Recapitulation of oral mucosal tissues in long-term organotypic culture. (41/3513)

To test the influence of fibroblasts on epithelial morphology and expression of keratinocyte proteins and barrier lipids, we bioengineered homotypic and heterotypic oral mucosae and skin using cultured adult human cells. Fibroblasts were allowed to modify collagen type I gels for 2 weeks before keratinocytes were added. The organotypic cultures were then grown at the air-liquid interface for 4 weeks. In homotypic combinations, epithelial morphology and protein expression closely mimicked those in vivo. In heterotypic combinations, the morphology resembled that in vivo and keratinocytes expressed their typical markers, except when skin keratinocytes were recombined with alveolar fibroblasts; they expressed K19, K4, and K13, which is similar to oral mucosal epithelia rather than to the epidermis. Morphologically, the stratum corneum layers were typical for the epithelial tissues. Grafting the bioengineered cultures to the backs of Nude mice did not change the results, suggesting that our findings are not merely a culture phenomenon. Lipid profiles of the homotypic combinations mimicked the profiles found in the normal epithelial tissues, except that the engineered alveolar epithelium expressed more ceramide 2 than that in vivo. In the heterotypic combinations, keratinocytes appeared to control the lipid profile, except in the combination of skin keratinocytes with alveolar fibroblasts, wherein the ceramide profile appeared to be partly that of alveolar epithelium and partly that of epidermis. These results suggest that cultured adult fibroblasts and keratinocytes are sufficient to recapitulate graftable oral tissues, and, except for alveolar fibroblasts, the type of fibroblast had little influence on keratinocyte differentiation.  (+info)

Inhibition of cartilage degradation: a combined tissue engineering and gene therapy approach. (42/3513)

OBJECTIVE: To determine if tissue-engineered cartilage can be protected from cytokine-induced degradation using a gene therapy approach. METHODS: Chemical and pantropic retroviral gene transfer methodologies were compared for their ability to introduce a luciferase reporter gene into adult bovine cartilage chondrocytes grown in monolayer. Pantropic retrovirus was then used to transduce these cells with human tissue inhibitor of metalloproteinases 1 (TIMP-1), and the stability of expression in monolayer or pellet culture was monitored for 6 weeks. Untransduced and TIMP-1-transduced cells were also used to tissue engineer 3-dimensional cartilage constructs that were then challenged with interleukin-1 (IL-1) for 4 weeks. Conditioned media and residual cartilage were collected for analysis of matrix components, including type II collagen and proteoglycans, and for TIMP-1 production and matrix metalloproteinase (MMP) activity. RESULTS: Chemical transfection of adult bovine chondrocytes gave rise to short-lived reporter expression that was virtually undetectable after 4 weeks of culture. In contrast, pantropic retroviral transduction gave rise to stable expression that persisted at a high level for at least 6 weeks. Pantropic transduction of the cells with TIMP-1 gave rise to similar long-term expression, both in monolayer and pellet cultures. TIMP-1-transduced tissue-engineered cartilage also retained TIMP-1 expression for an additional 4 weeks of culture in the presence of IL-1. Compared with control samples, TIMP-1-transgenic cartilage resisted the catabolic effects of IL-1, with MMP activity reduced to basal levels and a decreased loss of type II collagen. CONCLUSION: Pantropic retroviral transduction permits long-term expression of potentially therapeutic transgenes in adult tissue-engineered cartilage. While TIMP-1 transduction could be used to prevent collagen breakdown, alternative transgenes may be necessary to protect cartilage proteoglycans.  (+info)

Tissue-engineered bioprosthetic venous valve: a long-term study in sheep. (43/3513)

