OBJECTIVE: To investigate the changes of fibroblast growth factor (FGF) in burn wounds. METHODS: The FGF expression in the center of wound granulation, the edge of wound, the healed part of wound, the normal skin of patients, and the heal course of second degree burn wounds were detected by immunohistochemical methods. RESULTS: The expression intensity of FGF was different in the different sites of third degree burn wounds. The highest contents of FGF was in the center granulation of burn wounds, the less was in the borderline of wound and healed skin, and the least was in the healed skin. FGF expression mainly concentrated in the middle layer of wound, and almost no FGF expression in normal skin. The most FGF expression was occurred at 14 days after injury in second degree of burn wound. CONCLUSION: The changes of FGF in wounds are closely related to the wound healing, and rational use of FGF can promote wound healing.
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.
Objective To establish a method of constructing skin-equivalents (SE) by the hair follicle stem cells (HFSC) and the fibroblasts. Methods The K19 immunostainning was employed to localize the HFSC in the human scalp from the cosmetic surgery. The isolated HFSC through the enzyme digestion were seeded on the dermal equivalent (DE) formed by polymerization of the fibroblasts and collagen. After being cultured between the air-liquid interface for 14 days, SE were harvested and used for an evaluation. Results HFSC were located mainly in the outer root sheath in the hair follicle. Based on DE, the growing HFSC could build a fullydeveloped and multilayered epidermis with the basal membrane formedb etween the epidermis and the dermis. The fibroblasts were active and spread evenly in the collagen matrix. Conclusion The hair follicle stem cells located in the outer root sheath can be successfully used to construct skin-equivalents in vitro and have a promising clinical use in the treatment.
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 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 fabricate artificial human skin with the tissue engineering methods. METHODS: The artificial epidermis and dermis were fabricated based on the successful achievements of culturing human keratinocytes(Kc) and fibroblasts (Fb) as well as fabrication of collagen lattice. It included: 1. Culture of epidermal keratinocytes and dermal fibroblasts: Kc isolated from adult foreskin by digestion of trypsin-dispase. Followed by comparison from aspects of proliferation, differentiation of the Kc, overgrowth of Fb and cost-benefits. 2. Fabrication of extracellular matrix sponge: collagen was extracted from skin by limited pepsin digestion, purified with primary and step salt fraction, and identified by SDS-PAGE. The matrix lattice was fabricated by freeze-dryer and cross-linked with glutaraldehyde, in which the collagen appeared white, fibrous, connected and formed pores with average dimension of 180 to 260 microns. 3. Fabrication artificial human skin: The artificial skin was fabricated by plating subcultured Kc and Fb separately into the lattice with certain cell density, cultured for one week or so under culture medium, then changed to air-liquid interface, and cultured for intervals. RESULTS: The artificial skin was composed of dermis and epidermis under light microscope. Epidermis of the skin consisted of Kc at various proliferation and differentiation stages, which proliferated and differentiated into basal cell layer, prickle cell layer, granular layer, and cornified layer. Conifilament not only increased in number, but also gathered into bundles. Keratohyalin granules at different development stages increased and became typical. The kinetic process of biochemistry of the skin was coincide with the changes on morphology. CONCLUSION: Tissue engineered skin equivalent has potential prospects in application of repairing skin defect with advantages of safe, effective and practical alternatives.
