| Objective Distraction osteogenesis (DO) is an endogenous bone tissue engineering technique for promoting bone regeneration by mechanically stretching healing bone callus after bone damage or osteotomy and has been one of the study foci in the field of oral and maxillofacial surgery in recent years worldwide. It has demonstrated its advantages and application prospect in the overall reconstruction of bone deformities and defects due to congenital abnormal craniomaxillofacial development, tumor resection, osteomyelitis and trauma with its related basic and clinical researches ongoing. However, long distraction, consolidation periods and ensuing related complications have been the main reasons for restricting its wide clinical applications in despite of its abovely-mentioned advantages over conventional surgical protocols. How to promote new bone formation and regeneration quality, to shorten hospitality and treatment times, and to decrease related complications during DO has recently been the edge-cutting focus in the field. This study aimed to investigate: (1) the extraction and amplification of human BMP-2 gene fragment from human osteosarcoma by RT-PCR technique, the construction of pcDNA3.1-hBMP-2 eukaryotic expression vector by enzyme-cutting technique, and its transfection into rabbit mesenchymal stem cells (MSCs) and ensuing expression. (2) the in vivo ostetogenesis process and characteristics after transplanting autogenous MSCs modified with human BMP-2 gene into the distraction sites.Methods (1) Six-milliliter bone marrow was extracted from bilateral Greater trochanter of femur of 2- to 3-month-old healthy New Zealand's white rabbits. It was isolated by adherence methods into rabbit's MSCs. Then it was identified by cell morphology and cell surface antigen. Then the MSCs were cultured in the medium for inducing osteoblasts. and its alkaline phosphatase (ALP) expression and potential of calcific nodules was investigated by Gomori method and alizarin red staining, respectively.(2) Human BMP-2 gene fragments were extracted from human osteosarcoma tissue and amplified by RT-PCR technique, and then recombinated into pcDNA3.1 eukaryotic expression vector for pcDNA3.1-hBMP-2 eukaryotic expression plasmid by DNA recombination. And finally the recombinated DNA was authenticated by PCR amplification, electrophoretic analysis and DNA sequencing.(3) The pcDNA3.1-hBMP-2 eukaryotic expression plasmid was transfected into rabbit's MSCs by liposome-inducing eukaryotic transfection. Its mRNA expression was authenticated by RT-PCR, And its protein expression was authenticated by immunohistochemistry and western blot staining (IHC),respectively.(4) Thirty-six healthy New Zealand's white rabbits were randomed into three groups with twelve in each. The experimental group was injected with autogenous MSCs modified with human BMP-2 gene at P3 generation. The positive and negative control groups was injected with autogenous MSCs and saline solution, respectively. All the right mandibles of the objects was osteotomized as a DO animal model. After a 6-day latency the distractors were activated at a rate of 1.0 mm/d for two increments. The right mandibles were lengthened 7.0 mm totally before consolidation. At the second day of consolidation the experimental objects were injected with 200μl solution containing autogenous MSCs modified with human BMP-2 gene. The positive and negative control objects were injected with the same volumn of autogenous MSCs and saline solution, respectively. All the objects received radiological records at 2- or (and) 6-week of consolidation for evaluating bone regeneration and remodeling. Half the objects were sacrificed at 2- and 6-week consolidation, respectively. Half the newly regenerated bone in the distraction gap was harvested and prepared for gross, histological evaluation stained with hematoxylin-eosin (HE) and surfacial feature evaluation under scanning electronical microscopy (SEM). Successful transfection and protein expression was authenticated by RT-PCR,and immunohistochemistry staining on the other half respectively.Results (1)the isolation, culture and inducement of rabbit MSCs to osteoblasts: The MSCs were hard to identify for its mixture with blood cells at initial isolation and culture. The wall-adhesive cells demonstrated triangle, long-shuttle, multiangle or short- shuttle in shape at the third day for the first washing. After 10 to 20 days'culture fibroblastoid cells colonized on the bottoms. The subcultured cells proliferated faster and lined more orderly over ever. The MSCs would grow as colony in condition culture medium and then formed calcific nodules positively stained by alizarin red, which proved the MSCs had been induced to be osteoblasts. (2) The target gene fragment in the recombinated plasmid was confirmed as human BMP2-cDNA by PCR amplification, electrophoretic analysis and DNA sequencing. (3) Exogenous hBMP-2 gene could be successfully transfected into rabbit MSCs by liposome-inducing eukaryotic transfection. Extensive transcription of hBMP2 mRNA and protein expression in the pcDNA3.1-hBMP2-transfected rabbit MSCs were verified by RT-PCR , IHC and western blot respectively. (4) The gross specimen of both 2- and 6-week consolidations demonstrated that the stiffness of the experimental group was significantly higher than the positive and negative controls. And the positive control had no significant difference in stiffness with the negative control. The gray values of X-ray films in both 2- and 6-week consolidations was statistically higher than the positive and negative controls by related software (P<0.01). And the positive control had no significant difference in gray value with the negative control in both 2- and 6-week consolidations (P>0.05). Eectrophoretic analysis showed that the relative optical density (ROD) of BMP2mRNA at 2- and 6-week consolidation was statistically higher than the positive and negative controls by related software (P<0.01). And And the positive control had no significant difference in ROD with the negative control (P>0.05). IHC testing showed that the sections of regenerated bone of both 2-and 6-week consolidations were strongly positively stained. Its average gray value s were significantly lower than the positive and negative controls (p<0.01). And the positive and negative controls had no statistically significant difference (P>0.05).Conclusions (1) The adherence separation method is a simple and applicable one for the isolation and culture of whole blood marrow. Highly-purified rabbit MSCs could be isolated using this method and induced into osteoblasts by culture and subculture. (2) The successful construction of eukaryotic expression plasmid pcDNA3.1-hBMP-2 would have based for sequent researches. (3) Exogenous hBMP-2 gene could be successfully transfected into rabbit MSCs by liposome-inducing eukaryotic transfection and got stable expression. (4) The transplantation of autogenous MSCs modified by BMP-2 gene could promote bone regeneration of rabbit mandibular DO, which would provide an experimental base for clinically shortenning consolidation and thus treatment time.
|
PDF Full Text Request