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PubMed1992

Tissue engineering in the USA.

Tissue engineering is the application of the principles and methods of engineering and the life sciences towards the development of biological substitutes to restore, maintain or improve functions. It is an area which is emerging in importance worldwide. In the USA it has been actively fostered by the National Science Foundation, both through research grants and the sponsorship of a series of workshops starting in 1988. This brief review of activities in the USA focuses on cell culture technology as a foundation for tissue engineering and then discusses examples of applications. These include artificial skin and the use of encapsulated cells in the development of bioartificial organs. Also discussed is the reconstitution of a blood vessel in culture, both for use in basic research and for implantation as an artificial blood vessel in bypass surgery. In conclusion, other potential applications are mentioned as well as generic areas of technology for future development.

Artificial Organs↗

PubMed2000

[Study of human tissue engineering cartilage].

OBJECTIVE: To investigate the technique of human tissue-engineered cartilage and to study the medical collagen membrane of guided tissue regeneration (GTR) as the carrier of in vitro chondrocytes culture. METHODS: Tissue engineering technique was used to make human Tissue-engineered cartilage, which was examined histologically and functionally. RESULTS: It was found that the chondrocytes seeded on the medical collagen membrane of GTR grew well. A layer of milk white and cartilage-like tissue grew on the surface of medical collagen membrane of GTR after 1 week. It was demonstrated that the cartilage-like tissue was strong enough to be transferred after being implanted for 8 weeks. The cartilage-like tissue was proved to be human tissue engineered cartilage by HE stain and Alcian blue-poncean S stain. The chondrocytes could secrete chondroitin sulfate as proved by Lev-Spicer stain. CONCLUSIONS: The medical collagen membrane of GTR has characteristics of three-dimensional structure and cell reticular function, and it has the possibility to be developed as a natural scaffold for tissue engineering. The results indicate that it is possible to make human tissue-engineered cartilage with tissue-engineering technique.

Animals↗

PubMed1999

Poly(alpha-hydroxyl acids)/hydroxyapatite porous composites for bone-tissue engineering. I. Preparation and morphology.

Tissue engineering has shown great promise for creating biological alternatives for implants. In this approach, scaffolding plays a pivotal role. Hydroxyapatite mimics the natural bone mineral and has shown good bone-bonding properties. This paper describes the preparation and morphologies of three-dimensional porous composites from poly(L-lactic acid) (PLLA) or poly(D,L-lactic acid-co-glycolic acid) (PLGA) solution and hydroxyapatite (HAP). A thermally induced phase separation technique was used to create the highly porous composite scaffolds for bone-tissue engineering. Freeze drying of the phase-separated polymer/HAP/solvent mixtures produced hard and tough foams with a co-continuous structure of interconnected pores and a polymer/HAP composite skeleton. The microstructure of the pores and the walls was controlled by varying the polymer concentration, HAP content, quenching temperature, polymer, and solvent utilized. The porosity increased with decreasing polymer concentration and HAP content. Foams with porosity as high as 95% were achieved. Pore sizes ranging from several microns to a few hundred microns were obtained. The composite foams showed a significant improvement in mechanical properties over pure polymer foams. They are promising scaffolds for bone-tissue engineering.

Adhesiveness↗

PubMed2000

Tissue engineering of vascular grafts.

The challenge of tissue engineering blood vessels with the mechanical properties of native vessels, and with the anti-thrombotic properties required is immense. Recent advances, however, indicate that the goal of providing a tissue-engineered vascular graft that will remain patent in vivo for substantial periods of time, is achievable. For instance, collagen gels have been used to fabricate a tissue in vitro that is representative of a native vessel: an acellular collagen tubular structure, when implanted as a vascular graft, was able to function, and to become populated with host cells. A completely cellular approach culturing cells into tissue sheets and wrapping these around a mandel was able to form a layered tubular structure with impressive strength. Culture of cells onto a biodegradable scaffold within a dynamic bioreactor, generated a tissue-engineered vascular graft with substantial stiffness and, when lined with endothelial cells, was able to remain patent for up to 4 weeks in vivo. In our experiments, use of a non-degradable polyurethane scaffold and culture with smooth muscle cells generated a construct with mechanical properties similar to native vessels. This composite tissue engineered vascular graft with an endothelial layer formed using fluid shear stress to align the endothelial cells, was able to remain patent with an neointima for up to 4 weeks. These results show that tissue engineering of vascular grafts has true potential for application in the clinical situation.

Biocompatible Materials↗

PubMed1999

Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization.

Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair. We previously demonstrated that contractile cardiac cell-polymer constructs can be cultivated using isolated cells, 3-dimensional scaffolds, and bioreactors. In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle. Constructs seeded under well-mixed conditions with rat heart cells at a high initial density ((6-8) x 10(6) cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm(2)). Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p < 0.05). Advantages of culturing constructs under mixed rather than static conditions included the maintenance of metabolic parameters in physiological ranges, 2-4 times higher construct cellularity (p &le 0.0001), more aerobic cell metabolism, and a more physiological, elongated cell shape. Cultivations in rotating bioreactors, in which flow patterns are laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks. After 1-2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2-6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue.

Animals↗

PubMed2001

Tissue-engineered heart valve leaflets: an animal study.

BACKGROUND: Tissue-engineered heart valve leaflets are a promising way to overcome the inherent limitations of current prosthetic valves. The aim of this study was to compare the biological responses of an autologous cell seeded scaffold and an acellular scaffold implanted in the pulmonary valve leaflet in the same animal. METHODS: Myofibroblasts and endothelial cells were isolated and cultured from an ovine artery. A synthetic biodegradable scaffold consisting of polyglycolic acid and polylactic acid was initially seeded with the myofibroblasts, then coated with endothelial cells. Cells were seeded using a medium containing collagen and cultured. A tissue-engineered construct and a plain scaffold were implanted as double pulmonary valve leaflet replacement in the same animal in an ovine model (n=3). Additionally, the tissue-engineered construct (n=2) and the plain scaffold (n=2) were implanted as single valve leaflet replacements for long-term analysis. After sacrifice, the implanted valve leaflet tissues were retrieved, analyzed visually and using light microscopy. RESULTS: Three animals that underwent replacement of two valve leaflets with a tissue-engineered construct and a plain scaffold, survived only a short-time (12, 24, 36 hours). The death was attributed to heart failure caused by severe pulmonary insufficiency. Animals that underwent single valve leaflet replacement survived longer and were electively sacrificed at 6 and 9 weeks after operation. The analysis of the leaflets from the short-term survivors showed that the tissue-engineered constructs contained less fibrins and protein exudates than the plain scaffold. In contrast, leaflets obtained from animals surviving 6 and 9 weeks showed similar well organized granulation tissues in the tissue-engineered constructs and the plain scaffolds. CONCLUSION: This animal experiment demonstrates that in the early phase of implantation, the tissue-engineered construct shows a better biological response in terms of antithrombogenicity than the plain scaffold, although both of them have similar results in the later reparative phase.

Animals↗

PubMed1998

Tissue engineering.

Wound care has become one of the first fields to see the benefit of a new technology: tissue engineering. Tissue engineering involves the development of new materials or devices capable of specific interactions with biological tissues. In wound care, these materials may be based entirely on naturally occurring tissues and cells, or may be materials that combine synthetics, usually polymers, with biological layers. Both wound dressings and skin substitutes are available. The complexity of the materials depends on the end uses. Generally, synthetics made from polymeric materials such as Tegaderm and Opsite are used as wound dressings over relatively simple and shallow wounds or as coverings over more complex dressings. Their function is one of protection from water loss, drying, and mechanical injury. More complex dressings vary from dermal replacements made of reconstituted collagen and chondroitan sulfate backed by a polymer layer such as Integra(R) to the complex Apligraftrade mark that contains collagen and seeded cells. This last is designed as a complete skin replacement or skin substitute. Ultimately, engineered skin will contain all of the components necessary to modulate healing and provide the desired response: a wound closed with limited scar tissue that retains all of the characteristics of natural skin.

Biocompatible Materials↗

PubMed2001

Tissue engineering research in oral implant surgery.

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.

Absorbable Implants↗

PubMed2001

Tissue engineering: the biophysical background.

Tissue engineering is the construction, repair or replacement of damaged or missing tissue in humans and other animals. This engineering may take place within the animal body or as tissue constructs to be made in a bioreactor for later grafting into the animal. The minimal set of materials for this are the appropriate types of cell. Usually, however, non-living substrata are used as well. These substrata may be nothing more than materials that bulk up any voids in the damaged tissue and provide the mechanical strength that has been lost when the tissue is damaged or removed. They may serve a similar pair of functions in the bioreactor. They can do much more in terms of pattern formation. The orientations and morphology of the cells, the arrangement of intercellular material as it is laid down and the relationships between different cell types in the repairing or construct tissue are all of importance, for these should resemble the correct normal tissue as closely as possible. Most of these requirements are ones involving pattern formation. This review discusses the various ways in which tissue pattern can be engineered chiefly from a biophysical standpoint. Unpatterned cells are effectively not tissue. This engineering includes the use of topography on the substrata, chemical patterning of adhesive and other cues for the cells, mechanical force application to cause cell orientation and appropriate synthetic responses and electrical fields. The review also discusses the methods used to impart the appropriate cues to and through the materials which are often biodegradable polymers. The article gives particular attention to regions of research and practice where the involvement of the physicist or biophysicist is of importance.

