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 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.
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.
ObjectiveTo investigate the expression and relation of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in rats with diabetic retinopathy.MethodFifty-five Wistar rats were randomly divided into the control group (10 rats), and 1, 3, and 5-month-diabetes group (15 rats in each diabetes group), and the diabetic models were set up. The expressions of VEGF and bFGF were detected by situ hybridation and immunohistochemistry on retinal paraffin sections.ResultsThe results of situ hybridation showed that expression of bFGF was found in 3-month-deatbtes group with the percentage of 77.8%, and 88.9% in 5-month-deatbtes group; the positive expression of VEGF was not found in 3-month-deatbtes group but in 5-month-deatbtes group with the percentage of 66.7%. Immunohistochemistry indicated that the positive expression of bFGF started in 3-month-deatbtes group with the percentage of 55.6%, and 88.9% in 5-month-deatbtes group; the percentage of the expression of VEGF was 33.3% in 3-month-deatbtes group and 88.9% in 5-month-deatbtes group.ConclusionThe expression of VEGF occurs after the expression of bFGF in rats with DR.(Chin J Ocul Fundus Dis, 2005,21:37-40)
Objective To explore the regulator factor of osteogenes is induced by the fibroblast in vitro so as to provide enough seeding cells for the bon e tissue engineering. Methods The fibroblasts were isolated and purified from granu lation of New Zealand rabbits, and they were incubated in the media offibronectin (FN) 10, 20, 40, 60 and 80 μg/ml, respectively, in the experimenta l grou ps 1- 5，but there was no FN in the control group. The markers for osteogenic features were investigated by fibroblast morphogenesis,calcium nodules formationratios,labeling of tetracycline fluorescence, labeling of 3H-TdR, determination of o steocaline, and labeling of 3H-proline within 2 weeks. Results The morphologic al changes of the fibroblasts were manifested as transference from a long spindle to a round or multiple form, shifted nucleus increased in number, confluenced and formed multilayered structure. There was a piling-up of calcium crystals that were gradually merged into foggy substances. The foggy substances increased and formed nodules. The calcium nodules formation ratios were as follows: 15.35％± 3.45％in the control group, and 53.73％± 9.49％, 75.21％± 9.80％, 98.34％± 15.2 0％, 61.83％± 10.04％, and 45.11％± 8.70％ in the experimental groups 1.5 ,respectively. There was a significant difference between the control group and the 5 experimental groups at 14 days (Plt;0.05), and a significant differenc e be tween the experimental group 3 and the other experimental groups at 14 days (Plt;0.05). The histochemical study on the nodules with the specific labeling of tet racycline fluorescence indicated that the nodules were composed of new bones. Conclusion Fibronectin can stimulate the fibroblast to prolifer ate, secrete osteocaline, and synthesize collagen fibrils. Fibronectin, in an optimal dose of 40 -60 μg/ml, is capable of inducing the fibroblast to form the bone.
OBJECTIVE: To explore the effect of Fas/Apo-1 and Bcl-2 gene expression on mechanism of scar formation. METHODS: Immunohistochemical method was applied to defect the expression of Fas and Bcl-2 protein in fibroblasts from 10 cases with normal skin, 10 cases with hypertrophic scar and 10 cases with keloid. RESULTS: The positive expression rate of Bcl-2 protein in keloid was 83.2%, significantly higher than that in hypertrophic scar (38.6%), (P lt; 0.01), and the positive expression rate in hypertrophic scar and keloid was higher than that in normal skin (6.78%), (P lt; 0.01). But the positive expression rate of Fas/Apo-1 protein was 78.4% in normal skin 80.4% in hypertrophic scar, 84.4% in keloid respectively, which showed no significant difference among them (P gt; 0.05). CONCLUSION: Bcl-2 gene but Fas gene may take part in the formation of pathologic scar.