초록 ▼

· 년차별 세부목표에 따라 전복패각으로부터 바이오세라믹 수산화인회석 및 베타-제 3인산칼슘 합성방법 확립.
· 전복패각으로부터 합성된 수산화인회석 및 베타-제 3인산칼슘으로 구성된 이상 인산칼슘계열의 크기 500 μm이하, 내·외부 표면화 처리된 합성골 이식재 합성 방법 확립.
· 전복 패각 유래 합성골 이식재에 대한 한국기계전자시험연구원의 의료기기의 생물학적 안전에 관한 공통기준규격에 따른 시험 평가 결과에 세포독성: 2등급, 피내반응: 음성, 유전독성 (복귀돌연변이시험): 음성, 감작성 시험: 음성, 급성독성시험: 무

· 년차별 세부목표에 따라 전복패각으로부터 바이오세라믹 수산화인회석 및 베타-제 3인산칼슘 합성방법 확립.
· 전복패각으로부터 합성된 수산화인회석 및 베타-제 3인산칼슘으로 구성된 이상 인산칼슘계열의 크기 500 μm이하, 내·외부 표면화 처리된 합성골 이식재 합성 방법 확립.
· 전복 패각 유래 합성골 이식재에 대한 한국기계전자시험연구원의 의료기기의 생물학적 안전에 관한 공통기준규격에 따른 시험 평가 결과에 세포독성: 2등급, 피내반응: 음성, 유전독성 (복귀돌연변이시험): 음성, 감작성 시험: 음성, 급성독성시험: 무독성, 무균시험: 이상 없음으로 평가됨에 따라 치과의료용 합성골이식 재로써 기술 및 안전성 측면에서 신뢰성 확보.
· 특허출원 3건 (특허등록 1건; 등록번호 10-1572023), 학회 발표 4건 (우수학술상 수상 3건), 기술실시계약 3건, 인력양성 3명 (이학석사 2명/치의학박사 1명) 등의 연구 성과.
· 따라서, 본 연구는 해양 폐기물로 간주되고 있는 전복 패각을 고부가가치 의료 소재화를 통한 폐기물 처리비용 완화에 기여할 뿐만 아니라 전복생산관련 지역내 새로운 치과의료기기 관련 사업유치를 통한 일자리 창출 및 지역성장 동력산업화가 가능하리라 사료됨.
· 또한, 전복패각으로부터 바이오세라믹 합성 및 이를 활용한 치과의료용 합성골 이식재 합성기술을 개발함으로써 구강 질환 환자 치료를 위한 치과 골이식 분야, 합성골 이식재 생산원료 가공 분야, 치과의료용 합성골 이식재 생산 분야 등에서 고가 수입 치과 의료용 골이식재를 대체함으로써 치과 의료비용 경감을 통한 국민 구강 보건 건강 증진에 기여할 것으로 사료됨.

(출처 : 보고서 요약서 3p)

Abstract ▼

RESULTS

1. Synthesis and physiochemical characterisitcs of β-TCP synthesized from abalone shells

1-1. Synthesis of β-TCP synthesized from abalone shells

To determine the optimal sintering temperature for CaO synthesis from abalone shells, sintering was performed in an electric

