Reparation Of Rabbit Bone-defect By Implantation Of Marrow Stromal Cells Expressing Human Bone Morphogenetic Protein-7

In 1965, Urist made the key discovery that demineralized bone segments and extracts of demineralized bone induce bone formation when implanted subcutaneously or intramuscularly in animals. In 1990, bone morphogenetic protein-7 (BMP-7), a member of the TGF- β gene superfamily, was cloned and following investigations suggested that BMP-7 is a dimeric molecule involved in the growth, differentiation and repair of a wide variety of tissues, especially the skeletal system. Recent studies have shown that human BMP-7, generated by recombinant DNA technology, is capable of inducing new cartilage and bone formation in vitro and in vivo, and the quantity of the new bone formation is in positive correlation to the embedded.Use of BMP-7 for bone regeneration represents one of the most promising emerging therapies for bone regeneration and is a viable alternative to autologous and homologous bone grafts. BMP-7 has the unique ability to alter the differentiation pathway of mesenchymal cells toward chondrogenic and osteogenic lineages with the ultimate induction of endochondral bone at ectopic or orthotopic sites. Till now, there are some reports about the expression of BMP-7 in E.coli or in insect cells in China, but we all know that the exprssion products in E.coli have not proper activity and the insect cell products are not safe enough because of using the baculoviridae expression system. There are also some reports of recombinant human BMP-7 expression in CHO cell line in America and Europe. This expression system has the capacity for proper post-translational modifications, including proper protein folding and glycosylation. BMP-7 protein therapy, wherever recombinant or naturally sourced, which been surgically implanted in a defect, has recently been approved for limited use. Reasons for failure may be related to a lack of optimal matrixes for controlled, sustained BMP delivery at the site of implantation, short biological half-life, and inability of recombinant molecule presentation after implantation to mimic the route of administration in vivo by a BMP-producing cell. Thus, there is still clearly a need for alternative approaches to their use in bone regeneration.There are various therapeutic measures to induce the bone formation in bone-defect models including protein therapy with using BMP-7, cell therapy that some cells can differentiate into osteoblasts, and implantation with suitable carriers to hold the defected skeleton regeneration. Although each approach shows some promises in enhancing bone regeneration in animal and human studies, it is unlikely that any singe strategy will successfully regenerate bone in all situations. Several bone regeneration strategies are being development to restore congenital or acquired loss of bone structure and function. These strategies rely on three approaches: the development of bioactive factors to induce bone regeneration; the implantation of host cells capable of differentiation into the osteoblasts; and the development of implanted carriers that can support cell attachment, proliferation and differentiation, as well as deliver bioactive factors and host cells. The cooperated effect of genes, cells and carriers is required to mimic what an organism uses naturally in the growth and repair of bone and the consensus. It is at least two of these methods must be used concurrently for successful new bone regeneration. Integrating the three factors related with bone formation, BMP-7 gene therapy become the alternative route for fulfill the bone-inductive activity and realize new bone regeneration.Gene therapy was originally considered as a means of correcting hereditary disorders. However, more recent work is applying the same gene transfer technologies to situations where there is no underlying genetic defect, but, instead, a need to produce sustained amounts of a biologically active molecule. Those studies suggest that a further refinement of BMP-7 gene therapy might be the use of gene transfer technology to convert marrow stem cells at a designated localized site for bone regeneration into minireactors to produce sustained levels of BMP-7.Adenovirus is one of the most commonly vectors, widely used in gene therapy in various models. It has several characteristic making them well suited to this type of gene therapy, the most important of which is that it cannot integrate their DNA into the host genome which is particularly important for regenerative therapies because transgene expression can cease with cell proliferation or death of the originally implanted cell, limiting the period of protein production. The loss of function inimplanted cells within a few weeks of transduction may be disadvantageous in treating monogenic hereditary disorders, but may be beneficial in local treatment of skeletal defects in which only a short period of osteogenic stimulation appears to be necessary to induce skeletal regeneration. In the present study, we describe a novel approach that combines the efficiency of adenovirus-mediated gene delivery with cell-based therapy.Part I Clone the full length cDNA of BMP-7 and construct expression vectors1. Human BMP-7 full-length cDNA clone. Total RNA was extracted from the human osteosarcoma cell line U-2 OS. The full length of human BMP-7 cDNA was obtained