Experimental Research On Regenerative Effects Of Tissue Engineering Artificial Bone On Rabbit Mandibular Defect

Objective:Because of the exposed location and special anatomical structure and function,damages to oral and maxillofacial bones severely disrupt appearance,pronunciation,ingress,chewing and other functions.Therefore,repairing maxillofacial bone defect and restoring facial anatomical structure,function as well as appearance have been a conundrum for maxillofacial surgeon and orthopedics.Tissue engineereing bone takes advantages of unlimited source,low antigenicity,pre-controlled shape and potent vitality over autografts and allografts.Preparing tissue engineering artificial bone by integrating seeding cells,scaffold materials and growth factors has achieved great progress to realize bone defect repairing.The present study prepared VEGF/BMP-2sustainably releasing-nHAC/PLGAs scaffolds and inoculated ADSCs to detect osteogenic activity of this active artificial bone in vitro and in vivo,and further investigated the molecular mechanism of crosstalk between BMP-2 and VEGF enhancing ADSCs osteodifferentiation,which more realisticly simulated the microenvironment of bone defect repair.Hoping to provide theoretical and experimental basis for designing tissue engineering scaffolds,innovating bone defect repairing treatments and deepening related mechanism research.Methods:1.Rat tail collagen was extracted and BMP-2/VEGF loaded PLGA microspheres were prepared to detect the release characteristics and activity of BMP-2and VEGF from microspheres.2.Three-dimensional porous nHAC/PLGAs scaffold was prepared to detect its pore size,porosity,water absorption,mechanical properties and degradation characteristics.3.hADSCs were inoculated into the nHAC/PLGAs scaffold to detect their proliferative activity and observe their morphology on the scaffold surface.4.hADSCs were inoculated into the BMP-2 and VEGF respectively or simultaneously releasing-nHAC/PLGAs scaffold to detect the expression of osteogenic indicators col 1,ALP activity and mineralized nodule formation,and osteogenic differentiation factors RUNX-2,Dlx-5 and osterix,and phosphoration of signaling pathway related protein Smad,Akt and p38.5.hADSCs were inoculated in the BMP-2 and VEGF respectively or simultaneously releasing-nHAC/PLGAs scaffold under the treatment of Akt inhibitor LY294002,p38 inhibitor SB203580 or lentivirus containing osterix shRNA to detect the expression of osteogenic indicators.6.Rabbit ADSCs were isolated and cultured to detect their proliferative characteristics,surface molecular markers expression and multi-differentiative ability.7.Rabbit mandibular critical bone defect model was prepared and rADSCs loaded nHAC/PLGAs-BMP-2/VEGF scaffold were implanted,then mageological examination,general observation and histological observation were conducted at 4^th,8^thh and 12^thh week post-operatively.Results:1.The encapsulation efficiency of BMP-2 and VEGF in PLGAs microspheres was 68.94%±2.00%and 53.94%±3.29%respectively.The amount of BMP-2 and VEGF released during the first 7 days was about 58.94%and 55.17%of the total encapsulation amount respectively,that was 28.74%and 27.95%respectively during 8^thto 17^thday,and that was only 6.19%and 9.85%respectively during the next 11 days.The released BMP-2 enhanced the ALP activity of rat BMSCs,and released VEGF promoted the proliferation of Eahy926 cells.2.The nHAC/PLGAs scaffold occupied a three-dimensional porous structure with a diameter ranging from tens of?m to nearly300?m,and the diameter of PLGA microspheres was about 9.95?m.The porosity of the scaffold was 79.46%and the water absorption rate was 561.51%.The elasticity modulus of the scaffold was 0.72±0.17 MPa,and compressive strength was 2.85±0.49 MPa.In vitro degradation process,the degradation rate of the scaffold was rapid in the first 2weeks,then slightly slowed down;the three-dimensional porous structure of scaffold maintained within 28 days,then completely collapsed within 56 days;PLGA microspheres completely degraded at 6^thh week.3.Cellular proliferative activity of hADSCs in the scaffold increased with time and was similar to that of control group.hADSCs showed normal morphology and biological behavior on the scaffold surface under SEM observation.4.VEGF alone can promote hADSCs proliferation,but had no significant effects on collgen I expression,ALP activity and mineralized