An Experimental Study Of Allogenous Periosteal-derived Cells Transfected With Adenovirus-mediated BMP-2Repairing The Critical-sized Defects Of Mandible In Rabbits

Objective:1. The purpose of the first part of this thesis was to investigate the biological properties and characteristics of two different mesenchymal cells cultured in vitro, periosteum cells derived from lateral periosteum of rabbits’mandibular body and bone marrow stromal cells (BMSCs) from the femoral marrow of the same animal.2. The purpose of the second part was to investigate the effects of adenovirus-mediated bone morphogenetic protein-2(Ad-BMP-2) on the proliferation and differentiation of periosteum cells and BMSCs, and to choose suitable target cells for gene therapy.3. The purpose of the third part was to observe the effect of Ad-BMP-2on the ectopic bone formation around the lateral periosteum of mandible in rabbits.4. The purpose of this study was to investigate the biocompatibility of the bioactive glass ceramics (BGC) combined with cultured periosteum cells in vitro, and to select a favorable scaffold for bone tissue engineering.5. In the present study, we used the periosteum cells expressing BMP-2gene as the seeded cells, and porous bioactive glass ceramics as cell scaffolds to repair bone defects. The purpose of this study was to investigate the ability of gene modified tissue-engineered bone in repairing mandibular critical-sized defects in rabbits, and to have a preliminary comprehension on the mechanism of bone regeneration by the approach of tissue engineering and gene therapy. Methods:1. Periosteum cells were isolated by the methodology of enzyme digestion, while BMSCs were acquired by another technique of density-gradient centrifugation. Both cells were cultured and subcultured in vitro in Dulbecco’s Modified Eagle’s Medium (DMEM), including10%fetal bovine serum(FBS).The growth traits and biological characteristics of cultured cells were respectively examined by morphological observation、measurement of proliferation ability and colony-forming efficiency(CFE)、assaying the activity of alkaline phosphatase (ALP) and the ability of mineralization in vitro.Cultured cells of each rabbit were injected into the back muscle packs of the same animal in order to observe the cells’ability of ectopic bone forming in vivo.2. The replication-defective adenoviral vector encoding BMP-2was constructed and amplified by in vivo homologous recombination in293cells, and then the concentration of purified Ad-BMP-2was titered in293cells by serial dilution and plaque-formation assay.The transfection efficiency was evaluated by in situ hybridization for BMP-2mRNA. The expression of BMP-2protein in transfected cells was detected by the method of enzyme-linked immunosorbent assay (ELISA). The proliferation ability of transfected cells was tested by MTT method and the activity of alkaline phosphatase was assayed by enzymology. Statistical analysis was used to compare the difference between transfected cells and uninfected cells.3. Eighteen New Zealand White rabbits were randomly divided into three groups,and each group had six animals.About2×109pfu of Ad-BMP-2vectors (0.2milliliter lysate)were injected into the site around the lateral periosteum of mandible on the right side of each rabbit, and the left side had the same volume of phosphate buffered saline(PBS) injected into the periosteum to serve as the control group. Six animals were sacrificed at2、5and10weeks postsurgery. At sacrifice, the new tissues along with adjacent native bone and muscle were harvested and immediately fixed in4%paraformaldehyde for24hours. All the samples were then respectively examined by X-ray and histological chemistry observation to investigate the temporal and spatial characteristics of new bone formation.4. Periosteum cells isolated from the mandible of a young adult rabbit were cultured with the supernate of BGC, and at different time points cell proliferation ability and alkaline phosphatase activity were examined respectively by MTT method and enzyme assay. The morphological characters of cells seeded into the porous BGC were observed by scanning electronic microscope (SEM).5. Eighteen New Zealand White rabbits were randomly divided into two groups, and each group had nine animals. The critical-sized defects (about10mm×6mm)were made on both sides of each rabbit’s mandible, and bone defects were respectively implanted with group (A)Ad-BMP-2gene modified tissue-engineered bone,(B)unmodified tissue-engineered bone,(C)single bioactive glass ceramics,(D)defects were left without any implantation as the control group. Six animals were respectively sacrificed at4,8and12weeks after operation. All specimens were obtained by surgical process and examined by X-ray and histochemical staining. New bone formation in each grafted defects was evaluated by statistical analysis to determine if there was significant difference between different groups.Results:1. Both periosteum cells and BMSCs cultured in vitro had the similar figures as the fibroblast-like cells. Periosteum cells had higher