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PubMed2001

Construction and biological characterization of an HB-GAM/FGF-1 chimera for vascular tissue engineering.

OBJECTIVE: Cardiovascular tissue engineering approaches to vessel wall restoration have focused on the potent but relatively nonspecific and heparin-dependent mesenchymal cell mitogen fibroblast growth factor 1 (FGF-1). We hypothesized that linking FGF-1 to a sequence likely to bind to cell surface receptors relatively more abundant on endothelial cells (ECs) might induce a relative greater EC bioavailability of the FGF-1. We constructed a heparin-binding growth-associated molecule (HB-GAM)/FGF-1 chimera by linking full-length human HB-GAM to the amino-terminus of human FGF-1beta (21-154) and tested its activities on smooth muscle cells (SMCs) and ECs. METHODS: Primary canine carotid SMCs and jugular vein ECs were plated in 96-well plates in media containing 10% fetal bovine serum and grown to approximately 80% confluence. After being growth arrested in serum-free media for 24 hours, the cells were exposed to concentration ranges of cytokines and heparin, and proliferation was measured with tritiated-thymidine incorporation. Twenty percent fetal bovine serum was used as positive control, and phosphate-buffered saline was used as negative control. RESULTS: In the presence of heparin the HB-GAM/FGF-1 chimera stimulated less SMC proliferation than did the wild-type FGF-1 with a median effective dose of approximately 0.3 nmol versus approximately 0.1 nmol (P <.001). By contrast, the chimera retained full stimulating activity on EC proliferation with a median effective dose of 0.06 nmol for both cytokines. Unlike the wild-type protein, the chimera possessed heparin-independent activity. In the absence of heparin, the chimera induced dose-dependent EC and SMC proliferation at 0.06 nmol or more compared with the wild-type FGF-1, which stimulated minimal DNA synthesis at 6.0-nmol concentrations. CONCLUSIONS: The HB-GAM/FGF-1 chimera displays significantly greater and uniquely heparin-independent mitogenic activity for both cell types, and in the presence of heparin it displays a significantly greater EC specificity.

Animals↗

PubMed1996

Tissue engineering and autologous transplant formation: practical approaches with resorbable biomaterials and new cell culture techniques.

The engineering of living tissues in vivo requires new concepts in cell culture technology. In contrast to conventional cell cultures, the development of tissues depends on a three-dimensional arrangement of cells and the formation or synthesis of an appropriate extracellular matrix. Special emphasis is given to the major role of the extracellular matrix and cell differentiation in an artificial tissue. New technical approaches of in vitro tissue engineering are compared to the natural development of tissues in vivo. Current methods using resorbable biomaterials, tissue encapsulation and perfusion culture are discussed. Major consideration is given to scaffold structures of biomaterials that define a three-dimensional shape of a tissue or guide matrix formation. The different goals of tissue engineering such as in vitro models and transplant production are taken into account in the described techniques. Practical concepts comprising cell multiplication and differentiation in subsequent steps for future clinical applications are outlined.

Animals↗

PubMed1999

Bioreactor development for tissue-engineered cartilage.

The development of tissue engineered cartilage is emerging as a potential treatment for the repair of cartilage defects. By seeding chondrocytes onto poly-glycolic acid (PGA) biodegradable scaffolds within a convective-flow bioreactor, the synthesis of tissue-engineered articular cartilage has been recently demonstrated. The ability to cultivate and manipulate this cell-polymer construct to possess specific dimensions, as well as biochemical and biomechanical properties is critical for potential application as an in vivo therapy of damaged articular surfaces. Bioreactor design requirements for stages from research to development to commercialization are discussed. Advantages and limitations to various bioreactor designs are critiqued. These studies illustrate the ability to synthesize tissue-engineered cartilage under convective-flow conditions for potential human tissue repair.

Bioreactors↗

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↗

PubMed2001

A tissue engineered cell-occlusive device for hard tissue regeneration--a preliminary report.

