Objective To supply references to tissue-engineered skin cl inical appl ications with autogenic BMSCs composited collagen membrane to repair swine full-thickness cutaneous deficiency. Methods Twenty mL bone marrow were obtained respectively from 4 swine, autogenic BMSCs were cultured and passed to the 3rd passage. The fresh bovine tendontreated by means of chemically cross-l inked was made 5 cm diameter collagen I (Col I) membrane. The 2 × 107/mL P3 swine autogenic BMSCs labeled DAPI were planted to sterile Col I membrane for 24 hours incubation, then the tissue-engineered skin was constructed. The five full-thickness skin defect of 5 cm diameter was excised to the muscle from forward to backward on the back midl ine two sides of swine. The tissue-engineered skin were implanted in the experimental group, while Col I membrane was implanted in control group. After 3 and 8 weeks of implantation, the two swine wound surface heal ing circumstance was observed and further evaluated with histology analysis and TEM. After 3 weeks of implantation, the experimental group were observed with fluorescence microscopy and staining for glycogen. Results After 3 weeks of implantation, the wound surface of control group were observed nigrescence, scab and putrescence, and after 8 weeks of implantation, also evident putrescence and scar. The wound surface of experiment group was al ive after 3 weeks implantation, appearance was leveled off and flexible without evident scar. The wound surface recovered well after 8 weeks of implantation, wound surface heal ing rate was significantly difference between the two groups (P lt; 0.01). After 3 weeks of implantation, control group were observed acestoma hyperplasia and no epidermal coverage by histology analysis. The experimental group was showed integrity epidermis and dermis structure. The basal layer was crimson and continuously positive with glycogen staining. After 8 weeks of implantation, the experimental group and control group were emerged normal skin structure. After 3 weeks of implantation in control group, a lot of neutrophil ic granulocytes and fibroblasts were noticed, but no epidermal structure was observed under TEM. In the experimental group, a lot of epidermal cells were observed, dermatome connection among epidermal cells and hemidermosome connection between basilar membrane cells and basal membrane were observed in epidermis. In the dermis experimental group, blood capillary endothel ial cells were noticed. Furthermore, considerable collagen fiber deposit was found in the surrounding tissue of fibroblasts. After 3 weeks of implantation, BMSCs labeled with DAPI were located reconstructed epidermal basement membrane and dermis by fluorescence microscopy. Conclusion Tissue-engineered skin which is composited with autogenic BMSCs as seed cells and collagen membrane were potential prospects in appl ication of repairing swine full-thickness cutaneous deficiency.
【Abstract】 Objective To compare the properties of collagen membranes before and after crossl inked and to establ ish the foundation of appl ication of collagen membranes. Methods Fresh bovine tendons were separated and collagen was extracted by washing, smashing and acetic acid dissolving. The collagen protein was determined by ultraviolet spectrophotometer and its characteristics were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), wavelength scanning and amino acids detecting. Collagen membranes were produced by lyophil ization. And then the biocharacteristics of the membranes before and after glutaraldehyde crossl inked were compared. BMSCs separated from volunteer’s bone marrow were seeded on collagen membranes before and after crossl inked by 2×103 in 100 μL medium, seven days after culture, the absorption spectrum of BMSCs was examined, and BMSCs were observed by scanning electron microscope (SEM). Results The contents of collagen protein were 2 mg/mL. The maximum absorption wave length appeared at about 230 nm. SDS-PAGE suggested that molecularweight of main bands was more than 66.2×103, the same as collagen marker from calf skin. There were 21.47% glycine, 12.04% pral ine and 10.18% hydroxyprol ine. No tryptophan was found. Before crossl inked, collagen membranes were in shape of white sponges and with big holes and the range of pH value was from 4.5 to 5.0. SEM showed reticular conformation and pore structure of collagen membranes, but the bore diameter was bigger. Their water-absorbing capacity was 61 times as much as their weight. The mechanical strength was 210 g/cm3. The dissolution time of collagenase was 90 minutes. After crossl inked, collagen membranes became thin, colorless, semi-transparent and compact with better tenacity. Under SEM, compact collagen fiber appeared reticular. There was lower water-absorbing capacity and pH value ranged from 6.5 to 7.0. The mechanical strength was 3 400 g/cm3 and the dissolution time of collagenase became longer. BMSCs could grow better either on before-crossl inked collagen membranes or on after-crossl inked ones. Conclusion As biomaterial scaffolds, after crossl inked collagen membranes were better than before-crossl inked ones.
