이종골로부터 하이드록시아파타이트 제조 및 조골세포와의 생체 친화성

Abstract

Hydroxyapatite (HA) is well known as biocompatible and bioactive materials. HA has achieved the significant applications as bone graft materials in the range of medical and dental fields. When HA is implanted into osteogenic tissue, such as bone defects or medullary cavities, bone formation can occur on the surface and then the HA directly and strongly bonds to the bone. An alternative for treatment of bone defects is the use of xenogenous bone, which is morphologically and structurally similar to human bone. Also, it is easy to obtain and available in unlimited supply with low production cost. Bovine bone is composed of organic and inorganic components. The organic part contains mainly collagen and proteins, whereas the inorganic component is mainly hydroxyapatite with small amounts of other elements such as carbonate, magnesium and sodium being incorporated in the structure. The present study evaluates the biocompatibility of HA ceramics for supporting osteoblast cells growth and cytotoxicity using the MG-63 cell line model in vitro. We used two types of HA. One was the commercially-obtained artificial HA, noted as AHA. Another was the bovine bone derived HA (BHA) which was obtained as follows. The fresh bovine bones were cut into small pieces and cleaned well to remove macroscopic adhering impurities. The bone pieces were boiled in distilled water for easy removal of the bone marrow and tendons. The boiled bones were dried at 80˚C. The resulting product was crushed into small particles and they were ball-milled for 48 h. The BHA powder was calcined at 800˚C for 1 h. The AHA and BHA disks were prepared by uniaxial pressing and cold isostatic pressing, followed by sintering at 1200 ˚C for 1 h under moisture protection. MG-63 cells, a human osteoblast-like cell line were used to assess the cellular response to the AHA and BHA ceramics. The samples were placed in 24-well plates for MG-63 cells implantation at a set density of 1×104 cells/cm2. The MG-63 cells were further incubated in DMEM(dulbecco's modified eagle's medium) solution supplemented with 10% FBS and 1% PS for 1, 3 and 5 days. Following incubation of cells with two types of HA for indicated time in 24-well plate, cells were washed once with PBS(phosphate buffer saline). And then 35 ㎛l of MTS solution and 350 ㎛l DMEM solution of nothing excluded was added to each well. After incubating the plates at 37˚C for 90 min, absorbance in control and the treated wells was measured in a plate reader at 490 nm. The samples were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). For the FE-SEM(field emission scanning electron microscope) observation, the samples were dehydrated in a graded series of ethanol and were treated with isoamyl acetate. In chapter 3, We investigated the behavior of human bone cell on two types of hydroxyapatite (HA) ceramics. One was the AHA, the other was the BHA. Porous hydroxyapatite from bovine bone was biocompatible in the tests performed and any cell morphological change was not observed by light microscopy. FE-SEM observation showed that most of MG-63 cells was stellar shape and formed intercellular matrix containing fibers on porous BHA. The cells were well attached and grown up over the BHA surface meaning that there was no toxicity. In chapter 4, The response of MG-63 cell to HA 1100 and HA 1200 from bovine bone was compared. The samples differentiated the thermal annealing temperature at 1100 and 1200°C in moisture protection for 1 h. The samples have the density of 65% and 82%. And then MG-63 cell line were used to assess the cellular response to sample. We found that cells adhere to the surface of a sample after the cell culture for one day. Cytoplasm had been spread around the nucleus after the cell culture for 3 days. And then most of cells were extended to large area. Until 3 days, the cell viability was nearly same each other the growth of a cell after 5 days drastically increases in the porous BHA at FE-SEM and MTS assay.