The Animal And Clinical Study Of Bone Defect Repairing By Bone Regeneration Strategy Based On BMMNCs

Background and ObjectivesRepair of bone defects is a great challenge in clinical treatment.Due to limited source and dornor site mobidities,the traditional autologous bone graft have many limitations.The development of bone tissue engineering has opened up a new way for bone defect treatment.However,in vitro culture of tissue engineered bone is complex and expensive.There has been only a small number of clinical trials being carried out.Therefore,tissue engineered bone is not yet routinely used in clinical practice.On the other hand,bone marrow blood,which is rich in bone marrow mesenchymal stem cells(BMSCs),is often used to assist the repair of bone defects.Concentration of bone marrow mononuclear cells(BMMNCs)improves the level of stem cells and could further enhance the osteogenisis ablility.In recent years,there have been quite a lot of clinical reports about the use of fresh concentrated bone marrow for bone defects repair.However,there is no reports on the comparison between the classic tissue engineered bone and the BMMNCs,especially the detailed large animal experimental study.To this end,we constructed a segmental bone defect model in large animals,and using p-Tricalcium Phosphate(TCP)as a scaffold to compare BMMNCs and BMSCs in the repair of bone defects in one year period of time.We used a variety of advanced techniques to study the morphology,chemical composition and biomechanical properties of regenerated bone tissue formed under the two methods.We also found the difference between regenerated bone tissue and autologous normal bone tissue.In addition,we used BMMNCs combined?-TCP to repair the clinical alveolar bone defect.During the one-year follow-up,we compared this strategy with traditional autologous bone graft(ICBG).Our study has laid an important foundation for further clinical application of BMMNCs.Methods1.To prepare BMMNCs/?-TCP and BMSCs/?-TCP grafts:aspirate 15 ml bone marrow from iliac crest of Beagle dogs;harvest BMMNCs by density gradient centrifugation method;adjust the volume of BMMNCs suspension to 500 ?l,then seed the cells onto the scaffold.Wright-Giemsa stain was used to observe the changes of bone marrow aspirates before and after processing.Canine cytokine array kits were used to detect the cytokines of BMMNCs suspension,bone marrow aspirate and peripheral blood plasma.For BMSCs/?-TCP graft preparing,the same volume of bone marrow was aspirated;BMSCs were isolated and expanded to passage 1;adjust the cell concentration to 20×106/ml then seed 500 ?l cell suspension onto the same scaffold used above;the constructs were further cultured for 14 days in osteogenic differentiation medium.2.To repair the tibia segmental defect in Beagle dogs:tibia segmental defect model(15 mm)was created under general anesthesia;the defects were repaired by BMMNCs/?-TCP graft or BMSCs/?-TCP graft;the ?-TCP only group and blank group were set as control.X-rays were taken at 2 weeks,3 months,6 months and 12 months after surgery.The graft region were further evaluated by SPECT-CT,micro-CT and hard tissue histology examination.3.Micro-structure,chemical component and biomechanical analysis:both BMMNCs/?-TCP graft and BMSCs/?-TCP graft were used to repair the segmental bone defect in Beagle dogs.12 months after surgery,TPEF and SHG were used to detect the collagen and vascular vessels in regenerated bone tissue and contralateral tibia bone.Raman spectroscopy was used to explore the chemical component of bone tissue.Nano indentation was used to test micro biomechanical properties.4.Clinical alveolar cleft repair by BMMNCs combined with ?-TCP granules:3D printing models and CAE software were used to calculate the volume of defect region so as to determine the amount of graft needed.The alveolar cleft were repaired either by BMMNCs combined with ?