RESULTS

1. Synthesis and physiochemical characterisitcs of β-TCP synthesized from abalone shells

1-1. Synthesis of β-TCP synthesized from abalone shells

To determine the optimal sintering temperature for CaO synthesis from abalone shells, sintering was performed in an electric furnace with the temperature controlled at 900℃, 950℃, and 1000℃, for 3 h. The composites synthesized at each defined sintering temperature were analyzed by FT-IR and XRD. FT-IR results revealed a sharp band at 3656 cm^-1, two broad weak bands centered at approximately 3822 and 3388 cm^-1, a medium doublet centered at around 1444 cm^-1, and a very strong absorption below 600 cm^-1 for all the synthesized composites. The band profile of a composite obtained by sintering at 950℃ was matched with that of commercial CaO (Junsei Chemical Co., Ltd., Tokyo, Japan) used as a control. Furthermore, to verify the conversion from abalone shell to CaO, the composite synthesized by sintering at 950℃ was analyzed by XRD. XRD spectra of commercial (control) CaO were observed at 32.24°, 37.4°, and 53.92°. The XRD spectra of CaO derived from abalone shell by sintering at 950℃ matched the peaks of commercial CaO. These results indicated that CaO was successfully synthesized from abalone shell by sintering at 950℃ for 3 h.

Next, to synthesize CaCO₃ from abalone shell-derived CaO, CO₂ gas was injected into Ca(OH)₂ solution that was prepared by re-suspending CaO in distilled water. The resulting composite was analyzed by FT-IR and XRD to verify the CaO to CaCO₃ conversion. FT-IR profile of commercial CaCO₃ used as a control showed bands at 713, 875, and 1418 cm^-1, corresponding to asymmetrical stretching vibration peaks of O–C–O, respectively. The seare well known characteristic peaks of calcite. CaCO₃ synthesized by the infusion of CO₂ gas into Ca(OH)₂ derived from abalone shell had the same band profile as commercial CaCO₃. Furthermore, the XRD spectrum of commercial CaCO₃ used as a control showed characteristic diffraction peaks at 23.09°, 29.40°, 36.0°, 39.43°, 43.18°, and 48.52°. All the peaks indicated the formation of a calcite phase. The XRD spectra of abalone shell-derived CaCO₃ matched the peaks of commercial CaCO₃. Thus, these results indicated that CaCO₃ was successfully synthesized from abalone shell by CO₂ infusion of Ca(OH)₂ solution. In addition, ~4.5 g CaCO₃ were obtained from 5 g CaO derived from abalone shell.

CaHPO₄ was synthesized by a chemical reaction of H₃PO₄ and abalone-shell derived CaCO₃. To determine the optimal pH conditions, chemical reactions of H₃PO₄ and CaCO₃ were performed at pH 6.0, 7.0, and 8.0. Thereafter, chemical properties of the synthesized composites were examined by FT-IR and XRD. The broad absorption peak between 2400 to 3500 cm^-1 results from the O–H stretching vibration. H–O–H bending leads to absorption at 1653 cm^-1. Absorptions at 1219 and 1134 cm^-1 are caused by (P=O)-associated stretching vibrations. The absorption at 1057 cm^-1 is caused by P=O stretching vibrations. P–O–P asymmetric stretching vibrations lead to absorption at 987, 876, and 791 cm^-1. The seare well-known characteristic peaks of CaHPO₄. The pH of chemical reaction of H₃PO₄ and CaCO₃ is a critical factor in CaHPO₄ synthesis. Peak positions of ceramic products synthesized at pH 7.0 or 8.0 were different from the characteristic dicalcium phosphate dehydrate peaks used as a control. On the other hand, the positions of peaks of ceramic products synthesized at pH6.0 matched the characteristic peaks of CaHPO₄. These data indicated that the optimal pH for the synthesis of CaHPO₄ by a chemical reaction between H₃PO₄ and abalone shell-derived CaCO₃ is pH 6.0. XRD analysis was performed to verify CaHPO₄ synthesis under the optimized conditions. The XRD spectrum of commercial CaCO₃ used as a control showed characteristic diffraction peaks at 11.5°, 20.7°, 23.2°, 29.3°, and 31.4°. Moreover, the XRD spectra of CaHPO₄ derived from abalone shell matched the XRD spectra of commercial CaCO₃. Thus, these results indicated that CaHPO₄ was successfully synthesized through the chemical reaction between H₃PO₄ and abalone shell-derived CaCO₃ at pH 6.0. Finally, β-TCP was synthesized by sintering a mixture composed of CaO and CaHPO₄ derived from abalone shell. To determine the optimal conditions for β-TCP synthesis, sintering was performed at 950℃, 1000℃, 1050℃, and 1100℃, for 3 h. Next, chemical properties of the synthesized composites were analyzed using FT-IR and XRD. β-TCP used as a control is easily identified by a broad band between 900 and 1200 cm^-1, and by the presence of a peak at 724 cm^-1. The peak at 1211 cm^-1 is characteristic of non-degeneratede formation of hydrogen groups, such as H–OPO₃, O–PO₃, and HPO₄^2-. However, the absence of a band at 460 cm^-1 characteristic of α-TCP, is indicative of β-TCP. Peak positions of composites synthesized at 950 – 1050°C matched positions of peaks of β-TCP used as a control. These results indicated that β-TCP is successfully synthesized by sintering at 950–1050°C from a mixture of CaO and CaHPO₄ derived from abalone shell. However, peak positions of composites synthesized at 1100°C were different from peak positions of control β-TCP. Taken together, these results indicated that the optimal sintering conditions for synthesizing β-TCP from a mixture of CaO and CaHPO₄ derived from abalone shell are 950 – 1050°C for 3 h. XRD was also performed to verify the synthesis of β-TCP under optimized sintering conditions. The diffraction pattern of control β-TCP corresponded to the peaks of β-TCP or whitlockite. Furthermore, the electron microscopic morphology of the synthesized β-TCP showed the bigger size (approximately 105 μm) more than control β-TCP (approximately 52 μm). The diffraction pattern of β-TCP synthesized under optimized sintering conditions from a mixture composed of CaO and CaHPO₄ derived from abalone shell matched the diffraction pattern of control β-TCP. Thus, β-TCP obtained from a mixture of abalone shell-derived CaO and CaHPO₄ was successfully synthesized by sintering at 950 – 1050°C for 3 h.

