臺灣博碩士論文加值系統

本研究目的是以不同晶粒大小的氫氧基磷灰石(HA)塊材做為基材，探討人類類骨母細胞(HOS)與人類纖維母細胞(HSF)初期之增殖情況。本實驗以濕式法合成HA粉末，藉由不同的製備參數：滴定速率、滴定溫度、熟化時間及不同塊材燒結溫度，並透過X光繞射(XRD)鑑定其結構及計算晶粒大小，以了解影響晶粒大小之因素。此外，利用高解析分析電子顯微鏡(HR-AEM)觀測粉末型態和利用表面粗度測定儀量測塊材表面粗糙度。實驗結果顯示，在滴定溫度25℃和80℃下製備出的HA之晶粒大小分別為11和22nm，HA塊材經燒結400、800和1200℃後，晶粒大小分別為20、32和47nm。利用MTT測量細胞增殖情況，藉以評估細胞相容性，實驗結果發現晶粒愈大之HA塊材，HOS細胞增殖情況愈好；另一方面，HSF細胞在不同晶粒大小的HA塊材上，經一、三天培養後各有不同的增殖情形，且HA基材對於HSF細胞之增殖似乎有抑制的作用，因此，綜觀上述結論，HA塊材之晶粒大小可能會影響此兩種細胞的增殖表現，也因為不同的細胞種類對於基材的反應不同，造成細胞增殖上的差異。

The purpose of the study is to investigate the influence of hydroxyapatite grain size on initial proliferation of human osteoblast-like cells (HOS) and human skin fibroblasts (HSF). In this study, HA powders were prepared using wet methods. Reactant addition rate, reaction temperatures, aging time and sintering time would be changed respectively throughout the reactant process and examined using X-ray diffraction (XRD) to identify the phase and estimate the grain size. Besides, the morphology of HA powders was displayed by Ultrahigh Resolution Analytical Electron Microscope (HR-AEM) and the surface roughness of HA bulks was evaluated by Surface Roughness Measurement Instrument. The results revealed the grain size of HA powders reacting at temperature 25 and 80℃ were 11 and 22 nm, respectively. The average grain size of HA bulks increased to 20, 32 and 47 nm when sintering at 400, 800 and 1200℃, respectively. Further, cell proliferation was measured by the Methylthiazoletetrazolium (MTT) test. The results of the study provided that HOS proliferation was significantly greater on the larger grain size of HA bulks than the smaller ones. On the other hand, HSF cells showed disparate cell proliferation on different grain size of HA bulks after culturing 1 and 3 days; moreover, HA seemed to have the function of restraining the proliferation of HSF cells. Therefore, it has been observed that HA grain size may affect the proliferation of HOS and HSF cells. Also, the cell types react distinctively according to various materials, and thus caused a variety of cell proliferation.

中文摘要.............................................. I
Abstract.............................................. II
誌謝...................................................III
表目錄.................................................VI
圖目錄.................................................VII
第一章 緒論............................................1
第二章 文獻回顧與理論基礎..............................3
2.1 HA之組成與應用.....................................3
2.2 HA之製備方式.......................................4
2.3 影響HA晶粒大小之因素...............................5
2.4 骨母細胞與纖維母細胞...............................6
2.4.1 骨母細胞.........................................6
2.4.2 纖維母細胞.......................................7
2.5 細胞生長於HA的反應.................................8
第三章 實驗材料與方法..................................11
3.1 HA粉末之製備與相關鑑定.............................13
3.1.1 合成HA粉末.......................................13
3.1.2 改變合成參數.....................................14
3.1.3 HA粉末的相鑑定及晶粒大小量測.....................14
3.1.4 HA粉末的型態觀察.................................15
3.2 HA生胚之製備與燒結.................................15
3.2.1 HA生胚的壓製.....................................15
3.2.2 HA塊材之燒結.....................................17
3.2.4 HA塊材的表面粗糙度量測...........................18
3.2.5 燒結後HA塊材的相鑑定.............................19
3.2.6 HA塊材的滅菌處理.................................19
3.3 細胞培養...........................................19
3.3.1 HOS人類類骨細胞..................................20
3.3.2 HSF人類纖維母細胞................................20
3.4 細胞相容性評估.....................................20
3.4.1細胞增殖( Cell proliferation)實驗.................20
3.5 統計分析方法.......................................21
第四章 結果............................................22
4.1 材料性質鑑定.......................................22
4.1.1 合成粉末之相鑑定與晶粒大小測定...................22
4.1.2 合成粉末之型態觀測...............................26
4.1.3 HA塊材之體密度...................................28
4.1.4 HA塊材之表面粗糙度量測...........................30
4.1.5 燒結後HA塊材之相鑑定與晶粒大小測定...............32
4.1.6 HA塊材之孔隙度量測...............................34
4.2 細胞生長週期.......................................35
4.2.1 HOS細胞生長週期測試..............................35
4.2.2 HSF細胞生長週期測試..............................36
4.3 細胞相容性評估.....................................36
4.3.1 HOS細胞增殖實驗..................................36
4.3.1.1 一天後HOS細胞增殖情形..........................37
4.3.1.2 三天後HOS細胞增殖情形..........................39
4.3.2 HSF細胞增殖實驗..................................41
4.3.2.1 一天後HSF細胞增殖情形..........................41
4.3.2.2 三天後HSF細胞增殖情形..........................43
第五章 討論與結論......................................45
5.1 討論...............................................45
5.1.1 不同參數製備之粉末性質探討.......................45
5.1.2 HA塊材性質探.....................................46
5.1.3 不同晶粒大小之HA塊材對細胞增殖之影響.............48
5.1.3.1 HOS細胞增殖探討................................48
5.1.3.2 HSF細胞增殖探討................................49
5.2 結論...............................................51
參考文獻...............................................52
自 述...............................................55

1.Kokubo T, Kim HM, Kawashita M: Novel bioactive materials with different mechanical properties. Biomaterials 2003, 24:2161-2175.

