ObjectiveTo study the effect and feasibility of poly-lactide-co-glycolide (PLGA) loaded with recombinant human bone morphogenetic protein 2 (rhBMP-2) on repairing articular cartilage defect in rabbits. Methods PLGA was made into cylinders which were 4 mm in diameter and 3 mm in thickness. rhBMP-2 was fully homogenated before used. PLGA combined with 0.5 mg rhBMP-2 under the condition of vacuum(700 mmHg),and then lyophilized, packed ,sterilized with ethylene oxide and reserved. Defects of 4 mm in diameter and reaching medullary cavity were made in femoral condyles of 72 two-month-old New Zealand white rabbits. The 36 right defects were repaired with PLGA-rhBMP-2 composites as the experimental group, the 36 left defects with PLGA only as PLGA group, the other 36 left defects were left untreated as control group, and the other 36 right defects with PLGA-MSCs composites as cell group. At 4, 8, 12, 24, 36 and 48 weeks after operation, macroscopical and microscopical observations were made, and the histological grade wasdone.Results After 4 weeks of operation: In the experimental group and cell group, defects were filled with white translucent tissue which appeared smooth and soft; the matrix around chondrocytes was weakly metachromatic, the newly formed cartilage tissue was thicker than normal cartilage tissue; there was no formed tissue in the PLGA group and the blank control group. After 8 weeks of operation: In the experimental group and cell group, the new tissue was white, translucent, tenacious and smooth. The boundary with normal cartilage became vague. New cartilage cells distributed evenly. The cells of the surface layerparalleled, but the deeper layer lost directivity. The matrix dyed weakly. The new cartilage gradually became thinner, but it still thicker than the normal cartilage ones. The PLGA degraded besides some drops.In the blank control group and PLGA group, a little white membrane formed at the bottom of the defect. After 1224 weeks of operation: In the experimental group and cell group, defects were filled with new tissues which were white, translucent, tenacious and smooth. The boundary disappeared.The thickness of the new cartilage was similar to that of the normal ones. The cells of the surface layer paralleled to each other,but the cells of the deeper layer tended to arrange vertically. The matrix around chondrocytes was metachromatic,but the color was lighter than that of the normal cartilage. Bone under the cartilage and the tide mark recovered. The new cartilage linked with nomal cartilage finely.In the blank control group and PLGA group, there was a little fibrous tissue at the bottom of the defect withe obvious boundary. After 36 weeks and 48 weeks of operation：in the experimental group and the cell group, the new cartilage was slightly white，continuous and less smooth. The boundary disappeared. There was no proliferated synovial membrane.The thickenss of the new cartilage was thinner than that of the normal ones. The matrix around chondrocytes was weakly metachromatic. In the blank control group and PLGA group, the defect still existed, but became smaller.At the bottom of the defect, fibrous tissues formed. Some cartilage denudated and became less smooth.Some bone under cartilage exposed,and the synovial membrane became thick. The histologic grade of the repair tissue at 12 weeks and 24 weeks of operation in experimental group and cell group was significantly different from that at 4, 8 and 48 weeks of operation(Plt;0.01). There was also significant difference in the experimental group and cell group compared with the blank control group and PLGA group at each time after operation(Plt;0.01). But there was no significant difference between the experimental group and the cell group. Conclusion In the course of degradation。。。。。。.
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 extract and purify the cytoplasmic neurotrophic proteins from bone marrow stromal cell and to test their neurotrophic activity. Methods Bone marrow stromal cells were collected from rat femur,after ultrasonicgrind and ultracentrifugation, the supernate was ultrafiltrated and concentrated,the proteins that molecular weight was greater than 10 ku were collected. The neurotrophic active substance was extracted and purified by Sephadex G-100 gel chromatography and high performance liquid chromatography (HPLC). Then their neurotrophic activity was tested in cultured spinal cord motoneuron with MTT method and morphous observation. Results After supernate was analyzed by Sephadex G-100 chromatography, peak Ⅱ could promote the growth of neuron. A further analysisof Peak II with HPLC showed that peak A could promote the growth of neuron. The SDS-PAGE analysis of peak A indicated that a main protein zone with molecular weight of 13 ku was obtained. Conclusion The protein of 13 ku in MSCs has neurotrophic activity.
