| With the development of modern life style, the clinical patients with trauma and diseases are increasing. And also with the rapid development of medical technology, many orthopedic diseases which were difficult to treat in old times now have new treatment methods. With regards to an old problem with orthopaedic patients, it is vey necessary to find a new bone graft source to meet new requirements. Orthopaedic diseases such as fracture healing, nonunion, spinal fusion, bone tumor resection defects, multiple joint replacements required more bone stock with out immunogenicity. If patient can not provide such bone tissue, what can orthopaedic surgons do? At this stage, the patient’s own iliac bone graft is still the gold standard for treatment of this disease. The patient’s own iliac bone contains bone scaffold, growth factors and bone formation cell. However, harvesting bone autografts have its’ complication, such as bleeding, infection, prolonged operation time. And application of allografts also has disadvantages, such as high price, the immune reaction and the risk of disease spreading.Currently AAV is thought to be the safest virus particle for the clinical application internationally. When compared to the conventional adenovirus, AAV can be deliberately inserted into the desired position of human chromosome which could prevent both accidentally inactivation of cancer suppressor genes and activation of oncogenes by randomly insertion. It did not show any signs of endanger human wellbeing. At this moment, it is applied in many ongoing clinical trials in United States. AAV particle has many special characteristics, such as their quite small size, high transduction efficiency, very mild immune rejection, long time expression and high transduction potential for both dividing and non-dividing cells. Due to the above advantages, AAV is thought to be one of the most potential gene delivery vectors and applied world widely in the field of gene therapy and vaccine development. BMPs can induce mesenchymal stem cells differentiate into chondrogenic and osteogenic lineage, When BMPs gene are constructed into the virus vector, and they can be inserted into the genome of cells by virus transduction in vitro. The inserted gene then can be transcribed and translated into proteins, which could be secreted out of cells and induce surrounding cells differentiate into chondrogenic and osteogenic lineage. These results demonstrated that the constructed AAV-BMP4 virus has good bioactivity. The histological assay also showed that the ectopic process is endochondral bone formation.Bone tissue engineering includes three elements which are cell, growth factor and scaffold. The common used scaffolds are hydorxyapatitie(HA), decalcified bone matrix(DBM). HA is the major inorganic part of human bone tissue. It has certain rigidity, porous character on the surface which facilitates cell attachment, bone conductivity. However, it doesn’t have bone inductivity. DBM is made by decalcified bone matrix of allograft or xenograft. The manufacture process is to decrease the immune rejection potential of bone matrix as a graft. The main inorganic part of type I collagen which contains many bioactive growth factors which enable it to have the potential of bone inductivity. The composite of AAV-BMP4 plus Gelfoam has a very good potential of bone induction. When the composite is implanted into the muscle pocket, the AAV-BMP4 can transduce the cells in the skeletal muscle and induce the new bone formation, which have obvious bone induction.The research includes three parts. The first part is the construct and bioassay of adeno-associated virus containing bone morphogenetic protein-4(AAV-BMP4). The second part is the investigation of ectopic bone formation induced by AAV-BMP4. The third part is the investigation of bone tissue induced by AAV-BMP4 for the treatment of skull defect.Part 1 The construct and bioassay of adeno-associated virus containing bone morphogenetic protein-4(AAV-BMP4)The aim of present study is to construct BMP4 gene into AAV vector, and make virus particle by co-transduction of 293 packaging cell with three plasmids. The cells then are collected and broken by cyclic frozen and thaw. The virus particles are collected and purified by Cscl gradient centrifugation. After the purification and knowing the concentration of AAV-BMP4 particles, they were absorbed in the Gelfoam and surgically transplanted into muscle pocket of SCID mice. The results of experiment showed that the density of AAV-BMP4 particles can reach 5×1012 vp/ml after purification. Once they are implanted into the skeletal muscle pocket of SCID mice, they can induce ectopic bone formation in the skeletal muscle. These results demonstrated that the constructed AAV-BMP4 virus has good bioactivity. The histological assay also showed that the ectopic process is endochondral bone formation.Part 2 The investigation of ectopic bone formation induced by AAV-BMP4The experimental design includes two ways. The first one is to apply whole piece of skeletal muscle to wrap up the composite of AAV+Gelfoam, and then three of them were implanted into the subcutaneous area of SCID mice back. The second one is to pre-implant the minced skeletal muscle into subcutaneous area of SCID mice back, and 4 weeks later the composite of AAV-BMP4+Gelfoam was implanted into the same site. The results showed that HA group has a high signal in both subcutaneous area and skeletal muscle pocket under x-ray examination at week 4, 8 12 after surgery. DBM group has no high signal and AAV-BMP4 has high signal only in the skeletal muscle pocket. Histological examinations by both H.E staining and Von Kossa staining showed that both DBM and AAV-BMP4 group have new bone formation in the skeletal muscle pocket, however, there is no bone formation in the subcutaneous area for both groups. In HA group, there is no bone formation under histological examination. The composite of AAV-BMP4+Gelfoam wrapped with whole piece of skeletal muscle did not yield any bone formation in the subcutaneous area. The negative finding was also confirmed by both X-ray and Micro-CT examination. Histological examination showed that no new blood vessels were found in the implanted skeletal muscle in the subcutaneous area and skeletal muscle cells had signs of necrosis. The pre-implanted minced skeletal muscle with AAV-BMP4+ Gelfoam showed there was a little bit bone tissue formed in the subcutaneous area. The positive histological findings were also confirmed by both X-ray and Micro-CT examination.Part 3 The investigation of bone tissue induced by AAV-BMP4 for the treatment of skulThe methods of the present study is to use the gelfoam to resorb AAV-BMP4 virus in vitro and implant the composite of AAV+Gelfoam into the skeletal muscle pocket of SCID mice. At week 12 after implantation surgery, the ectopic formed bone tissue was harvested and transplanted into a skull defect site. The results showed that the transplanted bone tissue had no sign of overgrowth over time. The bone graft had a gradual modification with time and merged with host bone very well. The rough shape of transplanted bone tissue was changed into smooth shape gradually with time. The small holes at the beginning of transplantation were gone with time. This change was observed by Micro-CT examination at different time points after surgery.Conclusion:1. The constructed AAV-BMP4 in vitro has its bioactivity. Once it is implanted into the skeletal muscle pocket, it can induce ectopic bone formation. And the process of ectopic bone formation is through endochondral bone formation.2. Among three implants, AAV-BMP4 has the strongest bone induction potential in the skeletal muscle pocket, DBM is the second and HA has no bone induction potential. Three groups all have no bone induction potential in the subcutaneous area. AAV-BMP4+Gelfoam wrapped up with whole piece of skeletal muscle cannot yield any bone tissue in subcutaneous area. The pre-implanted minced skeletal muscle with AAV-BMP4+Gelfoam showed there was a little bit bone tissue formed in the subcutaneous area.3. The ectopic formed bone tissue induced by AAV-BMP4 implantation in the skeletal muscle can successfully repair the skull defect of SCID mice. The bone graft had no signs of overgrowth at the site of skull defect with time, and it merged with host bone well. |
PDF Full Text Request