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PubMed2003

Adipose tissue engineering based on human preadipocytes combined with gelatin microspheres containing basic fibroblast growth factor.

Gelatin microspheres containing basic fibroblast growth factor (bFGF) were prepared for the controlled release of bFGF. Co-implantation with the gelatin microspheres enabled preadipocytes to induce adipose tissue formation at the implanted site. Preadipocytes isolated from human fat tissue were suspended with the gelatin microspheres containing bFGF and incorporated into a collagen sponge of cell scaffold. Following subcutaneous implantation of the collagen sponge incorporating human preadipocytes, and gelatin microspheres containing 1 microg of bFGF into the back of nude mice, adipose tissue was formed at the implanted site of collagen sponge within 6 weeks postoperatively although the extent depended on the number of preadipocytes transplanted and the bFGF dose. The formation of adipose tissue was significant compared with the implantation of collagen sponge incorporating human preadipocytes and 1 microg of free bFGF. The area of adipose tissue newly formed was increased with the number of preadipocytes transplanted until to 1.0 x 10(5) cells/site and thereafter leveled off. The maximum area was observed at the bFGF dose of 1 microg/site. The area was significantly smaller at the bFGF dose of 0.5 microg/site or larger than 1 microg/site. Immunohistochemical examination indicated that the adipose tissue newly formed was composed of human matured adipocytes. No adipogenesis was observed at the implanted site of collagen sponge incorporating either gelatin microspheres containing bFGF or human preadipocytes and the mixed gelatin microspheres containing bFGF and human preadipocytes. We conclude that combination of gelatin microspheres containing bFGF and preadipocytes with the collagen sponge is essential to achieve tissue engineering of fat tissue.

Adipocytes↗

PubMed2004

Development of a tissue-engineered human oral mucosa: from the bench to the bed side.

The main objective of this publication is to make the reader aware of the complexity and steps that are necessary to make a Food and Drug Administration (FDA)-approved laboratory produced cell-based device, for use in clinical trials for reconstructive surgery. Most tissue-engineered cell-based devices are considered as 'human somatic cell therapy' and fall under the auspices of the Center of Biologic Evaluation and Research (CBER) and are considered a combination product by the FDA. We have illustrated the algorithm that is necessary to follow an Independent New Drug (IND) application by using our ex vivo produced oral mucosa equivalents (EVPOME), a tissue-engineered oral mucosa, as an example of a cell-based device that needs FDA approval prior to clinical application. By illustrating the experimental approach and presenting resulting data we attempt to explain each step that we address along the way.

Animals↗

PubMed2006

Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds.

Natural bone consists of cortical and trabecular morphologies, the latter having variable pore sizes. This study aims at engineering different bone-like structures using scaffolds with small pores (112-224 microm) in diameter on one side and large pores (400-500 microm) on the other, while keeping scaffold porosities constant among groups. We hypothesized that tissue engineered bone-like structure resulting from silk fibroin (SF) implants is pre-determined by the scaffolds' geometry. To test this hypothesis, SF scaffolds with different pore diameters were prepared and seeded with human mesenchymal stem cells (hMSC). As compared to static seeding, dynamic cell seeding in spinner flasks resulted in equal cell viability and proliferation, and better cell distribution throughout the scaffold as visualized by histology and confocal microscopy, and was, therefore, selected for subsequent differentiation studies. Differentiation of hMSC in osteogenic cell culture medium in spinner flasks for 3 and 5 weeks resulted in increased alkaline phosphatase activity and calcium deposition when compared to control medium. Micro-computed tomography (microCT) detailed the pore structures of the newly formed tissue and suggested that the structure of tissue-engineered bone was controlled by the underlying scaffold geometry.

Biocompatible Materials↗

PubMed2005

Ligament tissue engineering: an evolutionary materials science approach.

The anterior cruciate ligament (ACL) is important for knee stabilization. Unfortunately, it is also the most commonly injured intra-articular ligament. Due to poor vascularization, the ACL has inferior healing capability and is usually replaced after significant damage has occurred. Currently available replacements have a host of limitations, this has prompted the search for tissue-engineered solutions for ACL repair. Presently investigated scaffolds range from twisted fiber architectures composed of silk fibers to complex three-dimensional braided structures composed of poly (L-lactic acid) fibers. The purpose of these tissue-engineered constructs is to apply approaches such as the use of porous scaffolds, use of cells, and the application of growth factors to promote ligament tissue regeneration while providing mechanical properties similar to natural ligament.

Absorbable Implants↗

PubMed2003

Transglutaminase-mediated gelatin matrices incorporating cell adhesion factors as a biomaterial for tissue engineering.

