本研究目的是開發一個具藥物釋放功能之可注射性鈣矽複合骨水泥，以促進骨組織修復。使用明膠包覆中藥所形成的微粒與鈣矽組成之粉體結合成一複合材料。另外，本實驗中也探討加入抗生素後對於發炎反應的作用。我們針對此鈣矽複合骨水泥之藥物釋放、注射性、浸泡前後的徑向拉伸強度、型態、相組成以及生物性質作了一系列的測試。結果顯示加入明膠微粒之後會延長藥物釋放的時間，在注射性方面，單純的鈣矽骨水泥在五分鐘後只剩下不到40%可注射性，而包含有微粒的組別二十分鐘後還保有約一半的可注射性，三十分鐘後剩下20%。在細胞培養的部分，包含有續斷的微粒增進細胞的生長比單純微粒的高出1.35倍，而包含骨碎補的組別則高出約1.2倍。在基因表現上，此鈣矽複合材料具備有好的成骨特性，亦有許多對於發展牙科與骨科材料所應具備的優點，因此我們認為在骨科修復方面，此材料是具有發展潛力的。

The aim of this study was to develop novel injectabile calcium silicate-based composite bone cements with drug release for bone repair. The gelatin microspheres containing Chinese herbs were incorporated into calcium silicate powders to form the composite. Moreover, we investigated the effect of the inflammation after adding antibiotics. The drug release, injectability, before and after immersion behavior in physiological solution, including diametral tensile strength, morphology, and phase composition of various cements were evaluated, in addition to biology properties. The results indicated that the gelatin micropaticles may prolong drug release time. The results of injectability show that the pure calcium silicate cement only remained 20% after 5 minutes, however, the composite containing microspheres can extend injection time until 30 minutes. After 20 minutes, the composite containing microspheres can remain 40% injectability. This biocompatibility study indicated the composite containing drug significantly enhanced cell proliferation and differentiation. After Xu-Duan release in the medium could promote cell viability and have 1.35 times higher than the group without drug. Gu-Sui-Bu had about 1.2 times than pure microspheres. Gene expression showed these calcium silicate composites with high osteogenecity. It is concluded that the composite cement are a potential material for bone defect repair.

目錄
致謝 II
中文摘要 III
Abstract IV
圖目錄 IX
第一章 文獻回顧及理論基礎 1
1-1前言 1
1-2 生醫骨科材料的種類及發展 1
1-2-1 金屬材料 2
1-2-2 高分子材料 2
1-2-3 陶瓷材料 3
1-2-4 複合材料 3
1-3 骨水泥 4
1-4 天然高分子在生醫材料的應用 7
1-4-1 明膠(gelatin) 7
1-5 藥物 8
1-5-1骨碎補( Gu-Sui-Bu ) 9
1-5-2 續斷( Xu-Duan ) 9
1-5-3 抗生素(gentamicin) 9
1-6 研究動機與目的 9
第二章 材料與方法 11
2-1 材料製備 11
2-1-1 鈣矽粉體製備 11
2-1-2 微粒製備 11
2-2 物理性質分析 12
2-2-1 硬化時間測試 12
2-2-1 浸泡實驗 12
2-2-2-1 成分分析 12
2-2-2-2 徑向拉伸強度 12
2-2-2-3 重量損失 13
2-2-2-4 掃描式電子顯微鏡觀察 13
2-2-3 可注射性 13
2-2-4 藥物釋放測試 13
2-3 生物性質分析 14
2-3-1 不同藥物濃度的細胞培養 14
2-3-2 微粒釋放的細胞培養 14
2-3-3 直接培養 14
2-3-4 Ca2+染色 15
2-3-5 掃描式電子顯微鏡觀察 15
2-3-6 光學顯微鏡觀察細胞 15
2-3-7 基因測試 16
2-3-7-1 RNA萃取 16
