Objective To evaluate the effects of cryopreserved cultured allogenic dermal fibroblasts on angiogenesis and fibroplasia while artificial dermis grafting by spraying the cells on the graft bed.Methods Full thickness skin defect was made on the back of Wistar rat, fibroblasts mixed into fibrin glue (fibroblast group) and same amount fibrin glue (control group) were sprayed separately between the wound bed and artificial dermis in cell density of 1.0×105 cells/cm2 before the artificial dermis was grafted. On day 5 after grafting, the graft and surrounding tissue were examined histologically for angiogenesis and fibroplasia in the dermis and wound bed with hematoxylin eosin stain, VEGF antibody stain, Masson’s trichrome stain and India ink stain. Evans blue perfusion methodwas also used for detecting the angiogenesis quantitatively.Results In the fibroblast group, the angiogenesis of graft bed was significantly accelerated onday 5 after grafting; the numbers of the newly formed capillaries were 9.64±2.36/HP in the fibroblast group and 3.88±1.62/HP in the control group (P＜0.05). And on day 10 after grafting the angiogenesis was accelerated not only in graft bed but also in the artificial dermis when compared with control group, the newly formed capillaries network was clearly observed in the artificial dermis. Otherwise, the synthesis of collagen was increased in the dermis on day 10 after grafting in the fibroblast group when compared with control group. The immunoreactivity of VEGF antibody in the fibroblast group also showed a ber expression than that in control group on day 5 after grafting, the numbers of positive cells were 46.04±8.90/HP in the fibroblast group and 30.08±7.76/HP in the control group(P＜0.05).Conclusion Transplantation of cryopreserved dermal fibroblasts while artificial dermis grafting can accelerate the angiogenesis and fibroplasia in the artificial dermis and graft bed, thereby accelerate the formation of dermallike tissue in the artificial dermis.
Objective To compare the efficiency of epidermis cell culture between big graft method and small strip method. Methods The big graft method was to cut the skin tissue reticularly from dermis layer while the epidermis were not cut off. After it was digested fully in trypsin, theepidermis was separated from skin and was used to culture epidermal cells. The small strip method was routine. The time to cut the skin and to separate the epidermis was recorded, and the number and quality of cells were compared between two methods. Results It took 8-10 minutes to cut an area of 5 cm2 skin into small strips and 1-2 minutes into big grafts. It took 10-15 minutes to separate the epidermis from the same area skin by small strip method and 2 minutes by big graft method. The cells showed better vigor and its number was more by big grafts than by small strips.The chance of fibroblast contamination was reduced obviously. Conclusion The big graft method is simpler than the small strip method and can culture more epidermis cells with less chance of fibroblast contamination.
Objective To investigate the influence of lipopolysaccharide(LPS) on the proliferation and collagen synthesis of normal human skin fibroblasts so as to elucidate its relation with skin wound healing. Methods Fibroblasts wereisolated and cultured in vitro, and then exposed to different doses of LPS(0.005, 0.010, 0.050, 0.100, 0.500, and 1.000 μg/ml) from E.coli055∶B5 respectively. Then the absorbance (A) value of fibroblasts was determined with the colorirneteric thiazolylblue (MTT) assay, and the cell number was counted under inverted phase contrast microscope from the 1st day to the 9th day after LPS administration, and collagen synthesis of fibroblasts in culture medium was measured with the method of pepsin digestion after incorporation of 3Hproline into stable, single-layered, confluent fibroblasts at 7 days after LPS administration. Results Compared with control group, A value increased with the increasing concentration of LPS (0.005 μg/ml 0.500 μg/ml) and LPS of 0.100 μg/mlgroup had the best effect. The difference was remarkable from the 5th day to the 9th day(P＜0.05). A value decreased when challenged with the LPS of 1.000 μg/ml and the difference was remarkable from the 3rd day to the 9th day(P＜0.05). Cell number increased with theadministration of LPS of different concentrations (0.005 μg/ml 0.500 μg/ml) and LPS of 0.100 μg/mlgroup had the best effect. The difference was remarkable from the 1st day to the 6th day(P＜0.05). Cell number decreased remarkably when challenged with LPS of 1.000 μg/ml and the difference was remarkable from the 2nd day to the 9th day(P＜0.05). Collagen synthesis increased when challenged with LPS of different concentrations (0.005 μg/ml 0.500 μg/ml) and the 0.100 μg/ml group had the best effect. However, when the dose of LPS reached 1.000 μg/ml, it inhibited collagensynthesis. Conclusion LPS could promote the proliferation andcollagen synthesis of fibroblasts within a certain range of low doses, but over-high dose ofLPS might inhibit the proliferation and collagen synthesis of fibroblasts, suggesting that LPS of certain concentrations might contribute to wound healing, while excessive LPS has negative effect on wound healing.
