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 investigate the phenotypic change and proliferation of fibroblasts in human inflammatory strictured bile duct wall. Methods We observed the density and ultrastructure of fibroblasts, and the histologic structure in human normal bile duct wall and inflammatory strictured bile duct wall by light and electron microscope.Results The results showed that fibroblasts were the main source of extracellular matrix production in bile duct wall. The phenotype of fibroblasts in inflammatory strictured bile duct wall changed obviously, quiescent fibroblasts were activated and transformed to myofibroblasts, with massive proliferation. Conclusion These data suggest that massive proliferation of activated fibroblasts and myofibroblasts is the main source of extracellular matrix overproduction which results in inflammatory bile duct stricture.
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 determine whether fibroblasts can be used to promote endochondral bone formation in vivo by transfer of human bone morphogenetic protein-2(hBMP-2) into fibroblasts. Methods pcDNA3-hBMP-2 was constructed by use of gene clone and recombined technique.NIH3T3 fibroblasts were transfected with pcDNA3hBMP-2. The positive cell clones were selected with G418. In NIH3T3 fibroblaststransferred with pcDNA3-hBMP-2, the expression of hBMP-2 was determined by in situ hybridization and immunohistochemical analysis; alkaline phosphatase activity was measured. hBMP-2producing fibroblasts were implanted into nude mouse muscle to observe endochondral bone formation in vivo. Results pcDNA3-hBMP-2 was successfully constructed. In NIH3T3 fibroblasts transfected with -pcDNA3-hBMP-2,the BMP-2 expression was stable; alkaline phophatase activity was much higher than that in nontransfectedNIH3T3 cells. Endochondral bone formation invivo was observed at the site of implantation 4 weeks later.Conclusion Fibroblasts transfected by hBMP-2 gene can be used to promote endochondral bone formation in vivo.
Objective To explore the osteogenic potential of cervical intervertebral disc fibroblasts in vitro, to investigate the regulatory factors of recombinant human bone morphogenetic protein 2(rhBMP-2) and tumor necrosis factor α(TNF-α) on osteogenic phenotype of fibroblasts and to discuss the condition that facilitates osteogenesis of fibroblasts. Methods Theannulus fibroblasts cell lines of experiment goats were established in vitro and the biologicspecificity was found. According to different medias, 4 groups were included in this experiment: control group, TNF-α group ( 50 U/ml TNF-α), rhBMP-2 group (0.1 μg/ml rhBMP-2) and TNF-α+rhBMP-2 group (50 U/ml TNF-α+0.1 μg/ml rhBMP-2). Thefibroblasts were incubated in the media for about 3 weeks,and then the markers for osteogenic features were investigated by biochemistry, histochemistry observations. Results rhBMP-2 and TNF-α had no effect on the proliferation of fibroblasts from the experiment goats. rhBMP-2 or TNF-α could stimulate fibroblasts to secrete alkaline phosphatase and collagen type Ⅰ. The combined use of rhBMP-2 and TNF-α or the single use of rhBMP-2 could make fibroblasts to secrete osteocalin and the morphological changes of the fibroblasts were very obvious. Histochemical study of the nodules with specific new bone labeler(Alizarin red S) revealed positive reaction, denoting that the nodules produced by the fibroblasts werebone tissues. There was statistically significant difference(Plt;0.05) inALP activity between 3 experimental groups and control group and in secretion of osteocalcin between rhBMP-2 group, TNF-α+rhBMP-2 group and control group. Conclusion The results point out clearly that rhBMP-2 can induce theosteogenic potential of annulus fibroblasts in vitro.
Taking Wistar rats and pigs as models, the basic fibroblast growth factor (bFGF) was studied on wound healing. Five rats and five pigs were used in the experiment. Each rat had 2 roundshapedwound (1.8cm in diameter) and each pig had 4 wounds of the same size as that ofthe rat. One wound of each rat and 2 wound of each pig were dressed with bFGF saline (60U/cm2). While the other wounds of the rats and pigs were dressed with normal saline as control. The wounds treated with bFGF were completely covered with granulation tissue on the 7th day after injury, and in 14 days the wounds were almost completely covered by epithelium. The bFGF would enhance the growth of theepithelial cells, reepithelization of the wound and the tensile strength of thetissue. It was concluded that the bFGF could promote skin regeneration, whichmight be the direct action of bFGF on the bFGF receptors in the basic cells of skin.
