| BackgroundA challenge for orthopedic surgeons remains with respect to the repair of large bone defects caused by trauma, tumors or severe inflammation. Patients with large bone defects never heal spontaneously and the damaged sites have limited potential to totally recover. Autogenously osteochondral grafts and allografts are the most commonly procedures. However, these procedures carry certain problems. The former procedure is limited with the amount of available, bone donor-site morbidity and increased operation time. While the latter is limited in usage owing to immunological rejection, possible transmission of pathogens and bone nonunion. Currently, efforts to provide engineered tissue as an alternative to the traditional treatment that can meet the clinic needs are being pursued.Typically, tissue-engineered bone possesses three essential components:a) a cell source, b) appropriate osteoinductive factors, and c) an appropriately designed scaffold. Bone marrow-derived mesenchymal stem cells (MSCs) are ideal progenitor cells and have been widely used for bone tissue engineering. They are easily obtained and potentially can be used to develop bone, muscle, cartilage, adipose or fibrous tissue. In many reports, the ultimate fate of undifferentiated stem cells is determined by the local environment that it is located. Exogenous bone morphogenetic proteins (BMPs) are known to stimulate osteoblastic differentiation. Among the BMP subtypes, BMP-2, in particular, possesses high bone induction activity and stimulatory effects on MSCs to differentiate into osteoblasts. To maximize the osteogenic capacity of MSCs, the application of exogenous BMP-2 has frequently been used to promote the osteogenic differentiation of MSCs and in vitro BMP-2 gene therapy. For example, autologous MSCs, genetically modified to express BMP-2 via in vitro, enhance bone formation.To construction of a genetic tissue-engineered product, the selection of an appreciate scaffold is critical. Scaffolds, such as, poly (L-lysine) coated polylactide (PLA), poly (DL-lactic-co-glycolic) acid (PLGA), alginates andβ-TCP, have been used. It has been reported that a 3-D porous polymeric matrix could enhance bone regeneration and facilitates progenitor cell adhesion, proliferation and differentiation. Nano-hydroxyapatite/collagen/poly (L-lactic acid) (nHAC/PLA), as a biodegradable 3-D porous scaffold, appears to be a good candidate as a carrier for gene-modified cells. The nHAC/PLA developed by biomimetic synthesis exhibits features of natural bone both in the main composition and hierarchical microstructure.In this study, we transferred the BMP-2 gene into MSCs by electroporation. These gene-modified MSCs were then seeded onto nHAC/PLA scaffolds to develop cell/scaffold construct. The in vitro attachment and the growth of BMP-2 transfected cells in the scaffold were investigated. In vivo, we observed the repair of segmental bone defects after the implantation of cell/scaffold construct.Objective1. To investigate the isolation and culture of bone marrow mesechymal stem cell (MSCs), and explore their proliferation ability and biological characteristics. To demonstrate the multilineage differentiation potentials of MSCs and explore their capability of osteogenic differentiation and adipogenic differentiation. To valuate the feasibility of using MSCs as a cell source for bone engineering.2. To investigate the infection rate of BMP-2 gene transfection. To examine the expression of BMP-2 and its effect on osteogenic differentiation of MSCs.3. To investigate the in vitro attachment, growth, proliferation and differentiation of BMP-2 transfected MSCs in the nHAC/PLA scaffold. To evaluate the compatibility of nHAC/PLA scaffold with BMP-2 transfected MSCs.4. To investigate the repair of rabbit radius segmental bone defects (15 mm in length) after the implantation of BMP-2 transfected-MSCs/nHAC/PLA constructs.Methods1. MSCs were derived from 3-month-old New Zealand White rabbits. Briefly, bone marrow was obtained by aspiration from the tibias and femur. Then the MSCs were isolated by density gradient centrifugation and expanded in vitro. There biological characteristics were observed under inverted phase contrast microscope. The osteogenic medium was used to induce MSCs to osteogenic differentiation, and the osteogenic phenotype was examined by means of the alkaline phosphatase (ALP) staining and alizarin red staining. The adipogenic medium was used to induce MSCs to adipogenic differentiation, and oil-red O staining was used to verify the formation of neutral lipid vacuoles.2. The second-passage MSCs were transfected with pIRES2-EGFP-hBMP-2 by electroporation. Then the transfer efficiency was determined through the expression of EGFP. And RT-PCR and Western blot analysis were used to assess the expression of BMP-2 at mRN A and protein level, respectively. ALP activity assay was used to analyze the osteogenic differentiation of MSCs.3. BMP-2 transfected or mock transfected MSCs were seeded on nHAC/PLA scaffolds to generate a 3-D cell/scaffold constructs. SEM and MTT were used to analyze the adhesion and proliferation of MSCs cultured in the scaffolds. Quantitative measurement of ALP activity was used to analyze the osteogenic differentiation of MSCs cultured in the scaffolds.4. The cell/scaffold constructs obtained were allografted into the 15 mm critical-sized segmental bone defects in the radius of New Zealand White rabbits for 12 weeks. The rabbits were divided into four groups.Group A for