Experimental Study Of KLD-12 Peptide/rhBMP-2 Fiber Gel And BMSCs Inducing Osteogenesis Of Transverse Spinal Process

Objective: To investigate Osteogenesis at the transverse process space of rabbits with KLD-12 selfassembling peptide / recombinant human bone morphogenetic protein 2(rhBMP-2)fiber gel and rabbit bone marrow mesenchymal stem cells(BMSCs).Methods:(1)Culture of BMSCs.BMSCs are closely packed,and most of the cells are spindle-shaped after attachment.BMSCs had higher cell viability at 3 passages,and BMSCs were density dependent.Higher planting density may promote cell proliferation.(2)KLD-12 peptide sustained release experiment.The rhBMP-2 solution was diluted at different concentrations and mixed with KLD-12.New PBS buffer was added above the mixture at 1: 1,and the supernatant PBS solution was replaced and collected every day for 10 days,and the supernatant rhBMP-2 content was detected by the Elisa method.(3)Proliferation experiment of complex KLD-12 peptide BMSCs.Cell counting kit-8 method was used to detect the proliferation activity of BMSCs after complexing with the gel.(4)Animal experiments.New Zealand white rabbits were randomly divided into four groups: group A experimental group,group B positive control group,group C negative control group,and group D sham operation group,10 animals in each group.The experimental materials were implanted into the transverse process space.Group A is the experimental group(KLD-12 peptide / rhBMP-2 / BMSCs);Group B is the positive control group(KLD-12 polypeptide / rhBMP-2);Group C is the negative control group(KLD-12 self-assembling peptide fiber gel);Group D is a sham operation group(trauma treatment at the transverse process space).X-ray examination will be performed 4,8 and 12 weeks after modeling at the transverse process gap.Micro-CT,gross measurement observation,and histomorphology examination will only be performed after 12 weeks of modeling at the transverse process gap.Results:(1)KLD-12 peptide sustained release experiment: KLD-12 peptide has the effect of sustained release of rhBMP-2.(2)Proliferation experiment of complex KLD-12 peptide BMSCs: BMSCs grew well in KLD-12 peptide.(3)X-ray: There was no obvious osteogenesis in the four groups at 4 weeks.A and B groups had slight osteogenesis after 8 weeks of modeling in the transverse process space.Group A was slightly stronger than group B;there were no obvious osteogenesis images in groups C and D.Both groups A and B had obvious osteogenesis regions after 12 weeks of modeling in the transverse process space.The difference in gray levels showed that the effect of bone formation in group A was stronger than that in group B;there was no obvious osteogenesis image in the two groups.(4)Micro-CT results: At 12 weeks,the bone volume fraction,the number of trabeculae,the thickness of trabeculae,and the bone mineral density of group A were greater than those of group B positive control group.Bone volume fraction BV / TV(%): 25.566 ± 0.024 in group A,22.141 ± 0.68 in group B;number of trabecular bone TB.N(pieces / mm3): 7.239 ± 0.56 in group A,5.996 ± 0.35 in group B;tissue bone density TMD(mg / cm3): 523.363 ± 10.53 in group A,412.366 ± 3.733 in group B;trabecular thickness Tb.Th(mm): 0.165 ± 0.022 in group A,0.103 ± 0.009 in group B.Micro-CT index data showed statistically significant differences between groups A and B(P <0.05).(5)Observation of general specimens: The rigidity of the clamps was higher in the A and B groups after 12 weeks of modeling at the transverse process space,and the area of the osteogenic tissue was larger in group A than in group B;no significant results were found in groups C and D.Bone tissue.(6)HE staining: Bone cells(bone pits),osteoblasts and osteoclasts are visible.(7)Giemsa staining: Bone cells and bone pits,as well as osteoclasts at the edge of trabecular bone.Conclusion: KLD-12 peptide / rhBMP-2 / BMSCs nanofiber gel is well boned after implantation in the transverse process space,and rhBMP-2 compounded in KLD-12 peptide can be slowly released.While KLD-12 reduced the local rhBMP-2 concentration,the synergistic effect of BMSCs and rhBMP-2 compensated for the lack of osteogenic effects caused by the low concentration of rhBMP-2 after sustained release of KLD-12 peptide.