치조골 재생을 위한 약물 서방 방출형 다공성 수산화인회석

Abstract

Many studies in bone tissue engineering have been reported to promote the ability of bone regeneration. The limits of autografts, allografts, and xenografts have inspired the use of alloplastics. In recent years, strategies using synthetic bonegraft materials with biological factors have been come into the spotlight and hydroxyapatitite(HA) is generally considered of choice due to similarity in composition to human bone as well as teeth and excellent osteoconductivity. Biological factors, including growth factors for bone tissue engineering are very diverse, and considerable efforts have been made purify, isolate and identify the osteoinductive proteins presenting an osteoinductivity. Although many researches have been reported that optimize the combination of alloplatic bonegrafts and growth factors, it seemed that there is still no ideal solution. Tailored drug release from the inside of the hollow HA through the pores in the shell was investigated in this study. Needle-shaped HA nanoparticles, 20 nm in width and 100 nm in length, were synthesized by coprecipitation using 0.3M aqueous solution of (NH4)2·HPO4 and 0.5 M aqueous solution of CaCl2 with vigorous stirring at 10,000 rpm. The pH was adjusted to pH 10 and the temperature was maintained at 60℃. The coprecipitated HA was filtered, washed and freeze-dried. HA slurry was prepared by dispersing the synthesized HA powders into the distilled water with various organic additives such as 3% polyvinyl alcohol, 1% polyethylene glycol, 1% methyl cellulose, and 5% dimethyl formamide in order to improve the dispersing effect and prevent the crack formation during drying. Then amount of HA powders were varied from 30 to 70%(H groups), and followed by addition of sodium chloride up to 15%(N groups). Alginate beads were prepared by dropping 2% alginic acid solution into 1% calcium chloride solution. They were coated with HA slurry and subsequently dropped into liquid nitrogen. The HA-coated beads were freeze dried, and then organics were burned out during sintering. Vancomycin as a selected drug in this study was encapsulated with poly(lactic-co-glycolic acid)[PLGA] which is one of the representative biodegradable polymers by a double emulsion method. Vancomycin HCl injection was mixed with PLGA solution with dichloromethane(DCM). Through the evaporation of DCM under vacuum, ultracentrifugation at 10,000 rpm and subsequent lyophilization, vancomycin-laden PLGA nanoparticles were obtained. In order to insert vancomycin-laden PLGA nanoparticles into the porous and hollow HA granules, vacuum pressure was applied for a minute after dispersing them together in distilled water. Drug release rates from HA/PLGA complex bone-graft granules were monitored in vitro. They were immersed in phosphate-buffered saline and stored at 37℃ under 100 rpm under shaking. The amount of the released vancomycin was determined by the spectrophotometric plate reader. The phase composition of the synthesize HA powders was analyzed with an X-ray diffractometer. The shape and size of HA powder and PLGA particles were observed by scanning electron microscope and transmission electron microscope. The size and distribution of the HA powder and PLGA particles were measured by dynamic light scattering. The total amount of drug on the surface of the hollow HAp granules was calculated using simultaneous thermogravimetric analysis. The fabricated HA granules had an overall spherical geometry with 1.5 mm in diameter. The size could be controlled by the injector size as well as dropping speed into liquid nitrogen. Internal empty space was formed by the burning out the alginate beads during sintering process. Pores in the shell offered an connection between internal empty space and external surface. The thickness of the shell, the pore size and porosity were depends on the composition of HA slurry. In H groups, increasing the ratio of HA powder and distilled water, the thickness of the shell was increased from 20 to 100 μm in this study, while the pore size and porosity was decreased from (0.902±0.035) to (0.454±0.020) mL/g. This phenomena could be explained that pore was formed as water frozen and subsequent sublimation of ice inside the shell during lyophilization. The addition of sodium chloride in HA slurry did not affect pore size and porosity in groups N. However, irregular-shaped pores were caused because the sodium chloride remained after lyophilization. Nearly all the residual sodium chloride could be burned out during sintering process up to 1300℃. The structure of the pore channels is believed to have played a role in delay the initial burst out of the drug. It could be concluded that how to tailor the structure of pore channels connecting internal empty space and external surface was the key to control the sustained drug release rate from the internal empty space.