Fabrication Of Porous Spherical Calcium Phosphate Nano-Ceramics And Experimental Study In Rabbit Spinal Fusion Model

Objective:With the development of regenerative medicine,traditional biphasic calcium phosphate（BCP）bioceramics are facing many challenges in meeting the clinical requirements of bone tissue regeneration and repair,and their biological properties are limited due to the large grain size in comparison with nanocrystalline of natural bone mineral.It is generally believed that the micro-nano structure on the surface and interface of calcium phosphate ceramics has an important effect on the osteoinductive properties of the material.The surface of the calcium phosphate nanostructure can change the RGD sequence conformation of the protein,promote the adsorption of the material to the protein,affect cellular behaviors such as cell adhesion,spreading,migration,proliferation,and differentiation,which is conducive to the differentiation of osteoblasts and increase the amount of new bone formation.The purposes of this study were to:（1）fabricate new type porous BCP ceramic spheres with nanocrystalline by combining alginate gelatinizing with microwave hybrid sintering methods;（2）evaluate the in vitro cellular biological compatibility of MC3T3E-1 cells on three BCP ceramic materials,and explore the effect of three BCP ceramics to induce osteogenic differentiation of MC3T3 cells;（3）evaluate the ectopic bone formation ability of three kinds of BCP ceramics after intramuscular implantation in beagle dogs;（4）observe whether the porous spherical nano-BCP ceramic prepared by the new method can increase the posterolateral spinal fusion rate in rabbits,and provide the experimental basis for future clinical applications.Materials and Methods:（1）Fabrication and characterization of porous spherical BCP nanoceramics.BCP powder（HA/β-TCP=40/60）was prepared by a liquid-phase precipitation method.The bubbly slurry was slowly dripped and gelled in Ca Cl2 solution and hydrogel spheres were formed instantaneously,and the spheres were then hardened in Ca Cl2solution,washed,and dried.The dried spheres were presintered at 750°C to decompose most organic residue for 2 h.Then,the dried spheres sintered at 1050°C for 6 min via a novel microwave hybrid sintering method（heating rate of 150°C/min）to obtain BCP ceramic spheres with nanocrystalline（BCP-N）.BCP ceramic spheres with microcrystalline（BCP-G）were sintered by the conventional muffle sintering method（1050°C for 2 h）.The BCP-I was fabricated by crushing BCP porous bioceramic blocks into irregular granules.Scanning electron microscopy was used to study the morphology of three kinds of porous BCP granules.The phase composition was certified by X-ray diffractometry.The chemical functional groups of the three porous BCP ceramics were analyzed using Fourier transform infrared spectroscopy.The specific surface area of the three BCP ceramics was tested by a surface area analyzer,and their porosities and pore size distributions were characterized by mercury porosimetry.The samples were immersed in Tris-HCl buffer solution for 120 days to evaluate their degradation abilities.During the soaking process,a little amount（0.5m L）of the immersed solution was taken to assess and quantify the concentration of Ca²⁺and PO₄^3-ions by an inductively coupled plasma optimal emission spectrometer and was replaced with equivoluminal fresh Tris-HCl buffer solution.At each determining point,the samples were taken out,dried to constant weight,weighed,and then put back in fresh Tris-HCl solution.（2）Study on in vitro biological properties of porous spherical BCP ceramics.The Bone-like-apatite-forming ability of the three kinds of porous BCP granules was investigated by immersing them in the simulated body fluid（SBF）for 3 days at 37°C.Protein adsorption abilities of BCP granules were investigated by a BCA Protein Assay Kit.MC3T3-E1 cells were subcultured and seeded on three kinds of porous BCP ceramics in the exponential growth phase.The adhesion,spreading,and proliferation of MC3T3E-1 cells on the three different porous BCP ceramics were assessed by CCK-8,SEM,and cytoskeleton immunofluorescence staining.The osteoinductive properties of calcium phosphate ceramic materials were evaluated by ALP staining and activity measurement,alizarin red staining.Osteogenesis-related markers（such as genes and proteins）were detected by immunofluorescence staining,Western Blot,and real-time quantitative PCR.（3）Research on the performance of ectopic bone formation in porous BCP ceramics:The model of intramuscular implantation in the back muscle of four beagle dogs was used and the BCP ceramics should be completely wrapped by the muscle and does not contact the bone tissue.Two beagle dogs were sacrificed on 45 and 90 days respectively,and the BCP ceramics implanted in the muscle pocket and surrounding tissues were processed to evaluate the ectopic osteogenesis.