize signaling molecules such as growth factors to restore lost tooth support due to periodontitis, the most common bone disease affecting humans. This review will provide information on the status of growth factor therapies being applied in periodontology to treat advanced alveolar bone loss.

  14. Vascular Tissue Engineering: Building Perfusable Vasculature for Implantation

    PubMed Central

    Gui, Liqiong; Niklason, Laura E.

    2014-01-01

    Tissue and organ replacement is required when there are no alternative therapies available. Although vascular tissue engineering was originally developed to meet the clinical demands of small-diameter vascular conduits as bypass grafts, it has evolved into a highly advanced field where perfusable vasculatures are generated for implantation. Herein, we review several cutting-edge techniques that have led to implantable human blood vessels in clinical trials, the novel approaches that build complex perfusable microvascular networks in functional tissues, the use of stem cells to generate endothelial cells for vascularization, as well as the challenges in bringing vascular tissue engineering technologies into the clinics. PMID:24533306

  15. Optical Coherence Tomography in Tissue Engineering

    NASA Astrophysics Data System (ADS)

    Zhao, Youbo; Yang, Ying; Wang, Ruikang K.; Boppart, Stephen A.

    Tissue engineering holds the promise for a therapeutic solution in regenerative medicine. The primary goal of tissue engineering is the development of physiologically functional and biocompatible tissues/organs being implanted for the repair and replacement of damaged or diseased ones. Given the complexity in the developing processes of engineered tissues, which involves multi-dimensional interactions among cells of different types, three-dimensionally constructed scaffolds, and actively intervening bioreactors, a capable real-time imaging tool is critically required for expanding our knowledge about the developing process of desired tissues or organs. It has been recognized that optical coherence tomography (OCT), an emerging noninvasive imaging technique that provides high spatial resolution (up to the cellular level) and three-dimensional imaging capability, is a promising investigative tool for tissue engineering. This chapter discusses the existing and potential applications of OCT in tissue engineering. Example OCT investigations of the three major components of tissue engineering, i.e., cells, scaffolds, and bioreactors are overviewed. Imaging examples of OCT and its enabling functions and variants, e.g., Doppler OCT, polarization-sensitive OCT, optical coherence microscopy are emphasized. Remaining challenges in the application of OCT to tissue engineering are discussed, and the prospective solutions including the combination of OCT with other high-contrast and high-resolution modalities such as two-photon fluorescence microscopy are suggested as well. It is expected that OCT, along with its functional variants, will make important contributions toward revealing the complex cellular dynamics in engineered tissues as well as help us culture demanding tissue/organ implants that will advance regenerative medicine.

  16. Myocardial tissue engineering: toward a bioartificial pump.

    PubMed

    Sekine, Hidekazu; Shimizu, Tatsuya; Okano, Teruo

    2012-03-01

    Regenerative therapies, including cell injection and bioengineered tissue transplantation, have the potential to treat severe heart failure. Direct implantation of isolated skeletal myoblasts and bone-marrow-derived cells has already been clinically performed and research on fabricating three-dimensional (3-D) cardiac grafts using tissue engineering technologies has also now been initiated. In contrast to conventional scaffold-based methods, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cell sheets to construct 3-D cell-dense tissues. Upon layering, individual cardiac cell sheets integrate to form a single, continuous, cell-dense tissue that resembles native cardiac tissue. The transplantation of layered cardiac cell sheets is able to repair damaged hearts. As the next step, we have attempted to promote neovascularization within bioengineered myocardial tissues to overcome the longstanding limitations of engineered tissue thickness. Finally, as a possible advanced therapy, we are now trying to fabricate functional myocardial tubes that may have a potential for circulatory support. Cell sheet-based tissue engineering technologies therefore show an enormous promise as a novel approach in the field of myocardial tissue engineering.

  17. Mycoplasma detection and elimination are necessary for the application of stem cell from human dental apical papilla to tissue engineering and regenerative medicine.

    PubMed

    Kim, Byung-Chul; Kim, So Yeon; Kwon, Yong-Dae; Choe, Sung Chul; Han, Dong-Wook; Hwang, Yu-Shik

    2015-01-01

    Recently, postnatal stem cells from dental papilla with neural crest origin have been considered as one of potent stem cell sources in regenerative medicine regarding their multi-differentiation capacity and relatively easy access. However, almost human oral tissues have been reported to be infected by mycoplasma which gives rise to oral cavity in teeth, and mycoplasma contamination of ex-vivo cultured stem cells from such dental tissues and its effect on stem cell culture has received little attention. In this study, mycoplama contamination was evaluated with stem cells from apical papilla which were isolated from human third molar and premolars from various aged patients undergoing orthodontic therapy. The ex-vivo expanded stem cells from apical papilla were found to express stem cell markers such as Stro-1, CD44, nestin and CD133, but mycoplama contamination was detected in almost all cell cultures of the tested 20 samples, which was confirmed by mycoplasma-specific gene expression and fluorescence staining. Such contaminated mycoplasma could be successfully eliminated using elimination kit, and proliferation test showed decreased proliferation activity in mycoplasma-contaminated cells. After elimination of contaminated mycoplasma, stem cells from apical papilla showed osteogenic and neural lineage differentiation under certain culture conditions. Our study proposes that the evaluation of mycoplasma contamination and elimination process might be required in the use of stem cells from apical papilla for their potent applications to tissue engineering and regenerative medicine.