1-2. Biological safety of β-TCP derived from abalone shell

To determine the biological safety of abalone shell-derived β-TCP, its cytotoxicity was assessed using NHOKs and human MG-63 osteosarcoma cells. NHOKs viability in the presence of an eluent prepared from abalone shell-derived and control β-TCPs was 88 ± 4% and 93 ± 5%, respectively, compared with non-treated control. Although cell viability in the presence of the eluent prepared from β-TCP derived from abalone shell was lower than both that of non-treated control and in the presence of control β-TCP, the difference was not significant. Furthermore, viability of human MG-63 osteosarcoma cells in the presence of eluents prepared from abalone shell-derived β-TCP and control was 107 ± 8% and 102 ± 2%, respectively, compared with non-treated control.
To verify the effect of β-TCP derived from abalone shell on cell viability, live and dead cell assay was performed with NHOKs and human MG-63 osteosarcoma cells. Dead cells, fluorescing red upon ethidium homodimer-1 staining, were not observed in either NHOKs or human MG-63 osteosarcoma cells treated for 24 h with eluents prepared from abalone shell-derived and control β-TCP, or in non-treated control. To verify whether the eluents prepared from β-TCP derived from abalone shell induced apoptosis in the two cell lines, DAPI staining was performed to visualize morphological alteration of the nuclei, a typical apoptotic phenomenon. No cells with morphologically altered nuclei were observed in either NHOKs or human MG-63 osteosarcoma cells treated for 24 h with eluents prepared from abalone shell-derived and control β-TCPs, or in non-treated control. Taken together, these data clearly demonstrate biological safety of β-TCP derived from abalone shell.