（4）Study on the effect of porous BCP ceramics on rabbit posterolateral spine fusion:According to three different porous BCP ceramic materials,36 New Zealand rabbits were randomly assigned for rabbit lumbar posterolateral spine fusion.X-ray was performed at 6 weeks and 12 weeks postoperatively,and the biomechanical changes of the fusion segment were detected by manual palpation and three-point bending test at 12 weeks after the posterolateral spinal fusion surgery.Micro-CT was performed to examine the spinal fusion and material morphology changes.Fluorescence of calcein and tetracycline was used to mark new bone formation.Histological observation of non-decalcified specimens was performed to analyze the bone formation around and inside the material and to evaluate the spinal fusion status.Results:（1）SEM showed that all of the three porous BCP ceramics had an interconnected pore structure,but the pore sizes of BCP-G and BCP-N were smaller than that of BCP-I.The grain size of BCP-G ceramics is between 350 and 800 nm,with an average of about 380 nm;the BCP-I ceramic particles have the largest grain size,about 825 nm;while the BCP-N ceramics’grain size is about 105nm and there are abundant micropores between the grains.XRD found that the phase composition of the three ceramics was all the composite of HA phase andβ-TCP phase,and no other impurity phases were introduced during the whole synthesis process.The three BCP samples also have high porosity.The porosities of BCP-G,BCP-N,and BCP-I are 70.92%,75.04%,and 77.97%,respectively,and the microporosity is 19.22%,24.32%,and11.24%,respectively.Tris-HCl buffer solution（p H=7.4）was employed to evaluate the degradation abilities of the three kinds of BCP granules and found that c(Ca²⁺)and c(PO₄^3-)of the three groups increased as the degradation process progressed,and c(Ca²⁺)and c(PO₄^3-)releasing from BCP-N were significantly higher than those released from BCP-G and BCP-I.Moreover,c(Ca²⁺)releasing from BCP-G was lower than that released from BCP-I,but c(PO₄ ^3-)releasing between the two groups was similar.The three groups of porous BCP ceramics exhibited a sustained weight loss in the degradation process and of which BCP-N was the highest.（2）The surface morphology of BCP-G,BCP-N,and BCP-I after immersing in SBF for 3 days showed that the bone-like apatite formation ability was of the order BCP-N>BCP-G>BCP-I.The results of BSA and fibronectin protein adsorption showed that BCP-N with nanocrystalline could adsorb the most protein.CCK8,SEM,and CLSM test results show that BCP-N can promote cell adhesion,spreading,and proliferation.ALP staining,activity measurement,and alizarin red calcium nodule staining found that BCP-N ceramics can significantly promote the osteogenic differentiation of MC3T3 cells,which is significantly better than the BCP-I and BCP-G groups（P<0.05）.When MC3T3 cells were co-cultured with three porous BCP ceramics for 14 days,the OPN and OCN immunofluorescence analysis of the cells on the surface of the BCP-N ceramic material were significantly higher than those of the BCP-I group,while there is no significant statistical difference between the BCP-N and BCP-G samples.For COL-1,BCP-N is higher than BCP-G and BCP-I groups,and there is no significant difference between the BCP-I group and BCP-G groups.For RUNX2,there was no significant difference in the expression of the three groups.Real-time quantitative PCR results show that BCP-N can promote the expression of osteogenic related genes ALP,COL-1,OPN,OCN.（3）H&E staining demonstrated that obvious ectopic bone formation could have occurred in all three kinds of BCP granules after intramuscular implantations,accompanied by many osteoblasts and blood vessels.Conclusions:（1）We fabricate new type porous BCP ceramic spheres with nanocrystalline by combining alginate gelatinizing with microwave hybrid sintering methods.The new porous spherical nano-BCP ceramics have high porosity and interconnected microporous pores,and the crystal grain size reaches the nano-and submicron level.（2）The three kinds of BCP ceramics have good cell compatibility in vitro without cytotoxicity.BCP-N ceramic particles promote the adhesion,spreading,and proliferation of MC3T3 cells.The ability of BCP-N ceramic to induce osteogenic differentiation of MC3T3 cells is stronger than BCP-G and BCP-I ceramic particles.（3）The three kinds of porous BCP ceramics have good biocompatibility and no visceral toxicity.BCP-N with nanocrystalline has stronger ectopic bone formation ability than BCP-G and BCP-I.The spinal fusion rate of the BCP-N group in the rabbit posterolateral lumbar spine fusion model is higher than that of the BCP-G and BCP-I groups,which provides a new option for bone substitutes in the spinal fusion procedure.Micro-CT results showed that compared with the BCP-I and BPC-G samples,the BCP-N group had a higher BV/TV and a higher spinal fusion rate at 12 weeks after surgery.The results of fluorescent labeling of bone tissue showed that the BCP-N group had a higher fluorescent labeling area at 12 weeks.The staining results of methylene blue/basic fuchsin hard tissue sections showed that BV/TV in the BCP-N material group was significantly higher at 12 weeks（p<0.05）,and the integration of bone tissue and scaffold was better.