OBJECTIVE: to develop a graft bearing an immunologically tolerated tissue-engineered venous valve (TE graft) that will be incorporated into a native vessel, and restore normal valve function for the treatment of chronic venous insufficiency. METHODS: twenty-four TE grafts were grown using decellularised allogeneic ovine veins as donor matrix, which was subsequently repopulated with the future recipient's myofibroblasts (MFB) and endothelial cells (EC). TE grafts were implanted into the external jugular vein. Animals were sacrificed at 1, 6, and 12 weeks (n=4, each). Autografts served as controls (1 week, n=4; 6 weeks, n=4). Specimen for histology and immunohistochemistry were taken. RESULTS: the matrix was successfully repopulated with MFB and EC (n=8). Patency on venography in the TE graft-group was44,44, and 34 at 1, 6, and 12 weeks, and44 (44) in autografts at 1 (6) weeks, respectively. Except for 2 TE grafts after 12 weeks, valves were competent (duplex ultrasound). Patent TE grafts were merely distinguishable from autografts with minor inflammatory reactions. Reflux was caused by neo-intima formation related to the basis of the TE graft. CONCLUSION: acellularisation and consecutive in vitro autogeneic re-seeding of valved venous conduits can lead to immunologically acceptable, patent, and competent implants in sheep.  (+info)

Improving endothelial cell retention for single stage seeding of prosthetic grafts: use of polymer sequences of arginine-glycine-aspartate. (44/3513)

OBJECTIVE: single stage seeding within the timeframe of a typical vascular operation has not been successful. One reason for this is poor cell adherence to the graft lumen once exposed to pulsatile blood flow. In this study we have carried out investigations with the use of two different fibronectin-based peptides, fibronectin-like engineered protein polymer (FEPP) which contains multiple copies of arginine-glycine-aspartate (RGD) and fibronectin adhesion promoting peptide (FAPP) to improve cell adherence. MATERIALS AND METHODS: FAPP and FEPP were coated onto native polyurethane and heparinised polyurethane grafts. The grafts were then seeded for either 1 or 2h with human umbilical vein endothelial cells (HUVEC). After the incubation period the cells were washed off and cell retention was calculated. Cell metabolism was measured using Alamar Blue, and confirmed with scanning electron microscopy (SEM). RESULTS: heparinised grafts coated with FEPP showed the best cell retention after both 1 and 2h seeding (80+/-4% vs 81+/-3%). This graft had no significant difference in cell retention after both times whilst all the other grafts had better cell retention after a 2h seeding. The Alamar blue and SEM results confirmed cell viability and function for all graft types. CONCLUSION: heparinised graft coated with FEPP allows significant cell retention after only 1h of seeding and shows promise for single stage seeding.  (+info)

Gene therapy of bone morphogenetic protein for periodontal tissue engineering. (45/3513)

BACKGROUND: The reconstruction of lost periodontal support including bone, ligament, and cementum is a major goal of therapy. Bone morphogenetic proteins (BMPs) have shown much potential in the regeneration of the periodontium. Limitations of BMP administration to periodontal lesions include need for high-dose bolus delivery, BMP transient biological activity, and low bioavailability of factors at the wound site. Gene transfer offers promise as an alternative treatment strategy to deliver BMPs to periodontal tissues. METHODS: This study utilized ex vivo BMP-7 gene transfer to stimulate tissue engineering of alveolar bone wounds. Syngeneic dermal fibroblasts (SDFs) were transduced ex vivo with adenoviruses encoding either green fluorescent protein (Ad-GFP or control virus), BMP-7 (Ad-BMP-7), or an antagonist of BMP bioactivity, noggin (Ad-noggin). Transduced cells were seeded onto gelatin carriers and then transplanted to large mandibular alveolar bone defects in a rat wound repair model. RESULTS: Ad-noggin treatment tended to inhibit osteogenesis as compared to the control-treated and Ad-BMP-7-treated specimens. The osseous lesions treated by Ad-BMP-7 gene delivery demonstrated rapid chrondrogenesis, with subsequent osteogenesis, cementogenesis and predictable bridging of the periodontal bone defects. CONCLUSION: These results demonstrate the first successful evidence of periodontal tissue engineering using ex vivo gene transfer of BMPs and offers a new approach for repairing periodontal defects.  (+info)

Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: engineering cell-invasion characteristics. (46/3513)

