Nano-hydroxyapatite - Chitosan Composite Cell Adhesion Behavior Of A Preliminary Observation

Hydroxyapatite (HA) is one of the major constituents of the inorganic in human and animal hard tissues (bone and tooth), and it takes 60 percent in bones. The artificial HA is similar with apatite crystal of nature bones. Because of its good bioactivity and biocompatibility, it can form a compact bond with surrounding tissues after implanting that supplying good physiological scaffold for deposition and regeneration of new bone. As an attractive bone substitution material, HA has widely used in medical application. Recently, nanoscale particle of hydroxyapatite (nHA) plays a significant role in various biomedical applications owing to its unique functional properties of high surface area effect and volume effect and so on. However, these are some limitations in use of the material due to its brittleness and slow absorption. Composite is defined two or multidifferent materials or out-of-phase in the range of microscopy or micro. Composite can remain major property of native material and obtaining other function by combination, so its application area is increasing gradually. A new composite that combining HA and nature or artificial polymers is similar with nature bone, on the other hand the composite can prevent the brittleness and migration of particulate into surrounding tissue upon post-implantation. Chitosan (CS), a significant alkalinity nature polycose, is obtained from chitin through its deacetylation. Its final metabolites are N-acetylgluco- samine and glucidamin, which were nature metabolites. Favourable moulding and workability of chitosan provide its composite good physical and chemical property. It has used in operation sutural line, wound dressing, medicine retarder, filler and scaffold of tissue engineer.The present study prepared and detected nHA and a composite of nHA/CS. We assessed the biocompatibility of the composite by co-culture the composite and NIH3T3 cells.PartⅠPreparation and analysis of nanoscale particle of hydroxyapatite/chitosan compositeMaterials and methods The HA were prepared by sol-gel method using Ca(NO₃)₂·4H₂O and (CH₃O)₃PO as Ca and P precursors, respectively. The nHA were prepared by adding C₆H₈O₇ in precursors. The composite of nHA/CS were prepared by lyopyilization and studied by using X-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FT-IR), scanning electron microscope (SEM), transmission electron microscope (TEM).Results 1. The XRD peaks of HA and nHA prepared were based on hexagonal crystal system of stoichiometric HA. 2.The results of TEM showed HA particles size in composite was in range of nano levels, electronogram showed crystal structure of HA. 3.The SEM micrograph exhibited the pure chitosan scaffolds and composite scaffolds were similar in their morphology, which indicated that adding the HA in the system did not influence the porous structure of CS. EDAX showed changes of chemical composite. The nHA particles dispersed uniformity in chitosan. 4. The IR spectrum exhibited the major function groups of prepared HA and chitosan. There were no extraneous new functional groups detected from the composites. The above results confirmed that chitosan and nHA were intact well in the composite and no evident chemical reaction.Conclusions The nano HA can be prepared by adding citric acid as chelator using sol-gel method. Chitosan and nHA were intact well in the composite obtained by lyopyilization and no evident chemical reaction in composite.PartⅡThe adhesive observation of fibroblast to the nanoscale particle of hydroxyapatite /chitosan compound membraneMaterials and methods 2%CS/nHA compound membrane and 2%CS/HA compound membrane were prepared and then co-cultured with NIH3T3 cells. The adhesive quantity and behavior of NIH3T3 cells on the two compound membranes were observed in upside-down microscope.Results 1. In upside-down microscope, cells adhered and spread well after co-cultured. There were cells growth on two compound membranes and by the side of membranes, and cells morphous showed fusiform or multi-angle and body transparence.2. After 3 days, the quantity of cells adhesion on nHA/CS and HA/CS were no difference, after 5 and 7 days, the quantity of cells adhesion on nHA/CS was higher than on HA/CS.Conclusions nHA/CS compound membrane and HA/CS compound membrane were both semitransparent, which can observe cells growth condition in upside-down directly. In this study, cells adhered and spread well on both semitransparent membranes. After 5 and 7days, the quantity of cells adhesion on nHA/CS was higher than on HA/CS. Compared with HA, nHA can promote ability of cells proliferation.PartⅢThe adhesive experiment of fibroblast to the nanoscale particle of hydroxyapatite /chitosan composite scaffoldMaterials and methods The nHA/CS composite scaffold was prepared by lyopyilization, and dense HA and composite scaffold were prepared the same size and shape of disk. After 5, 7 and 9days co-cultured NIH3T3 with dense HA and 2%CS/nHA, cells adhesion quantitative were determined and morphology was examined with SEM and spectrum analysis. Results 1. In upside-down microscope, culture fluid was clarified and cells morphous showed fusiform or multi-angle and body transparence surrounding materiel in test and control groups. There were manipulus HA particles liberated from disk shaped HA in control group, but HA particles did not impact cells adhesion and proliferation. 2. The results of SEM in dense HA group showed cells adhered on the surface of material with ecphyma and spread well. 3. The results of SEM in nHA/CS composite scaffold group showed cells adhered on the surface of scaffold with ecphyma and spread well and crawled to surface of porous. In high power lens spectrum analysis, chemical composite change showed on the no cells surface of materials and cells surface of materials C content increased from 47.07% to 57.81%, however Ca content decreased from 10.27% to 1.09%. 4. The new scaffold material, nHA/CS can promote ability of cells proliferation after5, 7 and 9 days co-cultured with NIH3T3 cells.Conclusions NIH3T3 cells can adhere, grow and proliferate on nHA/CS composite and dense HA. Compared with dense HA, nHA/CS composite scaffold can promote ability of cells proliferation.