The Role Of Porous Calcium Phosphate Ceramics With Different Micro-nano Structures In Inducing Osteogenic Differentiation Of BMSCs

Objectives:Biphasic calcium phosphate(BCP),as a biologically active bone substitute material,has been widely used in clinical practice.However,it still has disadvantages such as poor mechanical properties and insufficient osteoinductive activity in bone tissue engineering applications.This affects the regeneration and repair of bone defects to some extent.In this experiment,two new types of BCP porous ceramics,which whisker-reinforced BCP(BCPw)with micro-pore structure and nanoparticles(NPs)composite whisker-reinforced BCP(BCPw-NPs)with micro-nano pore structure were prepared using porous BCP as the ceramic matrix.It is expected that the modified new porous ceramics will have more abundant micro-nano pores structure.By examining the effect of different materials on the growth,proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)influence and role,and evaluation of its in vitro biological effects.Methods:(1)Preparation and characterization of BCPw and BCPw-NPs ceramic materials:Using BCP as the ceramic matrix,through a series of modification experiments,BCPw and BCPw-NPs ceramic materials were prepared respectively,using scanning electron Scanning electron microscope(SEM),X-ray diffractometer(XRD),Archimedes drainage method and specific surface meter were used to characterize the composition,micro-nano structure,porosity,water absorption and specific surface area of BCP,BCPw and BCPw-NPs.(2)Isolation,culture and identification of rabbit BMSCs:The primary cells in the bone marrow cavity of the femur,tibia and humerus of newborn New Zealand rabbits were isolated and cultured under sterile conditions.The morphological characteristics,proliferation ability,phenotype and multi-directional differentiation ability of cells were identified by comprehensive use of light microscope,CCK8 experiment,immunofluorescence staining experiment and directional induced differentiation experiment.(3)Study on the osteogenic induction effect of materials on BMSCs:BMSCs were inoculated on BCP,BCPw,and BCPw-NPs ceramic materials for co-culture,and the cell culture plate was used as the control group.By performing fluorescein diacetate(FDA)/propidium iodide(PI)staining experiment,CCK8 experiment,real-time quantitative polymerase chain reaction(qRT-PCR)experiment for each group at specific time points,to detect the survival,proliferation and differentiation of BMSCs on different ceramic materials,and to evaluate the cellular biological effects of the different materials.Results:(1)Preparation and characterization of materials:The SEM results show that the three materials,BCP,BCPw,and BCPw-NPs,all have a honeycomb-like porous structure,with scattered macropores and small pores,and the macropore size is in the range of 100～500μm.Some large pores also have through-holes with a pore size of 10～100μm,indicating that the pores have good penetration.On the surface of BCPw,there are hexagonal columnar bodies(HA whiskers)with uniform size,uniform distribution and dense vertical growth.The surface of BCPw-NPs is composed of HA whiskers and nanoparticles with a particle size of less than 100 nm filled and covered between and on top of the whiskers.XRD results show that the surface components of the three porous ceramics BCP,BCPw and BCPw-NPs are all composed of HA phase andβ-TCP phase without other impurity phases.The porosity of the three kinds of porous ceramics,BCP,BCPw,and BCPw-NPs,were 74.8±1.55%,66.83±3.42%,83.05±1.06%,and the water absorption rates were 22.16±1.77%,32.38±1.09%,and 38.54±2.18%,respectively.The specific surface areas of the three porous ceramics BCP,BCPw,and BCPw-NPs were 1.24 m~2/g,1.45 m~2/g,and 1.93 m~2/g,respectively.(2)Isolation,culture and identification of BMSCs from newborn New Zealand rabbits:The cells isolated and extracted from rabbits were long spindle-shaped and swirled-like adherent growth under the light microscope;CCK8 experiment results showed that the cultured cells had good proliferation ability;The results of immunofluorescence staining showed that the cells expressed the mesenchymal stem cell phenotype CD44,but did not express the hematopoietic stem cell phenotype CD45.After 28 days of osteoblast differentiation,alizarin red S staining was performed,and dark red,massive calcium nodules of different sizes were observed.After 28 days of differentiation of adipocytes,oil red O staining was performed,and orange-red,round lipid droplets of different sizes were observed in the cells.After 28days of induced differentiation of chondrocytes,toluidine blue staining was performed,and it was observed that the cytoplasm was stained blue-purple.The results showed that the isolated and cultured cells were BMSCs and had multi-directional differentiation potential.(3)Research on the osteogenic induction effect of the materials on BMSCs:In the cell proliferation experiment,the cells of the three material groups proliferated slowly in the early stage of culture,and there was no significant difference between the groups(P>0.05).After 14 days of culture,the cells in the control group entered the proliferation plateau phase,and the cells in the three material groups proliferated rapidly.Finally,there was no significant difference between the control group,the BCP group and the BCPw-NPs group(P>0.05).The results of the FDA/PI staining experiment were consistent with the results of the cell proliferation experiment.In the osteogenic differentiation experiment,the expression levels of chondrogenesis-related genes(Sox9,Col Ⅱ,MMP13)and Col Ⅰ in the three material groups were lower than those in the control group and showed a decreasing trend.The expression level of Col X in BCPw-NPs group was significantly higher than other groups.The expression levels of osteogenesis-related genes(Runx2,Osterix,BMP2,OPN,OCN)in the three material groups were higher than those in the control group,and the increase trend was more significant in the BCPw-NPs group and could effectively maintain high expression.Conclusions:In this experiment,BCPw with micro-whisker surface structure and BCPw-NPs with micro-nano pore surface structure were designed and prepared using ordinary BCP as ceramic matrix.After co-culture of three materials with different micro-nano surface structures and BMSCs,BCPw-NPs with micro-nano pore surface structures showed a strong ability to induce BMSCs to differentiate into osteoblasts.