The goal of this work was to develop a novel biomaterial to be used for either wound dressing or as a scaffold for tissue engineering. The biodegradable hydrogels were prepared through cross-linking of gelatin with transglutaminase (TGase) in an aqueous solution. We found that the concentrations of 5 wt% gelatin and 1 unit/ml TGase were optimum for the proliferation of NIH/3T3 fibroblasts. Then, we investigated whether the cell proliferation was enhanced by incorporation of cell adhesion factors into the gelatin hydrogels. Since vitronectin and fibronectin can bind with gelatin by the action of TGase, we added these cell adhesion proteins into the gelatin hydrogels. The hydrogels incorporating these cell adhesion proteins significantly enhanced cell proliferation compared with the gelatin hydrogels without these proteins (p<0.05). Two types of synthetic Arg-Gly-Asp (RGD) peptides, RGDLLQ and RGDLLG were also added to the gelatin solution where RGDLLQ is a substrate of TGase by virtue of a glutamine (Q) residue with an epsilon-amino group and RGDLLG is not. These two RGD peptides enhanced cell proliferation, but RGDLLQ significantly enhanced the proliferation compared with RGDLLG (p<0.05). These results suggest that T-Gase-mediated incorporation of cell adhesion factors into gelatin matrices enhanced cell proliferation and this novel biomaterial is a potent tool for wound dressing or tissue engineering.

Journal Article↗

PubMed2001

Introduction of tissue engineering concepts into the field of endovascular grafts: an attempt to solve endoleakage problems of endovascular grafts implanted in aortic aneurysms.

To solve endoleakage problems of endovascular prostheses inserted in aortic aneurysms, a concept originating from tissue engineering was proposed. We proposed that in order to accelerate the thrombus inside the aneurysm, three major factors for successful tissue engineering, i.e., appropriate cells, extracellular matrices, and growth factors, should be introduced into a space between the endovascular grafts and the aneurysm. As a simulation model in animals, two kinds of fabric vascular prostheses were used. One was a small graft used as an endovascular graft and another was a large graft as an envelope for the small graft, used as the aneurysm wall. Adipose tissue fragments with fresh blood coagula were injected between the two grafts. The control was a preparation without tissue fragments. Two months and a half after surgery, the test group showed complete connective tissue formation in the space between the two grafts, but in the control, brownish thrombus remained. From this experiment, we speculated that tissue fragment transplantation will accelerate connective tissue formation of the thrombus, and this complete organization of the thrombus might reduce endoleakage.

Adipose Tissue↗

PubMed1999

In vitro analysis of biodegradable polymer blend/hydroxyapatite composites for bone tissue engineering.

Blends of biodegradable polymers, poly(caprolactone) and poly(D, L-lactic-co-glycolic acid), have been examined as scaffolds for applications in bone tissue engineering. Hydroxyapatite granules have been incorporated into the blends and porous discs were prepared. Mechanical properties and degradation rates in vitro of the composites were determined. The discs were seeded with rabbit bone marrow or cultured bone marrow stromal cells and incubated under physiological conditions. Polymer/ceramic scaffolds supported cell growth throughout the scaffold for 8 weeks. Scanning and transmission electron microscopy, and histological analyses were used to characterize the seeded composites. This study suggests the feasibility of using novel polymer/ceramic composites as scaffold in bone tissue engineering applications.

Animals↗

PubMed2004

Comparison of different fabrication techniques used for processing 3-dimensional, porous, biodegradable scaffolds from modified starch for bone tissue engineering.

3 dimensional, porous, biodegradable scaffolds were fabricated using modified starch of varying degree of substitution (DS) by extrusion processing. Freeze drying/lyophilization was also employed to fabricate scaffolds from modified starch. The research efforts have been focused on the comparison of the above-mentioned techniques by comparing the properties of the fabricated scaffolds in the paradigm of bone tissue engineering. The physicomechanical properties like porosity, compressive strength and modulus, pore size and microstructure were tested and analyzed by liquid replacement, mechanical testing and scanning electron microscopy respectively. The biodegradability of scaffolds was evaluated by soaking the samples in aqueous medium and Hank's balanced salt solution at 37-degree invitro. The cytotoxicity studies on these scaffolds were also conducted. The scaffolds have a 3D structure consisting of interconnected pores with good porosity, pore size, adequate compressive strength and modulus and exhibit good biodegradability as well as biocompatibility. After further optimization in the processing conditions and parameters they could be made useful for bone tissue engineering.

Absorbable Implants↗

PubMed1995

Tissue-engineered cartilage for implantation and grafting.

