To study the effect of the repair of rabbit articular cartilage defects by the composite of chondrogenic induction of autologous MSCs and autologous “two-phase” bone matrix gelatin (BMG). Methods Twentyfour healthy adult New Zealand rabbits weighing 2 to 3 kg were divided into group A, B and C with 8 in each. Autologous MSCsderived from group A were cultured in vitro and observed under inverted phase contrast microscope when enough cells through trypsinization transferring in vitro were obtained. Then the growth curves of 1, 3 and 5 passage culture of MSCs were drawn. The 3rd passage MSCs were induced into chondrogenic differentiation by adding TGF-β1 (10 ng/mL), IGF-1 (10 ng/mL) and vitamin C (50 ng/mL) in vitro. At 8 days after induction, the features of chondrocytes were observed under inverted phase contrast microscope, and immunohistochemical staining and Mallory staining were made. Getting out part of the il ium of group A and B, according to the method of Urist, the “two-phase” BMG was acquired. Chondrogenic induction of autologous MSCs was inoculated into the corresponding BMG to set up a composite of cell-carrier, and then it was observed through scanning electric microscope after 3 days of culture. The model of articular cartilage defects of rabbits was made: in group A, autologous cell-carriers were implanted; in group B, there only existed autologous BMG; in group C, there was nothing. At 8, 12 weeks after operation, the gross, HE staining and immunohistochemical staining were made, and grading scales were evaluated according to Wakitani histological grading method. Results Features of MSCs were as follows: the shape of primary cells was shotspindled and of passage cells was long. As to the growth curves of 1, 3 and 5 passage culture of MSCs, passage cells grew slowly for 3 days after being passaged and went into log-growth during the 3rd and the 7th days and into plateau later, but the 3rd passage cells grew best. Observation of MSCs after chondrogenic induction was performed: the shape of cells was ell iptical and the effect of induction was verified by the positive results of collagen type II, S-100 and Mallory staining. Under scanning electricmicroscope, the structure of BMG was good and cells were observed growing in it well. As far as repair of articular cartilage defects are concerned at 8, 12 weeks after transplantation, the defects in group A were repaired by the hyl ine-l ike tissue and the structures of the cartilage surface and normal cartilage were in integrity, and immunohistochemical staining of collagen type II was positive, while those in group B and C were repaired by the fibrous-l ike tissues and the surfaces were irregular. In Wakitanni histological score, at 8 weeks after operation, group A was (3.50 ± 1.51) points, group B was (10.00 ± 1.41) points and group C was (12.00 ± 0.93) points; at 12 weeks, group A was (1.13 ± 0.99) points, group B was (8.38±1.30) points, and group C was (10.13 ± 1.64) points. At different time points, group A was significantly better than group B and C, showing significant differences (P lt; 0.05). Conclusion Induced autologous MSCs and the composite with autologous “two-phase” BMG have the function to repair articular cartilage defects, and they are better than autologous BMG transplanted only or nothing transplanted.
