Three-Dimensional Printed Cobalt-Doped Bioceramic Scaffolds Mediate Osteogenic And Angiogenic Coupling To Direct Osteogenic Tissue Formation For Bone Repair

Part?.Preparation,characterization and cytotoxicity test of Co(0,1,2.5,5,10)scaffoldsObjective:To prepare porous bioceramic scaffolds doped with different cobalt content,investigate the effect of different amounts of cobalt on CLP microstructure,physical properties and chemical properties,and detect cytotoxicity for preliminary screening of biocompatibility.Methods:(1)The powders were synthesized by a solid phase sintering method.The corresponding chemical formulation was Ca_(10-x)Co_xLi(PO₄)₇,in which x%=0,0.1,0.25,0.5 and1mol%.The porous scaffolds were fabricated by SE-3D printing processes,named Co0,Co1,Co2.5,Co5,Co10 respectively.(2)Co(0,1,2.5,5,10)scaffolds were characterized by XRD,Raman and SEM.(3)The compressive strength of Co(0,1,2.5,5,10)scaffolds were measured by a universal testing machine.(4)The Mineralization of the Co(0,1,2.5,5,10)scaffolds in vitro were investigated by XRD and SEM.(5)The degradation property of the Co(0,1,2.5,5,10)scaffolds in vitro were evaluated by weight loss and p H value variation.(6)The cytotoxicity assessment of Co(0,1,2.5,5,10)scaffolds were evaluated by MTT assay.Results:(1)The addition of Co turned the color of CLP purple,and the color mutation occurred in the Co10 scaffold.(2)The XRD patterns of Co(0,1,2.5,5,10)scaffolds showed that with increasing cobalt doping content,the XRD profile moved towards a slight right shift as a whole.The Raman spectrum showed with increasing cobalt content,the bands at 950 cm^-1 and968 cm^-1shifted to the higher Raman direction.SEM surface morphology manifested the crystal size in Co(0,1,2.5,5)scaffolds decreased gradually with the addition of the cobalt dopant.When the doping percentage was up to 1 mol%,some melted crystals and microcracks were observed on Co10 scaffold surface.(3)The compressive strength tended to decrease gradually with increasing content of cobalt doping.Remarkably,the compressive strength sharply declined when the cobalt doping percentage exceeded 0.5 mol%.(4)The mineralization phases of Co(0,1,2.5,5)scaffolds were hydroxyapatite.However,the Co10 scaffolds showed none of the changes.(5)The weight loss of Co(0,1,2.5,5,10)scaffolds tended to increase over time.The p H value of Tris-HCl soaking Co(0,1,2.5,5,10)scaffolds increased synchronously.(6)Co(0,1,2.5,5)scaffolds did not show cytotoxicity,while the Co10 scaffold displayed obvious cytotoxicity.Conclusions:(1)Co²⁺ions were successfully doped into CLP.Doping was performed with the intention of replacing the Ca cations from the CLP with Co cations.The doping of Co²⁺has a significant effect on the structure of CLP,and the greater the doping amount,the more obvious the effect,especially when the doping content of cobalt is 1 mol%,the CLP structure has a significant mutation.(2)Although the compressive strength of Co(1,2.5,5)scaffolds is reduced,it is still within the range of the compressive strength of human bone trabeculae and can withstand external pressure and resist fracture.(3)Co(0,1,2.5,5,10)scaffolds can be degraded to a certain extent in vitro.(4)Co(0,1,2.5,5)scaffolds were mineralized in vitro,while Co10 scaffolds did not.(5)Co(0,1,2.5,5)scaffolds had no cytotoxicity,while Co10 scaffolds had cytotoxicity.Part?.Biocompatibility of C_O(0,1,2.5,5)scaffoldsObjective:To investigate the effects of Co(0,1,2.5,5)scaffolds and extracts on the adhesion and proliferation of rBMSCs.Methods:(1)BMSCs were isolated by bone marrow adherent method.Cell growth curve was plotted by MTT assay.Cell surface markers(CD44,CD45,CD90,CD20)were detected by flow cytometry,and osteogenic as well as adipogenic differentiation were applied to identify BMSCs.(2)Co(0,1,2.5,5,10)extracts were prepared,and the ion composition and concentration in the extracts were detected by ICP-OES.(3)Co(0,1,2.5,5)extracts were co-cultured with rBMSCs,and CCK-8 assay and live/dead cells staining assay were performed at preset time points.(4)Co(0,1,2.5,5)scaffolds were co-cultured with rBMSCs,and CCK-8,SEM and CLSM tests were performed at preset time points.Results:(1)P0?P7 rBMSCs were in the shape of long fusiform-shape and fibrous-like cells,and the cells were arranged in a“vortex”shape.P7 rBMSCs still maintained a better growth state.The positive rates of CD90,CD44,CD29,and CD45 detected by flow cytometry were 99.36%,99.77%,99.77%and 0.88%respectively.ALP staining and alizarin red staining were positive after osteogenic induction in vitro,and oil red O staining was positive after adipogenic induction.