Facial plastic and reconstructive surgeons are continually faced with the dilemma of what material to use for implantation and grafting. Tissue-engineered cartilage is a relatively new and exciting concept which utilizes chondrocytes and cultures them on a three-dimensional biodegradable template. This template/cell complex is first briefly incubated in vitro, then implanted into a recipient host. In situ the template resorbs and is replaced with new cartilage that is viable, compatible, and mature. This paper discusses the biochemical composition of cartilage, the concept of tissue engineering, advances in template quality, and cartilage immunogenicity. Future clinical applications of this type of graft research include microtia repair, facial reconstruction, rhinoplasty, and other facial cosmetic procedures.

Animals↗

PubMed1997

Tissue-engineered heart valve leaflets: does cell origin affect outcome?

BACKGROUND: We previously reported the successful creation of tissue-engineered valve leaflet constructs and the implantation of these autologous tissue leaflets in the pulmonary valve position in a lamb model. The optimal cell origin for creating these valve leaflets remains unclear. This study was designed to compare dermal with arterial wall myofibroblasts as the cells of origin for the leaflet constructs. METHODS AND RESULTS: Mixed cell populations of endothelial cells and fibroblasts were isolated from ovine femoral arteries or subdermis and then expanded in vitro. A synthetic biodegradable polymer scaffold was then seeded with the cultured cells. The tissue scaffold was composed of a polyglactin woven mesh sandwiched between two nonwoven polyglycolic acid mesh sheets, which measured 3x3 cm in size and 3.2 mm in thickness. The cell-seeded polymer construct was implanted to replace one pulmonary valve leaflet in the same juvenile animal from which the cells had originally been obtained. Using cardiopulmonary bypass, the right posterior leaflet of the pulmonary valve was completely resected and replaced with an autologous engineered valve leaflet. In group D (n=5), the cells were obtained from subdermis, and in group A (n=4), they were obtained from the arterial wall. Eight to 10 weeks after leaflet implantation, the animals were killed, and the implanted valve leaflets were examined histologically, biochemically, and biomechanically. The dimensions of each tissue-engineered leaflet (TEL) were compared with those of the two remaining native valve leaflets to obtain a growth index. A 4-hydroxyproline assay was performed to evaluate collagen content. Leaflet tensile strength was evaluated in vitro by using a Vitrodyne V-1000 mechanical tester. Factor VIII and elastin stains were performed to histologically assess the presence of endothelial cells and elastin, respectively. In all animals, the TEL persisted in the pulmonary valve position after 8 to 10 weeks, and all polyglycolic acid polymer had been degraded. Group A leaflets had a higher growth index (0.86+/-0.11) than group D (0.41+/-0.08) (P<.05). Macroscopically, the group D leaflets appeared thicker and contracted. Histologically, elastic fibers were more abundant in group A than in group D. Total collagen content and biomechanical testing showed no differences between groups. Leaflets from both groups had positive staining for factor VIII on the surface, confirming growth of endothelial cells to cover the TEL. CONCLUSIONS: Autologous TEL derived from vascular fibroblasts seem to develop functionally and morphologically like the native valve leaflets in the pulmonary circulation. Use of arterial myofibroblasts for the creation of TEL seems preferable to dermal fibroblasts with current tissue culture conditions.

Animals↗

PubMed2004

Development of novel tissue engineering scaffolds via electrospinning.

Electrospinning has recently been developed as an efficient technique to develop polymeric nanofibres. Various synthetic and natural biodegradable polymers have been electrospun into fibres with diameters in the nanometre range (< 1 microm). The fibre diameter, structure and physical properties of the nanofibre matrices can be effectively tuned by controlling various parameters that affect the electrospinning process. The dimension and structure of electrospun polymeric nanofibre mats resembles mostly the collagen phase of natural extracellular matrix. This, combined with excellent physical properties such as high surface area, high porosity, interconnective pores of the nanofibre matrices and appropriate mechanical properties, well-controlled degradation rates and biocompatibility of the base polymer, make biodegradable polymeric nanofibre matrices ideal candidates for developing scaffolds for tissue engineering. This article reviews the recent advances in the development of synthetic biodegradable nanofibre-based matrices as scaffolds for tissue engineering.

Animals↗

PubMed2004

Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement.

