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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↗

PubMed2000

[Bibliometric analysis on tissue engineering research literatures].

OBJECTIVE: To comprehend the progress of tissue engineering research and speculate its developmental trends. METHODS: MEDLINE search was conducted to retrieve the papers published between 1987 to 1999 under the main headings of tissue engineering. Years, nationalities, languages, journals, authors and heading frequencies of 314 papers were analyzed by bibliometrics. RESULTS: Since 1990, the number of tissue engineering research literatures had doubled, and papers between 1998 and 1999 made up 57.96% of the total papers. All papers came from 15 nations, in 6 languages and 140 journals; 64.97% came from United States and 25.79% from England, Netherlands and Germany; 93.95% was in English; 42.04% was published on 15 journals. Vacanti JP and 19 other authors presented 5 to 24 papers. Heading frequencies were cytology 22.89%, transplantation 13.30%, scaffolds and extracellular matrixes 11.72%, implanting 10.60%, polymers 8.91%, potential applications 8.91%, artificial substitutes 6.88%, tissue culture 6.70% and biogenetics 4.96%. CONCLUSION: Tissue engineering literatures mainly come from United States, England, Netherlands and Germany. English is the major language. J Biomed Mater Res and 14 other journals are important journals about tissue engineering research. Vacanti JP and 19 other authors are prolific authors. Cytology, transplantation, scaffolds and extracellular matrixes and implanting are hot topics and key points on tissue engineering research.

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↗

PubMed2002

Mechanical stimulation improves tissue-engineered human skeletal muscle.

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.

Biocompatible Materials↗

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↗

PubMed2002

Experimental and clinical experience with tissue engineering techniques for urethral reconstruction.

Tissue engineering has been proposed as a strategy for urethral reconstruction. This may involve matrices alone, wherein the body's natural ability to regenerate is used to orient or direct new tissue growth, or the use of matrices with cells. Acellular collagen matrices derived from donor bladder submucosa have been used both experimentally and clinically for onlay urethral replacement with good success at our center. If a tubularized urethral repair is needed, the use of cells on the collagen matrix is essential for adequate tissue formation. Tissue engineering techniques are useful for urethral reconstruction.

Biocompatible Materials↗

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↗

PubMed2003

Monitoring local cell viability in engineered tissues: a fast, quantitative, and nondestructive approach.

Assessment of cell viability is a key issue in monitoring in vitro engineered tissue constructs. In this study we describe a fully automated, quantitative, and nondestructive approach, which is particularly suitable for tissue engineering. The approach offers several advantages above existing methods. Living and dead cell numbers can be separately determined for both isolated cells and cells that form networks during tissue formation. Moreover, viability can be locally monitored in time throughout the three-dimensional tissue. The viability assay is based on a dual fluorescent staining technique using CellTracker Green (CTG) for detection of living cells and propidium iodide (PI) for dead cells. CTG and PI images are created with a confocal laser scanning microscope. To determine the number of living cells, CTG fluorescence intensity is determined from the CTG image. Thereby, novel image-processing techniques have been developed, normalizing for various undesired influences that alter measurements of absolute CTG fluorescence intensities. Dead cell numbers are determined from the PI image, using an improved computerized counting method. The approach was first evaluated on C2C12 monolayers, of which images were taken directly after probe addition and 24 h later. Results show that at both times, computed living and dead cell numbers highly correlate with manually counted cell numbers (r > 0.996). Next, the approach was applied for monitoring viability in three-dimensional engineered skeletal muscle tissue constructs, which were subjected to unfavorable environmental conditions. This example illustrated that local viability can be quantitatively, nondestructively, and locally monitored in three-dimensional tissue constructs, making it a promising tool in the field of tissue engineering.

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↗

PubMed2003

Cardiac tissue engineering, ex-vivo: design principles in biomaterials and bioreactors.

