Preparation And Related Research Of 3D Printing Magnesium/?-tricalcium Phosphate/rhBMP2-PLGA Composite Porous Scaffold

ObjectiveOsteonecrosis of the Femeral Head is a clinically common and intractable disease,and its incidence has been younger in recent years.Repairing the femoral head by hip-preserving surgery,retaining the hip joint of the patient,delaying or even avoiding artificial hip replacement has high clinical and social value.The bone defect area caused by the removal of necrotic bone tissue after hip-preserving surgery needs to be filled with bone implant material.However,various bone implant materials currently used in clinical practice have different degrees of disadvantages.In this study,a new type of bone tissue engineering scaffold,Mg-TCP/rhBMP2-PLGA(Mg-TCP/rhBMP2-PLGA)composite porous scaffold,was developed by 3D printing technology combined with ion-doped biomaterial modification and loaded controlled release drug-loaded microspheres.The composite porous scaffold was comprehensively evaluated from the aspects of physical and chemical properties,bone repair and reconstruction performance,and angiogenesis performance in vitro and in vivo.MethodsThis paper is composed of four experimental contents:(1)Preparation and physicochemical properties of 3D printed magnesium ion doped ?-tricalcium phosphate porous scaffold:Magnesium oxide was used as the source of magnesium ion,and different proportion of magnesium doping was set to modify ?-tricalcium phosphate by ion doping.According to the preset model,several groups of modified materials were prepared into porous scaffolds by using 3D printer and high temperature sintering.The morphological observation,porosity measurement,mechanical property measurement,phase analysis,elemental composition analysis and ion release behavior analysis in vitro degradation were used to comprehensively characterize the scaffold.The advantages of porous scaffolds in physicochemical properties were discussed.(2)Study on the biological properties of 3D printed magnesium ion doped ?-tricalcium phosphate scaffold:Using real-time extraction method,the effects of magnesium ions released during scaffold degradation on the behavior of hBMSCs and HUVECs were explored in vitro.The synthesis of magnesium phosphate was analyzed from cell proliferation,viability,morphology,osteogenic differentiation of hBMSCs and angiogenesis of HUVECs.The effect of tricalcium porous scaffold on cell behavior was initially explored to optimize the optimal incorporation ratio of magnesium ions.(3)Preparation and biological properties of 3D printed magnesium/?tricalcium phosphate/rhBMP2-PLGA composite porous scaffold:The rhBMP2PLGA sustained-release microspheres were prepared by double emulsion emulsification and volatilization method.The sustained release microspheres were combined with the initially selected porous scaffolds by gelatin coating to synthesize Mg-TCP/rhBMP2-PLGA composite porous scaffold.The morphology of sustained-release microspheres and composite scaffolds were observed by SEM.The encapsulation efficiency,drug loading and drug release performance of drug-loaded microspheres were detected by ELISA.The effects of Mg-TCP/rhBMP2-PLGA composite porous scaffold on bone tissue repair and angiogenesis were studied in vitro from cell proliferation,osteogenic differentiation of hBMSCs and angiogenesis of HUVECs.(4)In vivo bone repair performance of 3D printed magnesium/?tricalcium phosphate/rhBMP2-PLGA composite porous scaffold:Eight-week-old New Zealand white rabbits were selected as experimental subjects.Three groups of Mg-TCP/rhBMP2-PLGA composite porous scaffolds were implanted into the standard bone defect model of the lateral femoral condyle.At each time point,a comprehensive evaluation was made from the aspects of gross specimen observation,micro-CT and histology(pathological section).The effect of 3D printed magnesium/?