  18. [Human brown adipose tissue].

    PubMed

    Virtanen, Kirsi A; Nuutila, Pirjo

    2015-01-01

    Adult humans have heat-producing and energy-consuming brown adipose tissue in the clavicular region of the neck. There are two types of brown adipose cells, the so-called classic and beige adipose cells. Brown adipose cells produce heat by means of uncoupler protein 1 (UCP1) from fatty acids and sugar. By applying positron emission tomography (PET) measuring the utilization of sugar, the metabolism of brown fat has been shown to multiply in the cold, presumably influencing energy consumption. Active brown fat is most likely present in young adults, persons of normal weight and women, least likely in obese persons.

  19. Human glandular organoid formation in murine engineering chambers after collagenase digestion and flow cytometry isolation of normal human breast tissue single cells.

    PubMed

    Huo, Cecilia W; Huang, Dexing; Chew, Grace L; Hill, Prue; Vohora, Ambika; Ingman, Wendy V; Glynn, Danielle J; Godde, Nathan; Henderson, Michael A; Thompson, Erik W; Britt, Kara L

    2016-11-01

    Women with high mammographic density (MD) are at increased risk of breast cancer (BC) after adjustment for age and body mass index. We have developed a murine biochamber model in which both high MD (HMD) and low MD (LMD) tissue can be propagated. Here, we tested whether cells isolated by collagenase digestion and fluorescence-activated cell sorting (FACS) from normal breast can be reconstituted in our biochamber model, which would allow cell-specific manipulations to be tested. Fresh breast tissue was collected from women (n = 7) undergoing prophylactic mastectomy. The tissue underwent collagenase digestion overnight and, in some cases, additional FACS enrichment to obtain mature epithelial, luminal progenitor, mammary stem, and stromal cells. Cells were then transferred bilaterally into biochambers in SCID mice (n = 5-7) and incubated for 6 weeks, before harvesting for histological analyses, and immunohistochemical staining for cytokeratins (CK), vimentin, Ki-67, murine macrophages, and Cleaved Caspase-3. Biochambers inoculated with single cells after collagenase digestion or with flow cytometry contained glandular structures of human origin (human vimentin-positive), which expressed CK-14 and pan-CK, and were proliferating (Ki-67-positive). Glandular structures from the digested tissues were smaller than those in chambers seeded with finely chopped intact mammary tissue. Mouse macrophage infiltration was higher in the chambers arising from digested tissues. Pooled single cells and FACS fractionated cells were viable in the murine biochambers and formed proliferating glandular organoids of human origin. This is among the first report to demonstrate the success of formed human glandular organoids from isolated primary mammary cells in the murine biochamber model.

  20. Aloe Vera for Tissue Engineering Applications

    PubMed Central

    Rahman, Shekh; Carter, Princeton; Bhattarai, Narayan

    2017-01-01

    Aloe vera, also referred as Aloe barbadensis Miller, is a succulent plant widely used for biomedical, pharmaceutical and cosmetic applications. Aloe vera has been used for thousands of years. However, recent significant advances have been made in the development of aloe vera for tissue engineering applications. Aloe vera has received considerable attention in tissue engineering due to its biodegradability, biocompatibility, and low toxicity properties. Aloe vera has been reported to have many biologically active components. The bioactive components of aloe vera have effective antibacterial, anti-inflammatory, antioxidant, and immune-modulatory effects that promote both tissue regeneration and growth. The aloe vera plant, its bioactive components, extraction and processing, and tissue engineering prospects are reviewed in this article. The use of aloe vera as tissue engineering scaffolds, gels, and films is discussed, with a special focus on electrospun nanofibers. PMID:28216559

  1. Advancing tissue engineering by using electrospun nanofibers.

    PubMed

    Ashammakhi, Nureddin; Ndreu, A; Nikkola, L; Wimpenny, I; Yang, Y

    2008-07-01

    Electrospinning is a versatile technique that enables the development of nanofiber-based scaffolds, from a variety of polymers that may have drug-release properties. Using nanofibers, it is now possible to produce biomimetic scaffolds that can mimic the extracellular matrix for tissue engineering. Interestingly, nanofibers can guide cell growth along their direction. Combining factors like fiber diameter, alignment and chemicals offers new ways to control tissue engineering. In vivo evaluation of nanomats included their degradation, tissue reactions and engineering of specific tissues. New advances made in electrospinning, especially in drug delivery, support the massive potential of these nanobiomaterials. Nevertheless, there is already at least one product based on electrospun nanofibers with drug-release properties in a Phase III clinical trial, for wound dressing. Hopefully, clinical applications in tissue engineering will follow to enhance the success of regenerative therapies.