proliferation ability than BMSCs, while had lower CFE than the later. Histochemical staining and enzymology assay showed periosteum cells expressed higher (3～4times) activities of ALP than BMSCs. Cultured in induced DMEM (including10%FBS,10-8mol/l dexamethasone,10mmol/L B-glycerophosphate,50mg/L Vitamin C) for21days, both cells could form mineralized nodules which were stained positively with the method of red alizarin stain. Histological analysis of cells’autotransplantation into the back muscles of each rabbit indicated that new ossicles were formed in both induced cells,while non-induced cells could not form mineralized tissue.2. The viral lysate of the purified vectors was about5.5×1010plaque-forming units per milliliter (pfu/ml). The transfection efficiency of these two cells was positively correlative to the concentration of adenovirus vectors, and higher transfection efficiency (>95%) could be obtained when the multiplicity of infection (MOI) was over100. The BMP-2protein expression in the supernate of cultured infected cells was detected from the first day after Ad-BMP-2transfection, and the secretion activity of both cells kept higher levels for at least2weeks. Ad-BMP-2had no obvious negative effect on the proliferation activity of these two cells.The activity of ALP in cells was dependent on adenovirus BMP-2dose and directly related to the number of BMP genes transferred. The expression of ALP in transfected cells at MOI of100was significantly higher than those of uninfected cells, and gene transfection in BMSCs had higher positive effect on cell ALP activity than in periosteum cells.3. In vivo local delivery of Ad-BMP-2could result in a certain degree of immunological reflection, which was decreased with time went on. At two weeks,many native cells around the injection site began to proliferate and differentiate towards the cell line of osteoblast. At five weeks, osteoid tissue could be found and some new tissue began to be mineralized. At ten weeks, immature woven bone and marrow cavity-like structure were formed. There were some difference between animals in the ossification degree of new forming tissue.The process of ectopic bone formation occurred through two different mechanisms: intramembranous and endochondral bone formation at the early stage of transplantation, and through intramembranous osteogenesis at the later stage.4. The supernate of BGC had a little negative effect on the proliferation of cultured cells, and could stimulate the periosteum cells to secrete more ALP proteins. Most of the seeded cells attaching on BGC grew well on the surface of most pores and could produce some extracellular matrix (ECM).5. All the animals grew well after operation and no skeletal or wound complications were noted during healing. The results of X-ray examination showed that group A had higher density in the central and peripheral part of grafted BGC materials than group B and C,and all the three experimental groups (A、B and C) formed osteal connection at12weeks postoperation. Histologically, in all the three experimental groups more new bone tissues were found in the peripheral part of grafted defects than in the central part. At any time point more bones were quantified within defects filled with gene modified tissue-engineered bone grafts than filled with tissue-engineered bone grafts (p<0.05) and single BGC grafts (p<0.01).However, in central part of grafts there was no significant difference between the new bone forming of group A and B (P>0.05), and the amount of bone of these two groups was significantly higher than group C (p<0.05). In the unfilled controls (group D) there was much fibrous connective tissue formed in the defects by12weeks and no full osteal healing was found.Conclusions:1. Although BMSCs were described as the most routinely used source of tissue engineering, periosteum cells had some advantages and could be widely used as seeded cells in the field of bone tissue engineering.2. BMSCs and periosteum cells transfected with Ad-BMP-2could both express high leveled target gene and protein, and Ad-BMP-2could induce these two mesenchymal cells to differentiate towards the cell line of osteoblasts.It suggested that both of these two mesenchymal cells could be used as the excellent target cells of gene therapy.3. Ad-BMP-2could induce ectopic bone formation around the lateral periosteum of mandible and at some degree could augment the thickness of cortical bone.4. BGC had an excellent biocompatibility and osteoconduction with cultured periosteum cells in vitro, and could be used as cell scaffold in bone tissue engineering.5. Periosteum cells expressing BMP-2gene could be served as the seeded cells of bone tissue engineering.Gene modified tissue-engineered bone grafts had higher osteogenic potential than tissue-engineered bone and single BGC grafts.Ex vivo adenovirus-mediated BMP-2gene transfer to allogenous periosteum cells could enhance bone formation in repairing mandibular segmental defects and gene modified tissue-engineered bone may become an ideal bone substitute for bone reconstruction in the future.