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 observe the effects of keratinocytes on proliferation and collagen secretion of fibroblasts. Methods The conditioned medium,collected from cultured keratinocytes, was added to the cultured fibroblasts as the tested groups(12.5％, 25% and 50％ groups) and DMEM as control group. The MTT, hydroxyproline coloricmetric method and flow cytometer were employed to measure the fibroblast proliferation, the collagen secretion andthe change of the cell cycle.Results In fibroblast proliferation, the absorbency(A) value of tested groups was significantly different from that of the control group (P＜0.01). A value increased as increasing concentration, there was statistically significant difference betweetheconcentrations of 25%,50% and the concentration of 12.5%(P＜0.01), but no statistically significant difference between the concentrations of 25% and 50%(P＞0.01). In collagen secretion, there was no statistically significant difference between the tested groups and the control group(P＞0.01), and between the tested groups(P＞0.01). In cell cycle, 50% of conditioned medium could make the fibroblast pass the limit of G1/S and S/G2 period, the cell rates of S,G2-M period increased. Conclusion The conditioned medium from keratinocytes can increase fibroblasts proliferation, have little effect on general collagen secretion.
ObjectiveTo review the research progress of tissue engineered ligament. MethodsThe literature in recent years on tissue engineered ligament in repair of anterior cruciate ligament (ACL) injury was extensively reviewed, including cell sources, scaffold materials, growth factors, and mechanical stimulation in tissue engineered ligament. ResultsTissue engineered ligament constructed by mesenchymal stem cells and ACL fibroblasts has been successfully used in animal experiments. It is crucial for qualified tissue engineered ligament to choose appropriate seed cells, scaffold, mechanical stimulation, and essential cytokines. To further optimize culture condition and how to realize the tissue engineered ligament in vivo better survival and prognosis need to be further studied. ConclusionEnormous progress has been made in tissue engineered ligament for repair and regeneration of ACL. With the development of biochemistry and scaffold materials, tissue engineered ligament will be used in clinic in the near future.
OBJECTIVE: To study the relationship between intracellular actin and scar contracture. METHODS: Fibroblasts from 10 cases of hypertrophic scar and 5 cases of keloid were cultured in vitro. Total actin, filamentous actin(F actin), globular actin (G actin) and the ratio of F to G actin(F/G) were measured by densitometry after differential extraction and separation by polyacrylamide gel electrophoresis in the presence of sodium sulfate. RESULTS: Total actin, F actin, G actin and F/G in hypertrophic scar fibroblasts were 2.38 ng/10(4) cells, 0.98 ng/10(4) cells, 1.42 ng/10(4) cells and 0.68 respectively, while in keloid fibroblasts were 1.68 ng/10(4) cells. 0.46 ng/10(4) cells, 1.26 ng/10(4) cells, and 0.36 respectively. There was significant differences between two tissues fibroblasts in the items of total actin, F actin, G actin, and F/G (P lt; 0.01), while no significant difference in G actin (P gt; 0.05). CONCLUSION: Total intracellular actin, F actin, and F/G may play an important role in the scar contracture. The hypertrophic scar and keloid can be distinguished by the contents of total intracellular actin, F actin and F/G.
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 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.
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 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 investigate the different expression of actin, myosin II in hypertrophic scars, keloids and normal skins, and to understand the relationship of actin, myosin II and the scar contracture. METHODS Fifteen cases with hypertrophic scars, 10 cases with keloids and 15 cases with normal skins were chosen randomly. The expression of actin and myosin II were detected by immunohistochemical method in the hypertrophic scars, keloids and normal skins. The fibroblasts isolated from three types of tissue were cultured in vitro, then actin and myosin II in three different fibroblasts were measured using flow cytometry. RESULTS The immunohistochemical staining of myosin II in hypertrophic scars was positive, while the staining in keloids and normal skins were negative. The positive rate of myosin II expression in hypertrophic scars, keloids and normal skins were (95.11 +/- 2.78)%, (16.86 +/- 7.11)%, and (5.31 +/- 1.79)% respectively. There were significant difference between keloids and the two others(P lt; 0.01). The actin expression in three difference tissues were positive, there were no significant difference in hypertrophic scars, keloids and normal skins(P gt; 0.05). The positive rate of actin expression in hypertrophic scars, keoids and normal skins were(77.77 +/- 15.43)%, (88.89 +/- 10.29)%, and (82.92 +/- 13.48)% respectively, and there were no significant difference(P gt; 0.05). CONCLUSION Myosin II may play an important role in the scar contracture. Actin is the contractile protein of cell, it plays
OBJECTIVE: To analysis the biological characteristics of human fibroblasts transfected by human telomerase reverse transcriptase (hTERT) eucaryotic expression plasmid pGRN145. METHODS: Fibroblasts from children’s foreskin were isolated and cultured in vitro, and the fibroblasts were transfected by pGRN145 with Lipofec-tAMINE PLUS Reagent. After strict screening of hygromycin B, the positive clones were subcultured. The telomerase activity was detected by RT-PCR and TRAP-PCR technique. The cell generation cycle and apoptosis rate were detected by flow cytometry to investigate the proliferative characteristics after transfection, and the chromosome karyotype of transformed cells was analyzed. The collagen secreted by transformed cells was detected by immunohistochemical staining. RESULTS: The morphological properties of fibroblasts did not change obviously after transfection. There were telomerase activity in transfected fibroblasts, while it could not be detected in pre-transfection fibroblasts. The cell generation cycle had no obvious changes between pre-transfection and post-transfection. However, the apoptosis rate of transfected fibroblasts were decreased compared with that of pre-transfection. The fibroblasts transfected by pGRN145 maintained the normal diploid karyotype, as well as the cells could normally secret type I and III collagen. CONCLUSION: The human fibroblasts transfected by pGRN145 has telomerase activity with prolonged life span of culture, which preliminarily proves the availability of establishing standard seeding cell lines of tissue engineering by hTERT plasmid transfection techniques.