(2)All Co-doped scaffolds desorbed a certain amount of Co²⁺,and the desorption of Co²⁺raised with ascending Co content,while no Co²⁺ions were detected in the Co0extract.Additionally,the release of Ca²⁺and Li⁺in Co(0,1,2.5,5,10)extracts remained relatively stable.(3)With the extension of culture time,the extracts of all groups could promote the proliferation of rBMSCs,and Co(0,1,2.5)extracts promoted the proliferation of rBMSCs significantly,while the proliferation in Co5 group was lower than that of Co(0,1,2.5)group at each time point,and the differences were statistically significant(P<0.05).The Co(0,1,2.5,5)groups were dominated by living cells(green),while the number of dead cells(red)was very few.With the extension of culture time,the views of dead cells increased slightly in each group,and the Co5 group was the most.(4)With the prolongation of culture time,all scaffolds could promote the proliferation of rBMSCs.Among Co(0,1,2.5,5)scaffolds,Co2.5 scaffold promoted the proliferation of rBMSCs significantly after culture for 5 d and 7 d,and the differences were statistically significant compared with Co(0,1,5)scaffolds(P<0.05).SEM showed that there were no significant differences in the cell morphology of rBMSCs on the surface of Co(0,1,2.5)scaffolds,showing typical fibroblasts and abundant filamentous pseudopods,while the number of rBMSCs on the surface of Co5 scaffold was relatively small and few protrusions of pseudopods.CLSM observation showed that the cell morphology of rBMSCs on Co(0,1,2.5,5)scaffolds was regular,presenting a typical long spindle shape,and the cell body was extended,extending pseudopods of different lengths to connect with neighboring cells.Conclusions:(1)rBMSCs were successfully isolated by bone marrow adherent method.(2)With the prolongation of co-culture time,Co2.5 extract and scaffold significantly promoted the adhesion,extension and proliferation of rBMSCs,showing good biocompatibility.Part?.The effect of Co(0,1,2.5,5)extracts on the osteogenic differentiation of rBMSCsObjective:To explore the property of Co(0,1,2.5,5)scaffolds to promote osteogenic differentiation of rBMSCs.Methods:(1)Co(0,1,2.5,5)extracts were prepared for culture of rBMSCs.(2)To investigate the osteogenic properties of rBMSCs induced by Co(0,1,2.5,5)extracts,ALP staining,ALP activity,alizarin red staining and calcium content were detected,and the relative expression of osteogenesis-related proteins(ALP,BMP-2,Runx2 and OCN)were determined by western blot at different preset times.Results:(1)When rBMSCs were induced by Co(0,1,2.5,5)extracts for 7 days,the cells in each group were stained dark blue.With the extension of the induction time,the ALP staining intensity of each group on day 14 was significantly higher than that on day 7,which was blue-black,and the staining intensity of Co2.5 group was significantly stronger than that of the other groups.The ALP activity in Co(0,1,2.5,5)groups was lower than that in OM group at 7d of induction,and the differences were statistically significant(P<0.05).After 14 d of induction,the ALP activity in Co2.5 group was significantly increased compared with that in other groups(P<0.05).(2)After 21days of induction,red or orange mineralized nodules were observed in the Co(0,1,2.5,5)groups.The number of mineralized nodules in Co(1,2.5)groups and OM group were more than that in Co(0,5)groups.The Ca content in Co2.5 group was significantly higher than that in Co(0,1,5)groups,the differences were statistically significant(P<0.05),but lower than that in OM group,the differences were not statistically significant(P>0.05).(3)After 14 days of induction,the expression levels of ALP and BMP-2 in Co2.5 group were higher than that in Co(0,1,5)groups,with statistical significance(P<0.05).Although the expression of OCN among Co(1,2.5,5)groups were no significant difference,the expression of OCN in the Co2.5 group was significantly higher than that in the Co0 group,with statistical significance(P<0.05).The expression of Runx2 in Co2.5 group was also higher than that in Co(0,5)groups,but slightly lower than that in Co1 group,with no statistical significance(P>0.05).Conclusions:Co(0,1,2.5,5)extracts all promoted the osteogenic differentiation of rBMSCs,secreted extracellular matrix,and mineralized the secreted extracellular matrix.The Co2.5 group had the best osteogenesis performance.Part?.Angiogenesis induced by C_O(0,1,2.5,5)extracts in vitroObjective:To investigate the biological function of Co(0,1,2.5,5)extracts in simulating hypoxia environment and the effect on promoting HUVECs proliferation and tube formation.Methods:(1)Co(0,1,2.5,5)extracts were prepared for culture of HUVECs.