| Bone defect is a common clinical disease,which often requires the auxiliary treatment of bone implants.Autologous bone graft is still the clinical golden standard,but suffers from limited supply.Thus,the application of artificial biomaterials as alternative bone grafts,which can induce bone regeneration,is the key direction of the development of regenerative medicine and biomaterials.Calcium phosphate(Ca P)ceramics have inorganic components similar to natural bone.Due to their good biocompatibility and bioactivity,Ca P ceramics have been widely used as surface active coating for metal implants and bone filling repair materials.At present,Ca P ceramics fabricated by the traditional hydrogen peroxide foaming method have achieved satisfactory results in clinical application,attributing to their good osteoinductive capacity.However,due to the limitation of mechanical strength and tailorability,the foamed Ca P ceramics are only used to repair bone defects in nonload-bearing sites,and they are difficult to match the repair of bone defects with complex structure.In addition,it is hard to precisely control pore structure and pore size of porous Ca P ceramics prepared by the traditional methods,which affects the further optimization of their material properties and the deepening of mechanism research.In recent years,the advances of 3D printing technology has greatly promoted its application in the field of orthopaedic biomaterials,as it shows a unparalleled advantage in realizing personalized customization and precise control of the threedimensional(3D)structure of the scaffolds,especially for the ones with irregular and complex structure.At present,the good bioactivity of 3D printed bioceramics has been widely reported,but its osteoinductive capacity needs to be further improved.Therefore,our research focused on the structural design and performance optimization of Ca P bioceramics,based on the concept of advanced material genetic engineering and high-throughput experimental technology.We optimized the formula of photocurable slurry and computer-aided design of 3D structure model,and then used DLP-based lithography 3D printing technique to design and fabricate Ca P ceramic scaffolds with different porous structures.Their biological activities were further evaluated to screen and optimize the pore structure of scaffolds.We also constructed the coating layer loaded with active bone growth factor on Ca P ceramic surface to further improve the osteoinductivity of scaffolds.Meanwhile,we explored the application of high-throughput experimental technology to screen and optimize the osteoinductive properties of bioceramic scaffolds through the integrated preparation and biological evaluation of multi-structure composite Ca P scaffolds.These studies hold promise in providing the experimental and methodological foundation to reveal the relationship between material features and osteoinductivity.Firstly,in this study,the preparation procedure of high-performance Ca P ceramic scaffold was established by using stereolithography-based 3D printing technology through a DLP system with high precision forming characteristics.In order to increase the solid loading of Ca P powder in photosensitive resin premix,according to the principle of similar solvent compatibility,5.5 % MAEP was adopted to modify ceramic powder,which effectively improved the affinity of ceramic powder and photosensitive resin.Then,the photocurable slurry with a low viscosity and a high solid loading was prepared.Considering that abundant microporous structure of ceramic scaffold is an important factor to achieve its good biological activity,we introduced edible toner as secondary pore-forming agent in the slurry.On the premise of ensuring the printability of the slurry,the content of carbon powder was increased as much as possible to raise the microporosity.Thus,the slurry was optimized by containing the high solid loading of 50 wt.% Ca P powders and 2 wt.% tonors.The photocuring performance of the slurry was tested by measuring the transmission depth and critical exposure intensity,which were the two important parameters of photocurable 3D printing.The parameters were determined to match the highprecision 3D printing with slice thickness of 50 μm.Combined with 3D modeling software,three kinds of representative porous structures were designed,including Cube,Octet-truss,and Inverse-fcc,and then fabricated by using DLP-based 3D printing.The size errors of green body and model design of the three model structures were all less than 1 %.After degreasing and sintering,there were no residual additives such as resin and carbon powder left in Ca P scaffolds.Compared with green body size,the shrinkage rate of the ceramics with three model structures were both ～ 30 % in the horizontal direction(X-Y axis)and vertical direction(Z axis).Printing layers were tightly bonded without delamination,which proved the stability and reproducibility of the DLP process.The observation of micro-morphology showed that there were abundant micropores with sizes in submicron to micron scale on the pore wall of the Ca P scaffolds,which combined with the macropores constructed by computer-aided design to form a 3D interconnective hierarchical pore structure.We compared the physical,chemical and mechanical properties of three Ca P scaffolds of different structures,finding that the Inverse-fcc scaffold with a porosity of 70.2 % had the widest macroscopic pore size distribution and the highest mechanical strength.Secondly,the biological properties of the above three kinds of printed Ca P scaffolds were evaluated in vitro and in vivo.The widely-used porous Ca P ceramic prepared by hydrogen peroxide foaming(Foam)was used as positive control.The results of simulated body fluid(SBF)immersion experiment showed that three groups of printed ceramics and Foam ceramic could form abundant bone-like apatite at day3,suggesting that all of Ca P ceramics had good biological activity.The scaffolds were co-cultured with mouse bone marrow mesenchymal stem cells(BMSCs)in vitro.The results showed that there was no significant difference in adhesion,proliferation and spreading of BMSCs on three groups of printed scaffolds,but the expression of osteogenesis-related genes in Inverse-fcc group was slightly higher than that in Cube and Octet-truss groups.However,the proliferation,spreading,and expression of osteogenic genes of BMSCs on Foam ceramic were better than those on three groups of printed ceramics.Later,four groups of ceramic samples were implanted into the thigh muscle of mice,and the