2-3-7-2 RT-PCR放大 16
2-3-7-3 Agarose gel 16
第三章 結果與討論 17
3-1 物理性質分析 17
3-1-1微粒表面形態觀察 17
3-1-2 浸泡實驗 18
3-1-2-1 X光繞射分析 18
3-1-2-2 徑向拉伸強度 22
3-1-2-3 重量損失 24
3-1-2-4 掃描式電子顯微鏡觀察 25
3-1-3 可注射性 28
3-1-4 藥物釋放測試 29
3-2 生物性質分析 32
3-2-1 不同藥物濃度的細胞培養 32
3-2-2 微粒釋放的細胞培養 34
3-2-3 直接培養 35
3-2-4 Ca2+染色 36
3-2-5 掃描式電子顯微鏡觀察 37
3-2-6 基因表現 40
3-2-6-1 發炎反應 40
3-2-6-2 成骨作用 43
第四章 結論 46
第五章 參考文獻 47

圖目錄
Fig. 1 經交聯後之微球表面型態 17
Fig. 2 不同骨水泥浸泡於SBF後所得之X光繞射分析結果 21
Fig. 3 骨水泥含不同藥物浸泡於SBF後所得之逕向拉伸強度 (a) 骨碎補 (b) 續斷 (c)gentamicin 23
Fig. 4 骨水泥浸泡於Tris buffer後之重量損失變化 24
Fig. 5 將不同骨水泥浸泡入(a) SBF中1天 (b) Tris buffer 7天後表面及斷面的顯微結構圖 27
Fig. 6 含有不同藥物骨水泥的注射性質(A) 骨碎補 (B) 續斷 28
Fig. 7 (a) 骨碎補濃度之標準曲線 (b) 骨碎補藥物之釋放曲線 30
Fig.8 (a) 續斷濃度之標準曲線 (b) 續斷藥物之釋放曲線 31
Fig. 9 藥物濃度對於細胞生長的影響 (A)骨碎補及續斷 (B) gentamicin 33
Fig. 10 明膠微粒包含藥物對於細胞生長的影響 34
Fig. 11 細胞培養於材料表面上的生長情形 35
Fig. 12 細胞培養於材料表面之鈣化程度 36
Fig. 13 細胞培養於(A) s55 (B) GM (C) GSB (D) XD表面1天之SEM圖 37
Fig. 14 細胞培養於(A) s55 (B) GM (C) GSB (D) XD表面3天之SEM圖 38
Fig. 15 細胞培養於(A) s55 (B) GM (C) GSB (D) XD表面7天之SEM圖 39
Fig. 16 細胞培養於骨水泥表面1及3天的基因表現（A）不同組別的基因表現圖，（B）TNF-alpha、（C）iNOS、（D）IL-1及（E）IL-10的基因表現量 42
Fig. 17 細胞培養於骨水泥表面3及7天的基因表現（A）不同組別的基因表現圖，（B）collagen、（C）ALP、（D）OC及（E）BSP的基因表現量 45

[1]Boyne PJ. Implants and transplants review of recent research in this area of oral surgery. JADA 1973;87:1074–1081.
[2]Oikarinen J, Korhonen LK. The bone inductive capacity of various bone transplanting materials used for treatment of experimental bone defect. Clin Orthop Relat Res 1979;140:208–214.
[3]Mulliken JB, Glowacki J, Kaban LB, Folkman J, Murray JE. Use of demineralized allogenic bone implants for corrections of maxillocraniofacial deformities. Ann Surg 1981;194:366–372.
[4]闕山璋,“骨科植入物生醫材料及器材”,科儀新知, 第十三卷, 第一期, 1991.
[5]Kline J. Handbook of biomedical engineering. Academic press, Inc., 1988.
[6]Lemons JE. Bioceramics: Is there a difference? Clinical Orthopaedics and Related Research 1990;261:153–158.
[7]Kitsugi T, Yamamura T, Kokubo T. Bone bonding behavior of MgO-CaO-SiO2-P2O5 glass. J Biomed Mater Res 1989;23:631–635.
[8]Ogino M, Hench LL. Formation of calcium phosphate films on silicate glasses. J Non-Cryt Solids 1980;38:673–677.
[9]Albee FH, Morrison HF. Studies in bone growth triple calcium phosphate as a stimulus to osteogenesis. Ann Surg 1920;71:32.
[10]Nakamura T, Yamamuro T, Higashi S, Kokubo T, Itoo S. A newglass-ceramic for bone replacement: evaluation of its bonding to bonetissue. J Biomed Mater Res, 1985;19:685–698.
