Biocompatibility Of Three-dimensional Printed Nano-hydroxyaptite/polycaprolactone Scaffolds

Bone tissue engineering can be used as a final choice for the treatment of skull defects.The concept of bone tissue engineering consists of three major parts: scaffolds,cells and growth factors.Among them,the scaffolds as the most important component can provide support and space for the cells,and promote cells proliferation,differentiation,and guide the surrounding tissue grow in.3D printing technology can produce a three-dimensional architecture with optimal pore size,pore distribution and sufficient mechanical strength.Many synthetic polymers,natural polymers and ceramic materials have been developed and used as bone tissue engineering scaffolds materials.At present,a single material is still difficult to meet all the harsh requirements of bone tissue engineering scaffolds.The combination of two or more materials can give full play to their respective advantages,which has become a trend in the study of bone tissue engineering scaffolds.Ceramic/polymer composites as a biodegradable material have good mechanical strength,osteoinduction,bone conduction and compatibility.Nano-hydroxyapatite and polycaprolactone,respectively,as a typical representative of ceramic materials and synthetic polymers,have been widely used in the research of bone tissue engineering scaffolds.Object: To study the biocompatibility of 3D nano-hydroxyapatite / polycaprolactone scaffolds preliminary,and to provide experimental basis for further study.Methods: SD rat bone marrow mesenchymal stem cells were isolated and cultured.The cell identification was carried out by a variety of methods: The expression of CD34 and CD44 on the cell surface was detected by flow cytometry and immunofluorescence.Alizarin red staining was also performed on the cells after osteogenesis differentiation.The 3D nano-hydroxyapatite / polycaprolactone scaffolds were cultured with bone marrow mesenchymal stem cells.The growth of the cells was observed by scanning electron microscopy.After the cells were cultured in scaffolds extract(experimental group),the morphological changes were observed under inverted phase microscope,and control group was DMEM complete medium.The CCK-8 assay was used to detect the cytotoxicity of the scaffolds extract(experimental group)on bone marrow mesenchymal stem cells,the control group was DMEM complete medium.Hemolysis test with fresh rabbit anti-coagulant blood was performed on scaffolds extract(experimental group),normal saline(negative control group)and double distilled water(positive control group).Results: The third generation cells were spindle-shaped,and grew by static adherence.Flow cytometry showed that the positive rate of CD34 was 0.62% and the positive rate of CD44 was 96.51%.Immunofluorescence staining showed the cells were negative for CD34 and positive for CD44.After inducing osteoblastswas alizarin red staining observed orange-red calcium nodules.So the isolated and cultured cells were identified as bone marrow mesenchymal stem cells.Scanning electron microscopy showed that the 3D nano-hydroxyapatite / polycaprolactone scaffolds possessed porous mesh structure,and bone marrow mesenchymal stem cells could adhere,grow,proliferate and migrate on them well.The results of inverted phase microscope showed that bone marrow mesenchymal stem cells grew well in the scaffolds extract,and there was no significant morphology difference between the experimental group and the control group.The results of CCK-8 assay showed that there was no significant difference between the absorbance value of experimental group and the absorbance value of the control group at different time(P> 0.05).The cytotoxicity level of the scaffolds was grade 1.Hemolysis test results revealed that the hemolysis rate of the scaffold extract was 1.16%.Conclusion: 3D nano-hydroxyapatite/polycaprolactone scaffolds have a good three-dimensional pore structure.Bone marrow mesenchymal stem cells can adhere,grow,proliferate and migrate on the scaffolds.3D nano-hydroxyapatite/ polycaprolactone scaffold has good biocompatibility,which can be used for further animal experiments to verify the specific effect of bone defect repair,and is expected to be a new type of skull defect repair material.