Tissue engineering is an emerging discipline that applies engineering principles to create devices for the study, restoration, modification, and assembly of functional tissues and organs from native or synthetic sources. In the field of guided bone regeneration (GBR), cellular matter engineering has been applied, more or less successfully, to the development of biodegradable and bioresorbable devices with chemical, physical, or mechanical properties, structure, or form that permit active tissue integration with desirable cell types and tissue components. The employment of synthetic and naturally occurring polymers as well as sophisticated manufacturing technologies allow the tissue engineering of matrix configurations so that the biophysical limitations of mass transfer can be satisfied. The configuration of such a hybrid matrix can also be manipulated to vary the surface area available for cell attachment, as well as to optimize the exposure of the attached cells to nutrients. A biodegradable and bioresorbable device made of synthetic and natural polymers was engineered specifically for GBR procedures. The degradation and resorption kinetics as well as the mechanical properties give the device the potential to function as a carrier for bone growth factors. This innovative device was applied as a GBR membrane in a clinical investigation in seven patients.

Absorbable Implants↗

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↗

PubMed2003

[The method of accelerating osteanagenesis and revascularization of tissue engineered bone in big animal in vivo].

OBJECTIVE: To study whether tissue engineered bone can repair the large segment bone defect of large animal or not. To observe what character the fascia flap played during the osteanagenesis and revascularization process of tissue engineered bone. METHODS: 9 Chinese goats were made 2 cm left tibia diaphyseal defect. The repairing effect of the defects was evaluated by ECT, X-ray and histology. 27 goats were divided into three groups: group of CHAP, the defect was filled with coral hydroxyapatite (CHAP); group of tissue engineered bone, the defect was filled with CHAP + bone marrow stroma cells (BMSc); group of fascia flap, the defect was filled with CHAP + BMSc + fascia flap. After finished culturing and inducing the BMSc, CHAP of group of tissue engineered bone and of fascia flap was combined with it. Making fascia flap, different materials as described above were then implanted separately into the defects. Radionuclide bone imaging was used to monitor the revascularization of the implants at 2, 4, 8 weeks after operation. X-ray examination, optical density index of X-ray film, V-G staining of tissue slice of the implants were used at 4, 8, 12 weeks after operation, and the biomechanical character of the specimens were tested at 12 weeks post operation. RESULTS: In the first study, the defect showed no bone regeneration phenomenon. 2 cm tibia defect was an ideal animal model. In the second study, group of CHAP manifested a little trace of bone regeneration, as to group of tissue engineered bone, the defect was almost repaired totally. In group of fascia flap, with the assistance of fascia flap which gave more chance to making implants to get more nutrient, the repair was quite complete. CONCLUSIONS: The model of 2 cm caprine tibia diaphyseal defect cannot be repaired by goat itself and can satisfy the tissue engineering's demands. Tissue engineered bone had good ability to repair large segment tibia defect of goat. Fascia flap can accelerate the revascularization process of tissue engineered bone. And by this way, it augment the ability of tissue engineered bone to repair the large bone defect of goat.

Animals↗

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↗

PubMed2002

Composition options for tissue-engineered bone.

The logical assembly of tissue-engineered bone is ultimately directed by the clinical status of the patient. The basic elements for tissue-engineered bone should include signaling molecules, cells, and extracellular matrix. The assembly of these basic elements may need to be modified by tissue engineers to account for patient variables of age, gender, health, systemic conditions, habits, and anatomical implant. Moreover, different regions of the body will have different functional loads and vascularity. This review discusses several basic options that may be necessary to engineer bone, including spatial and temporal assembly of signaling factors, cells, and biomimetic extracellular matrices. Moreover, the importance of the health care status of the patient who may be receiving the tissue-engineered composition is emphasized.

Bone Marrow Cells↗

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↗

PubMed2002

Autologous tissue-engineered trachea with sheep nasal chondrocytes.

