| Objective The present study aimed to investigate whether nano-hydroxyapatite (nHA) coating on porous Biphasic calcium phosphate (BCP) ceramics can enhance osteoinductivity of BCP.Methods(1)We used a hydrothermal deposition method to coat conventional porous BCP ceramics with nHA.By field emission scanning electron microscopy (FESEM), field emission transmission electron microscopy (FETEM) and X-ray diffraction (XRD), the morphology and phase composition of the scaffolds were characterized,and the effects of the coating on the physical and mechanical properties of the underlying BCP were assessed.(2) The cytotoxicity test and hemolysis test were induced to evaluate the biological safety of the scaffold.(3)Mesenchymal stem cells (MSCs) were isolated and purified by density gradient centrifugation combined with attachment culture method. The biological characteristics of MSCs were observed under an inverted microscope.Cell surface markers were assessed by flow cytometry, and the MSCs were identified with alkaline phosphatase (ALP) staining and Alizarin red staining after osteogenic differentiation. The MSCs were co-cultured with BCP and nHA-coated BCP scaffolds,and the effects of nHA coating on MSCs attachment, proliferation, viability and osteogenic differentiation were investigated.(4) A total of20rabbits were randomly divided into four groups with5rabbits(10sides of anterior limb) in each group, including goup A, B,C and D.For all rabbits,1.5cm bone defects were created on both sides of radius, and then these bone defect regions were implanted with corresponding composites:group A was implanted with BCP, group B with nHA-coated BCP, group C with BCP/MSCs while group D with (nHA-coated BCP)/MSCs. At12th weeks after operation,10specimens from each group were obtained. The new bone formation were assessed by X-ray film examination, biomechanical test, histology and immunostaining. Results(1)Hydrothermal deposition method can be used to synthesize the nHA crystal,and the synthesized nHA had a rod-like shape with lengths ranging from～50-200nm and diameters from～15-30mm. XRD shows that hydroxyapatite formed a deposited layer on the BCP surface.FESEM showed that the nHA crystal layer was deposited onto the BCP surface.The nHA coating did not significantly affect the density, porosity, flexural strength, or compressive strength of the underlying BCP (P>0.1).(2) In cytotoxicity test, the cytotoxicity of the two experimental groups was class0.The highest rate of hemolysis of BCP group was at3.7%,and the highest rate of hemolysis of nHA-coated BCP group was at3.56%.(3)The adhered cells by primary culture were spindle-and whirlpool-shaped. After passage, the cells were spindle-shaped and distributed orderly. Cultured cells were positive for CD44, but negative for CD34.After osteogenic induction, ALP staining and Alizarin red staining were positive.The densities of MSCs attached on BCP and nHA-coated BCP scaffolds were62±26cells/mm2and63±27cells/mm2(P>0.1)respectively after1day, and415±62cells/mm2and541±35cells/mm2(P<0.05)respectively after14days.According to the MTT assay, MSCs viability was higher on nHA-coated BCP scaffolds than on BCP scaffolds (P<0.05).In addition,MSCs on nHA-coated BCP scaffolds expressed more alkaline phosphatase, collagen I,and osteocalcin than MSCs on BCP scaffolds (P<0.05).(4) The X-ray showed that the bone defects in group A were basically repaired, whereas the bone defects in groups B, C and D were completely repaired. Biomechanical testing showed that the maximum bending load of the group A was significantly higher than that of the group B (P<0.05), group B was significantly higher than group C (P<0.05),and group C was significantly higher than group D (p<0.05).Histological observation indicated that lots of mature new bone could be seen in all the groups, and mature bone grew within the scaffold pores. The new bone formation percentage in group D was significantly higher than in group C (P<0.05), group C was significantly higher than group B (P<0.05),and group B was significantly higher than group A (P<0.05).Immunohistochemical staining showed BMP-2in group D was significantly higher than in group C (P<0.05), group C was significantly higher than group B (P<0.05), and group B was significantly higher than group A (P<0.05).Conclusion(1)The hydrothermal deposition method can be used to coat nHA to the BCP scaffolds, forming a dense layer of rod-like nHA crystals on the BCP surface. The nHA coating did not significantly affect the density, porosity, flexural strength, or compressive strength of the underlying BCP.(2) BCP and nHA-coated BCP porous ceramics have no cytotoxic effect, having good biocompartibility and not causing hemolytic reaction.(3) In this study, for the first time, we coated porous BCP ceramic scaffolds with nHA and then induced the osteogenic differentiation of rabbit bone marrow-derived MSCs seeded on these scaffolds. Our results demonstrated that BCP scaffolds coated with nHA were more conducive for MSCs adhesion, proliferation, and osteogenic differentiation than conventional, uncoated BCP scaffolds.(4) Both aspects, nHA coating and MSCs, can accelerate new bone formation in BCP, but MSCs have higher ability of new bone formation than nHA coating, and a combination of both can significantly enhance the rate of new bone formation in BCP.(5)In vitro and in vivo experiments indicated that nHA coating can accelerate the new bone formation by enhancing the osteoinductive potential of BCP ceramics, making this material more suitable for applications in bone tissue engineering. |
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