  2. Aloe Vera for Tissue Engineering Applications.

    PubMed

    Rahman, Shekh; Carter, Princeton; Bhattarai, Narayan

    2017-02-14

    Aloe vera, also referred as Aloe barbadensis Miller, is a succulent plant widely used for biomedical, pharmaceutical and cosmetic applications. Aloe vera has been used for thousands of years. However, recent significant advances have been made in the development of aloe vera for tissue engineering applications. Aloe vera has received considerable attention in tissue engineering due to its biodegradability, biocompatibility, and low toxicity properties. Aloe vera has been reported to have many biologically active components. The bioactive components of aloe vera have effective antibacterial, anti-inflammatory, antioxidant, and immune-modulatory effects that promote both tissue regeneration and growth. The aloe vera plant, its bioactive components, extraction and processing, and tissue engineering prospects are reviewed in this article. The use of aloe vera as tissue engineering scaffolds, gels, and films is discussed, with a special focus on electrospun nanofibers.

  3. Engineering more than a cell: Vascularization Strategies in Tissue Engineering

    PubMed Central

    Phelps, Edward A.; García, Andrés J.

    2010-01-01

    Summary Host integration and performance of engineered tissues have been severely limited by the lack of robust strategies to generate patent vascularization and tissue perfusion. This review highlights a selection of exciting developments in vascularization approaches for tissue engineering research. Current strategies for vascularization in tissue engineering are related to growth factor signaling and delivery, cell transplantation, bioactive smart matrix materials, and directed fabrication. Application of these techniques to in vivo models has resulted in a number of robust host vascular responses, especially with synergistic and engineered bioactive systems. The future outlook of the field includes refinement and development of new technologies for vascularization and combining these techniques with functional repair models for metabolically active tissues and relevant disease states. PMID:20638268

  4. [HUMAN ADIPOSE-DERIVED STEM CELLS COMBINED WITH SMALL INTESNITAL SUBMUCOSA POWDER/CHITOSAN CHLORIDE-β-GLYCEROL PHOSPHATE DISODIUM-HYDROXYETHYL CELLULOSE HYBRID FOR ADIPOSE TISSUE ENGINEERING].

    PubMed

    Zhang, Shu; Luo, Jingcong; Lü, Qing; Deng, Xueqin; Xiong, Bingjun

    2015-08-01

    To study the feasibility of human adipose-derived stem cells (hADSCs) combined with small intestinal submucosa powder (SISP)/chitosan chloride (CSCl)-β-glycerol phosphate disodium (GP)-hydroxyethyl cellulose (HEC) for adipose tissue engineering. hADSCs were isolated from human breast fat with collagenase type I digestion, and the third passage hADSCs were mixed with SISP/CSCl-GP-HEC at a density of 1 x 10(6) cells/mL. Twenty-four healthy female nude mice of 5 weeks old were randomly divided into experimental group (n = 12) and control group (n=12), and the mice were subcutaneously injected with 1 mL hADSCs+SISP/CSCl-GP-HEC or SISP/CSCl-GP-HEC respectively at the neck. The degradation rate was evaluated by implant volume measurement at 0, 1, 2, 4, and 8 weeks. Three mice were euthanized at 1, 2, 4, and 8 weeks respectively for general, histological, and immunohistochemical observations. The ability of adipogenesis (Oil O staining), angiopoiesis (CD31), and localized the hADSCs (immunostaining for human Vimentin) were identified. The volume of implants of both groups decreased with time, but it was greater in experimental group than the control group, showing significant difference at 8 weeks (t = 3.348, P = 0.029). The general observation showed that the border of implants was clear with no adhesion at each time point; fat-liked new tissues were observed with capillaries on the surface at 8 weeks in 2 groups. The histological examinations showed that the structure of implants got compact gradually after injection, and SISP gradually degraded with slower degradation speed in experimental group; adipose tissue began to form, and some mature adipose tissue was observed at 8 weeks in the experimental group. The Oil O staining positive area of experimental group was greater than that of the control group at each time point, showing significant difference at 8 weeks (t = 3.41 1, P = 0.027). Immunohistochemical staining for Vemintin showed that hADSCs could survive at

  5. 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.

  6. Cell sheet-based tissue engineering for fabricating 3-dimensional heart tissues.

    PubMed

    Shimizu, Tatsuya

    2014-01-01

    In addition to stem cell biology, tissue engineering is an essential research field for regenerative medicine. In contrast to cell injection, bioengineered tissue transplantation minimizes cell loss and has the potential to repair tissue defects. A popular approach is scaffold-based tissue engineering, which utilizes a biodegradable polymer scaffold for seeding cells; however, new techniques of cell sheet-based tissue engineering have been developed. Cell sheets are harvested from temperature-responsive culture dishes by simply lowering the temperature. Monolayer or stacked cell sheets are transplantable directly onto damaged tissues and cell sheet transplantation has already been clinically applied. Cardiac cell sheet stacking produces pulsatile heart tissue; however, lack of vasculature limits the viable tissue thickness to 3 layers. Multistep transplantation