OBJECTIVE: This study was designed to evaluate the ability of autologous tissue-engineered trachea shaped in a helix to form the structural component of a functional tracheal replacement. METHODS: Nasal septum were harvested from six 2-month-old sheep. Chondrocytes and fibroblasts were isolated from tissue and cultured in media for 2 weeks. Both types of cells were seeded onto separate nonwoven meshes of polyglycolic acid. The chondrocyte-seeded mesh was wound around a 20-mm-diameter x 50-mm-long helical template and then covered with the fibroblast-seeded mesh. In 2 separate studies the implants were placed either in a subcutaneous pocket in the nude rat (rat tissue-engineered trachea) or in the neck of a sheep (sheep tissue-engineered trachea). Rat tissue-engineered tracheas were harvested after 8 weeks and analyzed by means of histology and biochemistry. Sheep tissue-engineered tracheas were harvested from the neck at 8 weeks and anastomosed into a 5-cm defect in the sheep trachea. RESULTS: Sheep receiving tissue-engineered trachea grafts survived for 2 to 7 days after implantation. Gross morphology and tissue morphology were similar to that of native tracheas. Hematoxylin-and-eosin staining of rat tissue-engineered tracheas and sheep tissue-engineered tracheas revealed the presence of mature cartilage surrounded by connective tissue. Safranin-O staining showed that rat tissue-engineered tracheas and sheep tissue-engineered tracheas had similar morphologies to native tracheal cartilage. Collagen, proteoglycan, and cell contents were similar to those seen in native tracheal tissue in rat tissue-engineered tracheas. Collagen and cell contents of sheep tissue-engineered tracheas were elevated compared with that of normal tracheas, whereas proteoglycan content was less than that found in normal tracheas. CONCLUSIONS: This study demonstrated the feasibility of recreating the cartilage and fibrous portion of the trachea with autologous tissue harvested from single procedure. This approach might provide a benefit to individuals needing tracheal resection.

Animals↗

PubMed2002

Tissue engineering of the lymphatic system.

The field of tissue engineering has seen tremendous expansion in the last decade. In the last several years, tissue-engineering strategies to treat diseases of skin, cartilage, bone, bladder, blood vessel, tendon, and other tissues have been described. However, tissue-engineering approaches to treat diseases of the lymphatic system are currently nonexistent. We propose that acellular tissues, either native or engineered, could be exploited as a platform for the study of lymphatic biology, and for lymphatic tissue engineering. While speculative, this type of experimental model system could prove powerful for dissecting molecular and cellular events surrounding tumor invasion of lymphatics, as well as lymphangiogenesis. Scaffolds seeded with genetically engineered lymphatic cells could also be implanted to repopulate lymphatic vasculature. In the future, the lymphatic system will surely be added to the list of tissues and organs that prove amenable to tissue-engineering therapies.

Animals↗

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↗

PubMed2003

Biomimetic materials for tissue engineering.

The development of biomaterials for tissue engineering applications has recently focused on the design of biomimetic materials that are capable of eliciting specific cellular responses and directing new tissue formation mediated by biomolecular recognition, which can be manipulated by altering design parameters of the material. Biomolecular recognition of materials by cells has been achieved by surface and bulk modification of biomaterials via chemical or physical methods with bioactive molecules such as a native long chain of extracellular matrix (ECM) proteins as well as short peptide sequences derived from intact ECM proteins that can incur specific interactions with cell receptors. The biomimetic materials potentially mimic many roles of ECM in tissues. For example, biomimetic scaffolds can provide biological cues for cell-matrix interactions to promote tissue growth, and the incorporation of peptide sequences into materials can also make the material degradable by specific protease enzymes. This review discusses the surface and bulk modification of biomaterials with cell recognition molecules to design biomimetic materials for tissue engineering. The criteria to design biomimetic materials such as the concentration and spatial distribution of modified bioactive molecules are addressed. Recent advances for the development of biomimetic materials in bone, nerve, and cardiovascular tissue engineering are also summarized.

Amino Acid Sequence↗