Objective To investigate the feasibility of cartilaginous implantscontaining bone marrow stromal cells(MSCs) derived from chondrocytes in biological resurfacing procedures for repairing articular cartilage defect. Methods MSCs derived from chondrocytes were obtained with high initial cell density subculture. An implant was constructed by dispersing the chondrocytes in a acid soluble type Ⅰ collagen gel(5×106cells/ml, final cell concentration). A fullthickness defect 3 mm×5 mm was created in the trochlear groove of femur in 36 rabbits. A piece of cotton soaked in 0.5% trypsin was laid into the defect for 5 minutes, then the defect was filled with MSC/collagen gel implant on one side(n=36), filledwith a plain collagen gel on the other side(n=18),and left empty as controls on the other side(n=18). The animals were sacrificed at 4, 8, 12, 24, 32,and 48 weeks. The repaired tissue was examined and evaluated with Pineda gradingscale. Results In MSCs group, the implanted cells resembled well differentiated chondrocytes and were surrounded by metachromatic matrix and the reparative tissue resembled hyaline cartilage after 4 weeks; bone was formed at the base of the defects, the thickness of new cartilage was larger than tht of normal one after 8 weeks; the thickness was reduced proximally, approximating to that of normal cartilage, and chondrocyte columns was formed and subchondral bone and tidemark reappeared after 12 weeks; the thickness of the new tissue was about 55% of the normal tissue, with smooth surface and there were hypertrophic chondrocytes near the tidemark after 24 weeks; no hypertrophic chondrocytes were observed, indicating cessation of endochondral ossification after 32 weeks; the tissue architecture was the same as that at 32 weeks, hyaline-like cartilage persisting, with subchondral bone and tidemark in continuity after 48 weeks. The four layer cell orientation was not as clear as that of normal cartilage. The defects were partially filled with fibrous tissue in controls. At 32 weeks, erosive cartilage, naked subchondralbone and proliferative synovial membrane indicated the presence of osteoarthrosis. There were no statistical difference according to Pineda tissue scales in the specimens from the MSCs group between 24, 32, and 48 weeks, but there was significant difference between 4 weeks and 24, 32 and 48 weeks (Plt;0.05). The joint function recovered after 2 weeks in MSCs group, while it deteriorated progressively incontrols. Conclusion MSCs derived from chondrocytes improve repair of largefullthickness defect in articular cartilage. The reparative hyaline-like cartilage is stable differentiation after 24 weeks, maintains good joint function after 48 weeks.
OBJECTIVE To investigate the ectopic osteogenesis of bone marrow stromal cells (MSC) induced by bone morphogenetic protein(BMP) in vitro and in vivo, providing the experimental evidence for making an artificial bone with its own capacity of bone formation. METHODS MSC were separated and cultured from bone marrow of Wistar rats, MSC were co-cultured with BMP in vitro (cultured in plate and diffuse chamber). Artificial coral hydroxyapatites (CHA) with MSC and BMP were implanted into dorsal muscles of Wistar rats, their bone formation were observed by morphological examination, histochemistry and immunohistochemistry. RESULTS Only cartilaginous matrix were produced by MSC in vitro (cultured in plate and diffuse chamber), and both cartilaginous and bone matrix production within the combined grafts were seen. The bone formation of experimental groups (CHA + BMP + MSC) was ber than that of control A(CHA + MSC) and control B(CHA). CONCLUSION It may be possible to produce an artificial bone with its own capacity of bone formation by combined graft (CHA + BMP + MSC). There may be multiple factors as well as BMP inducing bone formation both in the whole body and the location of the implantation. Further research on these factors will have the significance for making the ideal artificial bone.
