Objective To search the most suitable concentration of calcium in the medium for the basement membrane reconstruction in tissue engineering skin in vitro. Methods Composite chitosan tissue engineering skin was prepared according to previous studies. Four groups were included according to the concentrationof calcium (1.00, 1.45, 1.65 and 1.95 mmol/L respectively). After 7 days and 15 days of culture, the histological manifestation of basement membrane in tissue engineering skin was observed by hematoxylin amp; eosin staining and PAS staining, and collagen Ⅳ of basement membrane was detected immunohistochemicallyatthe dermalepidermal junction. Results This tissue engineering skin shared some histological features of normal skin, including a welldifferentiated stratifiedepidermis and a dense dermis. The epithelium in the group of 1.95 mmol/L calcium differentiated better than those in other groups. PAS staining showing a regularly red dying strap domain at the dermal-epidermal junction. Collagen Ⅳ was positively stained immunohistochemically at the dermalepidermal junction inthe tissue engineering skin. Conclusion The above results suggest that the medium with 1.95 mmol/L calcium should be suitable for the growth of composite chitosan tissue engineering skin and the reconstruction of basement membrane.
Objective To construct a tissue engineering skin containing capillary-like network by employing tissue engineering method.Methods The numan umbilical vein endothelial cells(HUVECs) were isolated from a new-born umbilical cord. The keratinocytes and dermal fibroblasts were isolated from a new-born foreskin biopsy. After thecollagen gelwas prepared, the fibroblasts and the vascular endothelial cells were added in a ratio of 1 to 1 to construct a skin substitute containing capillary-like network. The skin substitute was observed by HE staining and immuno histochemical staining (Ⅷ factor). The reconstructed skin containing capillary-like network was used to repair the nude mice skin defects in the experimental group. The tissue engineering skin containing no vascular endothelial cells was used in control group.Results Capillary-like network could be observed inthe dermal layer of the tissue engineering skin, and the nude mice skin defectswere repaired by the skin substitutes in the experimental group. In control group, no capillary-like network was found.Conclusion The tissue engineering skin containing capillary-like network is successfully constructed in vitro and can be used to repair the full-thickness skin defects.
Objective To constitute a new collagen gel artificial skin by using ch ito san as one of the components. Methods Human fo resk in fibroblasts were incorporated into thechitosan-collagen-GAGs to constitute dermal equivalent(DE). The growth of fibroblasts incorporated in gels and several factors which influenced the contraction of the gel were observed. The influence of different chitosan contents on the growth of fibroblast and keratinocyte and on the antibacterial effect were studied. Keratinocytes separated from normal children foresk in were seeded on the matured DE to reconstruct artificial skin, which was immersed at the early stage of culture, then lifted to an air-liquid interface. The structure of the DE and artificial skin were analysed by histology and scanning electron microscope. Results The contraction rate of the DE was proportional to the number of fibroblasts, and the final size of the DE was inversely proportional to the concent ration of collagen protein. Fibroblasts incorporated into the gel showed the exponential growth from the 2nd day to the 9th day. Chitosan-collagen-GAGs had no inhibition effect on the growth of fibroblasts, but promoted the growth of eratinocytes. Staphylococcus aureus was inh ibited even more as chitosan content increased. Scanning electron micro scopy indicated that the DE had abundant porous fabrication. Artificial skin shared some histological features of normal skin, which consisted of a good strat ifiedepiderm is and a dense dermis. Conclusion Chitosan-Collagen-GAGs collagen gelart ificial skin is a new collagen gel living artificial skin which has certain antibacterial ability and stratified epiderm is and dense dermis structure like normal skin.
Objective To study the grafting effect of tissue engineered artificial rat skin equivalent on full thickness wounds. Methods Full thickness wounds(Φ20mm) were made on the backs of twenty four nude mice which be divided in artificial skin(AS) group, chitosan membrane(CH) group and control group. All wounds were covered with AS, CH and petrolatum gauze , respectively. The wounds were observed daily by infrared ray scanning and histological examination on the 3rd , 7th, 14th, and 21st days. Results The wounds in AS group healed better than those in CH group and control group. The artificial skin achieved a good adherence to wound and there were some crescent regenerative blood vessel appeared in the AS group on the 3rd day of grafting. Then, the epidermal cells in artificial skin proliferated and differentiated to form a new epidermis consisting of stratum basal, stratum spinosum, stratum granulosum, stratum corneum almost like the natural skin. Dermis of the sd extracellular matrix secreted by fibroblasts; the chitosan lattice was degraded and replaced by the extracellular matrix. On the 14th day of grafting, the wounds healed. The color of artificial skin grafted was very similar to the natrual skin and the formed scar was very smaal. Conclusion A kind of new reconstructive tissue engineering artificial skin has good histocompatibility and can be transplanted into the full-thickness wounds.