-tricalcium phosphate/rhBMP2-PLGA composite scaffold on the repair and reconstruction of bone defects in vivo was discussed.Results(1)The prepared magnesium-doped tricalcium phosphate porous scaffold exhibits a porous surface structure similar to the preset model and a completely penetrating internal pore structure.SEM images showed that the edge length of the square micropore on the top and bottom of the porous scaffold was about 400 ?m,the edge length of the rectangular micropore on the side was 300 ?m x 100 ?m,and the diameter of the scaffold fiber was about 450 ?m.The surface of the scaffold fiber is highly crystalline and the elements are evenly distributed on the surface of the fiber.The porosity of the prepared porous scaffold was between 61.23%and 65.02%,and there was no significant difference between the four groups(P>0.05).The mechanical properties of porous scaffolds doped with magnesium ions can be significantly improved.Compared with pure tricalcium phosphate scaffolds,the mechanical properties of the scaffolds doped with magnesium ions have been improved in varying degrees,and the scaffolds doped with 1 wt.%magnesium oxide exhibit the highest compressive strength and Young' s modulus(P<0.01).Phase analysis results show that the main crystalline form of the four groups of materials after high temperature sintering is?-TCP,and a small amount of hydroxyapatite(HA)is formed;a small part of tricalcium phosphate in pure tricalcium phosphate group undergoes ?to-alpha phase transition;and a new compound,calcium magnesium phosphate,is formed in the three groups doped with magnesium.In the process of simulated degradation in vitro,the release of calcium ion was relatively stable,and the release concentration of phosphorus ion showed an upward trend with time.The concentration of magnesium ion released by the three groups of magnesium-doped scaffolds was positively correlated with the mass ratio of magnesium oxide,and gradually decreased with the extension of degradation time.(2)The cultured hBMSCs and HUVECs induced by real-time extracts of four groups of Mg-TCP porous scaffolds exhibited Mg ion concentrationdependent cell behavior.?Cell proliferation:With the prolongation of culture time,the proliferation of hBMSCs showed an upward trend;there was no significant difference in the proliferation of hBMSCs cells between the four groups at the early stage of induction culture;the proliferation of hBMSCs cells in the 1 wt.%MgO-doped group was significantly higher than that in other groups at the middle and late stage of culture(P<0.01).HUVECs cultured in pure tricalcium phosphate group and 1 wt.%magnesium oxide doped group showed an increase in the number of cells at each time point,and 1 wt.%magnesium oxide doped group was the best(P<0.01);the proliferation of HUVECs cultured in 2 wt.%magnesium oxide doped group was stagnated in the middle and late stage of induction culture;it is worth noting that the proliferation of HUVECs cultured in 4 wt.%magnesium oxide doped group was inhibited.?Cell viability:Both kinds of cells showed good cell viability in the four groups of porous scaffold extracts,and only a small number of cell death occurred in early culture,indicating that the four groups of materials had no obvious acute cytotoxicity.?Cell morphology:hBMSCs and HUVECs showed good cell morphology spreading and skeleton protein production in scaffold extracts of each group.?Detection of osteogenic differentiation of hBMSCs:Qualitative and quantitative results of ALP activity showed that ALP activity increased with time.The ALP activity of 1 wt.%MgO-doped group was significantly higher than that of other groups(P(0.05),and the blue-purple substances stained by cells in this group were also significantly darker than those in the other three groups.The results of alizarin red staining showed that more calcium nodules and more orange-red substances were produced in the 1 wt.%MgO doped group.RT-qPCR detection of osteogenesis-related genes showed that the abundance of Runx-2,Col-I,ALP,OCN and BSP of hBMSCs in 1 wt.%MgOdoped group was significantly higher than that in other three groups after induction culture(P<0.05).?Detection of vascular differentiation in HUVECs:NO fluorescence probe labeling confirmed that the presence of magnesium ion could significantly increase the synthesis of endothelial nitric oxide,and the HUVECs cultured in 1 wt.%MgO-doped group produced the most N0.RT-q PCR detection of vascularization-related genes generally showed that HUVECs cultured in 1 wt.%MgO-doped group expressed higher levels of VEGF and eNOS at each time point.