Objective To fabricate a novel porous bioactivecomposite biomaterial consisting of poly lactic acid (PLA)bone matrix gelatin(BMG) by using the supercritical carbon dioxide fluid technique (SC-CO2) and to evaluate its osteoinductive activity. Methods The cortical bones selected from healthy adult donors were processed into BMG by the defatting, demineralizing, and deproteinizing processes. PLA and BMG were mixed at a volume radio of 3∶1; then, the PLA-BMG mixed material and the pure PLA material were respectively placed in the supercritical carbon dioxide reaction kettles, and were respectively added by the NaCl particles 100200 μm in diameter for theporosity of the materials so that the porous PLA-BMG composite material and the porous PLA composite material could be formed. The mouse osteoblastlike MC3T3-E1 cells were cultured in the dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum. Then, 20 μl of the MC3T3E1 cell suspensions containing 2 ×106 cells /ml were delivered into the culturing plate (24 wells/plate) made of the different materials, which were co-cultured for 2 weeks. In the PLA-BMG group, 100 μg of the crushed PLA-BMG material was contained in each well; in the PLA group, 100 μg of the crushed PLA material was containedin each well; and in the DMEM group, only DMEM was contained, which served as the control group. There were 6 wells in each group. The quantitative analysis onthe calcification area was performed by the staining of the alizarin red S. Theco-cultured cells were harvested and lysated in 1 ml of 0.2% Nonidet P-40 by the ultrasonic lysating technique. Then, the ALP activity and the Ca content were measured according to the illuminations of the reagent kits. Results The porous PLABMG composite material showed a good homological porosity with a pore diameter of 50-150 μm and a good connectivity between the pores. The ALP activity, the Ca content, and the calcification area were significantly greater in the PLABMG group than in the PLA group and the control group (325.59±70.40 U/gprot, 3.51±1.64 mmol/gprot, 42.98±4.44% vs. 63.62±30.01 U/gprot, 1.04±0.21 mmol/gprot, 9.55±1.94%, and 2.40±1.47 U/gprot, 0.70±0.24 mmol/gprot, 0.86±0.41%; Plt;0.05). Meanwhile, there was a statistically significant difference between the PLA group and the control group in the ALP activity and the calcification area (Plt;0.05). Conclusion The porous PLABMG composite material prepared by the use of SC-CO2 has a good steoinductive activity and can be used as a promising bone biomaterial and a bone tissue engineered scaffold.
Objective To investigate the effect of “two-phase” tissue engineered cartilage constructed by autologous marrow mesenchymal stem cells(MSCs) and allogeneic bone matrix gelatin(BMG) in repairing articular cartilage defects. Methods Thirty-twoNew Zealand white rabbits were involved in the experiment. “Two-phase” allogeneic BMG scaffold (one side of porous cancellous bone and the other side of cortical bone; 3 mm both in diameter and in thickness) was prepared from iliac bone and limb bone of 5 rabbits by sequentially chemical method. The MSCs wereseparated from 18 New Zealand white rabbits and induced to express chondrocyticphenotype. The chondrocyte precursor cells were seeded onto “two-phase” allogeneic BMG to construct tissue engineering cartilage. Masson’s trichrome staining, PAS staining and scanning electronic microscopic observation were carried out at 1, 3 and 5 weeks. The defects of full thickness articular cartilage(3 mm both in diameter and in depth) were made at both sides of femoral medial condyles in 27 rabbits(including 18 of separated MSCs and the remaining 9). The defects were repaired with the tissue engineered cartilage at the right side (group A, n=18), with BMG at the left side(group B, n=18), and without any implant at both sides in the remaining 9 rabbits as a control( group C, n=18). After 1, 3 and6 months, the 6 specimens of femoral condyles were harvested in 3 groups, respectively. Gross observation, Masson’s trichrome and Alcian blue staining, modified Wakitani scoring and in situ hybridization of collagen type Ⅱ were carried out to assess the repair efficacy of tissue engineered cartilage. Results The “two-phase” BMG consisted of the dense cortical part and the loose cancellous part. In cancellous part, the pore size ranged 100-800 μm, in which the chondrocyte precursor cells being induced from MSCs proliferated and formed the cell-rich cartilaginous part of tissue engineered cartilage. In cortical part, the pore size ranged 10-40 μm, on which the cells arranged in a layer and formed the hard part of subchondral bone. After 1 month of transplantation, the cartilage and subchondral bone were regenerated in group A; during observation, the regenerated cartilage graduallythinned, but defect was repaired and the structure of the articular surface ansubchondral bone was in integrity. In groups B and C, defects were not repaired, the surrounding cartilage of defect was abrased. According to the modified Wakitani scoring, the indexes in group A were significantly higher than those in group B and C(Plt;0.01) except the thickness of cartilage at 6 months. The positive cell rate of in situ hybridization for collagen type Ⅱ in group A was also higher than those in groups B and C(Plt;0.01). Conclusion “Two-phase” allogeneic BMG is a prospective scaffold for tissue engineered cartilage,which combines with autologous chondrocyte precursor cells induced from MSCs toconstruct the tissue engineering cartilage. The tissue engineered cartilage can repair defects of articular cartilage and subchondral bone.
