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Objective: To investigate the effects of silencing P75 neurotrophin receptor (P75NTR) and nerve growth factor (NGF) overexpression on the proliferative activity and ectopic osteogenesis ability of bone marrow mesenchymal stem cells (BMSCs) combined with demineralized bone matrix for heterotopic osteogenesis. Methods: BMSCs of Sprague Dawley (SD) rats were cultured and passaged by adherent isolation method. The third generation BMSCs were transfected with lentivirus mediated P75NTR gene silencing (group B), NGF overexpression gene (group C), P75NTR silencing and NGF overexpression double genes (group D), respectively, and untransfected cells as control (group A). After 7 days of transfection, the expression of fluorescent protein of the target gene was observed by fluorescence microscope; cell counting kit 8 method was used to detect the cells activity for 8 days after transfection; the expressions of P75NTR and NGF proteins in each group were detected by Western blot. The adhesion of BMSCs to demineralized bone matrix (DBM) was observed by inverted phase contrast microscope and scanning electron microscope after transfection of p75NTR silencing and NGF overexpression double genes. After transfection, BMSCs and DBM were co-cultured to prepare 4 groups of tissue engineered bone, which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model ( n=6). HE staining was performed at 4 and 8 weeks after operation. ALP staining was used to observe the formation of calcium nodules at 8 weeks after operation. The expressions of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by real-time fluorescent quantitative PCR. Results: At 7 days after transfection, there was no fluorescence expression in group A, red fluorescence expression was seen in group B, green fluorescence expression in group C, and red-green compound fluorescence expression in group D. The fluorescence expression rate of target gene was about 70%. Western blot detection showed that the relative expression of P75NTR protein in groups A and C was significantly higher than that in groups B and D, and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B ( P<0.05). With the passage of time, the cell proliferation activity increased in all groups, especially in group D, which was significantly higher than that in group A at 3-8 days ( P<0.05). The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM. In the subcutaneous ectopic osteogenesis experiment, HE staining showed that at 4 and 8 weeks after operation, the more bone tissue was formed in group D than in the other 3 groups. ALP staining showed that group D had the highest ALP activity and better osteogenic expression. Compared with group A, the relative expressions of Runx2, ALP, and OCN mRNAs in group D were significantly higher than those in group A ( P<0.05). Conclusion: Silencing P75NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability. By increasing NGF level and closing P75NTR apoptosis channel, it can not only improve cell activity, but also promote bone tissue regeneration.
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Objective To evaluate the clinical outcome associated with the core decompression in combination with the nano-hydroxyapatite/collagen composite rod combined with decalcified bone matrix in a consecutive series of patients with osteonecrosis of femoral head,especially the prevention of collapse of femoral head and its predisposing factors.Methods From August,2012 to May,2013,46 pationts (50 hips) who had undergone core decompression in combination with nano-hydroxyapatite/collagen composite rod insertion in corporated with decalcified bone matrix in our hospital were involved in this study.Postoperative care consisted of prophylactic intravenous antibiotic and anticoagulation therapy.Patients were instructed to be non-weight-bearing for 3 weeks,to partial weight-bear for the next 3 weeks,and to weight bear as tolerated thereafter.All patients were evaluated both clinically and radiographically.The primary clinical outcome of this study was functional improvement assessed with the Harris hip score.Serial radiograms of the pelvis were taken at 1,3,6,12 months post-operatively to analyze the process of osteonecrosis.Results All patients followed up for 12 months,no one suffer complications.The mean Harris score pre-operation was 65.6 ± 10.6,post-operation score was 87.5 ± 15.3,with a mean improvement of 21.8 ± 13.2 (P ＜ 0.05).According to Harris hip score system,excellent for 30 hips,good for 14 hips,fair for 2 hip and poor for 4 hips.Refer to the Kaplan-Meier survivorship curve,the success rate at 12 months post-operatively was 92％.Radiological changes coincided with clinical changes.Conclusion Core decompressionin combination with nano-hydroxyapatite/ collagen composite rod insertion in corporated with decalcified bone matrix provided a minimally invasive surgical treatment option to treat early stage osteonecrotic hips(stage Ⅰ and Ⅱ) and to prevent femoral heads from collapsing,with clinical outcomes and success rates priorto other commonly used surgical procedures.