2. Synthesis and physicochemical properties of HA synthesized from abalone shells

2-1. Synthesis of HA synthesized from abalone shells

CaO is an important ceramic material in the synthesis of bioceramics. Hence, to synthesize the pure CaO from abalone shell, sintering was performed in an electric furnace at 950℃ for 3 h. Thereafter, to verify the synthesis of CaO from abalone shell by sintering process, FT-IR and XRD was performed. FT-IR spectroscopy revealed a sharp band at 3650 cm^-1, two broad bands centered at approximately 3823 and 3390 cm^-1, a medium doublet centered at approximately 1445 cm^-1, and very strong absorption at below 600 cm^-1 for the CaO synthesized from abalone shell. The profile of the bands for CaO obtained by sintering at 950˚C for 3 h matched that of commercial CaO (Junsei chemical Co., Ltd., Tokyo Japan) used as a control. Furthermore, the results of XRD showed the XRD patterns of commercial CaO showed peaks at 32.2°, 37.4°, and 53.92° 2θ. The XRD patterns of the CaO synthesized from abalone shell followed by sintering at 950˚C matched the peaks of commercial CaO. These data indicate that CaO had been synthesized from abalone shell by sintering at 950℃ for 3 h. Sequentially, CaO synthesized from abalone shell was phosphorylated by the chemical reaction with H₃PO₄ to synthesize the HA. Thereafter, synthesized composites were sintered at 1230℃ temperature for 3 h. To verify the synthesis of HA, FT-IR and XRD were performed. The FT-IR spectrum of composite sintered at 1230˚C temperature was obtained in the range of 500 – 4000. Especially, the peaks at 1030 and 570cm^-1, which were attributed to PO₄^3-, indicated the presence of HA. The C-O vibration in the CO₃ ^2- vibration band disappeared and the spectrum obtained was characteristic of HA. Furthermore, the XRD patterns of HA synthesized by sintering at 1230˚C and commercial HA were resulted. The XRD patterns of commercial HA used as a control showed the characteristic XRD peaks at 26, 29, 31.7, 32.2, 33, 40, 46.5, 49 and 53.2° 2θThe XRD patterns of HA synthesized from abalone shells were similar. Therefore, HA was synthesized successfully from CaO synthesized from abalone shell through the phosphorylation with phosphoric acid at pH 10.5 and sintering process at 1230˚C for 3h.

2-2. SEM and EDS mapping analysis of the HA synthesized from abalone shell

To observe the electron microscopic structure of HA synthesized from abalone shell, SEM scanning was performed at an acceleration voltage of 5.5 kV. The abalone shell-derived HA had a flat sheet morphology with a particle size of 25-50 μm. Furthermore, EDS was performed to examine the elemental composition of the HA sintered at 1230℃. Elemental analysis revealed O, P, and Ca for calcium phosphates. 4B. EDS showed that the commercial HA and abalone shell-derived HA contained similar elements. The calcium to phosphorous ratio (Ca/P ratio) in the HA synthesized from the abalone shells was approximately 1.83.

2-3. Cell cytotoxicity of abalone shell-derived HA in MG-63 cells

The relative cytotoxicity of abalone shell-derived HA and commercial HA were estimated as 96 ± 7% and 88 ± 8% compared with control, respectively. Although the relative cytotoxicity of abalone shell-derived HA is less than commercial HA, but there was no significance. Furthermore, to verify apoptotic cell death in the MG-63 cells treated with the effluent prepared from abalone shell-derived HA, DAPI staining was performed to observe the cells with nuclear condensation. Cells with nuclear condensation did not observed in the MG-63 cells treated with the effluent prepared from both HA. Moreover, the live and dead cell viability assay was performed to determine the number of live and dead cells attached to the HA or abalone shell-derived HA. The live cells were stained with green fluorescence by green calcein AM and the dead cells were stained with red fluorescence by ethidium homodimer-1 were visualized and counted under an inverted fluorescent microscope. The MG-63 cells in the presence of either commercial or abalone shell-derived HA were stained with green fluorescence through the cleavage of the membrane permeable calcein AM by the cytosolic esterase in living cells. Furthermore, dead cells with red fluorescence due to ethidium homodimer-1 staining were not observed in the MG-63 cells cultured with the abalone shell-derived HA for 24 h. This suggests that abalone shell-derived HA has an excellent biocompatibility as a material for bone grafting.

