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 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.
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.
OBJECTIVE This paper aims to explore the new method of continuous delivery of epidermal growth factor to wounds by transfected fibroblasts to promote wound repair. METHODS It was constructed a novel chimeric expression plasmid in which the biologically active portion of the human epidermal growth factor (EGF) gene was fused in-frame to the human granulocyte colony-stimulating factor signal sequence. RESULTS Clonally selected human fibroblasts transfected with this construct could secrete biologically active EGF. After the transplantation of irradiated gene-transfected fibroblasts suspended in fibrin glue to murine full-thickness wounds, EGF could be demonstrated for at least seven days in the wounds, slowly decreasing from initially 470 ng/L to 140 ng/L in 7 days. No EGF was found in the wound at 14 days. CONCLUSION A single application of irradiated EGF gene transfected fibroblasts to wounds can continuously deliver the transgene in vivo and can be used to administer drugs to the wound bed during the crucial first seven days of wound-healing.
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 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.
Objective To const ruct art ificial derm is on co llagen2chondront in sulfate (CS) scaffo ld. Methods Co llagen w as compounded from CS and 1-ethyl-3-(13-dimethyl am inop ropyl) carbodiim ide (EDC) used as a cro sslinker. Physical and chem ical p ropert ies of the scaffo ld w ere characterized by elect ron spect ro scopy fo r chem ical analysis (ESCA ) , scanning elect ron m icrograph (SEM ) , HE staining, and mechanical p roperty test. Derm is fibroblasts w ere iso lated from human embryo and w ere cultured on the scaffo lds. Th rough h isto logical test ing, immunoh istochem ical test ing and biochem ical p roperty test ing, the p roperty of co llagen-CS art ificial derm is w as compared w ith that of colla gen spongy art ificial derm is. Results Co llagen-CS had th ree2dimension st ructure w ith po rous. Compared w ith co llagen scaffo ld, themechanical p roperty of co llagen2CS scaffo ld imp roved. There w eremo re po lar group s on the surface of co llagen-CS scaffo ld. The fibroblasts on the co llagen-CS scaffo ld grew w ell, and art ificial derm is w as const ructed. Conclus ion Co llagen-CS art ificial derm is has mo re excellent bio logical and mechanical p ropert ies. F ibroblasts at tach and p ro liferate bet ter on co llagen2CS scaffo ld than on co llagen scaffo lds.
Objective To study the effects of dermal template on the biological behaviors of fibroblasts during wound healing. Methods A total of 120 rats were made fullthickness wound modes on the dorsum and divided into 4 groups,in group 1, the wounds were allowed to heal by contraction（ConT）；in group2, the wounds covered with fullthickness skin grafts( FTSG); in group 3, the wounds were with split thickness skin grafts (STSG); and ingroup 4, the wounds were covered by dermal regeneration template with overlying thin splitthickness autograft (ADMT).The specimens were obtained at one week, two weeks, four weeks, six weeks,and twelve weeks respectively. The expressions of α smooth muscle actin(αSMA，characteristic of MFB）,fibronectin(FN),integrin α2,β1 and transforming growth factor β1(TGF-β1) were examined by immunohistochemical analysis. Results Positive expression of α-SMA、FN、integrin α2β1 and TGF-β1 in ADMT groups was significantly lower than that in STSG group and ConT group, but higher than that in FTSG group（P＜0.05）. Conclusion Dermal regeneration template can inhibit the transformation of FB to MFB and restrain the expressionof FN,integrin α2,β1,and TGF-β1 in fibroblasts which might reduce thepossibility of hypertrophyic scaring, and improve wound healing.
In order to investigate the inhibitory effect of salvia miltiorrhiza (SM) and tetramethyl pyrazine (TP) on scartricial fibroblast, the hypertrophic scar tissue of chest was chosen for culture of fibroblasts, and the influence of SM and TP on fibroblasts was observed, The effect of the drugs on the growth of fibroblasts, on DNA synthesis of fibroblasts and on mitosis index of fibroblasts were all determined quantitatively. The results showed: 1. SM and TP could inhibit significantly the growth of the fibroblasts, the inhibitory effect was irreversible when the concentration of the drugs reached 5 mg/ml and 500 micrograms/ml respectively; 2. SM and TP could inhibit the absorption of 3H-TdR and this effect was correlated positively to the dosage of the drugs and; 3. SM and TP could reduce the mitosis index of fibroblasts. It was concluded that SM and TP had definite depressive effect on growth of fibroblasts which was correlated positively with the concentration of drugs and duration of application. The inhibitory effect of the drugs on fibroblasts was mainly through inhibition of synthesis of DNA.
Objective To investigate the effects of chitosan on the cell cycle of the human fibroblasts and on the Ki-67 antigen expression in vitro and to investigate the mechanism of chitosan preventing the postoperative tissue adhesion. Methods The cultured fibroblasts were treated for 48 hours with 0，0.01，0.1，1.0，10.0 mg/ml of chitosan, respectively;then, the cell cycle of the fibroblasts was measured by the flow cytometry. The cultured fibroblasts were treated for 24 hours with the chitiosan at the above concentrations; then, the Ki-67 antigen in the cell nucleus was detected with the immunohistochemical staining toobserve its expression. Results The growth of the fibroblastswas obviously suppressed by chitosan, especially in the cell morphology. When the concentrations of chitosan were 1.0 mg/ml and 10.0 mg/ml, the percentages of the fibroblasts in the proliferation stage were 32.3%±5.2% and 14.7%±2.9%, respectively,which were significantly smaller than the percentage of the fibroblasts when the concentration of chitosan was 0 mg/ml (the control group) (41.9%±5.8%， P＜0.05). When the concentrations were 0.01 mg/ml and 0.1 mg/ml, the percentages of the fibroblasts in the proliferation stage were 39.0%±6.0% and 35.5%±3.4%, respectively, which were smaller than that of the control, but not significantly different from that of the control (P＞0.05). When the concentrations of chitosan were 0.1 mg/ml,1.0 mg/ml and 10.0 mg/ml, the percentages of the fibroblasts that had the positiveKi-67 antigen were 37.3%±3.4%, 30.5%±6.2% and 17.8%±3.0%，respectively, which were significantly smaller than that of the control (57.6%±8.9%， P＜0.05). When the concentration was 0.01 mg/ml, the percentage of the fibroblasts that had the positive Ki-67 antigen was 54.1%±8.0%, which was smaller than that of the control, but not significantly different from that of the control (P＞0.05). ConclusionChitosan can inhibit the proliferation of the fibroblasts and increase the percentage of the fibroblasts in the quiescent stage, which can be considered as one of the mechanisms that chitosan can prevent the postoperative tissueadhesion.