Objective To evaluate the potential of bioresorbable collagen membrane in a combination with bone marrow stromal cells (BMSCs) or platelet rich plasma (PRP) in repairing alveolar bone defects. Methods The first and second premolars were extracted from the bilateral maxillary and mandibular bone and fouralveolar intrabone defects (8 mm in height, 5 mm in width,15 mm in length) werecreated in 3 male mongrel dogs. The experiment included 4 groups: group A (nothing was used as control group), group B (only Bio-Gide® group C (Bio-Gide® BMSCs) and group D (Bio-Gide®／PRP). The macroscopic, radiographic and histological observations were performed at 4, 8 and 12 weeks after surgery. Results The cells were circle or short spindleshape after 1 day of coculture; and the cellswere polygon and long spindleshape with process after 3 days. The macroscopic observation: after 4 weeks in the defect region, obvious excavation and organization of hematoma were seen in group A; and new bone formation and little organization of hematoma were seen in groups B, C, D. After 8 weeks, excavation was not obvious, fibrous tissue was seen at the top of defect, organized hematoma wasgradually replace by new bone in group A; the edge of membrane broke and adhered to deep tissue and needle could pierce the surface ofdefect in groups B, C, D. After12 weeks,excavation disappeared in 4 groups and fibrous tissue at top of alveolar ridge in group A was thicker than that in groups B, C, D. The radiographic observation: defect was full of new bone. In groups A, B, C and D, the grey values were 68, 50, 56 and 49 after 4 weeks; 46, 30, 24 and 30 after 8 weeks; and 24, 17, 15 and 20 after 12 weeks respectively. The histological observation:after 4 weeks, a lot of fibrous connective tissues granulation tissues were seen no obvious new bone formed in group A; and the collagen structure of membrane remained and new bone formed in medial surface in groups B, C, D. After 8 weeks, new bone trabecula displayed clump and web in group A; the collagen structureof membrane were not of integrity, and many bone islands and few fibrous connective tissue formed in groups B, C, D. After 12 weeks, defect was filled with newbone in 4 groups. Conclusion Guided bone regeneration (GBR) treatment with collagen membranes may significantly enhance bone regeneration within 8 weeks. Theinfluence of GBR in combination with BMSCs or PRP in accelerating the repair of alveolar bone defects shoud be further investigated.
Objective To compare the effect of guiding boneregeneration between l-ethyl-3(3-diaminopropyol)-carbodiimide(EDAC)crosslinked acellular bovine pericardium (ABP) and medical collagen membrane (CM). Methods Defects of 7 mm×7 mm×5 mm were created in both mandibles of 24 rabbits, which weighted 2.6～3.5 kg. One side defect was covered with EDAC-crosslinked ABP(EDAC-crosslinked ABP group), the other side defect with medical CM as control(CM group). The ability of bone defect repair and change ofboth membrane materials were evaluated by gross observation, histological study and computer graphic analysis in the 4th, 8th, 16th and 24th weeks after operation. Results The surface of bone defects was even, consistent with adjacent normal bonein EDACcrosslinked ABP group, while that of bone defects was of no evenness in CM group in the 16th and the 24th weeks. The histological observation showed that bone trabecula formed in the EDAC-crosslinked ABP group and fibrous connective tissue was seen in CM group in the 16th and the 24th weeks. There were no significant differences in new bone percentage of bone defects between 2 groups inthe 4th and the 8th weeks(P＞0.05). In the 16th week new bone percentage of bone defects was 81.99%±3.92% in EDAC-crosslinked ABP group and 76.35%±4.29% in CM group, showing significant difference (Plt;0.05). The average percentage of absorption in EDAC-crosslinked ABP group was 16.57%, 27.94%, 65.61% and85.72% in the 4th, 8th, 16th and 24th weeks respectively, while that in CM group was more than 50% in the 4th week and completely degraded at the end of 8 weeks. Conclusion EDAC-crosslinked ABP has a better effect on guiding bone regeneration than CM in the repair of bone defects.
