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 investigate the effect of leptin on fibroblast proliferation and collagen synthesis as to elucidate that fibroblasts play a role in leptin’s effect on wound healing. Methods Purified dermal fibroblasts were derived from sucking wistar rat skin and exposedto leptin at concentration of 0, 10, 50, 100, 200, and 400 ng/ml. The survived fibroblasts were assessed by the colorimetric thiazolyl blue (MTT) assay. Replication of fibroblast was quantified by the incorporation of 3H-thymidine. Collagen synthesis of fibroblast cell was measured by the incorporation of 3H-proline into collagenasesensitive protein. Results The absorption of fibroblast exposed to leptin at concentration of 200 and 400 ng/ml 0.082±0.013, 0.091±0.018 was higher than that of control group 0.063±0.010, P＜0.05. The incorporations of 3H-thymidine of fibroblast exposed to leptin at concentration of 200 and 400 ng/ml 379±101 cpm,326±33 cpm were significantly higher than those of control group 219±56 cpm, P＜0.05. The incorporations of 3H-proline of fibroblast exposed to leptin at concentration of 200 and 400 ng/ml 911±55 cpm, 1 072±259 cpm were significantly higher than that of control group 679±176 cpm, P＜0.05. Conclusion Leptin can promote rat cutaneous fibroblast proliferation and collagen synthesis in vitro. This suggests that cutaneous fibroblast plays a role in leptin’s promoting skin wound healing and it may be one of the main mechanisms by which leptin enhances skin wound healing.
Objective To analyze the molecular composition of type IV collagenous fibres in internal limiting membrane (ILM) of human retina. Methods ILM was surgically removed from retina and identified under phase-contrast and transmission electron microscopes. Monoclonal antibodies against different αchains (α1-α6) of type IV collagen were immuno-localized. Results α3, α4, and α5 chains of type IV collagen were immuno-localized in human retinal ILM, while α1, α2, and α6 chains could not be immuno-localized. Conclusion Type IV collagenous fibres in human retinal ILM are composed of α3, α4, and α5chains. (Chin J Ocul Fundus Dis，2004，20：364-368)
In order to explore the effects of clenbuterol on intramuscular collagen metabolism in denervated skeletal muscles, a randomized, double-masked and placebo-controlled group were studied. Seventy-one patients with complete function loss in muscularcutaneous nerve resulted from brachial plexus injury were administered clenbuterol or placebo 60 micrograms Bid for more than 3 months. Biopsies of the biceps brachia muscle were performed at the beginning and end of this study. The biopsied muscles were processed with anti-collagen I and IV immunohistochemical stains and image analysis as well. The result showed that the collagen proliferation of both type I and IV was much reducible in the clenbuterol-treated group than that of the placebo-treated group (P lt; 0.05). It was concluded that clenbuterol could inhibit partially the proliferation of intramuscular collagens in denervated skeletal muscle.
OBJECTIVE: To study the effect of collagen/hydroxyapatite(CHA) instead of autogenous bone transplantation on repairing the mandibular defects. METHODS: Ten Chinese experimental minipigs were made 2 cm bone defects in diameter in the mandible. The experimental group was implanted CHA, while the control group was implanted autogenous bone. The basic parameters of bone dynamics were determined by bone metrology. RESULTS: There was remarkable difference between the two groups in the mean distance and mineralization apposition rate of double label bands marked by tetracycline(P lt; 0.05), while the mean osteoid seam width and mineralization lag time had no remarkable difference(P gt; 0.05). It suggested that CHA had good osteogenesis. The collagen in CHA offered the condition of bone mineralization, and the mineralization peak of experimental group was present at 4 weeks earlier than that of control group (8 weeks). CONCLUSION: CHA may be a substitute of autogenous bone transplantation in repairing the mandibular defects, and the second operation for offering the implanting bone is avoidable, therefore, CHA may be an ideal material to repair bone defects.
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.
Objective To investigate the effect of hepatitis C virus (HCV) F protein on proliferation and collagen expression of hepatic stellate cells. Methods After pcDNA3.1-f plasmid containing HCV f gene or empty pcDNA3.1 plasmid was transfected hepatic stellate cells LX2 by liposome, LX-f or LX-p cells were obtained by G418 screening. The proliferation of LX-f or LX-p cells was analyzed by MTT, and the contents of collagen type Ⅰand Ⅲ secreted by LX-f or LX-p cells were detected by ELISA. Results After 24 h cultivation, the proliferation rate of LX-f cells was higher than that of LX-p cells at each time point （Plt;0.01）. After 48 h cultivation, the contents of collagen typeⅠand Ⅲ secreted by LX-f were (25.89±0.42) ng/ml and (18.21±0.49) ng/ml, which was significantly higher than those of LX-p cells 〔(22.65±0.49) ng/ml and (15.29±0.62) ng/ml〕, Plt;0.01. Conclusion HCV F protein is able to promote proliferation of hepatic stellate cells, and up-regulate the excretion of collagen type Ⅰand Ⅲ in those cells, which induces hepatic fibrosis.