OBJECTIVE: To explore the effects of nandrolone phenylpropionate (NP) on the expression level of pro alpha 1 (I) collagen after burn in rats and the possible mechanism involved in the process. METHODS: Thirty-two Wistar rats with a deep second-degree scald injury and 20% of total body surface area were randomly divided into two groups to receive either 5 mg/kg NP(NP group) or normal saline (control group) every other day. We analyzed the mean integrated optical density(mIOD) of androgen receptor (AR) to determine the distribution and expression of AR in fibroblasts by immunohistochemistry, and measured expression level of pro alpha 1 (I) collagen mRNA by quantitative fluorescent RT-PCR to find the relation between expressions of AR and pro alpha 1 (I) collagen mRNA. The total specimens were obtained from the scalded rats after 4, 7, 14 and 21 of after burn. RESULTS: The expression of pro alpha 1 (I) collagen mRNA in NP group was significantly higher than that in control group on the 7th, 14th and 21st days(P lt; 0.05), but there was no significant difference on the 4th day. The density of AR in fibroblasts had significant difference (P lt; 0.05) between the two groups after 4, 7, 14 and 21 days. A positive relationship existed between the expression of pro alpha 1 (I) collagen mRNA and quantity of AR in fibroblasts(r = 0.836). CONCLUSION: The nandrolone phenylpropionate increased the expression of pro alpha 1 (I) collagen mRNA and enhanced the density of AR in fibroblasts. The higher expression of pro alpha 1 (I) collagen mRNA had a relation with the change of quantity of AR in fibroblasts.
ObjectiveTo investigate the histological changes and vascularization of the porcine acellular dermal matrix (P-ADM) processed with matrix metalloproteinase 7 (MMP-7) (P-ADM-pm) after implanted into rats. MethodsSixty-two pieces of porcine reticular layer dermis which were from the pig abdominal skin and obtained by using a mechanical method, were randomly divided into group A (n=31) and group B (n=31). The porcine reticular layer dermis in 2 groups were treated with decellularization (P-ADM), then the P-ADM in group B were treated with processing by MMP-7 (P-ADM-pm). Thirty adult male Wistar rats were selected. P-ADM (group A) and P-ADM-pm (group B) were subcutaneously transplanted into the left and right fascia lacuna, respectively. The implants were harvested from 6 rats at 3, 7, 14, 21, and 28 days after implantation, respectively. Gross, histochemical, and immunohistochemical observations, and scanning electron microscopy (SEM) examination were performed to observe host cells, microvessels infiltration and histological changes in the implants. ResultsNo rat died in the experiment, incision healed well and no obvious inflammatory reaction was seen in all rats. Gross observation suggested that the implants of 2 groups were encapsulated by a thin layer of connective tissue at 7 days after implantation. With the time of implantation, the microvessels increased and coarsened, and the changes of group B were more obvious than those of group A. At 21 days, the microvessels of 2 groups decreased, and the implants of group B showed complete vascularization. The histochemical and immunohistochemical observations showed that group A had more severe inflammatory response than group B. Fibroblasts and microvessels in group B appeared in the superficial zone of implant at 3 and 7 days after implantation and they could be observed in the center zone of implant at 14 and 21 days. However, fibroblasts and microvessels in group A appeared in the superficial zone of implant at 3 and 14 days and they could not be observed in the center zone of implant at 28 days. Fibroblasts and microvessels of group B were significantly more than those of group A (P < 0.05). SEM examination showed that more fibroblasts and new collagen fibrils were observed in group B at 14 days. ConclusionThe host response to P-ADM-pm is similar to normal wound healing, and P-ADM-pm as implantable scaffold material plays a good template conduction role.
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.
In order to explore further the regulatory factors to the potentiality in inducing osteogenesis by fibroblasts, the fibroblasts were isolated, and purified from human skin, and were grown in incubation in the media of EGF, IL-6, TNF-alpha and BMP2 at different concentrations for two weeks, then, the markers for osteogenic features were investigated by biochemistry, histochemistry and electron microscopic observations. It was found that the combined use of TNF-alpha and BMP2 could stimulate fibroblasts to secrete alkaline phosphatase, osteocalcin and collagen, and the morphological changes of the fibroblasts were also very striking. In the extracellular matrix, the collagen fibrils, with or without periodicity, were arranged regularly or randomly oriented, and numerous minute calcium granules were interspersed among them. The fibroblasts were interwoven one on top of another in the form of multilayer structure and on the surface, there were secreting granules and piling up of calcium crystals which coalessed steadily and increased in size in forming bony nodules. It was considered that TNF-alpha and BMP2 were capable of inducing the fibroblasts to form bone.