Biomimetic Mineralization Preparation And Properties Of Calcium Phosphate Implant Coatings

Calcium phosphate（CaP）is frequently used as coating materials for bone implants to promote osseointegration.Different surgical conditions or different skeletal bone sites usually have different requirements for the osseointegration of CaP coatings.The micro-and nano-scale structure of CaP can significantly affect its physicochemical properties,including degradation rate and its effect on cell biological behavior.Therefore,it is expected to prepare the CaP with customized osseointegration properties via regulating the micro-nano structure of CaP coating.However,commercial CaP coatings via plasma spraying display similar microstructures,and it is difficult to control the micro-nano structure of the coating.Taking into account the mild conditions and limited raw materials of natural biominerals,the non-classical nucleation growth process can effectively control its crystal size,morphology,orientation,phase and other micro-nano structure characteristics.Herein,inspired by the formation of natural biominerals with various morphologies mediated by amorphous precursors,CaP coatings with tunable microstructures were fabricated.The microstructures of the coatings were precisely controlled by both polyaspartic acid（p Asp）and Mg²⁺.In vitro degradation experiments showed that the degradation rates of CaP coatings with different microstructures were different.The MC3T3-E1 cell biological behaviors on the CaP coatings with different microstructures exhibited significant differences.Furthermore,in vivo experiments demonstrated the osseointegration in different types of rats and skeletal bone sites indeed favored different CaP coatings.We anticipated this biomimetic strategy can be used to fabricate customized bone implants that can meet the specific requirements of different surgery conditions or different skeletal bone sites,and finally realize the precision of implant surgery.Part Ⅰ Calcium phosphate coatings with different microstructures on the titanium substrateObjective:In this experiment,calcium phosphate coatings with different microstructures were fabricated on titanium substrates via biomimetic mineralization.Methods:（1）The type Ⅰ collagen solution was spin-coated or dip-coated on the hydrophilic treated substrate surface,and the calcium phosphate coating with different microstructures can be obtained by regulating the ingredient,time,pH and other factors of the mineralizing solution during in-situ mineralization.（2）The surface morphology of the calcium phosphate coating and the cross-sectional thickness of the coating were observed by scanning electron microscopy（SEM）.（3）Energy-dispersive X-ray analysis（EDX）and mapping were performed to examine the chemical composition of the coatings.（4）Chemical surface analysis of the coatings was performed via Fourier transform infrared spectroscopy（FTIR）and X-ray diffraction（XRD）.（5）Further,composite coatings（vertical section thickness of approximately50–70 nm）,which were prepared via focused ion beam（FIB）microscopy,were characterized via high resolution transmission electron microscopy（HRTEM）.Results:（1）Through biomineralization technology and layer-by-layer mineralization strategy,four CaP coatings with different microstructures were prepared in this chapter,namely crystalline fHAP coating with flower-like morphology;loosely arranged tHAP coating with 500 nm particles;cCaP coating with a bilayer amorphous-crystalline phase structure;cHAP coating with a bilayer crystalline structure;the thickness of these four coatings was about 2-3μm.（2）By SEM observation,the fHAP coating surface presented a flower-like morphology,which was vertically arranged on the substrate.The tHAP coating consisted of loosely intertwined HAP nanospheres with a diameter of approximately 500 nm.Further,the cCaP coating consisted of two distinct layers.The top thicker layer consisted of large nanoparticles that grew loosely on a collagen scaffold,whereas the bottom thin layer consisted of closely packed tiny granules.The cHAP coating also has a two-layer structure,with crystalline HAP particles permeating the fHAP pattern surface and in the interstices.（3）EDX-Mapping confirmed the presence of calcium,phosphorus,and nitrogen in the coatings.（4）Further,the FTIR spectra of all the coatings showed absorption band characteristics corresponding to theν3 asymmetric stretching(1046cm^-1)andν4 antisymmetric bending(602 and 561 cm^-1)of PO4,which can be attributed to crystalline HAP.Furthermore,the XRD patterns of the coatings all showed a main peak at approximately 2θ=31.7°as well as some other peaks that were characteristic to HAP,indicating that the crystalline phase in the coatings was essentially HAP.