| PrefaceSpinal fusion is one of the most common ways to treat spinal diseases . Transverse process fusion is the frequently used spinal fusion method. Currently, autologous bone or allogenic bone are usually used to spinal fusion for most clinical situations. However, autologous bone cant provide enough bone to spinal fusion and has a number of major disadvantages: more hemorrhage when operating; postoperative pain at donor site; the ability to fabricate a functional shape is limited et al. Allogenic bone transplantation has disadvantages such as immune response and diseases transmitted et al.Tissue engineering bone includes three essential elements: (1) ideal seed cell, (2) appropriate carrier, (3) osteogenic inducter. Marrow Stromal Stem Cells are also named Mesenchymal Stem Cells ( MSCs) , which are the ideal seed cells of tissue engineering. Bone Morphogenetic Protein ( BMP) can induce MSCs osteogenic differentiation. Fibrin Glue ( FG) is the appropriate carrier of MSCs and BMP.FG,MSCs and rhBMP2 (BMP) is used to construct tissue engineering bone in our experiment and that is applied for rabbit transverse process fusion. Radiographic analysis,biomechanical test and histologic examination are used to check spinal fusion condition. We hope our study can provide more efficient method for spinal fusion.Methods1. The source of animals1 month old (weight0.5 -1 Kg) and 1 year old (weight 3 -5 Kg) healthy Japanese rabbits (no female or male limited) , which are provided and raised by China Medical University.2. MSCs culture in vitro and inductionSeparating and harvesting immature rabbit femur MSCs by density gradient centrifugation, seeding to culture flask by 1 × 106/ml, changing culture fluid for the first time three days later and changing culture fluid on alternate days after the first time. When primary culture cells grow into monolayer, 1:3 subcultu-ring. Conditioned medium is used when subculturing including 10% fetal bovine serum, VitC 50μmol/L, β -sodium glycerophosphate 10mmol/L, dexametha-sone 10-8 mol/L. Observing MSCs morphous and growth condition by inverted microscope. To subculturing cells, HE dyeing is carried out after 3 days culture; Gomori dyeing is carried out after 14 days culture, observeding ALP expression and calculating positive ratio; Von kossa dyeing is carried out after 28 days culture; draw the growth curve of the third subculturing cells.3. Compatibility evaluation of MSCs and FGMSCs and FG co - culture for 3 days, evaluating compatibility of them by SEM.4. Animal models construction40 one - year - old rabbits are equally divided into 4 groups randomly: FG/MSCs/BMP, FG/MSCs, FG/ BMP, FG. Spinal fusion operation is taked, burnishing the cortex, revealing the marrow cavity of transverse process, putting the carrier to the L5 - L6 transverse process. All the animals are sacrificed 6 weeks after operation and take the spine specimens out. To the specimens, the examination is carried out including: 1) observing by eyes; 2) manual palpation ; 3 ) radiographic observation; 4 ) gray analysis; 5 ) biomechanical test; 6 ) histologic examination.Results1. MSCs culture in vitro and inductionPrimary culture cells survival ratio can reach 98% by trypan blue dyeingexamination. Inverted microscope observation shows primary cells form colonies 3 days after culture, cells grow into monolayer 12 -14 days later. Subculturing cells proliferate more rapidly, reaching monolayer 9-10 days after culture. Because inducers are added, more cells take on polygon. HE dyeing shows basophilic nucleus of MSCs, acidophilic cytoplasm , fusiform or polygon body. Go-mori dyeing shows black particles in positive cells and positive ratio is 83.7%. Von kossa dyeing shows mineralization nodules are dark brown. Cell growth curve demonstrates cells enter logarithm proliferation period firom the second day after culture and enter platform period from the fifth day.2. Compatibility evaluation of MSCs and FGMSCs and FG co - culture for 3 days, SEM shows MSCs completely spread on the surface of FG, polygon or fusiform, connecting each other.3. The results of animal model spinal fusionWe can see connective mass exist between the left L5,L6 transverse processes of all the specimens in FG/MSCs/BMP. 2 specimens of FG/MSCs have connective mass. 4 specimens of FG/BMP have connective mass. There is nothing between the left L5,L6 transverse processes of all the specimens in FG.Manual palpation shows fusion ratio of FG/MSCs/BMP is 70%. Radiographic observation shows fusion ratio of FG/MSCs/BMP is 80%. There is no statistical difference between FG/MSCs/BMP and FG/BMP. Manual palpation and Radiographic observation show there is no fusion specimen in FG/MSCs group and FG group. CT scan and 3D - CT show there is continous bone mass between L5,L6 transverse processes.Gray analysis shows bone density and area of the specimens in FG/MSCs/ BMP are significantly higher than other three groups.Biomechanical test shows fusion strength of the specimens in FG/MSCs/ BMP are significantly more superior than FG/BMP.HE dyeing shows there are mature bone tissues in bone mass between transverse processes of the fusion specimens in FG/MSCs/BMP and FG/BMP.Masson dyeing shows collagen tissues are abundant in the trabecula of the specimens in FG/MSCs/BMP.Discussion1. Choose and separation, culture, amplification of seed cellsMSCs include determined osteogenic precursor cells and inducible osteogenic precursor cells. The former can still differentiate to sclerotomal cell even there are not inducing factors. Besides, MSCs have others conditions which the ideal seed cells should have. Those are in favor of making MSCs cultivated to be the seed cells. Density gradient centrifugation is a good method to separate MSCs.2. Choosing carrier and the compatibility of the carrier and seed cellsFG can become appropriate carrier because many advantages, such as good plasticity, adjustable clotting time, deliverying a lot kinds of factors which can promote MSCs to adhere and generate, suitable degradation speed, etc.3. Osteoinductive factors and the relationship between osteoinductive factor and carrierVitamin C, β- sodium glycerophosphate, Dexamethasone, BMP can promote MSCs to osteogenic differentiation. Stronger adhesive power of FG is favorable for the slow delivery of BMP, and the macromolecule proteinum character of FG can avoid BMP non - specificity proteolysis.4. The application of tissue engineering bone to rabbit transverse process spinal fusionAlthough there is not statistical difference between the FG/MSCs/BMP group and FG/BMP group, the former is better than the latter on bone formation density, area and biomechanical test. Further, FG/MSCs/BMP group is better than FG/MSCs group and FG group on all the examination. These demonstrate the compound has the best bone formation ability, which has all three tissue engineering essential elements.Conclusion1. FG is appropriate tissue engineering carrier.
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