(2)The effect of Co(0,1,2.5,5)extracts on HUVECs proliferation was detected by CCK-8 assay.(3)The effect of Co(0,1,2.5,5)extracts on tubule formation of HUVECs was detected.(4)The effects of Co(0,1,2.5,5)extracts on HIF-1?protein and VEGF protein expression were detected by Western blot.Results:(1)With the extension of culture time,Co(0,1,2.5,5)extracts could promote the proliferation of HUVECs.After 7 days of culture,Co2.5 group significantly promoted the proliferation of HUVECs,followed by Co1 group,Co0 group and Co5 group,the differences were statistically significant(P<0.05).(2)Co2.5 group kept better tubular structure formation than Co(0,1,5)groups.As can be seen from the quantitative analysis of the total branching length and number of branches,the values of the Co(1,2.5,5)groups were higher than that of the Co0 group at each time point,especially the Co2.5 group,with statistical significance(P<0.05).(3)Compared with Co0 group,the expression levels of HIF-1?and VEGF proteins in Co(1,2.5,5)groups were significantly upregulated,with statistical significance(P<0.05).Among Co(1,2.5,5)groups,Co2.5 group significantly increased the expression of HIF-1?and VEGF proteins.Conclusions:Co(0,1,2.5,5)extracts promoted the proliferation,migration and sprouting of HUVECs to form vascular-like structures.Compared to Co(0,1,5)groups,the number of vascular-like structures formed in Co2.5 group was more and the structure morphology was intact.In comparison with the Co0 group,Co(1,2.5,5)groups stabilized the expression of HIF-1?protein in rBMSCs to simulate hypoxia environment,and then upregulated the expression of VEGF protein to promote angiogenesis,among which the Co2.5 group had the excellent angiogenesis performance.Part?.Repair of cranium bone defects by Co2.5 scaffolds in vivoObjective:To investigate the feasibility of Co2.5 scaffold in repairing cranium bone defects,and evaluate the capacity of promoting new bone formation and blood vessels formation in vivo.Methods:(1)A single cranium bone defect model with a diameter of 5 mm was prepared in 18 SD rats.They were randomly divided into three groups:blank group,Co0 group and Co2.5group,with 6 bone defects in each group(n=6).Cranium specimens were harvested at 8w after operation.(2)General observation,micro-CT,histological and immunohistochemical methods were applied to analyze the in vivo blood vessel formation and bone regeneration of the Co2.5 scaffolds selected on the basis of the in vitro assessment.Results:(1)One SD rat in the Co0 group died of overdose of anesthetic after operation,and was immediately supplemented.The incision of all SD rats was healed without redness,swelling,exudation,purulent,skin rupture and necrosis.(2)Micro-CT analysis showed that bone volume fraction in the Co2.5 group was superior to that in the Co0 group and the blank group,and the differences among groups were statistically significant(P<0.05).BMD in each group from high to low was Co2.5 group,Co0 group and blank group respectively,and the differences among groups were statistically significant(P<0.05).(3)HE staining analysis showed that the percentage of new bone formation area from high to low was Co2.5 group,Co0 group and blank group,and the differences between groups were statistically significant(P<0.05);Masson staining displayed that there was a small amount of immature new bone tissue in the defect edge of blank group,while the mature new bone tissue dominated in CO0 group and CO2.5 group,and the amount of new bone tissue in Co2.5 group was significantly more than that in Co0group;(4)CD31 immunohistochemical analysis showed that the number of blood vessels in Co0 group and Co2.5 group was more than that in blank group,and the differences were statistically significant(P<0.05).The number of blood vessels in Co2.5 group was more than that in Co0 group,and the differences were statistically significant(P<0.05);Col-?immunohistochemical analysis revealed that Col-?was negative in blank group,while positive in Co0 group and strong positive in Co2.5 group?Conclusions:The Co2.5 scaffold can significantly promote new bone formation and blood vessels formation in vivo,and the capacity to promote repair of bone defect in rats is significantly better than that of Co0 scaffold and blank group.