| Backgrounds:Although considerable progress has made in recent years, reconstruction of large bone defects remains one of the major problems for orthopedic surgeons. the ultimate goal is to achieve healing of bone defects, while restoring limb function. At present, the most common ways for the treatment of limb segmental defects are autogenous cancellous bone graft, bone transport technique and vascularized fibular grafting technique, which have achieved great success in the treatment of bone defect, but there are some limitations or shortages. Autogenous cancellous bone grafting generally applies to rebuild bone defects no more than 4-6cm, otherwise the bone graft may be absorbed. bone transport technique is restrict by its long treatment period, vascularized fibular graft to repair large bone defects has certain advantages, but requires higher equipment and technical requirements, while prone to bone nonunion and other complications [1-3]. In 2000, Masquelet et al [4] first reported treatment of limb segmental bone defects with induced membrane technique, gradually being more and more popular by orthopedic surgeons for its high success rate, fewer complications, and easy to operate. Implanted with polymethyl methacrylate(PMMA) bone cement spacer in the bone defects after debridement, formation of a biologically active membrane, which is known as the induced membrane, then filled with autologous cancellous bone to repair bone defect. Early studies showed that success rate using induced membrane to treat bone defects is between 88% and 100%, and reported that up to 25 cm bone defects can be successfully repaired [5-7]. Induced membrane can not only prevent the bone graft to be absorbed by the surrounding tissue, but also providing the necessary blood supply and growth factors for bone graft, such as bone morphogenetic protein-2(BMP-2), vascular endothelial growth factor(VEGF), etc., the structure and function of the induced membrane as well as the similarities and differences with the periosteum is not very clear. In this study, we aims to inducing of an induced membrane in animal models, observed the structure and growth factor levels changed over time, then protein extract from the membranes cultured with mesenchymal stem cells to observed the proliferation and osteogenic differentiation, implanted with grafts in a closed induced membrane to explore the feasibility, aims to study the role and mechanism of induced membrane technique to repair bone defects.Objective1. To investigate the role of induced membrane on inactivation allograft. 2. To detect the structure and major growth factors in induced membrane, then compared with normal periosteum; 3. Culture the protein extracts from the membranes with mesenchymal mesenchymal stem cells, to observation stem cell proliferation and osteogenic differentiation.Methods1. 32 healthy rabbits acquired from Third Military Medical University Experimental Animal Center, average weight(2.35 ± 0.12) kg, 1.5cm bilateral radial bone defects were built, placement of polymethyl methacrylate(PMMA) bone cement, removed the bone cement after 2,4,6,8 weeks, carefully separating induced membrane, periosteum and epimysium, observation and testing at different time points as follows: ①HE staining to observe the membranes changes over time and compared with the periosteum. ② ELISA to detect the proteins extraction from the membranes, Vascular endothelial growth factor(VEGF), Angiotensin II(ANG II), Bone morphogenetic protein2(BMP2), Fibroblast growth factor2(FGF2), prostaglandin E2(PGE2) were performed, ③Proteins isolated from total cell lysates were cultured with mesenchymal stem cells to test the cell proliferation and alkaline phosphatase activity using epimysium as a control, while isolation and identification of the adherent cells.2. 1.5cm radius bone defect were built in 24 rabbits, implanted with polymethyl methacrylate(PMMA) bone cement, removed bone cement after 6 weeks, divided into three groups according to a random number table, experimental group, model group and control group were treated differently. Experimental group reservations induced membrane and implanted with inactivated autologous cancellous bone, model group reservations film, but not grafting, remove the membranes in control group and implanted with inactivation bone. New bone formation was detected by radiological and histological detection after 8 and 12 weeks.Results:1. HE staining showed that induced membranes and periosteum contained two distinct layers: the inner layer, which is rich in blood vessels, and the outer layer, which is comprised of fibroblasts and collagen. The thickness of induced membranes decreased over time, at any time point assessed, the induced membranes were thicker than the periosteum, while endochondral ossification was observed in the induced membrane.2. ELISA test results showed that BMP-2, VEGF, ANG-II and FGF-2 levels were lower in the epimysium at all time points. BMP-2 peaked in induced membranes after 6 weeks and then began to decline. The concentration of BMP-2 in induced membranes was significantly higher than the periosteum at 6 weeks(P < 0.05), but it was similar at all other time points. The VEGF concentration in induced membranes was similar to the periosteum at week 6 but was lower at the remaining points in time. FGF-2 and ANG-II peaked in the induced membranes after 4 weeks, then began to decline. FGF-2 concentration in the periosteum was always high, and the levels were similar to induced membranes after 4 weeks. ANG-II level in induced membrane was higher than in the periosteum after 2, 4 and 6 weeks. PGE2 levels in the induced membranes did not change significantly over time, and there were no significant differences between induced membranes, periosteum and epimysium(P > 0.05).3. This study demonstrated that mesenchymal stem cells in a culture medium containing total cell lysates isolated from the epimysium proliferated at a lower level as measured by absorbance at 570 nm. The ability of an induced membrane to promote mesenchymal stem cells was similar to the periosteum at weeks 2 and 8(P> 0.05). The ability of induced membranes to stimulate cell proliferation was greater than the periosteum at weeks 4 and 6(P < 0.05). Mesenchymal stem cells in osteoblasts were evaluated using alkaline phosphatase activity. The ALP activity in induced membrane was lower than in the periosteum at weeks 2 and 8(P < 0.05). The ALP activity in induced membranes at weeks 4 and 6 was similar to the periosteum. However, the stem cell proliferation generated by induced membranes was the strongest and the ALP activity in the induced membranes was the highest after 4 weeks compared to other time points. We got isolated and cultured adherent CD44 + cells showed osteogenic differentiation under inducing agent.4. Radiological detection showed that the new bone formation in experimental group better than the model group and the control group(P <0.01), the model group have a little callus formation, the control group had no osteogenesis; 8 and 12 weeks Histological showed the formation of osteoblasts and chondrocytes in experimental group, better than the model group and the control group(P <0.01), no bone cells and cartilage cells showed in control group.Conclusions1. The content of the growth factors in induced membrane reach the peak at 4-6 weeks, matured induced membrane has the similar structure and ability to promotion of mesenchymal stem cells differention into bone with periosteum. 2. Induced membrane promotion of inactivation allograft to repair bone defects by offer grows factors and cells. |
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