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Objective To observe the biocompatibility of rabbit bone marrow mesenchymal stem cells (BMSCs)combined with allogeneic decalcified bone matrix(DBM)after transfecting adenoviral recombinant human bone morphogenetic protein-2(Ad-rhBMP-2).Methods The rabbit allogeneic DBM material was prepared according to the Ursit method.After transfecting Ad-BMP-2 on rabbit bone marrow mesenchymal stem cells,the immunohistochemical was used to detect the expression of BMP-2 in the transfected cells;after 48 h of transfection,the cells were planted on the allograft DBM,then the scanning electron microscopy was used to observe the cell growth and adhesion condition on material,and the proliferation condition of BMSCs was detected by MTT. Results After 48 h of adenoviral transfection,BMSCs could express BMP-2 successfully.The scanning electron microscopy showed that the cells after transfection adhered well and massively proliferated on DBM material.The MTT assay showed that the prolifer-ation condition of the cells after transfection planted on DBM was normal,which showed no statistically significant difference when compared with the control group (P >0.05).Conclusion The Ad-BMP-2 transfection on BMSCs is well biocompatible to allogene-ic DBM.
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10.3969/j.issn.2095-4344.2013.24.001
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@#Objective To evaluate the antibiotics release characteristic and osteogenesis ability of anti-tuberculosis composite graft by observing the healing effect of segmental radial defect in rabbits.Methods Rabbit decalcified bone matrix(DBM) was made under the method of Urist and was combined with rifampicin-gelatin solution. The antibiotics release characteristics were observed by PBS release test in vitro and K-B disc diffusion test in vivo. The osteogenesis ability was measured by critical radius bone defect of rabbit.Results It was detected in release test that the antibiotic releasing time could maintain at least 28 days in vivo and in vitro and the release ratio was 87%. The rabbit critical radius bone defect could be repaired by this anti-tuberculosis composite graft perfectly.Conclusion Good antibiotics release characteristics and bone formation ability showed during this anti-tuberculosis composite graft and it definitely could be used widely in further clinics.
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[Objective]To introduce the surgical technique and its rationale and to evaluate the early and mid-term clinical outcome of routine core decompression and insertion of a biomaterial loaded allograft threaded cage(ATC)in the treatment of necrotic femoral head．[Methods]Seventy-six patients(78 hips)with femoral head necrosis were allocated to a program of either core decompression or core decompression and implantation of ATC．[Results]At review all patients had a minimum follow-up of 24-months(24 to 68 months)．In the control group，no significant improvement in Harris hip score(HHS)was found，and 13 of the 22 hips had deteriorated to stageⅢ．In the treatment group．the mean HHS was improved from 62．8 to 81．6．Collapse was seen in 1 hip，and this collapse Was progressive in 3 hips．[Conclusion]It，s thus evident that the technique is attractive as a salvage procedure．which shows encouraging Success rates and early clinical results.
