Objective To investigate the effects of transformin growth factor-beta (TGF-beta;) and interferon-gamma(IFN-gamma;)on collagen synthesis in human retinal pigment epithelial cells(RPE)． Methods TGF-beta;(0.01～10 ng/ml),recombinant IFN-gamma;(100～10000 Ｕ/ml)or a combination of two were added to cultures of RPE and collagen synthesis of the cells were measured by3 H-proline incorporation assay,indirect immunofluorescence staining and dot-blot hybridization. Results TGF-beta; at 10 ng/ml increased cell uptake of 3 H-proline to 130．87% of controls．It intensified Type IV,I and Ⅲ collagen fluorescent staining as well as mRNA expression．IFN-gamma; at 10000 Ｕ/ml caused 54．72% inhibition of 3 H-proline uptake by RPE,and decreased TypeⅣ collagen fluorescent staining as well as mRNA expression of Type Ⅳ,I and Ⅲ collagens. Conclusion TGF-beta; and IFN-gamma; stimulated and inhibited collagen synthesis of human RPE,respectively．The combination of two had antagonistic effects．IFN-gamma; can be used for inhibition of collagen synthesis of RPE. (Chin J Ocul Fundus Dis, 1999, 15: 245-248)
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 develop a scaffold material containing collagen Ⅰ and sodium hyaluronate for the cartilage tissue engineering and to evaluate its biocompatibility by using the rabbit chondrocytes derived from amandibular condylar process. Methods The porous matrices containing collagen Ⅰ and sodium hyaluronate were fabricated by the freezedrying technique and were crosslinked by using 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC). The microstructure of the scaffold was observed under thescanning electron microscope (SEM), and the enzymatic degradation test was performed to compare the ability of the scaffold resistance to collagenase before and after the crosslinking. The chondrocytes from the rabbits’ condylar process were isolated and cultured before they were seeded into the scaffold, and cell attachment and proliferation were measured by the cell count 1, 3, 5, 7 and 10 daysafter the cell being seeded; then, the biocompatibility of the scaffold was evaluated by the light microscopic examination, histological examination, and the SEM exmination. Results The porous structure of the scaffold facilitated the penetration and attachment of the seeded cells. The porosity was 83.7% and the pore size was 100-120 μm. The cell number increased from 3.7×104 per scaffold 1 day after the cell being seeded to 8.2×104 per scaffold 10 days after the cell being seeded. The crosslinking treatment could significantly enhance the scaffold resistance to the collagenase activity. The examinations under the light microscope and SEM indicated that the chondrocyte adhered and spread well on the scaffold, and the extracellular matrices were also observed around the chondrocytes. Conclusion The porous scaffold composed of collagen Ⅰ and hyaluronan has anappropriate structure and a good biocompatibility for the attachment and proliferation of the chondrocytes, which can facilitate it to become a useful scaffold in the cartilage tissue engineering.
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 repair esophageal defects with an artificial prosthesis composed of biodegradable materials and nonbiodegradable materials, which is gradually replaced by host tissue. METHODS: The artificial esophagus was a two-layer tube consisting of a chitosan-collagen sponge and an inner polyurethane stent with a diameter of 20 mm and a length of 50 mm. We used the artificial esophagus to replace 5 cm esophageal defects in group I (five dogs) and in group II (ten dogs), and nutritional support was given after operation. The inner polyurethane stent was removed after 2 weeks in group I and after 4 weeks in group II endoscopically and epithelization of the regenerated esophagus was observed by histologic examination and transmission electron microscope. RESULTS: In group I, the polyurethane stent was removed after 2 weeks, and partial regeneration of esophageal epithelial was observed; and constriction of the regenerated esophagus progressed and the dogs became unable to swallow after 4 weeks. In group II, the polyurethane stent was removed after 4 weeks, highly regenerated esophageal tissue successfully replaced the defect and complete epithelization of the regenerated esophagus was observed. After 12 weeks, complete regeneration of esophageal mucosa structures, including mucosal smooth muscle and mucosal glands and partial regeneration of esophageal muscle tissue were observed. CONCLUSION: Esophageal high-order structures can be regenerated and provided a temporary stent and support by polyurethane stent and an adequate three-dimensional structure for 4 weeks by collagen-chitosan sponge.
Abstract: Objective Using Amplex red fluorometric assay to detect the lysyl oxidase (LOX) enzyme activity in tissue engineered heart valve (TEHV). Methods Porcine aortic valves were decellularized with trypsin+ethylene diaminetetraacetic acid(EDTA), TritonX-100, and RNaseⅠ＋DNaseⅠ, then they were seeded by myo-fibroblasts that harvested from rats. Then they were fed with Dulbecco’s modified Eagle medium (DMEM) which contained high glucose for 27 days, they were fed with phenol red-free and serumfree DMEM for 24 hours, and the medium was harvested and used for LOX enzyme activity assays with the Amplex red fluorometric assay. And reverse transcription-polymerase chain reaction (RT-PCR) technique was used to analyze the expression of LOXmRNA in TEHV. Results All the samples produced measurable amounts of active LOX enzyme. The fluorescence units were 45.60±1.66, and the corresponding concentration of LOX enzyme were 0.123±0.003μg/ml. At the same time, all the samples expressed LOXmRNA. The expression of LOXmRNA was corresponding to the results of the Amplex red fluorometric assay. Conclusion It is feasible to detect the LOX enzyme activity in TEHV with the Amplex red fluorometric assay. And this assay gives a way to reflect that LOX plays an important role in collagen cross-linking of extracellar matrix in TEHV.
