| Bone defects and deformity are common diseases in clinical dentofacial surgery and orthopaedics.The reconstruction of mandibular defect is very difficult in the field of dentofacial surgery. There are many strategies to fill the bone defects with advantages and disadvantages respectively.The biological artificial bone fabricated with seed cells, scaffolds and growth factors according to tissue engineering methods have been thought to be one of the most effective techniques which could be used for the treatment of bone defects and bone remodeling in the future. Recently, the sources of seed cells for tissue engineering include bone, periosteum, marrow and ectosteal tissues with merits and shortcomings respectively. Increasing attention has been paid to the seed cells derived from bone marrow. It has been proved that theses bone marrow stromal cells have the advantages, such as widespread sources, easily isolation, stably growing, rapid multiplication, favourable directional differentiation, easy survival after subculturing and the ability to transform into osteoplasts in some conditions. Moreover, the target genes can be easiliy transfected into these cells. Therefore, these cells can be used as the target cells for gene therapy to repair the bone defect. In this study, we choose bone marrow stromal cells as the seed cells to deliver bFGF gene. Tissue engineering material includesceramics, high molecule polymers and bio-derivative material. Many studies indicate that Bio-oss is an allergen-free natural bone substitute material obtained from ox bones whose organic composition has been removed to form anorganic porous material. Physico-chemical properties of Bio-oss are very similar to that of human bones because of its natural structures with the character of wonderful biocompatibility. The anorganic composition of Bio-oss scaffolds is similar to that of the tissues of hunman bones, whose ventage structures, natural ingredient and bone trabecula structure constitute the osteoconductivity frame for in-growth of osteoblasts. At the same time, the regeneration of new vessles can be ensured and the osteoblasts will be promoted to form new bone faster. Compared with other bone substitute materials, Bio-Oss can fuse with the bone tissues around earlier. Bio-oss is a kind of carbonate structure with characters of porosity and large inner surface area, which provides ideal frame structures for osteoblasts to grow. There are less hydroxide radicals and more carbonate in Bio-oss whose crystals are similar to that of human bones, therefore material absorbtion can be ensured without a second operation. In a word, Bio-oss has upstanding osteoconductivity and ability of bone formation, whose mechanical intensity and hardness are similar to that of bone, so Bio -oss was choosed as the scaffold material in this study. The basic fibroblast growth factor (bFGF) is a broad spectrum mitogen and has the property to promote mesoderm and ectoderm derived cell growth, whose target cells include fibroblasts, vascular endothelial cells, chondrocytes and osteoblasts et al. The main biological functions of bFGF are stimulating angiogenesis, promoting repair process of wounded soft tissues, cartilages, bones and nerve tissues. Previously, tissue engineering research of artificial bone was focused on the application of scaffold and growth factor individually. But, the results were not satisfaction. At present, the study direction gradually turns to combinated materials constructed with scaffold, growth factor and seed cells. This kind of combinated materials possess the advantages that pure anorganic materials, high polymer organic materials and natural biological materials have not, so it could bewidely applied in the future. However, it is hard for topically applied bFGF to produce market and lasting effects because it will be diluted and metabolized quickly, and there hasn't been ideal local slow-release systerm now. Repeated injection is expensive and can cause toxicity or immune reaction. In this study, in order to keep and prolong the bioactivity of the growth factor, gene transfection method was adopted to deliver the bFGF gene into BMSCs to obtain stable expression of the transfected gene. Research on reparing mandibular defect with natural Bio-oss graft combined with BMSCs modified bFGF gene approaches clinic, and the feasibility and necessary were discussed in cellular and molecular levels for clinical application.Objective: To evaluate the effects of bFGF on proliferation and differentiation bone mesenchymal stem cells (BMSCs); to construct the eukaryotic expression vector encoding human bFGF gene; to transfect recombinant pVAXl-bFGF into BMSCs by lipofectamineto identify the expressed product with indirect immunofluorescence to confirm whether the transfection was successful; to observe the structure of multi-pore Bio-Oss ossein bone and the growth of BMSCs on Bio-Oss ossein scaffolds by scanning electron microscope; to observe the repair and bone formation effect of rabbit BMSCs cultured in vitro on maximum mandibular defects,which were transfected by bFGF gene and