Synthetic hydrogels have been molecularly engineered to mimic the invasive characteristics of native provisional extracellular matrices: a combination of integrin-binding sites and substrates for matrix metalloproteinases (MMP) was required to render the networks degradable and invasive by cells via cell-secreted MMPs. Degradation of gels was engineered starting from a characterization of the degradation kinetics (k(cat) and K(m)) of synthetic MMP substrates in the soluble form and after crosslinking into a 3D hydrogel network. Primary human fibroblasts were demonstrated to proteolytically invade these networks, a process that depended on MMP substrate activity, adhesion ligand concentration, and network crosslinking density. Gels used to deliver recombinant human bone morphogenetic protein-2 to the site of critical defects in rat cranium were completely infiltrated by cells and remodeled into bony tissue within 4 wk at a dose of 5 microg per defect. Bone regeneration was also shown to depend on the proteolytic sensitivity of the matrices. These hydrogels may be useful in tissue engineering and cell biology as alternatives for naturally occurring extracellular matrix-derived materials such as fibrin or collagen.  (+info)

Adult mesenchymal stem cells and cell-based tissue engineering. (47/3513)

The identification of multipotential mesenchymal stem cells (MSCs) derived from adult human tissues, including bone marrow stroma and a number of connective tissues, has provided exciting prospects for cell-based tissue engineering and regeneration. This review focuses on the biology of MSCs, including their differentiation potentials in vitro and in vivo, and the application of MSCs in tissue engineering. Our current understanding of MSCs lags behind that of other stem cell types, such as hematopoietic stem cells. Future research should aim to define the cellular and molecular fingerprints of MSCs and elucidate their endogenous role(s) in normal and abnormal tissue functions.  (+info)

Repression of allo-cell transplant rejection through CIITA ribonuclease P+ hepatocyte. (48/3513)

AIM: Allo-cell transplant rejection and autoimmune responses were associated with the presence of class II major histocompatibility complex (MHC II) molecules on cells. This paper studied the effect of Ribonuclease P (RNase P) against CIITA, which was a major regulator of MHCII molecules, on repressing the expression of MHCII molecules on hepatocyte. METHODS: M1-RNA is the catalytic RNA subunit of RNase P from Escherichia coli. It were constructed that M1-RNA with guide sequences (GS) recognizing the 452, 3408 site of CIITA by PCR from pTK117 plasmid, then were cloned into the EcoRI/BglII or EcoR//SalIsite of vector psNAV (psNAV-M1-452-GS, psNAV-M1-3408-GS) respectively. The target mould plate (3176-3560) of CIITA was obtained from Raji cell by RT-PCR, and then inserted into the XhoI/EcoRIof pGEM-7zf(+) plasmid (pGEM-3176). These recombinant plasmids were screened out by sequence analysis. psNAV-M1-452-GS, psNAV-M1-3408-GS and its target RNA pGEM-3176 were transcribed and then mixed up and incubated in vitro. It showed that M1-3408-GS could exclusively cleave target RNA that formed a base pair with the GS. Stable transfectants of hepatocyte cell line with psNAV-M1-3408-GS were tested for expression of class II MHC through FCM, for mRNA abundance of MHCII, Ii and CIITA by RT-PCR., for the level of IL-2 mRNA on T cell by mixed lymphocyte reaction. RESULTS: When induced with recombinant human interferon-gamma (IFN-gamma), the expression of HLA-DR, -DP, -DQ on psNAV-M1-3408-GS(+) hepatocyte was reduced 83.27 %, 88.93 %, 58.82 % respectively, the mRNA contents of CIITA, HLA-DR, -DP, -DQ and Ii decreased significantly. While T cell expressed less IL-2 mRNA in the case of psNAV-M1-3408-GS(+) hepatocyte. CONCLUSION: The Ribonuclease P against CIITA-M1-3408-GS could effectively induce antigen-specific tolerance through cleaving CIITA. These results provided insight into the future application of M1-3408-GS as a new nucleic acid drug against allo-transplantation rejection and autoimmune diseases.  (+info)