ObjectiveTo investigate the antibacterial pretreatment protocol for primary fibroblast cell culture from transbronchial biopsies in patients with benign tracheal stenosis (BTS). MethodsFifteen specimens of intratracheal hyperplastic granulation tissue were obtained from 14 BTS patients by transbronchial biopsies. The tissues were divided into 3 groups according to different antibacterial pretreatment with 5 specimens in each group. An usual concentration of antibiotics pretreatment group (group 1) was pretreated by washing with PBS contained 1%-2% penicillin/streptomycin. A high concentration of antibiotics pretreatment group (group 2) was pretreated by washing with PBS contained 6% penicillin/streptomycin. An alchohol and high concentration of antibiotics pretreatment group (group 3) was pretreated by washing with 75% alcohol 3-4 seconds firstly,then by washing with 6% penicillin/streptomycin. After different pretreatment,all tissues were cultured with tissue culture method in the same condition. ResultsThe primary fibroblasts were successfully cultured from the tissue pretreated by method 2 and 3,but not cultured from the tissue pretreated by method 1 with large amount of bacteria. There were significant differences in the furthest radius of cell proliferation between different culture time points in three groups (P<0.01). The differences in the furthest radius of cell proliferation between three groups were not different at 24,48 or 72 h (P>0.05),but were significant between three groups at 96 h (P<0.05). ConclusionAn pretreatment protocol with high concentration of antibiotics or 75% alcohol is feasible in human primary fibroblasts culture from small specimens obtained by transbronchial biopsy.
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 investigale effect of subretinal fluld(SRF)on proliferalion of the cellular elements of PVR. METHOD:The effect of SRF of 28 patients with rhegmatogenous retinal detachment proliferation of the cultured human retinal pigment epithelial cells(RPE),retinal glial cells (RG),and fibroblast (FB)was observed and detected by the methods of cell-counting and 3H-TdR in DNA synthesis. RESULTS:The range of proliferatinn-stimulating activity was 52.5%~233.3%, 36.4% ~ 177.8%，55.4% ~277.8% above the baseline in 1:10 dilution of these 3 kinds ,of cellular elements,and there was no significant difference among them. CONCLUSION ;The stimulating effect of SRF on the cellular proliferation was thougt to be due to the actions from certain growth factors. （Chin J Ocul Fundus Dis,1996,12: 233-235）
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.
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.
ObjectiveTo summarize the research status and biological characteristics of stromal fibroblast in breast cancer. MethodsRelevant literatures about the breast cancer stromal fibroblasts published recently were collected and reviewed. ResultsIn addition to cancer cells, breast cancer included stromal cells. The fibroblasts were the major components of breast cancer stromal, which had significantly different biological characteristics from normal fibroblasts. The fibroblasts were characterized by α-SMA positive, p53 gene mutation, secretion of various cytokines or chemokines in addition to the production of collagen substances, involving in breast cancer growth, migration, invasion and metastasis through a variety of signaling pathways. ConclusionThe biological characteristics of stromal fibroblasts in breast cancer may reflect lesion properties, be of great importance to diagnosis and differential diagnosis and prognosis prediction of breast cancer. More attentions will be paid to the target therapy for stromal fibroblasts in breast cancer.
OBJECTIVE: To construct human bone morphogenetic protein-2(hBMP-2) expressing eukaryotic vector and observe whether it can be expressed in eukaryotic cells. METHODS: pUC19 was digested with Sal I and Xba I. The resulting Sal I-Xba I fragment (1.24 kb) which contains the full length of human BMP-2 cDNA was separated on agarose gel and ligated into eukaryotic expression vector pcDNA3 digested with XhoI and XbaI. The recombinant pcDNA3-hBMP-2 plasmid was transferred into fibroblasts cell line NIH3T3. The stable expression of hBMP-2 in the positive cells G418 selected was determined by in situ hybridization and immunohistochemical analysis. RESULTS: The two fragments digested from recombinant pcDNA3-hBMP-2 plasmid by EcoR I and Xba I represented 1.3 kb and 5.38 kb respectively by agarose electrophoresis, meanwhile the Xho I site was disappeared in pcDNA3-hBMP-2 indicating the successful construction of recombinant pcDNA3-hBMP-2 plasmid. Stable expression of hBMP-2 in pcDNA3-hBMP-2 transfected cells was confirmed by in situ hybridization and immunohistochemical analysis. CONCLUSION: hBMP-2 expressing eukaryotic vector is successfully constructed and can be expressed in eukaryonic cells.