the implantation of nHAC/PLA scaffolds combined with BMP-2 transfected MSCs, Group B for the implantation of nHAC/PLA scaffolds combined with mock-transfected MSCs, Group C for just nHAC/PLA scaffolds, Group D for blank control. The bone regeneration was assessed by radiographic and histological analyses at 8 weeks and 12 weeks post-operation.Results1. Primary MSCs demonstrated fibroblast colony-forming units by phase contrast microscopy. The passaged MSCs proliferated well and demonstrated two morphologically distinct cell types:spindle-shaped cells and large flat cells. However, most cells were spindle-shaped. After cultured in osteogenic medium for 2 weeks, the ALP expression of MSCs was detected by ALP staining, and the formation of mineral extracellular matrix was detected by alizarin red staining. Respectively, after cultured in adipogenic medium for 3 weeks, Oil Red O staining showed that MSC were positive for staining neutral lipid vacuoles.2. The quantification of BMP-2 gene transfer efficiency in the MSCs was determined by assessing the number of EGFP expressing cells under inverted fluorescent microscope. A transfer efficiency of 35.5±3.8% was achieved. RT-PCR and Western blot analysis confirmed the expression of the BMP-2. ALP activity assay indicated that BMP-2 gene therapy promoted the osteogenic differentiation of MSCs.3. By SEM observation, nHAC/PLA scaffold had a three-dimensionally porous structure with porosity no less than 75%. Two hours after cells seeding, the BMP-2 transfected MSCs started to attach and spread on the scaffolds. After 1 day, nearly all cells had spread on the scaffold, the BMP-2 transfected MSCs exhibited a highly flattened shape and attached to the pore wall of the scaffold with their pseudopodia with partially bridging over some pores. With prolonged periods in culture, much more cells were observed on the nHAC/PLA scaffolds. ALP activity assay indicated that the ALP expression of BMP-2 transfected MSCs was markedly higher than the control after 3 and 7 days of cell seeding.4. In vivo findings:Results of radiographic examination:Eight weeks after operation, a lot of bony callus filled in the defects of group A. In contrast, the rabbits in group B had only a cloudy mineralization in the defect site without reconnection between both ends of the radius segments. At 12 weeks post-operation, the defect site was nearly restored in all defects of group A, at least 90% of the defect site was filled with cancellous ossification and the medullary cavity partly recanalized. For group B, the bone mineral density in the defects area was much lower than that of group A, and the fracture line can also be observed. However, the result of group B at 12 weeks is obviously much better than that of group C. As for group C, the defects reconstructed with just scaffold appeared radiolucent in most part of the defect at 12 weeks. Moreover, no radiographic evidence of bone formation was presented in group D 12 weeks after operation. According to the Lane-Sandhu X-ray criteria, Lane-sandhu scores of group A were much higher than group B both at 8 weeks and 12 weeks post-operation (p<0.05).Results of histological examination:eight weeks after operation, histological examination of group A showed lots of trabecular bone formation in the pores of nHAC/PLA scaffolds, and lots of new bone which surrounds the trabecular bone. In contrast, group B showed lots of loose fibrous connective tissue in the pore space and a small amount of new bone generated in few pores of the nHAC/PLA scaffolds. At 12 weeks post-operation, group A showed that a discrete amount of highly cellular bone marrow-like tissue was filled the space among newly formed trabecular bone, and we also observed that the residual scaffold was surrounded by marrow-like tissue in the medullary cavity. While in group B, more trabecular bone was observed with the implantation prolonged, and we also observed lots of new bone which surrounds the trabecular bone. For group C, connective tissues and inflammatory infiltration were present in superficial as well as inner pores and no new bone formation was observed both at 8 and 12 weeks post-operation. In addition, the percentage of newly formed bone of group A was much higher than group B (p<0.05).Conclusion1. Bone marrow mesenchymal stem cells (MSCs) are easy to harvest, culture and expand in vitro. MSCs had the potentials of multilineage differentiation and could be induced to osteoblasts and adiposities under conditioned culture medium. It is an ideal choice to use MSCs as bone engineering seeding cells.2. MSCs can be successfully transfected by BMP-2 gene. Transfected efficiently by BMP-2, the MSCs express BMP-2 and showed stronger capability of osteogenesis. The in vitro study demonstrated the potentiality of BMP-2 gene therapy with MSCs in the future clinical applications.3. Novel nHAC/PLA scaffold showed multi-porous structures and good biocompatibility with the BMP-2 transfected MSCs in vitro. nHAC/PLA can be used as scaffolds in the gene bone tissue engineering.4. Tissue-engineered bone with BMP-2 transfected MSCs seeded on nHAC/PLA scaffolds accelerated bone defects healing. The mechanism as follow:BMP-2 was secreted by the BMP-2 transfected MSCs, and the BMP-2 induce osteogenic differentiation of both implanted MSCs and host MSCs via autocrine and paracrine effects. Then the osteoblasts can also express BMPs and then accelerate the process of bone defect repair.
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