then combined with Bio-oss ossein scaffolds; to observe synosteosis effects of bone, implant and tissue engineering bone on repaired maximum mandibular defects of dogs with BMSCs transfected by bFGF gene combined with implant +Bio-oss scaffolds.Methods: (1) Rabbit BMSCs were isolated in separating medium of lymph cells, amplified, and then induced to differentiate. In treatment group, bFGF was added in both culture and mineralization medium. MTT colorimetric assay was used to draw cell growth curve. (2) Total RNA from bone marrow stromal cell (BMSC) was extracted to perform RT-PCR amplification to acquire human bFGF cDNA. After the sequence was confirmed by sequencing analysis, eukaryotic expression vectors were constructed.Recombinant plasmids were transfected into BMSCs through lipofectamine and the expression product was detected by indirect immunofluorescence. (3) Scanning electron microscope was used to evaluate the structure of multi-pore mineralized Bio-Oss bone, Bio-Oss ossein bone, the growth, adhesion and stomal secretion of BMSCs on the surface of Bio-Oss ossein in vitro culture. (4) Liposome transfectiom technique was applied to transfect the recombinant plasmids pVAXl-bFGF into BMACs of rabbits. After transfection, these cells were induced with mineralizatation medium, mixed with gelatum and Bio-Oss ossein, then were applied to repair ultimate defects of mandibles. Macroscopy, radioactive ray, bone density measurement and histology techniques were used to evaluate the plerosis of bone defect at 8 weeks and 12 weeks after operation. (5) Implant + dog BMSC with bFGF gene transfection + gelatum + Bio-Oss ossein scaffords were used to repair ultimate defects of dog mandibles. Macroscopy, radioactive ray, bone density measurement and histology techniques were used to observe the plerosis of bone defect at 24 weeks after operation.Results: (l)As shown from MTT colorimetric assay, the proliferation of BMSCs in exogenous bFGF group was more active than that of BMSC group. (2)Human bFGF gene has been successfully transfected into eukaryotic expression vector pVAXl, whose nucleotide sequences encoding bFGF of pVAXl-bFGF were identical to that published in NCBI GenBank: its sequences were identical to 3 bFGF encoding nucleotide sequences and highly homologous to the bFGF encoding nucleotide sequences of Ml 7599.1 except one different basic radical. The indirect immunofluorescence result showed green fluorescence on the membrane of transfected cells. (3)The porosity of the multi-pore mineralized Bio-Oss bone is 65% and aperture is 700 urn . The pore is equably round or ellipse and stereo lattice spatial structure was formed. BMSCs were observed to be extense adequately and fibroblast-like adhering to the surface of Bio-Oss ossein. It had an overlapped growth after BMSCs+Bio-Oss ossein compounds were cultured for 7d. Bio-Oss ossein pores were filled by some cells andextracellular matrix. Many petty calcium nodules and collagen fibers adhered on the scaffolds.(4)8 weeks after the repair on rabbit maximum mandibular rdefect: In the groups simply treated with the Bio-Oss ossein without cells transplantation, newly formed osteoid tissues were found around the unabsorbed scaffolds with in-growth connective tissues and desmoplasia; In the groups of BMSC+gelatum+Bio-Oss ossein, large amount of aggregated and differentiated mesenchymals, immature woven bone and absorbed scaffolds were found; In the groups of BMSC transfected by bFGF gene+gelatum+Bio-Oss ossein, there is large amount of newly formed bone around the scaffolds and active osteoblasts which can be found on the surface of new bone.Mature bone tissues and new born capillaries appeared in some regions, and macrophages can be found around the materials. 12 weeks after the operation: In the groups simply treated with the Bio-Oss ossein, the defects were mainly filled with fibrous tissue, the peripheralbone formation is distinct and there was a small amounts of porosis in the center; In the groups with the mixture of BMSC, gelatum and Bio-Oss ossein, the defects were basically repaired with new born bone tissue; in the group with BMSC, gelatum,Bio-Oss ossein together with bFGF gene transfection, the defects were completely reconstructed with relatively matured new born gone tissue; (5) Treating maximum mandibular defects of dogs by implant combined with BMSCs tranfected by dFGF: In the groups with implant + BMSCs+ gelatum+ Bio-Oss ossein,the defects were basically repaired with new born bone tissue .Bone tissue density had a little nonuniform and bone conjunction formed basically between implant and surrounding bone tissue; In the groups with implant + BMSC with bFGF gene transfection + gelatum + Bio-Oss ossein scaffords, the defects were basically repaired with matured new bone tissue having a good moulding which had no distinct edge with surrounding bone tissue and integrating with implant bone companied by bone trabecula forming.Conclusions : (l)Proper dose of bFGF can induce the proliferation and promote directional differentiation of BMSCs.
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