2.Vance RJ, Miller DC, Thapa A, Haberstroh KM, Webster TJ: Decreased fibroblast cell density on chemically degraded poly-lactic-co-glycolic acid, polyurethane, and polycaprolactone. Biomaterials 2004, 25:2095-2103.

3.Bharati S, Basu D: Hydroxyapatite coating by biomimetic method on titanium alloy using concentrated SBF. Bull Mater Sci 2005, 28(6):617–621.

4.Wang M: Bioactive materials and processing. Biomaterials and Tissue Engineering 2004, editor: D. Shi, Berlin Heidelberg: Springer, p. 1–82.

5.Webster TJ, Ergun C, Doremus RH, Siegel RW, Bizios R: Specific proteins mediate enhanced osteoblast adhesion on nanophase ceramics. J Biomed Mater Res 2000, 51:475-483.

6.Evis Z, Sato M, Webster TJ: Increased osteoblast adhesion on nanograined hydroxyapatite and partially stabilized zirconia composites. J Biomed Mater Res A 2006, 78:500-507.

7.Ravaglioli A, Krajewski A: Bioceramics. Chapman & Hall, London 1992.


8.Webster TJ, Ergun C, Doremus RH, Siegel RW, Bizios R: Enhanced osteoclast-like cell functions on nanophase ceramics. Biomaterials 2001, 22:1327-1333.

9.Webster TJ, Massa-Schlueter EA, Smith JL, Slamovich EB: Osteoblast response to hydroxyapatite doped with divalent and trivalent cations. Biomaterials 2004, 25:2111-2121.

10.Porter AE: Nanoscale characterization of the interface between bone and hydroxyapatite implants and the effect of silicon on bone apposition. Micron 2006.

11.Wei M, Evans JH, Bostrom T, Grondahl L: Synthesis and characterization of hydroxyapatite, fluoride-substituted hydroxyapatite and fluorapatite. J Mater Sci Mater Med 2003, 14:311-320.

12.He Z, Ma J, Wang C: Constitutive modeling of the densification and the grain growth of hydroxyapatite ceramics. Biomaterials 2005, 26:1613-1621.

13.Christenson RH: Biochemical markers of bone metabolism: an overview. Clin Biochem 1997, 30:573-593.

14.Stein GS, Lian JB, Owen TA: Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation. Faseb J 1990, 4:3111-3123.

15.Aubin JE, Heersche JM: Osteoblastic cell lineage. Cellular and molecular biology of bone. New York academic Press 1993:1-45.

16.Schmidt C, Ignatius AA, Claes LE: Proliferation and differentiation parameters of human osteoblasts on titanium and steel surfaces. J Biomed Mater Res 2001, 54:209-215.

17.Baxter LC, Frauchiger V, Textor M, ap Gwynn I, Richards RG: Fibroblast and osteoblast adhesion and morphology on calcium phosphate surfaces. Eur Cell Mater 2002, 4:1-17.

18.Cerroni L, Filocamo R, Fabbri M, Piconi C, Caropreso S, Condo SG: Growth of osteoblast-like cells on porous hydroxyapatite ceramics: an in vitro study. Biomol Eng 2002, 19:119-124.

19.Hong JY, Kim YJ, Lee HW, Lee WK, Ko JS, Kim HM: Osteoblastic cell response to thin film of poorly crystalline calcium phosphate apatite formed at low temperatures. Biomaterials 2003, 24:2977-2984.

20.Jun IK, Jang JH, Kim HW, Kim HE: Recombinant osteopontin fragment coating on hydroxyapatite for enhanced osteoblast-like cell responses. J Mater Sci Mater Med 2005, 40:2891 – 2895.

21.Kumar R, Prakash KH, Cheang P, Khor KA: Temperature driven morphological changes of chemically precipitated hydroxyapatite nanoparticles. Langmuir 2004, 20:5196-5200.

22.Kothapalli CR, Wei M, Legeros RZ, Shaw MT: Influence of temperature and aging time on HA synthesized by the hydrothermal method. J Mater Sci Mater Med 2005, 16:441-446.

23.Gu YW, Loh NH, Kho KA, Tor SB, Cheang P: Spark plasma sintering of hydroxyapatite powders. Biomaterials 2002, 23:37-43.

24.Wang C, Duana Y, Markovicc B, Barbarad J, Howlettd CR, Zhanga X, Zreiqatd H: Proliferation and bone-related gene expression of osteoblasts grown on hydroxyapatite ceramics sintered at different temperature. Biomaterials 2004, 25:2949–2956.

25.Rouahi M, Gallet O, Champion E, Dentzer J, Hardouin P, Anselme K: Influence of hydroxyapatite microstructure on human bone cell response. J Biomed Mater Res A 2006, 78:222-235.

26.Schaffner P, Dard MM: Structure and function of RGD peptides involved in bone biology. Cell Mol Life Sci 2003, 60:119-132.