3. Synthesis and physicochemical properties of bone grafting materials composed of abalone shell-derived HA and β-TCP

All of bone grafting materials were synthesized as porous spherical particles with 0.5~1.0 mm using drop-wise into liquid nitrogen to format the micropore at both internal and external surface. After synthesis of bone grafting materials according to the defined mixing ration, they were performed the characteristic analysis according to the test guide line supplied from the Ministry of Food and Drug Safety, Republic of Korea. The electronic microscopic image analysis to observe the surface and to determine the pore distribution showed that the surface of synthesized bone grafting materials were similar with travecular bone with approximately 130 μm micropore size. The pH of synthesized bone grafting materials was pH 8.5~9.0. Each bone grafting materials synthesized by defined mixing ratio showed the low cytotoxicity and high cell attachment in both human normal oral keratinocyte and human osteoblast cell line MG-63. Especially, bone grafting material synthesized by mixing ratio 4:6 of HA and β-TCP showed the most bio-compatibility than that of other bone grafting materials. Furthermore, the Ca/P ration of bone grafting material synthesized by mixing ratio 4:6 of HA and β-TCP have a 1.64, that is a similar value of Ca/P of human bone and teeth.

(출처 : SUMMARY 24p)

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 보고서 요약서 ... 3
• 요 약 문 ... 5
• SUMMARY ... 18
• CONTENTS ... 34
• 목차 ... 35
• 제 1 장 연구개발과제의 개요 ... 38
• 제 1절 연구 개발 목적 및 필요성 ... 38
• 가. 연구개발의 개요 ... 38
• 나. 연구 개발대상 기술의 경제적, 산업적 중요성 및 연구 개발의 필요성 ... 38
• 제 2절 연구개발의 목표 및 연구개발 수행 내용 ... 43
• 1. 연구 개발의 최종 목표 및 주요 내용 ... 43
• 2. 최종목표의 성격 및 설정 근거 ... 45
• 3. 연차별 연구 개발의 목표 및 내용 ... 49
• 제 2 장 국내외 기술개발 현황 ... 52
• 제 1절 국내.외 관련분야에 대한 기술 개발 현황 ... 52
• 1. 국외 기술 동향 ... 52
• 제 2절 국내.외 기술개발현황에서 차지하는 위치 ... 67
• 1. 연구 개발 과제 및 대상 기술의 중복성 ... 67
• 제 3 장 연구개발 수행 내용 및 결과 ... 79
• 제 1절 1차년도 연구 결과 ... 79
• 가. 1차년도 연구개발 목표 및 내용 ... 79
• 나. 1차년도 연구 수행 내용 ... 79
• 제 2절 2 차년도 연구 결과 ... 112
• 가. 2 차년도 연구개발 목표 및 내용 ... 112
• 나. 2 차년도 연구 수행 내용 ... 112
• 제 3절 3 차년도 연구 결과 ... 138
• 가. 3 차년도 연구개발 목표 및 내용 ... 138
• 나. 3 차년도 연구 수행 내용 ... 138
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 326
• 제 1절 연구목표달성도 ... 326
• 가. 정량적 성과목표 달성도 ... 326
• 나. 계량형 성과목표 달성도 ... 329
• 제 2절 관련분야에의 기여도 ... 334
• 제 5 장 연구개발 성과 및 성과활용 계획 ... 350
• 제 1절 실용화 산업화 계획. ... 350
• 제 2절 특허, 논문 등 지식재산권 확보계획 ... 367
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 369
• 제 1절 해외특허 분석 결과 ... 369
• 제 2절 논문분석 ... 369
• 제 3절 제품 및 시장 분석 ... 370
• 제 4절 출발 물질로의 전복패각의 가치 ... 370
• 제 7 장 참고문헌 ... 372
• 끝페이지 ... 375