(3)The prepared rhBMP2-PLGA microspheres were milky white particles.The results of SEM observation showed that the particle size distribution of the microspheres was relatively uniform,about 30 ?m-100 ?m;there were some tiny cavities on the surface of the microspheres;the gap between the rhBMP2-PLGA/Mg-TCP composite scaffolds was filled with gelatin and rhBMP2?PLGA.Sustained release microspheres.The entrapment efficiency of rhBMP2PLGA sustained-release microspheres was 87.32±10.83%,and the drug loading was 3.49±0.43%.The rhBMP2 showed a burst release after 24 hours of degradation in vitro,and the concentration reached 6.35±0.21 ng/mL;The release concentration of rhBMP2 showed a gradual upward trend,and the release concentration of rhBMP2 was 12.30±0.20 ng/mL on the 28th day.Biological properties:? Cell proliferation:With the passage of time,the number of cultured hBMSCs and HUVECs cells induced by three groups of MgTCP/rhBMP2-PLGA scaffold extract increased continuously,and the cell proliferation of 1Mg-TCP/rhBMP2-PLGA group was the best(P<0.01).?Osteogenic induction:ALP activity in three groups of Mg-TCP/rhBMP2-PLGAinduced cultured hBMSCs increased with the prolongation of culture time;ALP activity in 1Mg-TCP/rhBMP2-PLGA group was significantly higher than that in the other two groups(P<0.05)and the blue-purple substance stained was the darkest;RT-q PCR results showed that 1Mg-TCP/rhBMP2-PLGA composite scaffold had better ability to induce osteogenic differentiation of hBMSCs.Vascularization induction:NO fluorescence labeling results showed that 1Mg-TCP/rhBMP2-PLGA group induced the most NO in cultured HUVECs,and the abundance of vascularization-related gene expression of HUVECs induced by the composite scaffolds in each time point was better than that of the other two groups(P<0.05).(4)After the implantation of Mg-TCP/rhBMP2-PLGA composite porous scaffold,the experimental animals were in good condition,and only 2 died due to infection in the operation area.According to the observation of gross specimens,three groups of Mg-TCP/rhBMP2-PLGA composite porous scaffolds could closely bind with autologous bone tissue in the host body,fully fill the bone defect area,and new tissue grew into the pore of the scaffold;the blank control group had no obvious signs of bone repair,and the defect area was depressed.Micro-CT images showed that the implanted Mg-TCP/rhBMP2-PLGA composite porous scaffolds were closely embedded in cancellous bone without obvious bone resorption around the scaffolds;there were different degrees of degradation and absorption around the scaffolds 8 weeks after operation;different degrees of bone tissue repair and reconstruction could be seen in the pore of the scaffolds in the implantation group;the regeneration of new bone in the 1Mg-TCP/rhBMP2PLGA composite porous scaffolds implantation group was the most significant.Histological observation showed that new fibrous tissue and bone tissue grew in the pore of scaffolds in each group;the reconstruction of bone tissue in the pore of scaffolds increased significantly after 8 weeks of scaffolds implantation compared with 4 weeks of scaffolds implantation;the number of new bone tissue in 1Mg-TCP/rhBMP2-PLGA group was relatively high.Conclusion(1)A new type of bone tissue engineering scaffold,Mg-TCP porous scaffold,was successfully prepared by low-temperature extrusion 3D printing technology and high-temperature sintering,which accurately designed and matched the pore and mechanical properties of natural cancellous bone.(2)Cell experiments in vitro confirmed the concentration-dependent cell behavior of magnesium ions in hBMSCs and HUVECs,?-tricalcium phosphate mixed with 0-2 wt%of magnesium oxide has good biocompatibility,and screened out that 1 wt%magnesium oxide in ?-tricalcium phosphate could promote the osteogenic differentiation of hBMSCs and the angiogenesis of HUVECs.(3)PLGA sustained-release microspheres can ensure the local sustained and stable release of rhBMP2.The loading of rhBMP2-PLGA sustained-release microspheres can further enhance the osteoporotic reconstruction and vascular regeneration of Mg-TCP porous scaffolds,and form Mg-TCP/rhBMP2PLGA composite porous scaffolds with the trinity of "inorganic scaffoldsorganic coatings-cytokine ".(4)In vitro and in vivo osteogenesis and angiogenesis studies confirmed that the porous composite scaffolds with 1 wt%magnesium oxide and 3D printed magnesium/?-tricalcium phosphate/rhBMP2-PLGA had the best osteogenesis and vascular regeneration induction performance.This study provides a theoretical and research basis for the transformation and clinical application of 3D printed magnesium/?-tricalcium phosphate/rhBMP2-PLGA composite porous scaffolds.