STUDY DESIGN: By the technique of tissue engineering, composite intervertebral disc implants were fabricated as novel materials for disc replacement, implanted into athymic mice, and removed at times up to 12 weeks. OBJECTIVES: The goal of this study was to construct composite intervertebral disc structures consisting of anulus fibrosus cells and nucleus pulposus cells seeded on polyglycolic acid and calcium alginate matrices, respectively. SUMMARY OF BACKGROUND DATA: Previous work has documented the growth of anulus fibrosus cells on collagen matrices and nucleus pulposus cells cultured on multiple matrices, but there is no documentation of composite disc implants. METHODS: Lumbar intervertebral discs were harvested from sheep spine, and the nucleus pulposus was separated from surrounding anulus fibrosus. Each tissue was digested in collagenase type II. After 3 weeks in culture, cells were seeded into implants. The shape of the anulus fibrosus scaffold was fabricated from polyglycolic acid and polylactic acid, and anulus fibrosus cells were pipetted onto the scaffold and allowed to attach for 1 day. Nucleus pulposus cells were suspended in 2% alginate and injected into the center of the anulus fibrosus. The disc implants were placed in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, 4 samples were stored in -70 degrees C for collagen typing and analysis of proteoglycan, hydroxyproline, and DNA. Other samples were fixed in 10% formalin for Safranin-O staining. RESULTS: The gross morphology and histology of engineered discs strongly resembled those of native intervertebral discs. Biochemical markers of matrix synthesis were present, increasing with time, and were similar to native tissue at 12 weeks. Tissue-engineered anulus fibrosus was rich in type I collagen but nucleus pulposus contained type II collagen, similar to the native disc. CONCLUSION: These results demonstrate the feasibility of creating a composite intervertebral disc with both anulusfibrosus and nucleus pulposus for clinical applications.

Animals↗

PubMed2002

Epidermal-dermal interactions regulate gelatinase activity in Apligraf, a tissue-engineered human skin equivalent.

BACKGROUND: Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) have important functions during skin development, repair and maintenance. MMP-2 and MMP-9 (gelatinase A and gelatinase B) are involved in regulating keratinocyte migration. OBJECTIVES: To analyse whether Apligraf, a bilayered tissue-engineered human skin equivalent (HSE), produces gelatinases and TIMPs and whether or not epidermal-dermal interactions regulate MMP activity. METHODS: The tissue distribution of MMP-2, MMP-9, TIMP-1, TIMP-2 and fibronectin was analysed by immunohistochemistry. Secreted MMP activity was quantified by a fluorimetric assay and gelatin zymography was used to monitor gelatinases in tissue culture supernatants. RESULTS: Apligraf expressed MMP-2 and MMP-9 and contained immunohistochemically detectable amounts of TIMP-1 and TIMP-2. The gelatinases were predominantly produced in the epidermis, whereas immunostaining of TIMP-1 and TIMP-2 was largely confined to the dermal component of the HSE. Fibronectin was expressed only in the dermis. Gelatin zymography demonstrated that intact Apligraf produced both MMP-2 and MMP-9, the latter predominantly in its latent form. Separation of the dermis from the epidermis resulted in an enhanced production and activation of MMP-9 by the epidermal layer, and secretion of latent and active MMP-2 by the dermal layer. Moreover, the incubation media of the separated epidermis demonstrated significantly stronger MMP activity than did intact Apligraf or its dermal component. CONCLUSIONS: These observations provide evidence that epidermal-dermal interactions suppress epidermal gelatinase activity. In addition, coexpression of TIMPs and fibronectin in the Apligraf dermis suggests that the product has the potential to counteract the imbalance between matrix production and degradation in chronic wounds and thus may support wound re-epithelialization.

Collagen↗

PubMed2004

Tissue-engineered skin substitutes: from in vitro constructs to in vivo applications.

The field of skin tissue engineering is a paradigm for the various efforts towards the reconstruction of other tissues and organ substitutes. As skin replacement, this biotechnological approach has evolved from simple cultured autologous epidermal sheets to more complex bilayered cutaneous substitutes. The various types of such substitutes are herein presented with their intended use. However, two integrative characteristics are analysed more specifically because of their critical role: neovascularization and re-innervation. Furthermore, the in vitro use of these various skin substitutes has shed light on various physiological and pathological phenomena. Thus, not only the in vivo application of these skin substitutes as grafts, but also their in vitro value as skin models, are presented.

Animals↗

PubMed2001

Molecular tissue engineering: applications for modulation of mesenchymal stem cells proliferation by transforming growth factor beta 1 gene transfer.