Objective To compare the attachment and growth of fibroblasts on the different porcine accellular dermal matrix (ADM) so as to find the suitable scaffold for tissue engineering skin. Methods Fibroblasts (5×10 5) were seeded on 4 kinds of ADMs which were crosslinked with glutaraldehyde, uncrosslinked, crosslinked with glutaraldehyde and removed basement membrane, corsslinked with glutaraldehyde and then meshed. The same density fibroblasts were seeded on petri dish as a control. Cell count was done on the 1st, 3rd, 5th days after seeding. The at tachment of fibroblasts on ADM sw as observed by HE staining. Results The grow th and at tachment of fibroblasts on cro sslinked and non2meshed ADM increasedmarkedly w hen compared w ith the o thers. There w as no obvious difference betw een the group s of w ith o r w ithout basement membrane. Conclus ion The above results indicate that non2meshed and co rsslinked w ith glutaraldehyde ADM ismo re suitable fo r the at tachment and grow th of fibroblasts than the o thers and that the modified ADM can be used fo r the scaffo ld of t issue engineering skin.
Objective To build artificial dermis by using the acellular dermis matrix(ADM), collagen membrane and collagen gel as scaffolds. Methods The fibroblasts were isolated by enzyme from infant skin and were cultivated in the DMEM medium. After 14 days when the fibroblasts were seeded into 3 different scaffolds, the autografts were detected by HE staining, transmission electron microscope and scanning electron microscope. Results ①The fibroblasts obtained from the fullskin by enzyme could be passaged in the Dulbecco’s modified Eagle’s medium 2high gluco se w ith 10% calf bovine serum. ②A layer of fibroblastsw ere found on the surface of th ree different scaffo lds, the fibroblasts could grow into the co llagen membrane and the co llagengel, but could no t be found in the inner of ADM. ③A rt ificial derm is cont racted slightly by inoculat ing fabricat ion on collagen membrane and ADM , and the fibroblasts on them w ere no t act ive in proliferat ing; but the art ificial derm is built by the collagen gel cont racted obviously. Conclus ion The art ificial dermis built by ADM , collagen membrane and collagen gel as scaffolds have a preferable structure for an ideal subst itute of sk n, and can beused as the graft in the next experiments.
Objective To investigate the possible mechanism of the fibroblasts inducing the vascularization of dermal substitute. Methods Fibroblasts were seeded on the surface of acellular dermal matrix and cultivated in vitro to construct the living dermal substitute. The release of interleukin 8 (IL 8) and transfonming growth factor β 1(TGF β 1) in culture supernatants were assayed by enzyme linked immunosorbent assay, the mRNA expression of acid fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) were detected by RT-PCR. Then, the living substtute was sutured to fullth ickness excised wound on BALBouml;C m ice, and the fate of fibroblast w as observed by using in situ hybridizat ion. Results Fibroblasts cultured on acellular dermalmat rix p ro liferated and reached a single2layer confluence. Fibroblasts could secret IL 28 (192. 3±15. 9) pgouml;m l and TGF-B1 (1. 105±0. 051) pgouml;m l. There w as the mRNA exparession of aFGF and bFGF. Fibroblasts still survived and proliferated 3 weeks after graft ing. Conclusion Pept ides secreted by fibroblasts and its survival after graft ing may be relat ive to the vascularizat ion of the dermal subst itute.
OBJECTIVE: To construct a tissue engineering skin containing melanocytes by employing tissue engineering method. METHODS: The keratinocytes, dermal fibroblasts and melanocytes were isolated and purified. Then the cells were used to construct a tissue engineering skin containing melanocytes. The location of melanocytes in the tissue engineering skin were detected by Dopa staining, transmission electron microscope (TEM) and S-100 immunohistochemical staining. RESULTS: Melanocytes can be detected in the basal layer of the constructed tissue engineering skin. The results of TEM showed that the melanocytes were in good conditions. CONCLUSION: The artificial skin containing melanocytes was successfully constructed in vitro and can be used to repair the full-thickness skin defects.
OBJECTIVE: To build the trestle of tissue engineering for skin with the collagen. METHODS: The collagen was obtained from the baby cattle hide pretreated by Na2S and elastinase and Protease M, then the collagen was dissolved in 0.5 mol/L acetic acid solution. The collagen was treated with Protease N to minimize its immunogenicity. The resulting collagen could be used to build the trestle of tissue engineering for skin because of good biocompatibility. The collagen molecular weight and structure were analyzed by SDS-PAGE. The bioactivity of trestle was tested in the experiment of the mice wound healing and the cell implantation. RESULTS: The SDS-PAGE result of the collagen treated by Protease M showed the typical spectrum of type I collagen. The built trestle was a collagen sponge matrix in which micropore size was 50-200 microns. It could accelerate wound healing and the implanted fibroblasts could proliferate well. CONCLUSION: The collagen treated by Protease N can get good biocompatibilily and is suitable for building the trestles of tissue engineering for skin with good bioactivity.