（5）The large nanoparticles in the top layer of the cCaP coating was determined to be HAP based on the fast Fourier transform（FFT）analysis of the corresponding HRTEM image.The bottom layer of the cCaP does not show any diffraction spots in the selected area electron diffraction（SAED）patterns,indicating that it was characterized by an amorphous structure,consisting of tiny granules.The fabrication of the cHAP coating involves an intermediate stage,in which an ultrathin ACP layer was overgrown on a thick flower-like HAP layer.The ACP layer crystallized after soaking in the inducing solution;thus,all the layers in the cHAP coating were crystalline.Conclusion:（1）The fHAP coating can be fabricated through the classical crystallization process.（2）ACP as a precursor can induce the formation of a partially transformed cCaP composite coating and a fully transformed tHAP coating.（3）Through a layer-by-layer mineralization strategy,bilayer crystalline cHAP coatings can be constructed.（4）CaP coatings with different microstructures can be prepared by regulating the factors such as the composition of mineralization and inducing solution,time and pH.Part Ⅱ The degradation rate of calcium phosphate coatings with different microstructures in vitroObjective:The degradation rate of CaP coatings with different microstructures were detected,and the viability of changing the properties of CaP coatings by microstructure design was verified.Methods:（1）The degradation kinetics of the CaP coatings with different microstructures were evaluated based on the release of calcium ions in an in vitro simulated environment.Specifically,the CaP coatings were soaked in phosphate-buffered saline（PBS）with pH=5 and without calcium and magnesium for30 days to detect calcium ion release and pH in the solutions at 27℃.（2）The mercury intrusion test was used to detect the specific surface area of the CaP coatings.（3）The morphology changes of calcium phosphate coatings before and after degradation were observed by SEM.Results:（1）It was observed that the calcium ion concentration and the pH of the solutions of all the CaP coatings exhibited a similar pattern,except for their final equilibrium values.On day 1,the calcium ion concentrations in all the solution increased rapidly.Thereafter,the increase was gradual until the maximum on the seventh day,after which they decreased slightly.Finally,a stable plateau was attained.The fHAP coating has the highest degradation rate,while the tHAP coating has the lowest degradation rate,with those of the cHAP and cCaP coating in between.（2）The largest volume of mercury intrusion corresponded to the fHAP coating（0.0181 m L）,followed by the cHAP coating（0.0057 m L）,cCaP coating（0.0033 m L）,and tHAP coating（0.0024 m L）.Statistical analysis showed that the differences in the specific surface areas of the four coatings were statistically significant（P<0.05）.Conclusion:（1）Even though the four CaP coatings has a similar composition,they exhibited quite different degradation rates.（2）The degradation rate of CaP coatings may be related to the specific surface area and radius of curvature conferred by different microstructures.The larger the specific surface area and the smaller the radius of curvature of the coatings,the faster the degradation rate of the coatings.Part Ⅲ Biological behavior of MC3T3-E1 cells on calcium phosphate coatings with different microstructuresObjective:By studying the biological behavior of MC3T3-E1 cells on CaP coatings with different microstructures,the viability of changing the interaction with osteoblasts through the design of implant microstructures was verified.Methods:（1）To observe the cell adhesion on the CaP coatings via SEM.（2）The spreading behavior of the MC3T3-E1 cells on the CaP coatings was further investigated by laser scanning confocal microscopy.（3）The biocompatibility of the CaP coatings was detected by fluorescent staining of live and dead cells.（4）The effect of the MC3T3-E1 cells on the proliferation ability of the CaP coatings was detected by CCK-8 kit.（5）To assess the osteogenic differentiation trends of the CaP coatings with different microstructures,alkaline phosphatase（ALP）activity analysis and alizarin red staining（ARS）were employed to quantify the differentiation levels of osteoblasts mineralization.