Construction Of PCDNA3.1/BMP-4 And Its Application To Treatment Of Bone Defects In Rabbits

The current practice for fractures including surgical methods and physical methods makes most of fractures treated effectively. However, many researchers are still making efforts to find alternative therapeutic strategies with purpose of ensuring proper healing of complex fractures. Substantial efforts also have been devoted to development of biological factors and their delivery to influence or control the cellular events associated with bone formation and repair. Recombinant proteins like the bone morphogenetic proteins (BMP) have shown particular promise as therapeutic alternatives. Unfortunately, direct use of these factors has been limited by its short half-life etc. Fracture healing lends itself well to application of gene therapy because of transient and localized need for enhancing factors, thus many studies were focused on the gene therapy of fracture healing in this study. The pCDNA3.1/BMP-4 was constructed and the integrity of BMP-4 gene was verified by sequencing. The BMP-4 gene was transcribed and translated when it was transferred into flbroblasts in vitro and its mRNA and protein were detected with RT-PCR, Western Blot, and the excretion of BMP-4 induced marrow stroma cells to differentiate into osteoblast. Based on those results, we designed two studies to explore the effect of BMP-4 gene therapy through direct injection of pCDNA3.l/BMP-4 liposome. The first experiment was the treatment of long segmental defects in rabbits with the model of ulna defect (Part One), and the second was thetreatment of firearm femur defect (Part Two).Part One: 48 adult rabbits were averagely divided into four groups randomly. Both the two ulnas of each rabbit were operated and removed 15mm section from the central part of ulnas. 12 animals were directly injected 40 g pCDNA3.1/BMP-4 liposome into the tissues between the two ends of defects 1 week after operation (Group A ); pCDNA3.1/BMP-4 liposome was directly injected into another 12 animals as in Group A, also, 20ng TGF was injected at the 2" and the 4th week respectively after operation (Group B ); 12 animals with only pCDNA3.l liposome injection or only operation were used as control (Group C and Group D). The ALP activity, calcium content, pathological and radiological changes of the local tissues was examined at the 2nd, the 4th,the 8th and the 12th week after operation. The main results and conclusions are as the following:1) The BMP-4 gene was transcribed and translated in local tissues between the two ends of ulna defects for six weeks.2) The local tissues started the cartilaginous ossification marked with the appearance of cartilage at the 4th week after operation in Group A and Group B. The ossification was nearly completed at the 12th week in Group B, while the ossification was still lasting in Group A. No ossification was observed in Group C and Group D.3) The X-ray examination showed that the new bone began to form at the 8th week and it stuffed the defects completely at the 12th week in Group A and Group B, but the quality and quantity of new formed bone were higher in Group B than that in Group A.4) The ALP activity reached its peak at the 8th week and reduced to arelatively stable level at the 12th week in GroupB, while it remained the same level in Group A. The calcium content at the 8th and 12th week was significantly higher in Group B than that in Group A.5) The results above indicated that the expression and secretion of BMP-4 after transfection can induce the differentiation of mesenchymal cells into osseous tissue and promote the fracture healing, and BMP-4 gene therapy combined with TGF can more effectively promote the fracture repair.Part Two: 24 adult rabbits were averagely divided into two groups randomly. The central part of one femur of each rabbit was made a 6-8mm defect with a fire of steel ball through the anterior muscular layer. After debridement, delayed suture and internal fixation, 12 animals were directly injected pCDNA3.l/BMP-4 liposome and TGF (Group A), and the other 12 animals w...