OBJECTIVE To testify the inductive osteogenesis of allogeneic bone matrix gelatin (BMG) in promoting intervertebral fusion. METHODS The gelatin sponge, allogeneic BMG, decalcified bone matrix (DBM) and alcohol conserved bone were implanted respectively into the intervertebral space of rabbit, whose intervertebral discs were removed before implantation. The intervertebral spaces were evaluated by X-ray and histological examination at 4, 8, and 12 weeks after operation. RESULTS No obvious immune rejection was observed. Amounts of new bone were formed in the intervertebral spaces at 4 and 8 weeks. And complete infusion of the intervertebral spaces were appeared at 12 weeks. CONCLUSION Allogeneic BMG can promote bone fusion of intervertebral spaces through osteoinduction, which suggests that allogeneic BMP is an ideal substitute for bone replacement.
OBJECTIVE To study the function of the composite of bone matrix gelatin(BMG) and plaster in the repairing process of bone defects. METHODS Sixteen New Zealand rabbits which were defected in corpus radii were made as implant zone of bone. Sixteen sides of radii were implanted with the composite of BMG and plaster as experimental group. Others were implanted with BMG(8 sides) and bone stored in alcohol(8 sides) as control groups. The repairing process in bone defects were observed by X-ray and histological examination. RESULTS There was an obvious osteogenesis in experimental group. The defects of radii were almost healed at 12th week after operation. There were osteogenesis in both control groups, but the repairing process was slower than that of the experimental group. CONCLUSION The composite of BMG and plaster is a good material for bone transplantation.
ObjectiveTo evaluate the biocompatibility of poly lactic acid/bone matrix gelatin (PLA/BMG) composite biomaterial so as to lay a foundation for bone defect repair. MethodsRats'MC3T3-E1 cells were cultured with leaching solution of PLA/BMG and PLA material respectively for 7 days. The cell proliferation rate was tested by MTT and cell toxicity grading was carried out everyday. The PLA/BMG and MC3T3-E1 cells were co-cultured, the cell shape and proliferation were observed by inverted phase contrast microscope at 1, 3, and 5 days and cell adhesion by scanning electron microscope at 5 days. The PLA and PLA/BMG were implanted subcutaneously in 15 Wistar rats. The histological observation was done, and the thickness of fibrous membrane, the number of inflammatory cells, and the vascularization area were measured at postoperative 2nd, 4th, and 8th week. ResultsThe tests for cytotoxicity in vitro showed that the cell proliferation rates were over 100% and the cell cytotoxic grades were grade 0 at 1-7 days in PLA/BMG group. While in PLA group, the cell proliferation rates were less than 100% and the cell cytotoxic grades were grade 1 at 2, 4, and 7 days. After co-culture of PLA/BMG and MC3T3-E1 cells, cells grew on the surface and in the pores of PLA/BMG, and the cellular morphology was triangle or polygon with abundant microvillus on the surface. After subcutaneous implantation, the rats survived to the end of experiment, and incision healed well. PLA was wrapped by connective tissue where there were a lot of lymphocytes and neutrophilic granulocytes. The cells and tissue grew slowly in PLA. The PLA/BMG materials were wrapped by little connective tissue where there were a few inflammatory cells. The connective tissue ingrowth was observed in the center of PLA/BMG. There was no significant difference in the thickness of fibrous membrane between 2 groups at each time point (P>0.05). The number of inflammatory cells of PLA/BMG group were significantly less than those in PLA group at 2, 4, and 8 weeks (P<0.05); the vascularization area was significantly larger than that in PLA group (P<0.05). ConclusionPLA/BMG composite biomaterials prepared by super critical-CO2 technique are good in cell and tissue biocompatibilty.