nodule formation,and RUNX-2,Dlx-5 and osterix expression;BMP-2 alone has no obvious effect on the proliferation of hADSCs,but increased collgen I expression,ALP activity and mineralized nodule formation,and RUNX-2,Dlx-5 and osterix expression;Except for RUNX-2,combination of BMP-2 and VEGF further enhanced the expression of osteogenic indicators.5.VEGF alone increased expression of pAkt and p-p38,but had no effect on pSamd.BMP-2 alone increased expression of pSamd and p-p38,but had no effect on pAkt.Enhancement of simultaneous BMP-2 and VEGF treatment on the pAkt and p-p38 expression was higher than that of BMP-2 or VEGF treatment alone.6.LY294002 treatment had no significant effect on collgen I expression and mineralized nodules formation of hADSCs.SB203580 treatment significantly inhibited collgen I expression and mineralization nodules formation,had no significant effect on the expression of Dlx-5 and osterix,but decreased nucleus/cytoplasm ratio of osterix.Collgen I expression and mineralized nodules formation in each group remarkably weakened after stable osterix knockout.7.rADSCs proliferated rapidly and expressed stromal stem cell markers CD90,CD44 and CD29.Multiple lipid droplets were observed in the enlarged cell after adipogenic induction,and numberous calcium nodules were observed in the extracellular matrix after osteogenic induction.rADSCs secreted a large number of sulphate mucoprotein that can be stained light blue by Alcian dye after chondrogenic induction.8.In the repairing process of rabbit mandibular critical bone defect,mageological examination indicated no narrowing of bone defect cavity of control group,only a small amount of new bone were observed in defect cavity of VEGF group until 12^thweek after operation;Obvious new bone formation was observed in BMP-2 and BMP-2/VEGF group at 4^thh week after operation,and the bone defect healed completely at 12^thh week,but the amount of new bone in BMP-2 group were less and the healing rate was slower than that of BMP-2/VEGF group.General observation of the samples indicated bone defect cavity of control group was filled with granulation tissue,scaffold of VEGF group degraded gradually and was replaced with fibrous connective tissue,a few thin callus in bone defect cavity were observed until 12^thweek after operation;Bone defect cavity of BMP-2 and BMP-2/VEGF group narrowed along with time,the scaffolds gradually degraded and infiltrated tightly into the surrounding tissues,a large amount of new bone callus grew into the material,and callus of BMP-2/VEGF group remodeled well to hardly distinguish the original defect location at 12^thh week after operation.Histological observation indicated bone defect cavity of control group was filled with cellulose and erythrocytes,numerous erythrocytes grew into the scaffold of VEGF group at 4^thh week after operation,the scaffold almost disappeared and was replaced with a large number of fibrous connective tissue,a few new bone and blood vessels at 12^thh week after operation.At 4^thh week after operation,scaffolds of BMP-2 and BMP-2/VEGF group infiltrated into the edge of defect cavity,new bone and blood vessels can be observed in the“fusion area”.At 12^thh week after operation,scaffolds of BMP-2 and BMP-2/VEGF group almost completely degraded and were replaced with a large number of new bone and blood vessels,and lamellar bone and regular bone trabecula dominated in BMP-2/VEGF group.Conclusion:1.PLGA microspheres can be successfully prepared by the modified double emulsification method to achieve sustainable release of BMP-2 and VEGF and maintain their activity.2.nHAC/PLGAs scaffold possessed excellent physical properties,favorable degradation characteristics,satisfactory biocompatibility,low toxicity and potent osteoinductive effect.3.In vitro experiments,VEGF alone significantly promoted hADSCs proliferation,BMP-2 alone significantly promoted hADSCs osteogenic differentiation in nHAC/PLGAs scaffold,osteoinductive effect of simultaneous VEGF and BMP-2 treatment further enhanced,which may be mainly through increasing nuclear translocation of osterix protein by activation of the p38 MAPK pathway.4.In vivo experiments,rADSCs loaded BMP-2/VEGF sustainably releasing-nHAC/PLGAs scaffold displayed different osteogenic effect,simutaneous application of VEGF and BMP-2 can greatly promote ossification and vascularization in rabbit mandibular critical bone defect.