Animals↗

PubMed2001

The design of scaffolds for use in tissue engineering. Part I. Traditional factors.

In tissue engineering, a highly porous artificial extracellular matrix or scaffold is required to accommodate mammalian cells and guide their growth and tissue regeneration in three dimensions. However, existing three-dimensional scaffolds for tissue engineering proved less than ideal for actual applications, not only because they lack mechanical strength, but they also do not guarantee interconnected channels. In this paper, the authors analyze the factors necessary to enhance the design and manufacture of scaffolds for use in tissue engineering in terms of materials, structure, and mechanical properties and review the traditional scaffold fabrication methods. Advantages and limitations of these traditional methods are also discussed.

Animals↗

PubMed2001

Fibrin gel -- advantages of a new scaffold in cardiovascular tissue engineering.

OBJECTIVE: The field of tissue engineering deals with the creation of tissue structures based on patient cells. The scaffold plays a central role in the creation of 3-D structures in cardiovascular tissue engineering like small vessels or heart valve prosthesis. An ideal scaffold should have tissue-like mechanical properties and a complete immunologic integrity. As an alternative scaffold the use of fibrin gel was investigated. METHODS: Preliminary, the degradation of the fibrin gel was controlled by the supplementation of aprotinin to the culture medium. To prevent tissue from shrinking a mechanical fixation of the gel with 3-D microstructure culture plates and a chemical fixation with poly-L-lysine in different fixation techniques were studied. The thickness of the gel layer was changed from 1 to 3 mm. The tissue development was analysed by light, transmission and scanning electron microscopy. Collagen production was detected by the measurement of hydroxyproline. Injection molding techniques were designed for the formation of complex 3-D tissue structures. RESULTS: The best tissue development was observed at an aprotinin concentration of 20 microg per cc culture medium. The chemical border fixation of the gel by poly-L-lysine showed the best tissue development. Up to a thickness of 3 mm no nutrition problems were observed in the light and transmission electron microscopy. The molding of a simplified valve conduit was possible by the newly developed molding technique. CONCLUSION: Fibrin gel combines a number of important properties of an ideal scaffold. It can be produced as a complete autologous scaffold. It is moldable and degradation is controllable by the use of aprotinin.

Aprotinin↗

PubMed2000

[Musculoskeletal tissue engineering with resorbable polymers].

Musculoskeletal tissues can present congenital or acquired defects as a result of disease, accidental trauma or iatrogenous causes. This loss of bony substance is traditionally treated by the replacement of bony tissue (grafts or flaps), or by synthetic materials. Each of these methods of treatment, however, entails its specific disadvantages, limitations and complications. The recent approach for treatment of musculoskeletal defects has been the development of the growing of neotissues derived from autogenous cells, and artificial biodegradable matrixes. This method assumed the name "tissue engineering" in the late 1980s. Tissue Engineering, or TE, has employed advances made in the area of cellular culture, intercellular matrix biology, and also, in the area of biomaterial science. TE is an multi-disciplinary approach. Musculoskeletal TE, although in its preliminary stages, should allow access to treatments of the future.

Biocompatible Materials↗

PubMed2001

Dynamics of extracellular matrix production and turnover in tissue engineered cardiovascular structures.

Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial requirements for their long-term patency and function. This complex process requires the deposition and accumulation of extracellular matrix molecules as well as the remodeling of this extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). In this study, we have investigated the dynamics of ECM production and the activity of MMPs and TIMPs in long-term tissue-engineered vascular conduits using quantitative ECM analysis, substrate gel electrophoresis, radiometric enzyme assays and Western blot analyses. Over a time period of 169 days in vivo, levels of elastin and proteoglycans/glycosaminoglycans in tissue-engineered constructs came to approximate those of their native tissue counter parts. The kinetics of collagen deposition and remodeling, however, apparently require a much longer time period. Through the use of substrate gel electrophoresis, proteolytic bands whose molecular weight was consistent with their identification as the active form of MMP-2 (approximately 64--66 kDa) were detected in all native and tissue-engineered samples. Additional proteolytic bands migrating at approximately 72 kDa representing the latent form of MMP-2 were detected in tissue-engineered samples at time points from 5 throughout 55 days. Radiometric assays of MMP-1 activity demonstrated no significant differences between the native and tissue-engineered samples. This study determines the dynamics of ECM production and turnover in a long-term tissue-engineered vascular tissue and highlights the importance of ECM remodeling in the development of successful tissue-engineered vascular structures.