OBJECTIVE: To study the effect of simvastatin on the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphates (ALP) activity in the primary cultured bone marrow stromal cells, and to elucidate the mechanism of the anabolic osteogenetic effect of simvastatin. METHODS: Bone marrow stromal cells in femur and tibia of adult mouse were cultured in vitro. after treated with different concentrations of simvastatin (0, 0.1, 0.2, 0.5 and 1.0 mumol/L) or recombinant human BMP-2 for 72 hours, ALP activity of bone marrow stromal cells was determined. BMP-2 expression of bone marrow stromal cells was analyzed by using immunocytochemistry and Western blotting. RESULTS: After treated with simvastatin for 72 hours, BMP-2 expression increased, while little BMP-2 expression could be observed in the control group. ALP activity also increased in a dose-dependent manner; t-test showed that ALP activity in the group which concentrations of simvastatin were 0.5 mumol/L (t = 2.35, P = 0.041), 1.0 mumol/L (t = 2.348, P = 0.041) had significant difference when compared with control group. CONCLUSION: Simvastatin lead to high expression of BMP-2 in bone marrow stromal cells, via the increased auto- or para-crine of BMP-2, and ALP activity increased. These may be parts of the mechanism on the anabolic osteogenetic effect of simvastatin.
OBJECTIVE: To investigate the effects of dexamethasone on the proliferation and differentiation of bone marrow stromal cells(MSC). METHODS: MSC were isolated and cultured in vitro. After treatment with different concentrations of dexamethasone (0, 10-10, 10-9, 10-8, 10-7 and 10-6 mol/L), the proliferation and alkaline phosphatase (ALP) activity of MSC were measured to evaluate the effect of dexamethasone on the biological characteristics of MSC. RESULTS: Dexamethasone inhibited cell proliferation. With the increase of concentration of dexamethasone, the effect was enhanced, which was more significant when the concentration of dexamethasone was over 10-8 mol/L. At the same time, dexamethasone promoted the activity of ALP. This effect was enhanced with the increase of concentration of dexamethasone, but the alteration was small when the concentration of dexamethasone was over 10-8 mol/L. The effects increased with the time. The activity of ALP was enhanced 2 to 4 times with the dexamethasone for 6 days. CONCLUSION: Dexamethasone inhabit the proliferation of MSC, while induce them to differentiate into osteoblasts. The appropriate concentration of dexamethasone was 10-8 mol/L.
OBJECTIVE To study the biocompatibility on bioactive glass ceramics (BGC) and polylactic acid (PLA) combined with cultured bone marrow stromal cells (BMSCs) in bone tissue engineering. METHODS BMSCs were cultured combined with BGC and PLA in vitro, and the morphological characters, cell proliferation, protein content, and alkaline phosphatase activity were detected. RESULTS: BMSCs could be attached to and extended on both BGC and PLA, and normally grown, proliferated, had active function. BGC could promote cell proliferation. CONCLUSION The results show that both BGC and PLA have good biocompatibility with BMSCs, they can be used as biomaterials for cell transplantation in tissue engineering.
OBJECTIVE: A rare huge desmoplastic fibroma on thoracic wall in 1 female case of 25 years old was resected, and the accompanying huge thoracic wall defect, ribs and soft tissues were repaired by tissue engineered bone and pedicled flap. The paper aims to explore the clinical results of early stage after operation. METHODS: Autogeneic bone marrow stromal cells (MSC) were obtained from bone marrow puncture of iliac bone and isolated and cultured according to the Houghton’s methods, MSC were directively induced and differentiated to osteoblasts. Allogeneic ribs were made to the bio-derived bone scaffold materials after treatment of decell, deantigen, decalcification and dry freezing. 5 x 10(6)/ml MSC were cocultured with the bio-derived bone for 6 days in vitro. After intact resection of tumor, the diaphragm flap was applied to repair pleural cavity, the three defect ribs were repaired by tissue engineered bone and the soft tissue defect was repaired by transfer of pedicled ipsilateral abdominal flaps. RESULTS: The patient recovered well with first intention. Followed up for 3 months, tissue engineered ribs were matured in vitro and the heart and pulmonary functions were improved markedly. CONCLUSION: The tissue engineered bone constructed by autogeneic MSC is advantageous in individual treatment.