Cardiac tissue engineering has emerged as a promising approach to replace or support an infarcted cardiac tissue and thus may hold a great potential to treat and save the lives of patients with heart diseases. By its broad definition, tissue engineering involves the construction of tissue equivalents from donor cells seeded within 3-D biomaterials, then culturing and implanting the cell-seeded scaffolds to induce and direct the growth of new, healthy tissue. In this review, we present an up-to-date summary of the research in cardiac tissue engineering, with an emphasis on the design principles and selection criteria that have been used in two key technologies employed in tissue engineering, (1) biomaterials technology, for the creation of 3-D porous scaffolds which are used to support and guide the tissue formation from dissociated cells, and (2) bioreactor cultivation of the 3-D cell constructs during ex-vivo tissue engineering, which aims to duplicate the normal stresses and flows experienced by the tissues.

Biocompatible Materials↗

PubMed2002

Regulatory barriers to tissue-engineered products.

Autologous tissue-engineering technology uses the body's own regenerative capacity to solve clinical problems. Human cellular- and tissue-based products include an array of medical products for repair, reproduction, replacement and other therapeutic purposes. However, the lack of European Union regulation for these products is putting the future of the technology at risk in Europe.

Costs and Cost Analysis↗

PubMed2002

Functional analysis of the tissue-engineered stomach wall.

We have established a method for in situ tissue engineering of the stomach in a canine model using an acellular collagen scaffold graft. The current study was conducted to evaluate the functional aspects of the tissue-engineered stomach wall. The anterior wall of the stomach in beagle dogs was replaced with a collagen sponge scaffold measuring 4 x 4 cm. At 16 weeks after implantation, the animals were sacrificed and the stomach specimens were evaluated immunohistochemically and physiologically. Regeneration of the proton pump and thin muscle layer, which are essential for mechanical and chemical digestion by the stomach, was observed in the tissue-engineered gastric tissue. However, acetylcholine-induced contraction was not observed in the tissue-engineered stomach wall. Although there is still room for improvement, the tissue-engineered stomach wall had a highly organized structure, and it is anticipated that this approach could eventually become an alternative for stomach reconstruction after gastrectomy.

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↗

PubMed2002

The suitability of cells from different tissues for use in tissue-engineered skin substitutes.

Tissue-engineered skin substitutes may be a future remedy for burn wounds and chronic wounds, as wound contraction and scar formation cannot be prevented with the current standard treatment. The aim of this study therefore was to identify readily available sources of fibroblasts suitable for dermal substitution. Three different tissues were studied: dermal tissue from split-skin graft, subcutaneous fat tissue and eschar tissue obtained through debridement of burn wounds. We determined the cellular profile and the cell numbers immediately after isolation and after 2 and 14 days of fibroblast culture using flow cytometry and cell counting with a cytometer. In addition, parts of the isolated cell suspensions were seeded directly into a porous collagen dermal substitute to investigate contraction over time. Various cell types were isolated from the three different tissues, but after 14 days of culturing predominantly fibroblasts (>90%) were detected. Keratinocytes, granulocytes and macrophages, if present, disappeared within 14 days. In the cell populations derived from dermal tissue, the percentage of myofibroblasts had decreased significantly by day 14 (from 8% to 3%, P=0.028). In contrast, this percentage had increased in the cell populations derived from fat and eschar (from 23% to 40% and from 20% to 38%, respectively). The fibroblast yield from dermal tissue after 2 weeks of culturing (50 x 10(6) cells/g of tissue) was significantly higher than the yield from fat and eschar tissue (2 x 10(6) cells/g of each tissue, P=0.029). Immunohistochemistry of collagen matrices seeded and cultured with fat- and eschar-derived cells revealed a high prevalence of myofibroblasts, whereas hardly any myofibroblasts were detected in the matrices seeded with dermal cells. The contraction of the eschar matrices was highest (74+/-6% remaining area), whereas dermal matrices contracted significantly less (92+/-7% remaining area, P=0.029) with intermediate contraction for fat matrices. We conclude that fibroblast cultures can be established from dermal tissue, fat tissue and eschar tissue. Dermis is the best fibroblast source for use in skin substitutes as it yields the highest numbers of fibroblasts with minimal numbers of myofibroblasts.

Adipose Tissue↗

PubMed2002

Tissue engineering--current challenges and expanding opportunities.

Tissue engineering can be used to restore, maintain, or enhance tissues and organs. The potential impact of this field, however, is far broader-in the future, engineered tissues could reduce the need for organ replacement, and could greatly accelerate the development of new drugs that may cure patients, eliminating the need for organ transplants altogether.

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↗