Objective To investigate the curative effects of homograft of the mesenchymal stem cells(MSCs) compbined with the medical collagen membrane of the guided tissue regeneration(MCMG) on the full thickness defects of the articular cartilage. Methods MSCs derived from New Zealand rabbits aged 3-4 months weighing 2.1-3.4 kg were cultured in vitro with a density of 5.5×108/ml and seeded onto MCMG. The MSC/MCMG complex was cultured for 48 h and transplanted into the fullthickness defects on the inboardcondyle and trochlea. Twenty-seven healthy New Zealand rabbits were randomly divided into 3 groups of 9rabbits in each. The cartilage defects in the inboard condyle and trochlea werefilled with the auto bone marrow MSCs and MCMG complex (MSCs/ MCMG) in Group A (Management A), with only MCMG in Group B (Management B)and with nothing in Group C (Management C). Three rabbits were killed at 4, 8 and 12 weeks after operation in each group, and the reparative tissue samples evaluated grossly,histologically and immunohistochemically were graded according tothe gross and histological scale. Results Four weeks after transplantation, the cartilage and subchondralbone were regenerated in Group A;for 12 weeks, the regenerated cartilage gradually thicked; 12 week after transplantation, the defect was repaired and the structures of the carticular surface and subchondral bone was in integrity.The defects in Group A were repaired by the hylinelike tissue and the defects in Groups B and C were repaired by the fibrous tissues. Glycosaminoglycan and type Ⅱcollagen in Groups A,B and C were reduced gradually.The statistical analysis on the gross at 12 weeks and the histologicalgradings at 4 weeks,8 weeks and 12 weeks showed that the inboardcondylar repairhad no significant difference compared with the rochlearepair(Pgt;0.05).Management A was significantly better than Managements B and C (Plt;0.05), and Management B was better than Management C(Plt;0.05). Conclusion Transplantation of the MSCs combined with MCMG on the full thickness defects of the articular cartilage is a promising approach to the the treatment of cartilage defects. MCMG can satisfy the demands of the scaffold for the tissue-engineered cartilage.
Objective To investigate the promotion effects of the collagen membrane incorporating bFGF impregnated microspheres on the wound healing of the pigskin losing its full-thickness layers. Methods The bFGF containing microspheres was added into the dry microspleres.The collagen membranes were prepared by incorporating bFGF-impregnated microspheres, and 6 York pig models of skin wounds with loss of their full-thickness layers were established for the ob servation of the effects on the wound healing. Results The healing time and the 28day healing rate were 27.30±1.14 days and 98.12%±1.97%, respectively.The healing rate was significantly higher and the healing time was significantl y shorter in the experimental group than in the control group (Plt;0.05). The histological examination showed that the proliferation condition of the epidermiswasalso much better in the experimental group. Conclusion Incorporation of bFGF-impregnated microspheres into the collagen membrane is a promising method of pro moting the healing of the wound with a loss of the fullthickness skin.
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.
ObjectiveTo observe the long-term outcome and biocompatibility of the porcine collagen membrane (DermalGen) after xenotransplantation in vivo.MethodsTwenty Sprague Dawley rats were randomly divided into 2 groups (n=10). DermalGen were implanted subcutaneously into the dorsum of rats in experimental group, and the rats in control group were treated with sham-operation. At 3, 7, and 15 days and 1, 3, 6, and 12 months after operation, the samples of experimental group were harvested and gross observation, histological observation, CD31 immunohistochemical staining, and transmission electron microscope observation were taken to observe the inflammatory reaction, angiogenesis, and collagen arrangement. The skin tissues of the control group at 12 months were observed and compared.ResultsAll incisions healed in experimental group, without obvious swelling and inflammatory reaction. The DermalGen was closely contact with the surrounding tissue without obvious rejection, and it was still legible at 12 months. Histological observation of experimental group showed that the infiltration of fibroblasts and inflammatory cells were seen at 7 days. More capillaries and fibroblast cells were seen and the inflammatory response gradually faded at 15 days and 1 month. There were abundant vessels and cells in the DermalGen at 3 months. The angiogenesis and fibroblasts decreased gradually, and the collagen started to format and margin blended simultaneously at 6 and 12 months. The inflammatory cells in experimental group at 15 days and 1 month were significantly more than that in control group (P<0.05), and no significant difference was found at 12 months between experimental group and control group (P>0.05). Immunohistochemical staining of experimental group showed that the angiogenesis changed obviously with the time, and the density of vessels decreased significantly at 12 months. Compared with control group, the possitive expressions of CD31 in experimental group at 15 days and 12 months after operation were significantly decreased (P<0.05), and were significantly increased at 1 month (P<0.05). Transmision electron microscope observation showed that the arrangement of collagen in grafted DermalGen had no obvious changed when compared with the DermalGen, and vascular endothelial cell, capillarypericytes and fibroblast cells could be seen inside.ConclusionThe DermalGen structure is stable after long-term xenotransplantation and with good tolerogenic property in vivo.