OBJECTIVE: To investigate the effects of bone morphogenetic protein (BMP) on the proliferation and collagen synthesis of skeletal muscle satellite cells. METHODS: Skeletal muscle satellite cells were harvested and cultured in vitro. The 0 ng/ml, 50 ng/ml, 100 ng/ml, 500 ng/ml, and 1000 ng/ml BMP were used to induce skeletal muscle satellite cells for 48 hours. Cell proliferation, rate of myotube formation and collagen-1 synthesis were measured. RESULTS: BMP promoted cell proliferation and reduced the rate of myotube formation. Collagen synthesis increased when skeletal muscle satellite cells were induced with more than 500 ng/ml BMP. And the higher the concentration of BMP was, the ber this effect became. CONCLUSION: BMP can enhance the proliferation of skeletal muscle satellite cells and change their differentiation from myoblasts to osteoblasts.
Objective To make an experimental research of the tissue engineered rat submandibular glands (SMG) cells growing on a collagensponge scaffold under an optimal culture condition. Methods The Wistar rat (8 days old) SMG cells of the second generation were seeded onthesurface of the collagen sponge scaffold (5 mm×5 mm×2 mm) and were cultured under a physiologically optimal condition for 3 weeks. At 1, 2 and 3 weeks, the cultured cells were observed on their shapes and structures by the histological examination and the scanning electron microscopy. The cultured cells underwentthe immunohistochemistry research (the cytokratin 813,CK8.13;αsmooth muscular actin,αSMA) staining performed at 3 weeks of the culture, and the amylaseactivity analysis (the Amano method) performed at 1 day, 1, 2 and 3 weeks of the culture for an evaluation on the secretion function of the cells; the ultrastructures of the cells were also observed by the transmission electron microscopy for an identification of their origins. Results The observatio n under the scanning electron microscope showed that at 1 week after the cellseeding, the seeded cells were attached to the collagen sponge scaffold surface, with no cell process formed; at 2 weeks the cells increased, with formation of the cell process that was anchored on the collagen sponge scaffold surface; and at 3 weeks, the scaffold surfaceattached cells increased, with formation of thefiliform fibers in the surface layer of the cells. The immunohistochemistry staining showed that the cultured epithelial cells of SMG were bly positive for the specific antibody of CK8.13, and the myoepithelial cells were positive forthe specific antibody of αSMA. The transmission electron microscopy showed that in the surface layer of the cultured epithelial cells of SMG the microvilli,plasm crease, and zymogen granules were observed, with a big and ovalshaped nucleus in the cell, and mitochondria and rough endoplasmic reticulum in the cytoplasm of the cell. The amount of amylase secreted by the cells cultured with thecollagen sponge scaffolds increased at a different degree with an extension of the culturing time. Conclusion The collagen sponge has a satisfactory cell compatibility, and the SMG cells cultured with this kind of collagen sponge can keep their abilities of proliferation and differentiation and theirfunction of secretion. Therefore, this kind of cultured SMG cells can be used as the tissueengineered cells seeded in the scaffold.
OBJECTIVE: To investigate the feasibility of the chitosan-collagen membrane (CCM) as a dermal substitute. METHODS: Fresh bovine tendo calcaneus collagen was dispersed in 0.5 mol/L acetic acid, co-precipitated with chitosan and lyophilized. Dry membranes were cross-linked in 0.05% glutaraldehyde for 24 hours. In vitro its degrading rate was measured by use of collagenase degrading test. The chitosan-collagen membrane was implanted to subcutaneous dorsal sites of SD rats. After implantation, histocompatibility, vascularity and degradation were observed in vivo. RESULTS: The chitosan-collagen membrane was yellowish, translucent, and porous. Pore size ranged 50-250 microns, and collagen fiber bundles were reticular arrangement in the membrane. It had slower degradation than pure collagen membrane by collagenase in vitro. Subcutaneous implantation test showed the minimal inflammation, good histocompatibility and earlier vascularization. The membrane degradation was slower in vivo. Eight weeks after implantation, organized collagen structure was retained. CONCLUSION: The chitosan-collagen membrane has better physical and biological properties, ideal histocompatibility, earlier vascularization and slower degradation. Therefore, It is an optimum substitute for dermal scaffold.