-TCP granules or standard iliac crest bone graft(ICBG).CT examinations were taken to evaluate the repair of cleft region and to observe the changes of grafts at 3 months,6 months and 12 months after surgery.Results1.The average concentration of BMMNCs acquired from 15 ml bone marrow were 33.0±8.8×106/ml.After processing,most of the red cells were removed and nucleated cells were retained.The cells have a round shape and were evenly distributed on the scaffold.The cytokine analysis revealed that IL-8 is higher in BMMNCs than in bone marrow and peripheral blood plasma,whereas RAGE and SCF were lower in BMMNCs and bone marrow than in peripheral blood plasma.2.In the tibia bone defect reparing,BMMNC group showed better results.80%of the animals in this group achieved bone union within 3 months;no refractures were observed after removing the internal fixations;12 months after surgery,bone callus remodeling,increased cortex density,formation of bone marrow cavity were observed;the transition between the graft and auto tibia was smooth.In the BMSC group,however,the osteotomy lines were radiolucent within 6 months;the internal fixation could not be removed on time;there are clear boundaries between the graft and auto tibia;most of the scaffold did not degrade.Nevertheless,considerable bone regeneration could be noted inside the scaffold.In the the scaffold-only group,the grafts were almost absorbed within 6 months.Compared to BMSC group,BMMNC group showed more radioactivity at the graft area in SPECT-CT exmaination.Micro-CT revealed that the average bone volume of the BMMNC group was 1,231.38 mm3,and the average bone volume fraction was 65.14%,which were significantly higher than in the BMSC group.3.High resolution SHG images showed the organization of the collagen network.Within the macro pores of the BMMNC group,the collagen fibers were either arranged concentrically around the vascular tissue or aligned in layers.Osteocytes were observed between the lamellae.However,the collagen fibers in the BMSCs were disorganized and had a lower density.The collagen network was focused onto the walls of the macro pores.Laser Raman spectroscopy showed that the autotibia,BMMNC,and BMSC specimens shared similar peak positions,but the intensities at certain Raman shifts were different.The BMMNC group showed a significantly higher mineral to matrix ratio than the BMSC group;the carbonate to phosphate ratio was significantly higher in the BMMNC group and the BMSC group compared to the autotibia;the collagen cross-linking ratio was decreased in the BMMNC group but not in the BMSC group;bone remodeling indicator amide I ratio was significantly higher in the BMMNC group than in other groups.Nanoindentation showed that,in the BMMNC group,the average indention modulus was 4.68 GPa,and the average hardness was 0.64 GPa,which was close to those of the autotibia and significantly higher than those in the BMSC group.4.In the preoperative evaluation of alveolar cleft pationts,the average defect volume measured from the simulated bone grafts by 3D-printed models was 1.52 ml,higher than the mean volume of 1.47 ml calculated by CAE software.No significant difference was found between the two methods.All patients involved in the study healed properly without any severe complications.8 of 10 patients in the BMMNC group achieved alveolar bone union.The mean bone volume of the BMMNC group was 724.5 ± 220.5 mm3 at 3 months after surgery,and it decreased to 612.2 ± 211.5 mm3 at 6 months after surgery.However,the mean bone volume at 12-month follow-up had no significant change(589.0 ± 180.5 mm3).In the ICBG group,the mean bone volume was 678.7 ±211.0 mm3 at 6 months after surgery.As similar with the BMMNCs/?-TCP group,the mean bone volume of ICBG group slightly decreased at 12 months after surgery(635.3 ±197.5 mm3),the difference between the two groups was insignificant.Conclusions1.In the segmental defect repair of Beagle dogs,fresh prepared BMMNCs combined with ?-TCP showed better bone formation than cultured BMSCs/?-TCP grafts.2.Compared to BMSCs/?-TCP graft,BMMNCs combined with ?-TCP induced more bone regeneration,and its chemical component and biomechanical properties were close to auto tibia.3.The mirror-reversed technique by CAE software is as accurate as the simulated operation on 3D-printed models in unilateral alveolar cleft patients.These findings further validate the use of 3D printing and CAE technique in alveolar defect repairing.4.Although considerable resorption was observed in BMMNCs/?-TCP grafting,it was radiographically equivalent to ICBG in alveolar cleft repairing.