（6）The effect of CaP coatings on the osteogenesis-related genes expression,such as ALP,OPN,OCN and RUNX2,was detected by RT-q PCR.Results:（1）The morphologies of MC3T3-E1 cells were observed for all the groups.After 24 h of culture,cells corresponding to the titanium control group were found to be elongated.Although the morphology of the cells on the fHAP coating and the control coatings were similar,CaP particles could be observed on the cells cultured on the fHAP coating.Furthermore,while the MC3T3-E1 cells cultured on all the CaP coatings showed better cell adhesion and more filopodia than those cultured on bare titanium,the cells cultured on the cCaP coating stretched much shorter filopodia than the others.（2）In this regard,confocal laser scanning microscopy results showed that,compared with the titanium control group,the cells grown on the surfaces of the CaP coatings spread more extensively and have numerous cell projections with randomly arranged cytoskeletal actin filaments,even though the cell morphologies of the various CaP surfaces were different.（3）In contrast to the titanium control group,the viability of the cells cultured on the CaP coatings after 3days were considerably higher,which was confirmed by the predominance of green（live）over red（dead）cells.Additionally,the live/dead cell ratios of the MC3T3-E1cells corresponding to all the groups were over 90%.（4）The results of CCK-8 assay showed that after culturing for 1,4,and 7 days,the MC3T3-E1 cells in all the CaP coating groups showed enhanced proliferation compared with those corresponding to the titanium control group.（5）The fHAP coating showed a statistical significantly higher ALP activity,followed by the cCaP,cHAP,and tHAP coatings.Further,the differences between the groups showed statistical significance（P<0.05）.（6）The ARS results indicated that significantly higher matrix mineralization nodule formation on all the CaP coatings than that on the control substrate.Spectra data further indicated that the cCaP coating showed the highest osteogenic differentiation of MC3T3-E1cells among the five groups（P<0.05）.（7）After the 3 and 7 days of cell cultivation on the coatings,the expressions of genes,including OCN,OPN,ALP and RUNX2,were all up-regulated compared with the control group.The relative expression level of ALP and RUNX2 were the highest in the fHAP coating,while the relative expression level of OCN and OPN were the highest in the cCaP coating.Conclusion:（1）Compared with the control group,CaP coatings with different microstructures were beneficial to the adhesion,proliferation and differentiation of MC3T3-E1 cells.（2）The CaP coatings with different microstructures have different promoting effects on the early osteogenic ability of MC3T3-E1 cells.（3）CaP coatings with different microstructures have different effect on the relative expression of osteogenesis-related genes.Part Ⅳ In vivo studies of calcium phosphate coatings with different microstructuresObjective:To investigate whether the CaP coatings with different microstructures can meet the different demands for different rats and skeletal bone sites in vivo osseointegration.Methods:（1）Six-week-old male SD and GK rats（24 animals for each type）were used in this study.Four implants（two oral and two femur）were inserted into each of the animals.At 2 and 4 weeks post-implantation,the rats were sacrificed.（2）The implants were divided along the longitudinal axis of the bone implants using an Exakt saw and ground to have thickness in the range 30-40μm.And then,staining using methylene blue–acid fuchsin was then performed to evaluate bone-implant contact（BIC）values.（3）3D images were reconstructed to measure new bone formation around the peri-implant bone interfaces（100μm）by Micro-CT.Furthermore,the bone volume/total volume（BV/TV）values were calculated based on a Micro-CT examination.（4）The mineral apposition rate（MAR）was calculated as the distance between the midpoint of the corresponding edge of the two labels divided by the time difference between the labeling periods using Image Pro Plus software.Results:（1）From the staining results,it was found that the portion stained in pink was newly formed bone tissue.The BIC of fHAP coating in the oral implant of SD rats was the highest,while the BIC of cCaP coating in the femur implant of SD rats was the highest.And the differences were statistically significant（P<0.05）.The BIC of cCaP coating was the highest in the oral implant of GK rats,and the BIC of tHAP coating was the highest in the femur implant of GK rats,and the differences were statistically significant（P<0.05）.（2）BV/TV and MAR results were consistent with BIC results.Conclusion:Different surgical conditions prefer different CaP-coated bone implants.CaP coatings with different microstructures mediated by ACP biomimetic strategies can provide an option for the preparation of personalized bone implants.