OBJECTIVE Influence of irradiation and phenytoin sodium on modulatory activities of wound fluid on proliferation of fibroblasts and collagen synthesis was studied. METHODS The male Wistar rats were used in this study. The rats were divided into irradiated and non-irradiated groups, and in each of them it was subdivided into phenytoin group and control. A 7 cm long incisional wound was made on the back of each rat, in which a polyvinyl alcohol sponge (PVAS) with a size of 1.0 cm x 0.4 cm was implanted into the wound and the wound was sutured up. The PVAS was prepared by rinsing in running water over night and then was boiled for 30 minutes. Before implantation, the sponge was immersed in phenytoin sodium solution (10 mg/l ml) or normal saline (as control). From each wound the wound fluid and fibroblasts were collected. The methods of incorporation of 3H were adopted to assess the proliferation of fibroblasts and synthesis of collagen. RESULTS It was shown that proliferation of fibroblasts and collagen synthesis were stimulated by wound fluid remarkably on 5 to 8 days after wounding, and that 6 Gy to total-body irradiation wound decrease this effect. It was also noted that topical phenytoin sodium increased the modulatory activity of wound fluid irrespective of being irradiated or not. CONCLUSION It could be drawn that, after total-body irradiation, stimulation of hyperplasia of fibroblasts and collagen synthesis by wound fluid was markedly lowered indicating the total-body irradiation resulted in changes of local conditions of the wound which was unbenefitted to repair of tissue cells, while phenytoin sodium could enhance the stimulating action of wound fluid on proliferation of fibroblasts and synthesis of collagen which was beneficial to wound healing.
ObjectiveTo investigate relationship between ultrastructural changes and expression of basic fibroblast growth factor of diabetic retinopathy in rats.MethodsDiabetes was induced in rats with a single injection of streptozotocin (STZ) and divided into normal control group and 1- , 3- and 5- month diabetes group. The paraffin slide was observed by in-situ hybridization and immunohistochemistry, and retinal ultrastructure was examined by transmission electron microscopy.ResultsNo change of retinal ultrastructure was found in the control group. Different degrees of ultrastructure lesion were found in 1-month diabetic rats with fragmental increase of thickness of basement membrane, swelling of endothelial cells and obvions fingerlike processes in the capillary cavity, disconcentration of heterochromatin both in endothelium and pericyte, and swelling and degeneration of mitochondrion. The edema of endothelial cells of 3-month diabetic rats was more serious than that of 1month ones, and the capillary cavity was nearly occluded. In 5-month diabetic rats, the basement membrane was unevenly thickened, or obviously split. The positive rate of in-situ hybridization in 3-month diabetic rats was 77.8% while the positive rate of immunohistochemical stain was 55.6%, which increased to 88.9% in 5-month diabetic rats.ConclusionsThe occurrence of the ultrastructural changes in STZ rats with diabetic retinopathy is earlier than that of the expression of bFGF.(Chin J Ocul Fundus Dis, 2003,19:348-351)
Objective To establish an effective way to cryopreserveprecartilaginous stem cells(PSCs) of neonate rat. Methods PSCs ［fibroblast growth factor-3(FGFR-3) positive cells］ were isolated and purified by magnetic cell sorting method. PSCs were cultured and amplified to the third generation. PSCs were preserved in liquid nitrogen. The biological properties of cryopreserved PSCs were investigated by reverse transcriptase polymerase chain reaction(RT-PCR), immunohistochemistry, and immunofluorescence. Results Immunohistochemical and immunofluorescent analysis showed widespread expression of FGFR-3 in cryopreserved PSCs. FGFR-3 could be dectected by RT-PCR in cryopreserved PSCs.Cryopreserved PSCs kept high cell viability, and phenotypic and proliferation characteristics of PSCs in vivo.Conclusion Cryopreservation of PSCs can supply adequate qualified cells for repairing the defects of epiphyseal growth plate by tissue engineering technique.
Porpose To investigate the optimal concentration of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on DNA synthesis and their synergism indensity arrested human retinal pigment epithelial (RPE) cells. Methods Growth factor effects in cultured human RPE of the 6th generation were assessed by [3 H]-thymidine incorporation and radioautography. Results EGF and bFGF were potent stimulators when used alone,and their optimal concentrations were 10ng/ml in DMEM and 1ng/ml in 2% serum DMEM.When used in combination (10ng/ml EGF and 10ng/ml bFGF),they caused a significant enhancement of [3 H]-thymidine incorporation about 2.96 times. Conclusion EGF and bFGF were potent stimulators in RPE cells,and demonstrated synergism in their action. (Chin J Ocul Fundus Dis,1998,14:98-100)