Animals↗

PubMed1999

An overview of tissue engineered bone.

Numerous important developments in tissue engineering of new bone during the last 10 years are reviewed. Early efforts to combine cells with biocompatible materials are described and applications of this technology are presented with particular focus on uses in orthopaedics and maxillofacial surgery. Basic principles of tissue engineering focusing on cell biology and materials science as used currently in the field are presented. Finally, future challenges are outlined from the perspective of integrating technologies from medicine, biology, and engineering in hopes of translating tissue engineering to clinical applications.

Animals↗

PubMed2000

Tissue engineering of heart valves: in vitro experiences.

BACKGROUND: Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. METHODS: We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent (1 x 15 x 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 microm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. RESULTS: It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. CONCLUSIONS: Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure.

Animals↗

PubMed2000

[Tissue engineering of vascularized bone and soft tissue transplants].

The utilization of in vitro angiogenesis in tissue engineering might be useful in order to establish an artificial vascular network. However, it remains unclear how far the in vitro preformation of vascular structures may contribute to the perfusion of larger artificial tissue aggregates regarding the improvement of oxygenation and nutrition. In an in vitro study, we developed a model of a vascularized tissue. Stromal cells of a target tissue, e.g., adipose tissue or bone tissue, were expanded in vitro and seeded onto microcarriers or microparticles. Densely covered microcarriers were brought into a fibrin matrix together with endothelial cells. In order to demonstrate the formation and stabilization of capillary-like structures, UEA-I labeled specimens were evaluated using laser scanning microscopy and digital image analysis. The stabilization of capillary-like structures was better with stromal cells from bone marrow than from adipose tissue. In one of the culture aggregates, the total length of capillary-like structures increased after 6 weeks of cultivation to up to 140 mm/mm3. Additional tests were performed utilizing hyperbaric oxygenation. In the oxygenation group, a significant increase in the length of capillary-like structures was found. The method implies the option of coculturing different tissue elements and of an in vitro preformation of vascularized tissues.

Bone Transplantation↗

PubMed1999

Joint cartilage regeneration by tissue engineering.

The research field of tissue engineering combines cells biology, biomaterial science, and surgery. Major long-term goals are tissue and organ replacement therapies using the patients' own cells. Our work is focused on the treatment of severe joint defects and on plastic surgery using in vitro engineered cartilage tissues. The practical approaches in cartilage engineering face problems with three-dimensional cell distribution or cell immobilization raising biocompatibility problems. The tissue engineering of cartilage is based on combining biocompatible cell embedding substances such as fibrin, agarose, alginate, hyaluronic acid and fiber fleece scaffolds of poly alpha-hydroxy acids (PLLA/PGLA). Different technical approaches were established: a) three-dimensional in vitro cultures of chondrocytes for the development of vital tissue transplants and b) interacting three-dimensional cultures consisting of different cell populations, such as BMP-transfected mesenchymal cells. The preshaped artificial tissue constructs were cultured in perfusion chambers to maintain a stable diffusion of nutrients during the in vitro pre-formation step. Subsequently, pre-formed tissues were implanted into nude mice and into 4 mm articular joint defects of rabbits. Transplants were found to produce cartilage typic morphological patterns and matrix. 80% of the transplants remained stable in vivo. However, 20% of the tissues are resorbed or replaced by a fibrous tissue. These results demonstrate that current artificial cartilage transplants are already feasible for plastic reconstruction. The treatment of severe joint defects, however, faces additional problems which are addressed in ongoing studies: (a) the fixation of engineered cartilage in joints, (b) the protection against chronic inflammatory degradation, and (c) the required enormous mechanical stability.

Animals↗

PubMed2000

[Cell transplantation in surgery--reality and prospects for tissue engineering].

Traditionally surgical repair of tissue defects and loss or failure of function has relied on mechanical means, medical (drug) treatment, autologous and allogenic transplantation, and alloplastic/synthetic devices. Tissue engineering represents a new interdisciplinary field of applied research combining engineering and biosciences together with clinical application (mainly in surgical specialities) to develop living substitutes for tissues and organs. The understanding of cell-cell interactions and chemical signalling (growth factors) and the selection of appropriate matrices (cell-matrix interaction) is the key for success. Gene therapy represents the logical combination with tissue engineering on the molecular biology level. Application of cultivated skin and cartilage has already become reality, engineering of vascularized, more complex organs remains a challenge for this century.

Cells, Cultured↗