samples were harvested for tissue section and immunohistochemical staining analysis at 60 and 90 days,respectively.The results confirmed that all groups of ceramics could induce ectopic bone formation,but Foam group exhibited significantly better osteoinductivity than three printed groups,as evidenced by more osteogenic incidence and larger new bone area.Among the three groups of printed ceramics,Inverse-fcc group had better osteoinductive capacity than the other two groups.The histological observation found that new bone tissue tended to grow along the curvature of circular hole.Therefore,these finding suggested that Inverse-fcc group was more favorable for osteogenesis,according to the analysis of the material characteristics and biological effects of 3D printed scaffolds.However,compared with Foam ceramic,the osteoinductive ability of 3D printed ceramic needed to be further improved.Furthermore,our study constructed a coating layer of sustained and controlled release of bone growth factor onto the surface of ceramic scaffold.The Inverse-fcc ceramic was modified by an active layer containing BMP-2 to obtain a composite scaffold with a sustainable release of BMP-2.A stable composite polymer coating(BCP/layer)was constructed on the surface of printed ceramics by using dopamine and dihydroxyphenylacetic acid.Then,Heparin/PEI nanogels loaded with BMP-2(NP/BMP2)were prepared by self-assembly technology.The Ca P scaffolds with BMP2-loading active layer(BCP/layer/NP/BMP2)were formed by constructing the polymer coating onto the ceramic surface through electrostatic attraction and amide bond bonding.The results of in vitro experiments showed that the loading amount of BMP-2 was positively correlated with its initial concentration.The loading efficiency of Heparin/PEI-BMP2 was higher than that of directly binding BMP-2 to the polymer coating(BCP/layer/BMP2),and continuous release of BMP-2 could be achieved for more than two weeks.Moreover,BCP/layer/NP/BMP2 showed a more stable sustained release ability than BCP/layer/BMP2 group.The results of ion release experiments showed that the construction of surface active layer only had a certain effect on the degradation and ion release of printed ceramic scaffolds in a short time,which tended to be consistent at day 7.There was little difference in the degree of degradation among different groups in the long time.Then,the in vitro and in vivo biological properties of the 3D printed Ca P scaffolds before and after the construction of active layer were systematically evaluated.In the in vitro experiment,four groups of materials(BCP,BCP/layer,BCP/layer/BMP2 and BCP/layer/NP/BMP2)were co-cultured with BMSCs,respectively.The results showed that BMSCs grown on the two groups loaded with BMP-2 showed better proliferation and spreading morphology,and the extract of BCP/layer/NP/BMP2 group could promote cell migration.The expression of osteogenic genes and marker proteins in BMSCs decreased from high to low rate as BCP/layer/NP/BMP2 >BCP/layer/BMP2 > BCP/layer > BCP.The heterotopic bone induction ability of these four groups was investigated by using the mouse thigh intramuscular implantation model.The results showed that ectopic bone formation was observed in the samples of the other three groups except for BCP after 60 days implantation.After 90 days of implantation,new bone formation was observed in all the four groups,but the BCP/layer/NP/BMP2 group had the most osteogenic incidence and largest new bone area,and the strongest expression of osteogenic marker protein OCN.In order to further investigate the bone regeneration and repair ability of printed samples before and after the construction of surface active layer,we constructed a rat femoral condyle defect model to investigate bone growth at the defect site.Micro-CT and histological analyses showed that the peripheral bone tissues grew along the scaffold after 4 weeks of implantation.The defect areas were regenerated well after 8 weeks of implantation.It was found that the bone growth in the defect area was generally consistent with the trend of intramuscular osteogenesis in mice,and the BCP/layer/NP/BMP2 group showed better repair effect than the other three groups.The above in vitro and in vivo experimental results indicate that the construction of controlled-release BMP-2 active layer was beneficial to improve the osteoinductivity and in situ bone regeneration ability of 3D printed Ca P ceramics.Finally,based on the idea of materials genome,in this paper,we creatively constructed a high-throughput experimental method to screen and optimize the osteoinduction of 3D printed Ca P scaffolds.We used DLP-based printing technology to prepare the integrated ceramic samples with twenty-four structure features including four different shapes of pores and six gradients of porosity.Then,we constructed a canine intramuscular implant model to investigate their osteoinductivity.Samples were collected after 6 months implantation,and evaluated by micro-CT and histological analysis.24 different structural parameters were analyzed at one time.The preliminary results showed that Ca P ceramic with diamond and its polyhedral composite unit structure and 70 % porosity exhibited the best osteoinductive capacity.In this study,the high-throughput experimental method was successfully used to conduct a preliminary exploration for the optimization of Ca P ceramic scaffold materials.The feasibility and effectiveness of this new experimental method in the biological evaluation of scaffold materials were proved,which laid a foundation for the subsequent systematic research and the optimization of scaffold materials for bone repair.In conclusion,our study developed a complete set of methods and techniques for the preparation of Ca P ceramic scaffolds based on stereolithography-based 3D printing technology through a DLP system,and also constructed surface active layer loaded with bone growth factor to improve the osteoinductivity of printed 3D Ca P ceramics.Meanwhile,3D printing technology and micro-CT analysis were combined to realize the integrated fabrication and evaluation of multi-structural scaffolds for high-throughput screening,which could greatly reduce the time cost and expense of traditional evaluation experiments for optimizing and screening the osteoinductivity of biomaterials.These findings provided a good experimental and methodological basis for the application of material genetic engineering in the development of novel biomaterials with desired clinic performance. |
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