Fabrication And Evaluation Of PAMAM Dendrimers Complexed RhBMP-2Gene Activated Matrices For Bone Regeneration

Purpose:Currently, tissue engineering has been widely studied to enhance bone regeneration. Although promising. results of inducing tissue regenerations have been presented by loading grow factors into materials, problems are still exist, which includes hard to control the release style, easy to deactivate and the high cost. The development of gene therapy provides opportunities to solve these problems. Coating transfectable gene vectors into the materials to build gene activated matrices has been considered a new strategy in regenerative medicine. As the matrices degrede, the vectors will be delivered to transfect cells and express the encoded growth factors. In the present study, G5dPAMAM/plasmid complexes were prepared, and subsequently loaded into the nano-β-TCP/type I collagen composite scaffolds to form newly builed GAMs. We examined the biocompabilitis, transfection effencies and the osteoinduction of stem cells. Morever, we applied these GAMs directly into freshly created segmental bone defects in rat femurs, and evaluated their osteogenic effencis. Through these researches, we wish to provide the applications of GAMs in treating bone defects the experimental basis in order to achieve the optimum curative result.Methods:1. The rMSCs were isolated from femoral and tibia bone marrow of adult male Sprague Dawley (SD) rats of<120g at5-7weeks. Cells at the third passage were subcultured in osteogenic medium or adipogenic medium for about3weeks. Osteogenic and adipogenic differentiation of rMSCs (passage4) were confirmed by alizarin red S or Oil red staining. Expressions of CD29, CD90, CD14, CD31, CD34and CD45on the cells were detected by flow cytometer.2. G5dPAMAM and plasmid hBMP-2were mixed at the weight ratio of4:1to form complexes in serum-free medium at4℃to prepare G5dPAMAM/plasmid complexes. Effects on the proliferations of rMSCs and COS-7cells were respectively examined by MTT assay. Transfection concentrations of non-cytotoxicity were discussed. Furthermore, in vitro transfections to COS-7cells were performed, and the results were observed by fluorescence microscope.3. Type I collagen and nano β-TCP powders were mixed in aqueous alkali solution at room temperature. The mixture was lyophilized to prepare porous nano β-TCP/Type I collagen scaffolds. G5dPAMAM/plasmid complexes were then loaded into the scaffolds to form GAMs. Quantity-related effects on cell proliferations of dPAMAM/plasmid complexes were examined by MTT assay to select the most proper loading quantity to the scaffolds. Porosities of GAMs and non gene loaded scaffolds were detected. Microstructures were observed by SEM, and the release style of plasmid was measured by Picogreen tests. After cocultured with rMSCs for5days, the growth and distributions of cells in the GAMs were observed by confocal laser scanning microscope.4. GAMs were fabricated following the method of step3. After being cocultrued with rMSCs for3days, the GAMs were removed. The nuclei of the MSCs were stained DAPI. The expressions of GFP and the staining of DAPI were observed by fluorescence microscope to calculate the transfection effencies. Media from the GAM-cell, pure scaffold-cell or blank groups were collected for rhBMP-2ELISA and ALP detection. The possible osteogenic applications of newly built GAMs in serum containing environment will be discussed.5. A total of45male SD rats were divided into three groups, each of which was prepared a5-mm-long segmental defect in the right mid femur under general anesthesia. GAM or B-TCP/type I collagen scaffold was implanted into the defect sites, and fixed at both ends using intramedullary stainless steel needles. For the control group, an intramedullary needle was fixed without any implant. After12weeks'follow-up, the survival rate and healing of the wounds were surveyed. The femoral bone defects were radiographically examined by X-rays. Serum hBMP-2concentration and ALP activity were assessed by ELISA. After the animals were sacrificed, H&E staining and immunohistochemistry was performed to detect the expressions of rhBMP-2in the defect sites.Result:1. The extracted cells appeared classical adherent cells. After subcultured in osteogenic medium or adipogenic medium for2to3weeks, osteogenic and adipogenic differentiations of the cells were confirmed by alizarin red staining and Sudan III staining respectively. Flow cytometry assays showed high expressions of CD90and CD29on the cells, but low expressions of CD14,CD31,CD34and CD45.2,G5dPAMAM/plasmid complexes showed superior biocompatibility. The complexes could transfect COS-7cells efficiently. High concentrations of the complexes showed negative effects on the proliferation of COS-7cells. The transfection effects could proceed in serum, and showed time related accumulation.3. The newly formed GAMs possessed high porosity. Delivery of plasmid complexes maintained for2weeks, with the amount of plasmid topped on the6th day. MTT showed superior biocompatibility of the GAMs. The rMSCs could proliferate rapidly in the GAMs and infiltrate into different depths of the GAMs.4. The plasmid complexes released from GAMs transfected the rMSCs efficiently, and the expression of rhBMP-2could be detected. After cultured for25days, GAMs group exhibited higher ALP activity than other groups, which suggests the enhancement of the osteogenic differentiation of the rMSCs in GAMs. 5. All the45rats survived with nomal healings of the wounds. As is showed by X-ray, the defect areas in GAM group were filled totally by newly formed bone in12weeks, whereas the connection in the defects still appeared to lack union in other groups. Serum ELISA showed significant expression of hBMP-2in serum from GAM group individuals at2weeks after implantation. In vivo ALP activities are was enhanced in GAM group compared with other group after the surgical procedure. HE staining showed the fastest osteogenesis procedure in GAM group animals, and the immunohistochemistry indicated the expressions of rhBMP-2in newly formed bone tissue in the GAM group during the first4weeks.Conclusions:1. The rMSCs extracted from the rat tibia and femurs possessed multilineage differentiation potentials. The cells could be induced to osteocytes. The flow cytometry confirmed a high purity of the rMSCs. The rMSCs could be used as seed cell in bone tissue engineering researches.2. G5dPAMAM/plasmid complexes were suitable for gene therapy for their superior biocompatibility and in vitro transfection efficiency. The complexes can transfect cells in serum containing environment.3. The nano β-TCP/Type I collagen scaffolds and newly prepared GAMs highly porous matrices. Both of them were suitable for the growth of rMSCs. The GAMs delivered plasmid complexes in sustained way.\4. The GAMs could release rhBMP-2plasmid complexes and cause transfections to rMSCs. The transfected cells expressed and secreted rhBMP-2, which induced osteogenic differentiations of rMSCs.5. The implantation of GAMs enhanced the bone augment in the gaps. The effective expressions of rhBMP-2lasted during the first4weeks postoperatively. This gene delivery system shows promising potentials in clinical application.