To observe the collagen-hydroxylaptite composite in the repair of bone defect, ten minipigs were chosen to make a mandibular dafect measuring 2 cm in diameter and the composite was implanted, while the use of autogenous bone graft and the blank wese served as control. On the 4, 8, 12, 24 and 48 weeks after the operation, the animals were sacrificed and the samples were examined under light microscope. The result showed that: no infection or necrosis occurred. The composite coalesced with host bone and the outcome was similar to that of the autogenous bone graft. No foreign body giant cells or vacuum left from osteonecrosis was observed. It was suggested that the composite had the advantage of abundant supply, easy to handle and no harm. The biocompatibility was good and might be hopeful as a bone substitute.
Objective To evaluate the cytocompatibility of collagenmembraneswith transitional cells of rabbit in vitro and to discuss the possibility of the collagen membranes as urologic tissue engineering scaffolds. Methods Primary cultured transitional cells isolated from New Zealand rabbits were implantedon collagen membranes at 1×105 cells/cm2. The changes of cell adhering were observed by inverted microscope and scanning electron microscope 2, 12 and 24hours later. The experiment was divided into 4 groups: non-cell group (black control) culture medium group(negative control), extract medium from Polyvinyl chloride group(positive control) and extract medium from collagen membranes group(experimental group). The cells of generations 2 to 4 were implanted in 96-hole-plank at 1×104 cells every hole. And every group had 5 holes. Then absorption coefficient were detected at the wave length of 490 nm by MTT assay. Then the cytotoxicity and cytocompatibility were evaluated by comparison of the numbers of absorptioncoefficient.Results The bladder transitional cells began to adhere to the collagen membrane 2 hours after implanting, and the number of the adhered cells increased with time.The actual absorption coefficient of experimental groups was 0.590±0.024,1.065±0.040 and 1.129±0.074 after 24, 72 and 120 hours. The actual absorption coefficient of negative control group was 0.639±0.068,1.022±0.044 and 1.087±0.111. The actual absorption coefficient of positive control group was 0.302±0.029,0.653±0.083 and 0.694±0.031. There was significantdifference between the experimental group and positive control (Plt;0.01), and no significant difference between the experimental group and negative control(Pgt;0.05).Conclusion Collagen membrane has good cytocompatibility withtransitional cells and no cytotoxity. It can be used as scaffolds of urologic tissue engineering.
Objective To investigate the preventive effect of carbachol on the formation of postoperative intra-abdominal adhesion. Methods Forty-four Wistar rats were randomly divided into sham operation group (SO group, n=12), operation group (n=16) and carbachol treated group (carbachol group, n=16, carbachol 50 μg/kg). Animal model of abdominal adhesion was established by rubbing the procussus vermiformis of cecum with dry sterile gauze, and by clamping and scuffing abdominal wall. Half of rats were separately killed on day 7 and day 14 after surgery, respectively. The degree of adhesion was evaluated according to Phillips 5-scale grade and the feature of this model. The histopathological changes of adhesive tissues were observed and the content of collagen type Ⅰ in the tissues was detected by immunohistochemistry. Results The scores of intra-abdominal adhesion were significantly lower in the carbachol group than those in operation group both on 7 d and 14 d (P＜0.01). Mild inflammatory changes and less fibrous proliferation were observed in carbachol group microscopically. The contents of collagen type Ⅰ detected by immunohistochemistry were significantly lower in the carbachol group than those in operation group both on 7 d and 14 d (P＜0.01). There was no significant difference of the score of abdominal adhesion and content of collagen type Ⅰ in the same group between 7 d and 14 d (Pgt;0.05). Conclusion Carbachol may take a significant role in the prevention of postoperative abdominal adhesion in rat.
Objective To review the appl ication of collagen and biodegradable polymer composite scaffolds in vascular tissue engineering, and describe the multi-layering vascular scaffolds of collagen-based material in recent years. Methods The l iterature concerning collagen composite scaffold production for scaffold of vascular tissue engineering was extensively reviewed and summarized. Results As one of the structural proteins in natural blood vessel, collagen is widely used in vascular tissue engineering because of good biocompatibil ity, biodegradabil ity, and cell recognition signal. The vascular scaffolds with biological activity and good mechanical properties can be made by collagen-polymer composite materials. In addition, the structure and function of the natural blood vessel can be better simulated by multi-layering vascularscaffolds. Conclusion Collagen-polymer composite material is the hot spot in the research of vascular scaffolds, and multilayering vascular scaffolds have a brill iant future.