[Influence of different sintering temperatures on mesoporous structure and ectopic osteogenesis of biphasic calcium...

To detect the difference in the osteogenesis ability of biphasic calcium phosphate (BCP) ceramic granular materials with different mesoporous diameters prepared at different sintering temperatures through in vivo and in vitro experiments, so as to provide evidence for screening BCP materials with better clinical application parameters.

Three kinds of BCP (materials 1, 2, 3) were prepared by mixing hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) at a ratio of 8∶2 and sintered at 1 050, 1 150, and 1 250℃ for 3 hours, respectively. The internal porosity and the diameter, volume, and area of the mesopore were measured by Brunauer-Emmett-Teller test (BET); the composition of the material was evaluated by X-ray diffraction (XRD); the microscopic surface morphology of the material was observed by scanning electron microscopy (SEM). The 3rd generation bone marrow mesenchymal stem cells (BMSCs) from Sprague-Dawley rats were co-cultured with the materials 1, 2, and 3 for 7 days in vitro respectively (groups A, B, and C), and the cells adhesion on the materials was observed by SEM and phalloidine staining, respectively. Cell proliferation activity was measured by cell counting kit 8 method. In vivo, 9 muscle bags were made in dorsal muscles of 9 beagles, respectively. The muscle bags were randomly divided into 3 groups (3 per beagle in each group) and materials 1, 2, and 3 were placed into the muscle bags of groups A, B, and C, respectively. After 1, 2, and 3 months of operation, 3 beagles were anesthetized and the samples were stained with HE, Masson, and Safranin, and the bone formation area ratio in the BCP gap was calculated. Real-time fluorescence quantitative PCR (qRT-PCR) was performed to detect the expressions of bone-related genes [including alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OC)].

The BET test showed that with the increase of sintering temperature, the internal porosity of the particles did not change significantly, but the diameter, volume, and area of the mesopores gradually decreased. The XRD detection showed that the XRD waves of HA and β-TCP could be seen in all 3 kinds of materials; SEM showed that there were widely distributed macropores on the surface of 3 kinds of BCPs, and the interpores connected with the others. In vitro, BMSCs adhered and proliferated on the surfaces of 3 kinds of BCPs, and the cell biocompatibility of the materials in groups B and C was better than that in group A. In vivo, obvious osteoid tissue deposition could be observed in the intergranular space of 3 kinds of BCPs from 2 months after implantation. The bone formation area ratio of each group increased with time. The bone formation area ratio in group A was significantly higher than that in groups B and C at 2 and 3 months after implantation, and in group A than in group B at 1 month ( P<0.05). qRT-PCR showed that the expressions of osteogenic related genes peaked at 2 months in group A, and gradually increased with time in groups B and C. The relative expressions of ALP and OPN mRNAs in group A were significantly higher than those in groups B and C at 1 month after implantation, the relative expression of OC mRNA in group A was significantly higher than that in groups B and C at 2 months after operation, the relative expression of ALP mRNA in groups B and C and the relative expression of OPN mRNA in group B were significantly higher than those in group A, all showing significant differences ( P<0.05); there was no significant difference in the relative expression of each gene among the other groups at each time point ( P>0.05).

The mesoporous diameter of BCP decreases with the increase of sintering temperature. Different mesoporous diameters lead to different ectopic osteogenesis of BCP materials. BCP material with mesoporous diameter of 12.57 nm has better osteogenic ability which can activate the osteogenic gene earlier. The mesoporous diameter is expected to be an adjustable index for optimizing the osteogenic capacity of BCP materials.