Regulation Of Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells By Hydroxyapatite Bioceramics With Micropattern And Nanostructure

Bone as the hardest tissue in the human body owns the excellent mechanical property,which is closely related to its own micro/nano-hierarchical structure.The synthesized hydroxyapatite(HA)has has been widely used in bone tissue engineering due to similar component with human bone,good biocompatibility and excellent osteoconductivity.However,the traditional HA lacked the osteoinductive ability to induce osteogenic differentiation and new bone formation.It has been proven that HA bioceramics with micropattern or nanostructure could stimulate the cell adhesion,proliferation and differentiation of mesenchymal stem cells(MSCs)and osteoblasts,which is essential for bone regeneration.However,the role that micro-and nano-structure play in the promotion of osteogenesis is still unclear,and in particular,the difference of the micro-and nano-structures of HA effect on osteogenic differentiation need to be further investigated.Whether the hybrid structure combining micro-or nano-structure will enhance more excellent osteogenic differentiation is the key for designing and optimizing bioactive bone repair materials.It is a great challenge to fabricate directly a micropatterned structure on the surface of HA bioceramics because of the brittleness.It is worth noting that bone repair materials will degrade after implanting into bone defect,whether the structures can stimulate cells enough activity before degradation should be considered in the design of bone implant materials.In this study,we prepared HA bioceramics with micropattern and nanostructure,and investigated the effect of the surface structure on osteogenic differentiation.Then we researched the different role of the micro-and nano-structures played in osteogenic differentiation and the mechanism of osteogenic differentiation induced by the micro/nano hybrid structure.Finally,the timeliness of the activation was investigated for exploring the effects of the structure on osteogenic differentiation before degradation.The related contents and results were as follows:HA bioceramics with micropattern structure were prepared by dry pressing using an ordered nylon mesh as templates,and the size of micropatterns could be facilely modulated though the selection of the nylon mesh.Compared with traditional samples with flat surfaces,the fabricated HA bioceramics with micropatterned surfaces possessed better wettability and higher surface energy,which significantly promoted the adhesion,proliferation,and osteogenic differentiation of MSCs.In particular,the micopattern with a size close to that of the cell size showed a better stimulation of cell response compared with larger pattern sizes.Our study suggested that the fabrication of micropatterned structured HA bioceramics is critical for designing optimal biomaterials for bone regeneration and cell culture substrate applicationsBefore introducing the nanostructure with the controllable feature,superior biological performance and fine structure on the basis of the micropattern surface,we have tried to prepare the nanostructure on the surface of HA bioceramics with smooth structure.According to the crystallization kinetics,the p H values of the deionized water were adjusted at 3,7,and 11,respectively.The HA bioceramics with the different structures such as irregular surface roughness,and nanorods were obtained using hydrothermal treatment.The results showed that the surface structure could stimulate the cell adhesion,proliferation and osteogenic differentiation of the osteoblasts.Besides,compared to the irregular roughness,the surface with nanorods possessed better ability to promote osteogenesis.Therefore,we would select the nanorod structure to combine with the micropatterned structure.In addition,we used EDTANa2 Ca as the calcium source to optimize the nanorod structure,and the optimized nanorod structurte was introduced into bioceramics with the micropattern as the secondary structure.The study would lay the foundation for the introduction of nanostructure on HA bioceramics with micropattern structure.Based on the previous researches,HA bioceramics with micro/nano hybrid structure were prepared through combining template method and hydrothermal method.It was found that the micropattern,nanostructure and micro/nano hybrid structure could enhance the selective adsorption of proteins,especially for micro/nano hybrid structure,which was closely related to integrins activation.Furthermore,the micropattern,nanostructure and micro/nano hybrid structure have been proved to stimulate the cell adhesion,proliferation and differentiation of MSCs.Compared to the single-scale micropattern and nanostructure,the micro/nano hybrid structure showed a synergistic effect on osteogenic differentiation.Interestingly,in the process of inducing osteogenic differentiation,the micro-and nano-structures played different roles in the activation of integrins,BMP2 signaling pathway and cell-cell communication,which may result in the synergistic osteogenic effect of the micro/nano hybrid structure.In addition,the reason of the synergistic effect of the micro/nano hybrid structure on integrins activation and osteogenic differentiation,was not only the direct activation mechanism of the structure when contacted with cells,but also the indirect effects of surface structure on the promotion of biological activity by promoting the selective adsorption of proteins.Among them,the direct mechanism could be summarized as follows: When the cells are implanted on the surface of biomaterials,the micro/nano-structure first activated integrins,and then further activated BMP2 signaling pathway and cell-cell communication,which the activated BMP2 could in turn activate integrins and Cx43-related cell-cell communication.According to the previous three parts,HA bioceramics with the micropattern,nanostructure and micro/nano hybrid structure were fabricated to explore the stimulate time of the micro-and nano-structures affect on cells,which could lead to the continuous osteogenic differentiation.In addition,how long the structure has been removed,the activated osteogenic effect could still exist.The results revealed that the micro/nano hybrid structure had an early activation effect on the osteogenic differentiation,and the osteogenic ability still had a sustained expression after removing biomaterials.In addition,our study also found that the longer the time of the surface structures effected on cells,the stronger the activation effect in the late period after removing the structure.Moreover,compared with the single-scale structure,cells were stimulated by the hybrid structure in the early stage,the continuous osteogenic differentiation expression was also the most obvious,and the difference between the micro-structure and nano-structure was not significant.Therefore,we need to consider the stability of the structure when designing materials with surface structures in the future.