The effect of transforming growth factor beta 1 (TGF-beta 1) gene transfection on the proliferation of bone marrow-derived mesenchymal stem cells (MSCs) and the mechanism was investigated to provide basis for accelerating articular cartilage repairing using molecular tissue engineering technology. TGF-beta 1 gene at different doses was transduced into the rat bone marrow-derived MSCs to examine the effects of TGF-beta 1 gene transfection on MSCs DNA synthesis, cell cycle kinetics and the expression of proliferating cell nuclear antigen (PCNA). The results showed that 3 microliters lipofectamine-mediated 1 microgram TGF-beta 1 gene transfection could effectively promote the proliferation of MSCs best; Under this condition (DNA/Lipofectamine = 1 microgram/3 microliters), flow cytometry and immunohistochemical analyses revealed a significant increase in the 3H incorporation, DNA content in S phase and the expression of PCNA. Transfection of gene encoding TGF-beta 1 could induce the cells at G0/G1 phase to S1 phase, modulate the replication of DNA through the enhancement of the PCNA expression, increase the content of DNA at S1 phase and promote the proliferation of MSCs. This new molecular tissue engineering approach could be of potential benefit to enhance the repair of damaged articular cartilage, especially those caused by degenerative joint diseases.

Animals↗

PubMedDate not supplied

In vivo tissue engineering of bone using poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) and collagen scaffolds.

Porous poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and calcium phosphate-loaded collagen (CaP-Gelfix) foams were seeded with rat bone marrow stromal cells and implanted into defects created in rat femurs to study in vivo bone formation and to test their suitability for use in bone tissue engineering. At 3 and 6 weeks, new bone formation was evaluated by macroscopy, radiography, dual-energy X-ray absorptiometry (DEXA), and quantitative computerized tomography (QCT). Atomic contents of the implants were further assessed by QCT. Some initial inflammation that significantly decreased with time was observed in the CaP-Gelfix group. PHBV inflammation was minimal at all stages. Fibrous tissue formation in the CaP-Gelfix group was more than in the PHBV group. Both cell-loaded and cell-free PHBV matrices elicited minimal fibrous tissue formation during the 6-week implantation duration. Macroscopic and radiological studies demonstrated better healing with PHBV matrices than with CaP-Gelfix in 3 weeks. Histologically, fibrous connective tissue establishment and inflammation scores were significantly higher in the CaP-Gelfix group when compared with the PHBV group at both time intervals. At 6 weeks, however, the extent of healing was almost the same with both implants. DEXA and QCT results indicated that there was an increase in bone mineral density in both PHBV and CaP-Gelfix implants at the end of 6 weeks. This study suggests that even though PHBV and CaP-Gelfix have different bulk and surface chemistries they both are promising cell carriers that may be suitable for use in bone tissue engineering.

Animals↗

PubMed2003

Tissue-engineered cartilage and bone using stem cells from human infrapatellar fat pads.

Multipotential processed lipoaspirate (PLA) cells extracted from five human infrapatellar fat pads and embedded into fibrin glue nodules, were induced into the chondrogenic phenotype using chondrogenic media. The remaining cells were placed in osteogenic media and were transfected with an adenovirus carrying the cDNA for bone morphogenetic protein-2 (BMP-2). We evaluated the tissue-engineered cartilage and bone using in vitro techniques and by placing cells into the hind legs of five severe combined immunodeficient mice. After six weeks, radiological and histological analysis indicated that the PLA cells induced into the chondrogenic phenotype had the histological appearance of hyaline cartilage. Cells transfected with the BMP-2 gene media produced abundant bone, which was beginning to establish a marrow cavity. Tissue-engineered cartilage and bone from infrapatellar fat pads may prove to be useful for the treatment of osteochondral defects.

Adipose Tissue↗

PubMed2003

In vitro development of a tissue-engineered model of peripheral nerve regeneration to study neurite growth.

A unique tissue-engineered model of peripheral nerve regeneration was developed in vitro to study neurite outgrowth. Mouse dorsal root ganglia neurons were seeded on a collagen sponge populated with human endothelial cells and/or human fibroblasts. Addition of nerve growth factor (NGF; 10 ng/ml) was not required for sensory neurons survival but was necessary to promote neurite outgrowth, as assessed by immunostaining of the 150 kDa neurofilament. A vigorous neurite elongation was detected inside the reconstructed tissue after 14 and 31 days of neurons culture, reaching up to 770 microm from day 14. Axons were often observed closely associated with the capillary-like tubes reconstructed in the model, in a similar pattern as in the human dermis. The presence of endothelial cells induced a significant increase of the neurite elongation after 14 days of culture. The addition of human keratinocytes totally avoided the twofold decrease in the amount of neurites observed between 14 and 31 days in controls. Besides the addition of NGF, axonal growth did not necessitate B27 supplement or glial cell coculture to be promoted and stabilized for long-term culture. Thus, this model might be a valuable tool to study the effect of various cells and/or attractive or repulsive molecules on neurite outgrowth in vitro.

Animals↗