by reverse transcript-polymerase chain reaction(RT-PCR) and cloned by pGEM-T ligation system. Sequencing analysis suggested that the sequence of the clone we obtained was coincided with the sequence reported in GeneBank.2. Construction of pll4-BMP-7 expression vector. hBMP-7 cDNA fragment was digested from pGEM-T-BMP-7 vector with Hindlll and Xbal, and ligated with the expression vector pi 14. pll4-BMP-7 was identified by restrict enzyme digestion assay.3. Recombinant Adenovirus(AdCMV-BMP-7) construction. AdCMV-BMP-7 was constructed by Cre/lox recombination using AdEasy-1? Vector System. The recombinant adenovirus AdCMV-BMP-7 and this recombinant adenovirus was transfected into 293 cells, the packed cell line, using Lipofectamine2000 method. Positive plaques were amplified in 293 cells and were purified by CsCl gradient ultracentrifugation. The titration of the AdCMV-BMP-7 was 3.6 X 1012VP/ml.Conclusion: In this part, we cloned the BMP-7 cDNA and constructed recombinant pll4-BMP-7 expession system and adenovirus system successfully.Part II Stable BMP-7-expressed CHO cell line generation and test biological activity of secreted BMP-7 in vitro test1. Generation of stable BMP-7-expressed CHO cell lines. The purifiedpll4-BMP-7 plasmids are transfected to CHO cells by Lipofectamine2000 serum-free method. The CHO cell clones were selected for expression DHFR and amplified using a stepwise protocol for resistance to 20um L-methionine sulfoximine(MSX).2. Detection of the correctly processed BMP-7 from CHO cells. Conditioned medium from transduced CHO cells was fractionated by 12% SDS-PAGE and then transferred to a PVDF membrane. Immunoreactivity was determined using the enhanced chemi luminescence method.3. Quantification of BMP-7 secreted from CHO cells. The amount of BMP-7 in supernatant was determined by enzyme linked immunosorbent assay(ELISA). BMP-7 concentration was determined by comparison to standard curve.4. Determination of BMP-7 activity in vitro. The activity of rhBMP-7 on the mouse stromal cell line W-20-17 was detected colorimetrically by measuring induced alkaline phosphatase (ALP) activity as compared to activity induced by a commercialized rhBMP-7 standard.Conclusion: In this part, we generated stable BMP-7-expressed CHO cell line, which could secret BMP-7 with the molecular weight of 32-36kDa, and the highest CHO-13-H5 BMP-7-expressed level was 15mg/L. In addition, the activity assay suggested that BMP-7 was able to stimulate the synthesis of ALP with the manner of dose-dependent.Part III Adenovirus expressing BMP-7 induced new bone formation in vivo in rabbit bone defect repair model1. Rabbit MSCs preparation. Percoll density gradient was used to isolate pure rabbit MSCs to eliminate unwanted cell types that were presented in the marrow. The 4th passage MSCs were used to be infected by AdCMV-BMP-7.2. BMP-7 expression level and activity in vitro determination. The BMP-7 expression level of AdCMV-BMP-7 infected MSCs was detected by western-blot and ELISA. The effects on MSCs by AdCMV-BMP-7 infection was examined by cell morphologic and cell growth rate comparison. The activity of secreted BMP-7 expressed in infected MSCs supernatant was determined colorimetrically bymeasuring induced ALP. Results showed that AdCMV-BMP-7 could not stimulate MSCs proliferation significantly and the secreted BMP-7 from infected MSCs was quantified as 0.5mg per 105 MSCs. MSCs could synthesize ALP which suggested MSCs were differentiated into osteoblasts after activated by AdCMV-BMP-7.3. Collagen biocompatibility test. An ideal delivery system would allow a slow release of the BMP-7, be biologically and immunoligically inert, quickly absorbed, and supportive of cell proliferation. In sterile condition, cut the collagen sponge into small pieces, then put them in 12-plate followed by supernatant MSCs were added. Results showed that collagen sponge has a good biocompatibility to be suitable for MSCs to grow in.4. AdCMV-BMP-7 induced new bone formation in vivo. In this part, we describe a novel approach that combines the efficiency of adenovirus-mediated gene delivery with cell-based therapy. At a word, the whole process is composed with four steps: first, prepare rabbit marrow stromal cells(MSCs), culture and expand it in vitro; second, construct the recombinant adenovirus expressing BMP-7(AdCMV-BMP-7) and then infect the expanded MSCs; third, search a suitable implanted carrier that can provide a three-dimentional space for MSCs to grow in and proliferate; forth, make a critical-defect model to test whether the ex vivo gene therapy method is effective. Control implants contained the same titer of control adenovirus.A unilateral ten-millimeter segmental defect was created in the radio-diaphysis in 48 skeletally mature New Zealand White rabbits. Bone formation was evaluated with radiograph at two-week intervals. The rabbits were randomly divided into five groups with different treatment respectively. In Group I, only simple lengthening without any treatments were performed; in Group II, implanted carrier was placed in the defect site; in group III, IV, and V, the defect was filled with implanted carrier with MSCs, implanted carrier with adenovirus control vector or AdCMV-BMP-7 respectively.The effect of AdCMV-BMP-7 on the healing of the segmental defect was clearly demonstrated by comparison of radiographs, made at two-week intervals from the day...