Autologous Bone Marrow Mesenchymal Stem Cells Combined With Gelatin Sponge To Repair Goat Intervertebral Disc Defects

Objective: 1.To fabricate a novel scaffold with acellular nucleus pulposus(NP)matrix and acellular cartilage matrix and verify the feasibility of it as a scaffold for NP tissue engineering through detecting physical and chemical properties.2.To establish an animal model of annulus fibrosus(AF)partial defect for the repairing of intervertebral disc(IVD)defect.3.To compare the effect of two different methods of autologous bone marrow mesenchymal stem cells combined with gelatin sponge in the repair of goat IVD defects.Methods: 1.The nucleus pulposus and the articular cartilage were crushed,acellular,frozen-dried and cross-linked to fabricate the scaffold.The general observation,physical and chemical detection of scaffolds was made.CCK-8 method and live/dead staining were used to detect the toxicity and biocompatibility of the scaffold.2.Using Image J 1.46 r software to measure the height of the intervertebral space on the X-ray film of goat.Axial split disc,to measure thickness of AF for the defect.An 11 blade was used to make a trapezoid defect of upper bottom 3mm,lower bottom 5mm,height 5mm and thickness 3mm on the IVD in vitro and in vivo.Through the measurement of the length of the trapezoid defect of AF and the weight of tissue from trapezoid defect,the trapezoid defect of AF was evaluated.3.8 goats were used as experimental animals.The X-ray and MRI of the lumbar spine were performed before and after the operation for 24 weeks.L1-2 disc of each goat was used as control group(group A)without any treatment.L2-3 IVD as a simple removal group(group B),only remove part of the nucleus pulposus without transplantation.L2-3 intervertebral disc as a simple removal group(group B),only part of the removal of the nucleus pulposus does not transplant operation.L3-4 IVD was used as the enrichment group(Group C),and the goat bone marrow and gelatin sponge complex was enriched by bone marrow concentrator.L4-5 IVD was used as autologous BMSCs culture group(Group D),and the third generation of autologous BMSCs and gelatin sponge were implanted.Animals were sacrificed at 24 weeks after operation,and were examined by biomechanics,histology and molecular biology.Results: 1.The scaffolds was milky white,like porous sponge.Electron microscopy and light microscope showed that the scaffold was network structure and pores connected to each other.Hochest33258 staining showed that the cells were completely removed.HE staining,safranine O staining and toluidine blue staining showed no cell residue in the scaffold,and the content of proteoglycans was abundant.The pore diameter of scaffold was 219.82 ± 46.21?m,the porosity was 95.43% ± 2.26%,and the water absorption was 1829.14% ± 273.97%.Live/dead staining showed that cells grew well on the scaffold.The results of CCK-8 showed that the scaffold extract had no adverse effect on cell proliferation.2.There were significant differences in the intervertebral space height of(4.45 ± 0.28)mm,the thickness of AF(4.08±0.50)mm and thickness(3 mm),height(5 mm)of trapezoidal defect(P < 0.05),respectively.There were no significant differences in AF trapezoidal defects in vitro on the upper bottom(3.03 ±0.09)mm,the lower bottom(5.03±0.09)mm,the height(4.97 ±0.10)mm,the thickness(3.02±0.06)mm and the trapezoidal defect predetermined value on the upper bottom 3mm,the lower bottom 5 mm,the height 5mm and the thickness 3mm(P >0.05).The weights of the tissue taken out from AF trapezoidal defects in vitro and vivo were(0.162±0.011)g and(0.166 ±0.014)g,and there was no significant difference between them(P > 0.05).3.All the animals survived after operation.24 weeks after operation,A group was no degenerative change,but B,C,D group had different degrees of degeneration.Imaging examination showed that compared with B group,DHI% and RGI of C?D group were significantly higher(p<0.05),and RGI in D group was higher than that in C group(p<0.05).Biomechanical test showed that C and D group had significantly better spinal mobility than B group(p<0.05),but there was no significant difference between C group and D group(p>0.05).The histological staining showed that compared with B group,the cells proliferation of C group and D group was active,the number of cells was significantly higher,the tissue structure was relatively better,and the content of proteoglycan and collagen in NP was high.The real-time PCR showed that compared with B group,proteoglycan,type II collagen and Sox-9 gene expression of C group and D group was significantly higher(p<0.05)and D group was higher than that of C group(p<0.05).Conclusion: 1.The NP-cartilage acellular scaffold had three-dimensional porous,uniform pore size,good biocompatibility,and wide source of raw materials,which make it a suitable a scaffold for NP.2.By the left side transverse retroperitoneal approach,using the method of making the defect in the experiment can be simple,safe and reliable to produce goat animal model with shape,size uniform AF trapezoid defect,which is expected to be an ideal animal model for the repair of AF defects.3.Transplanting gelatin sponge complex combined with enrichment of bone marrow and autologous BMSCs respectively into goat NP,which can effectively delay IVD degeneration,promote cell proliferation and secretion of NP extracellular matrix,maintain IVD height and biomechanical properties.Compared with autologous BMSCs culture group,although the repair effect of the enrichment group was weak,it was simple,safe and economical,so it still had good clinical application prospect.