Bone Marrow Mesenchymal Stem Cell Combined With Hydroxyapatite/Tricalcium Phosphate For The Spine Fusion

Stem cells can renew themselves for long periods through cell division,proliferate rapidly and have the remarkable potential to develop into many different types in the body. Serving as a sort of repair system for the body,they can theoretically divide without replenishing other cells as long as the person or animal is still alive.When a stem cell divides,each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function.Embryonic stem cells have many limitations in application because of ethical,religional and moral principles.So,recently many researchers have focused on the adult stem cells.Bone marrow mesenchymal stem cells which derivatived from many tissues can generate bone,cartilage,fat,vascular endothelial cells,pancreatic islet,muscle,even liver cell,neurocyte and so on.They are the main source of cells in bone tissue engineering.Hydroxyapatite / tricalcium phosphate are used extensively now.In this experiment we used bone marrow mesenchymal stem cells as seed cells and hydroxyapatite / tricalcium phosphate which is widely used as support stuff in spinal fusion,and provide new evidences and technical methods for the clinical application of spinal fusion.Part 1 Studies on bone marrow mesenchymal stem cells combined with the scaffold in vitroObjective To create a methods by which rabbit's marrow mesenchymal stem cells can be cultivated in vitro and its growth rule and morphology can be observed.To explore its biocompatibility with hydroxyapatite / tricalcium phosphate.Methods(1)Bone marrow are obtained from long bone of rabbit and cultivated by whole bone marrow culture for primary culture and serial subcultivation respectively. Observe the growth characteristic and growth curve of every generation,evaluate the feasibility of bone marrow mesenchymal stem cells as seed cell.(2)Characterize bone marrow mesenchymal stem cells through the ability of multi-directional differentiation.(3)Culture the bone marrow mesenchymal stem cells on the surface of matrix scaffold.The morphologic characters during culture on hydroxyapatite / tricalcium phosphate scaffold's surfaces were checked by light microscope,fluorescence microscope,and scanning electronic micro-scope,and tested the cell proliferationResults(1)It was feasible to culture bone marrow mesenchymal stem cells by whole bone marrow culture.The cultivation of bone marrow mesenchymal stem cells in vitro shows that they increased lastingly and has a fast multiplication speed in vitro.They can passage and amplified lots of generations.(2)Bone marrow mesenchymal stem cells obtained from bone marrow could be differentiated to be osteoblast cells,adipocyte and neuron-like cells.(3)Bone marrow mesenchymal stem cells showed good biocompatibility with hydroxyapatite / tricalcium phosphate scaffold.The cells growth into the micropore of hydroxyapatite / tricalcium phosphate scaffold could be seen under both fluorescence microscopy and scaning electron microscopy,and proliferated generously.Conclusion Bone marrow mesenchymal stem cells can be seed cells in bone tissue engineering,hydroxyapatite/tricalcium phosphate scaffold can be used as a vehicle for the cell transplantation,be good for seed cells overgrowth.Part 2 Enriched bone marrow mesenchymal stem cells combined with HA/TCP for spine fusionObjective To investigate the clinical results of spine fusion with the autologous enriched bone marrow mesenchymal stem cells(EBMSCs)combined with hydroxyapatite (HA)/tricalcium phosphate(TCP).Methods(1)Between January 2005 and February 2006,48 patients who underwent pedicle screw ins trumentation for treating thoracolumbar fractures were selected.(2)According to different bone substitutes,the cases were randomly divided into two groups:Group A was autologous EBMSCs plus composite material(n =24,13 males, and 11 females;aged 25 to 49 years);Group B was autologous iliac crest bone graft(n =24,15 males,and 9 females;aged 26 to 50 years).(3)Autologous EBMSCs suspens ion was obtained by gradient-centrifugation method,then combined with HA/TCP for 2 hours to serve as bone graft extenders.In the group A,autologous EBMSCs cultured with HA/TCP were used as bone substitute;In group B,autologous iliac crest bone harves ted was used as a bone graft extenders.(4)Spine fusion healing,clinical effects and correction ross of two groups were compared to as sess spine fusion extent.Results Forty-eight cases were all involved in the result analyses after a 12 months post-operative follow-up.(1)Effectiveness of marrow enrichment:The concentration of MSCs increased 8 times after enrichment.(2)In group A,pain in the location of harvested marrow diminished in 1 week after operation without infection;In group B,pain in the location of harvested iliac crest bone still remained in 3 months after operation.There was no complication such as infection,internal ins trumentation loosening or breakage.LBOS scores showed that the excellent rate in the group A was 71%(17/24), and group B was 79%(19/24).(3)Spinal fusion outcomes:X-ray and CT scanning indicated that spine fusion in all cases were good after a more than 6 months follow-up. The density of grafting substitutes was decreased and bilateral thin fus ion phenomenon was observed in 3 months,moreover,unilateral or bilateral autologous blocks of bone mas s were found in 6 months after operation.According to Lenke letter grades,there was no C or D grade spinal fusion.(4)Cobb angles' outcomes:Mean degree of correction loss in group A was 2.38°,and group B was 2.42°.Conclusion Autologous EBMSCs combined with HA/TCP is safe,simple and effective as a graft material for posterolateral spine fusion in treating thoracolumbar fractures,and has the same clinical effect as that of autologous iliac crest bone graft.