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 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 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.
This experiment was designed to observe the proliferative effects of platelet derived wound healing factor (PDWHF) of different concentrations on fibroblasts from rat wounds and on epithelial from human wounds. Cultured fibroblasts from rat wound and epithelia from human wound were randomly divided into three groups. (1) In blank control, the cells were treated with basic medium (BM, contains 1640/0.5% FCS); (2) the positive control, the cells were treated with 1640/10% FCS and (3) in the PDWHF group, the cells were treated respectively with BM/1% PDWHF, BM/3% PDWHF, BM/5% PDWHF, BM/7% PDWHF, BM/10% PDWHF, BM/12% PDWHF, respectively. The Cells were collected after 48 hours culturing with BM or PDWHF, and the cell proliferation was measured by MTT method according to the OD values. The result showed that the PDWHF could remarkably enhance the proliferation of fibroblasts and epithelial cells when its concentration was between 1% and 7%, which was obviously higher than that of the blank control (P lt; 0.01). When the concentration of PDWHF reached 10%, its proliferative effect was not remarkable when compared with the blank control, When the concentration of PDWHF reached 12%, it showed inhibitory effect on fibroblasts and manifested no obvious inhibitory effect on epithelial cells. It was concluded that the PDWHF was a combination of a variety of growth factors. In a certain range of concentration, the PDWHF might effectively promote the proliferation of fibroblasts and epithelial cells. Howerve, when its concentration reached to relatively higher level, its effect was not remarkable any more, or even showed inhibitory effect on cell proliferation.
Objective To investigate the effects of caveolin-1 scaffolding domain peptide ( CSD-p)on expressions of extracellular matrix and Smads in human fetal lung fibroblasts. Methods Human fetal lung fibroblasts were cultured in vitro and divided into four groups. A control group: the cells were cultured in DMEMwithout TGF-β1 or CSD-p. A CSD-p treatment group: the cells were cultured in DMEMcontaining 5 μmol /L CSD-p. A TGF-β1 treatment group: the cells were cultured in DMEMcontaining 5 μg/L TGF-β1 .A TGF-β1 + CSD-p treatment group: the cells were cultured in DMEM containing 5 μg/L TGF-β1 and 5 μmol /L CSD-p. Caveolin -1 mRNA was detected by RT-PCR. Caveolin-1, collagen-Ⅰ, α-SMA, p-Smad2,p-Smad3 and Smad7 proteins were measured by Western blot. Results Compared with the control group,the Caveolin -1 mRNA and protein expressions in the cells of TGF-β1 group significantly reduced ( mRNA:0. 404 ±0. 027 vs. 1. 540 ±0. 262; protein: 0. 278 ±0. 054 vs. 1. 279 ±0. 085; P lt; 0. 01) , and the expression levels of collagen-Ⅰ and α-SMA proteins significantly increased ( collagen-Ⅰ: 1. 127 ±0. 078 vs.0. 234 ±0. 048; α-SMA: 1. 028 ±0. 058 vs. 0. 295 ±0. 024) . Meanwhile, the expression levels of p-Smad2 ( 1. 162 ±0. 049 vs. 0. 277 ±0. 014) and p-Smad3 proteins ( 1. 135 ±0. 057 vs. 0. 261 ±0. 046) increased with statistical significance ( P lt; 0. 01) , but the expression level of Smad7 protein significantly reduced( 0. 379 ±0. 004 vs. 1. 249 ±0. 046, P lt;0. 001) . In the CSD-p group, CSD-p had no significant effects on the expressions of above proteins compared with the control group. But in the TGF-β1 +CSD-p group, the overexpressions of collagen-Ⅰ, α-SMA, p-Smad2 and p-Smad3 induced by TGF-β1 were obviously inhibited by CSD-p ( collagen-Ⅰ: 0. 384 ±0. 040 vs. 1. 127 ±0. 078; α-SMA: 0. 471 ±0. 071 vs. 1. 127 ±0. 078;p-Smad2: 0. 618 ±0. 096 vs. 1. 162 ±0. 049; p-Smad3: 0. 461 ±0. 057 vs. 1. 135 ±0. 057; P lt; 0. 01) .Otherwise, the up-regulation of Smad7 ( 0.924 ±0. 065 vs. 0.379 ±0. 004) was found. Conclusions CSD-p can reduce fibroblast collagen-I and α-SMA protein expressions stimulated by TGF-β1 , possibly through regulation of TGF-β1 /Smads signaling pathway. It is suggested that an increase in caveolin -1 function through the use of CSD-p may be an intervention role in pulmonary fibrosis.
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.
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 explore the expression of alpha-smooth muscle actin (alpha-SMA) induced by transforming growth factor beta 1 (TGF-beta 1). METHODS: Five samples of hypertrophic scars and three samples of normal mature scars were collected as the experimental and control groups respectively. The fibroblasts were isolated from scars, and cultured in 2-dimension or 3-dimension culture system. The immunohistochemical staining method of LSAB were used to investigate the expression of alpha-SMA in fibroblasts in the different concentration of TGF-beta 1. RESULTS: The expression of alpha-SMA in 3-dimension culture system were markedly lower than those in 2-dimension culture system with respect to the fibroblasts in the experimental group. The expression of alpha-SMA in fibroblasts were different in response to various TGF-beta 1 concentration, it was more effective at the concentration of 5 ng/ml. The expression of alpha-SMA in the fibroblasts from hypertrophic scars seemed to be more sensitive to TGF-beta 1 compared to that of the normal mature scars. CONCLUSION: There are concentration-dependent in the expression of alpha-SMA induced by TGF-beta 1 in scar fibroblasts in vitro. The biological characteristics of the fibroblasts from hypertrophic scars and normal mature scars and their sensitivity to the inducement of TGF-beta 1 were different. The inducement of TGF-beta 1 may be depressed by extracellular matrix components and that may decrease the expression of alpha-SMA.
ObjectiveTo explore the methods of separation， culture， and identification of breast cancer stromal fibroblasts (BCSFs), which could build up a good basis for the further research of function. MethodsBreast cancer tissues were obtained during breast cancer operation， and were cut into pieces with size of 1 mm×1 mm×1 mm under aseptic conditions， then the pieces of the tissues were digested by collagenase Ⅰ and hyaluronidase. Finally the cells separated from the tissues incubated at 37 ℃ with 5% CO2 and 95% air humidified incubator. Morphological characteristics of the fibroblasts were observed under light microscope. The certain proteins were examined by immunohistochemistry （using CK， Vimentin， α-SMA, and TE-7 antibody） and flow cytometric analysis (CD34 and CD45). ResultsThe separated cells begin to attach to the wall of flask within 24 h and reached almost confluency in about 7 d to 10 d . According to identification, the successful rate of separation and culture of BCSFs was 90%（18/20）, and the characteristics of cells showed that morphological characteristics of the fibroblasts was flat spindle， rich cytoplasm, and a flat ovoid cystic nuclear. The fibroblasts in breast cancer tissues showed negative staining for cytokeratin， positive staining for vimentin， alpha-smooth muscle actin, and TE-7， and negative for CD34 and CD45 by flow cytometric analysis. ConclusionsThe fibroblasts in breast cancer tissues could be easily obtained by tissues cuting combined enzyme digestion and rocking technology in vitro. The present study provide an experimental foundation for further studies on 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.