Experimental Study Of Bone Defect Repaired With Embryonic Periosteum Cell Suspension Compound By Calcium Alginate Gelatin

Objective: Bone defect is a thorny problem of clinical departments, and bone grafting is the primary method to solve it. Bone grafting is divided into autologous bone grafting, homologous or heterologous bone grafting and bone tissue engineering bone grafting which is popular in nowadays research. The first two methods have many morbidities including limited source, destruction of donorsites and immune response. Now using tissue-engineering technique construct bioartificial bone generates a new way of thinking and methods to overcome this tough problem. Tissue engineering is, according to its historic definition, an"interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function". Bone tissue-engineering include three key factors: signaling molecule (bone growth factor,bone-inducible factor), tissues scaffolds, seeding cell. The use of calcium alginate gelatin which is a perfect tissue engineering bracket material in repairing of orthopaedics wound is a hotspot at the research of tissue engineering. As tissues scaffolds the calcium alginate gelatin with the characteristics: (1)good biocompatibility; (2)good biodegradability; (3)the degradation rates of tissues scaffolds match the ability of bone formation; (4)good performance of porous scaffolds; (5)strong permeability. At present, it is used widely at bone (cartilage) tissue-engineering, drug delivery system, cell three-dimensional culture system. Periosteum is divided into fibrous layer and germinal layer (osteogenesis layer), which contains osteoprogenitor cells, osteoblast, osteoclast and vessels derived endothelial cell etc. The germinal layer in embryonic period consists of osteoprogenitor cells and osteoblast, which has the multiplication capacity and ability of bone formation. Embryonic periosteum is better proffer of germ cell for its immune rejection reactions are few, and it has self-ossifying ability. We compound embryonic periosteum cell suspension in calcium alginate gelatin, bring the osteoacusis the self- ossifyication and the bone induction into play sufficiently. In this experiment, we sum up the research experience of predecessor, and take stock of whether embryonic periosteum cell suspension compound with calcium alginate gelatin can ossify or not in self- entironment and the mode of ossifyication and find a new method which is used conveniently in clinic to therapy bone defect there by.Method: 10 pregnant rabbits which pregnant 26~28 day are open to get embryonic rabbits. Stripping the embryonic periosteum from embryonic rabbits, then put into cryopreservation tube with PBS, DMEM and DMSO. After using two-step freezing method we take it in liquid nitrogen which is -198℃for cryopreservation. Then rewarming at 37℃water-bath, embryonic periosteum preparate to embryonic periosteum cell suspension. 80 adult New Zealand rabbits are divided into 4 groups of 20 animals at random.All the animals are operated into standard bone defect of 15 mm in left radius. Group A are filled with calcium alginate gelatin/embryonic periosteum cell suspension; Group B are filled with calcium alginate gelatin; Group C are filled with embryonic periosteum cell suspension; Group D are taken as the blank control group. The animals are sacrificed at 2nd, 4th, 6th, 8th and 12th week postoperation, 4 animals of each group are sacrificed each time. After muscles are removed away from the specimens, results are revealed in gross observation, histology and X-radiography. Moreover, level of alkaline phosphatase(ALP), serum calcium are measured on each group animals. The complexion of bone forming is observed in macroscopical and microcosmic side, and has been evaluated by statistical analysis.Result: Gross observation, histological analysis, X-ray film and biochemical examination are taken at 2nd, 4th, 6th, 8thand 12thweek postoperation. the result are following: In group A, there are distinctive periosteum reaction and lots of callus growth in transplanting area, consistent callus finally bridging the gaps of defect, new bone contoured well and proliferated in the way of multi-center cartilage ossifyication, mature bone and bone marrow cavity are gradually formatted, bone defect healed completely. There is no phenomenon of immune rejection reaction. In Group B, there are part ossifyication at each end of the defect. There aren't bone link at the middle of defect area, and are hold by surrounding connective tissue. In Group C, most specimens show ossifyication by the side of ulna and can form bone connection, other place of defect are hold by surrounding connective tissue, new bone growth and reconstructing little and slowly, the other specimens show no ossifyication and the defect are hold by surrounding connective tissue completely. Also there is no phenomenon of immune rejection reaction. In Group D, the two stumps of all the specimens occur osteosclerosis, medullary cavity closed, and there aren't bone link at the middle of defect area, and are hold by surrounding connective tissue. Results of histologyical observation reflecte: In group A, before the degradation of calcium alginate gelatin, the ossifyication is fast, mainly growth to cartilage and stricke up the cell bracket, the calcium alginate gelatin begin degradation gradually after 4 week, and the cartilage transform to bone at the same time. At 8 week the bone defect has been repaired, rebirth blood vessel grows well, trabecular of bone arranges in order, mature bone tissue fills in completely, and there is no divideng line with defect end in evidence. At the juncture, there is diffusion lymphocytes and eosinophilic granulocyte occasionally, and disappear in 6 week. In group B, before the degradation of calcium alginate gelatin, there are partly sprawl and substitution, ossify from the two end to the middle of the defect, this process stop when the calcium alginate gelatin begin degradation gradually after 4 week and there are plenty of connect tissue in the middle of the defect area. In group C, the periosteum cells grow attached the periosteum of ulna, the periosteum of ulna is also incrassate and some small blood vessels grow to the defect. However there are filled in connect tissue in most part of defect area, so the bone growth are limited and the quantity of bone is little, trabecular of bone arranges no order like weaving bone. The Implanted cells growth well, shaped long spindle and cross links each other. There is lymphocytes and multinucleated macrophages occasionally, and disappear in 6 week. In group D, there aren't bone link at the middle of defect area, and are hold by surrounding connective tissue. X-ray observation reflecte: In group A, many callus exist distinctively in transplanting area, defect of bone are obscure and they are soon filled with new bone. In group B bone defect exist distinctively and there are bone proliferation to a certain extent in both end of defect. In group C, part defect is repaired. There is little periosteal reaction and callus growth. In group D, there is no callus exists distinctively in transplanting area, bone nonunion formed. Result of date in biochemistry reflects: all the three groups exists ossifyication before 4 week, but group A is higher than B and C in secum calcium and ALP. At 6 week, the date of group A, C is higher than B, group B has get back to the level of preoperation, A is also higher than C. At 8 week group A also get back to the level of preoperation, C is higher than A and B. At 12 week, all three groups get back to the level of preoperation.Conclusion:1 The embryonic periosteum cell is the dependable source of the bone seed cell. It has strong osteogenesis in accommodation carrier, and it proliferat in the way of multi-center cartilage ossifyication in calcium alginate gelatin.2 The embryonic periosteum cell suspensions by using the two-step freezing method have good histocompatibility with the transplantation host.3 Calcium alginate gelatin has good biology consistent quality,surface activity,biological degradation and plasticity, so it is a perfect tissue engineering bracket material.4 Embryonic periosteum cell suspension compound by calcium alginate gelatin takes an effective repairing to the bone defect, and there is no adverse reaction. The operation is easy and prone to mastery and appliance in clinic.5 Calcium alginate gelatin has the problem of poor mechanical property, it suitable for the region that no stress or less stress, or it can be used with effective immobilization.To sum up transplantation of calcium alginate gelatin compound with embryonic periosteum cell suspension bring the osteoacusis the self-ossifyication and the bone induction into play sufficiently, the operation is easy and prone to mastery and appliance in clinic...