The Experimental Study Of The Plasmid As A Vector Of RhBMP Gene Transfer Therapy Stimulating Reparative Dendtinogenesis

Objective: In order to augment odontoblast-like cell's efficacy in inducing reparative dentinogenesis in, this study was based on the new development of bone morphogenetic protein(BMP) in stimuting reparative dentin and gene transfer, we have cloned BMP cDNA to plasmid and put it on the exposed sites of dog's tooth pulps, then observing its efficacy in stimulating odontoblast-like cell's activitor ,speeding-up reparative dentinogenesis and sealing the aperture of exposed pulps in dogs. Then we evaluated the clinical feasibility of cDNA plasmid in order to explore a new therapeutic method for maintain the dental pulp's function.It is important for the Dentine- Pulp Complex (DPC) to maintain the aperture of dental pulp' function. The dental pulp tissue contains tissue stem cells, which retain the potential to replicate and differentiate into dentinforming odontoblasts (Nakashima et al., 1994a; Gronthos et al., 2000). Damaged odontoblasts can be replaced by newly generated populations of odontoblasts derived from residual cycling stem cells from pulp (Tziafas et al., 2000). After physiologic stimulation or injury such as caries and operative procedure, pulp stem cells may be mobilized to proliferate and differentiate into odontoblasts by morphogens secreted from surrounding dentin matrix. In addition, during minor insults, odontoblasts differentiate for secondary dentin (Tziafas et al.,2000).Bone morphogenetic proteins (BMPs) are morphogens implicated in embryonicand regenerative odontogenic differentiation. A single recombinant bone morphogenetic protein (BMP) can act on multiple steps such as chemotaxis, mitosis, differentiation, and apoptosis of cells, which is referred to as pleiotropy (Reddi, 1998). BMP also can play pivotal roles in the development of many organs and tissues including bones and teeth as well as in the establishment of the embryonic body plan (Reddi, 1998). BMP2 is a member of the BMP family, and is expressed in terminally differentiated odontoblasts (Nakashima et al., 1999). Recombinant human BMP2 induced the expression of dentin sialoprotein (Dsp), a differentiation marker for odontoblasts, in mouse dental papilla mesenchyma in organ culture, suggesting that BMP2 might stimulate differentiation of the pulp stem cells into odontoblasts in vitro. Dental pulp capping to stimulate odontoblast differentiation and reparative dentin formation is an important target for developing gene therapy strategies.Therapeutic reparative dentin formation can be achieved with BMP proteins (Rutherford et al., 1993, 1994, 1995; Rutherford, 2000; Nakashima, 1994b,c). During the conventional topical application of BMPs to induce reparative dentin formation, the half-life of the morphogen is limiting and high concentrations are required together with an optimal carrier (Nakashima, 1994b). An alternative approach using gene transfer may therefore have the advantage of transferring into specific cells with specific promoter and appropriate vectors to attain a sustained gene expression and more efficient way of delivering in vivo. For therapeutic genes such as BMPs to be effective, they must reach the pulp tissue stem cell nucleus. Gene expression and production of BMPs are obligatory. Both viral and nonviral methods are used. Recently successful gene transfers in mesenchymal cells with a recombinant adenoviral vector have been reported using BMP2 (Cheng et al., 2001, Lee et al., 2001, Olmsted et al., 2001), BMP7 (Franceschi et al., 2000, Krebsbach et al., 2000), or BMP9 (Varady et al., 2001); the gene-transferred cells can produce BMPs, differentiate into an osteoprogenitor line and induce bone formation both in vitro and in vivo. Using viruses to deliver genes, high levels of gene expression can be achieved. Immune response to viruses and a concern about insertional mutagenesis, however, can limit the effectiveness of gene therapy with a viral vector (Marshall, 1999; Somiaand Verma, 2000). The use of the nonviral methods has many advantages, although the efficiency of gene delivery is relatively low. Manipulation is easy and large inserted sequences can be delivered. The cDNA plasmids can be produced stably and inexpensively with a high level of purity with minimal risk of replication or incorporation. They are also weakly immunogenic and can be administered repeatedly to immunocompetent subjects (Ng and Liu, 2002). When considering an ideal pulp-capping gene therapy, it would be desirable to modify nonviral plasmid DNA transfection. In this study, we demonstrated that the BMP2 cDNA plasmid was transferred efficiently into pulp stem cells and induced the expression of BMPs, stimulating the differentiation of odontoblasts. As a pulp capping material it induce dental bridge much faster and much better than calcium hydroxid . Therefore, in this investigation, we have optimized techniques of gene transfer of BMP-2 to induce differentiation of pulp stem cells into odontoblasts and reparative dentin formation for the treatment of pulp tissue regeneration and dentin repair. Gene therapy has the potential to induce reparative dentin formation for potential pulp capping. We have optimized the gene transfer of recombined human bone morphogenetic protein(rhBMP) BMP2 plasmid DNA into dental pulp.Methods: In this study two group pf dogs were compared. One group was treated with rhBMP-2 gene (cDNA plasmid) as direct pulp capping, in addition, another group was treated with calcium hydroxid. histopathological and immunohistochemistry were performed 1 and 4 weeks after the operation. Resultant: Dental pulp tissue treated with cDNA plasmid showed significant efficiency of gene transfer and high level of protein production selectively in the local region, within exposed site of the pulp. The rhBMP cDNA plasmid implanted into dental pulp tissue, induced the expression of BMPs, rhBMP-2 gene (cDNA plasmid) as pulp capping can induce dental bridge much faster and much better than calcium hydroxid.Conclusion: These results suggest the possible use of rhBMP cDNA plasmid using as direct pulp capping material in gene therapy for endodontic dental treatment.