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Objective To analyze the radiographical and histological features of implanted composites of decalcified bone matrix(DBM) impregnated with calcium phosphate cement(CPC) and recombinant human bone morphogenetic protein-2(rhBMP-2) in bone defect,and evaluate its biodegradation properties.Methods By prefabricating the rabbit DBM,the CPC/DBM/rhBMP-2 composites were prepared in a DBM/CPC ratio of 2∶8 and the rhBMP-2 content of about 1.2mg/cm3.Twenty-four rabbits were divided into groups A and B(12 each).Animals in group A were implanted with the composites in the bone defects at femoral condyle and femoral muscle,and the rabbits in group B CPC was implunted in the same place.Animals were sacrificed at 6,12 and 24 weeks after operation(4 each),and the implants were examined radiographically and histologically.Results It was shown by radiography that,in group A,the margin of implants in femoral condyle defects became irregular 6 weeks after operation;the appearance of the implants appeared hazy and shrunken in size 12 weeks after operation;and most of the implant was absorbed and the medullary cavity became partly rediotransparent 24 weeks after operation.The density of the implants in muscles beoame less dense gradually during 6 to 24 weeks after operation.While in group B,the appearance and density of the implants in femoral condyle defects and muscles were not obviously changed during 6-24 weeks after operation.Histological observation showed that,in group A,the interface between the implants in the bone defects was filled with newly formed bone grown into the composites 6 weeks after operation.New bone formed inside the composites 12 weeks after operation,and the bone defect was repaired,and most of the implants were replaced by new bones,with a little portion of it remained 24 weeks after operation.Fibrous tissue was seen to grow into the composites in muscles during 6-12 weeks after operation,and only a little of the composites remained 24 weeks after operation.In group B,no fibrous tissues or new bones grew into composites implanted in femoral condyle defects or muscles 6-12 weeks after operation,and the bone defects was still filled with materials of CPC,and no new bone or fibrous tissue was found inside CPC 24 weeks after operation.Conclusion Compare with CPC,the DBM with an optimal formula is are easy biodegradable and can be replaced by autologous bone.
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one hundred and five Wistar rats were randomized into 3 groups and heterogenous decalcified bone matrix (HDBM) + CaCl_2+Na_2HPO_4+KH_2PO_4, HDBM + CaSO_4+Na_2HPO_4+KH_2PO_4, and HDBM + NaCl were implanted into the quadriceps muscle of both thighs of the animals respectively. They were monitored with ~3H-TdR and ~(45)Ca tracing to evaluate the efficacy of the 3 delivery systems to enhance osteogenesis. It was found that coadministration of CaSo_4, Na_2HPO_4, and KH_2PO_4 could significantly enhance the osteogenesis induced by HDBM. It is concluded that HDBM and the coadministration of Na_2HPO_4, KH_2-PO_4, and CaS0-4/CaCl_2 together will be much more effective to induce osteogenesis than HDBM is used singly.
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Objective To analyze the ultra microstructure and histological properties of implanted composites of decalcified bone matrix (DBM) impregnated calcium phosphate cement (CPC) with recombinant human bone morphogenetic protein-2 (rhBMP-2) in bone defect, and evaluate the osteoplastic efficiency of this composites. Methods The rabbit DBM was prepared beforehand. The composites of DBM impregnated CPC with rhBMP-2 was made at 0.2 proportion of DBM. The rabbit's bone defect of femur condyle was filled with implantation of the composites (group A, n=12) or CPC (group B, n=12) or bone cement (group C, n=12). Animals were sacrificed at 6, 12, or 24 weeks after operation, and the implants were examined by histological technique and scanning electron microscopy (SEM). Results In group A, at the 6th week after operation, the interface region between implants and bone tissue became fuzziness, and was crossed by the generated fibers of bone tissue, and filled with woven bone growing inside into the composites; at the 12th week after operation, blood vessels, osteoblasts and new bone were generated inside of composites; at the 24th week after operation, the bone defects were recovered and became solid union, most of the implants were replaced by new bones. In group B, at the 6th week after operation, the interface of CPC and bone tissue was sharp, and was not crossed by new bones; at the 24th week after operation, the bone defects were still filled with materials of CPC and no new bone was found inside CPC. In group C, at the 24th week after operation, the interface of bone cement and bone remained non-union. Higher osteoblastic activity,more neogenetic blood vessels and higher growth rate of woven bone were observed in group A compared with those in group B. Conclusions For bone defect, the implantation of composites with DBM proportion of 0.2 can stimulate the growth of osteoblast, blood vessel and woven bone. It is biodegradable and can be replaced by autogenous bone.