[11]Lin FH, Hon MH, Wu SC. Fabrication and biocompatibility of a porous bioglass ceramic in a Na2O-CaO-SiO2-P2O5 system. J Biomed Eng 1991;13:328–334.
[12]Ishizawa H, Ogino M. Formation and characterization of anodic titanium oxide films containing Ca and P. J Biomed Mater Res 1995;29:65–72.
[13]Ishizawa H, Ogino M. Characterization of thin hydroxyapatite layersformed on anodic titanium oxide films containing Ca and P by hydrothermal treatment. J Biomed Mater Res 1995;29:1071–1079.
[14]Takatsuka K, Yamamuro T, Nakamura T, Kokubo T. Bone-bonding behavior of titanium alloy evaluated mechanically with detaching failureload. J Biomed Mater Res 1995;29:157–163.
[15]Mittelmeier H, Katthagen BD. Clinical experience with the implantation of collagen-apatite for local bone regeneration. Zeitschrift fur Orthop und Ihre Grenzgebiete 1983;121:115–123.
[16]Chieerici G. Biological response to a lyophilised collagen gel implanted in parietal bone defects in a Rhesus monkey. Collagen Corporation Internal Publication, 1984, Report 3, pp. 116.
[17]Driessens FC. The mineral in bone, dentin and tooth enamel. Bull Chim Belg 1980;89:663.
[18]生醫陶瓷的現況及開發法，“工業技術研究院工業材料所編譯資料”,No.mr73-002,1984.
[19]Albee FH, Morrison HF, Studies in bone growth triple calcium phosphate as a stimulus to ostrogenesis. Ann Surg 1920;71:32.
[20]Hench LL, Lee TG, Allen WC, Piotrowski G. An investigation of a Prosthetic material. U.S. Army Med Res and Develop Command Contract No. OA 17-70-C-001, 1970–1975.
[21]Hench LL, Paschall HA. Direct chemical bond of bioactive glass-ceramic material to bone and muscle. J Biomed Mater Res 1973;4:27–25.
[22]Hench LL, Splinter RJ, Allen WC, Greenlee TK. Bonding mechanism at the interface of ceramic prosthetic materials. J Biomed Mate. Res 1971;2:117–141.
[23]Blencke BA, Bromer H, Deutcher K. Glass ceramic—A new bioactive implant material. Med Orthop Tech 1975;95:144–148.
[24]Kokubo T, Shigematsu M, Nagashima Y, Tashiro M, Nakamura T, Yamamuro T, Higashi S. Apatite- and wollastonitecontaining glass–ceramic for prosthetic application. Bull Inst Chem Res 1982;60:260–267.
[25]Jarcho M, Bolen CH, Thomas MB, Bobick J, Kay JF, Doremus RH. Synthesis and characterization of hydroxyapatite in dense polycrystalline form. J Mater Sci 1976;11:2027–2035.
[26]Balas F, Perez-Pariente J, Vallet-Regi M. In vitro bioactivity of silicon- substituted hydroxyapatite. J Biomed Mater Res 2006;66A:364–375.
[27]Gibson IR, Best SM, Bonfield W. Chemical characterization of silicon- substituted hydroxyapatite. J Biomed Mater Res 1999;44:422–428.
[28]Alexandra EP, Claudia MB, Maria AL, Jose DS, Serena MB, William Bonfield. Ultrastructural comparison of dissolution and apatite precipitation on hydroxyapatite and silicon- substituted hydroxyapatite in vitro and in vivo. J Biomed Mater Res 2004;69A:670–679.
[29]Alexandra EP, Serena MB, Bonfield W. Ultrastructural comparison of hydroxyapatite and silicon- substituted hydroxyapatite for biomedical applications. J Biomed Mater Res 2004;68A:133–141.
[30]Alexandra EP, Patel N, Skepper JN, Best SM, Bonfield W. Comparison of in vivo dissolution process in hydroxyapatite and silicon- substituted hydroxyapatite bioceramics. Biomaterials 2003;24:4609–4620.
[31]Sayer M, Stratilatov AD, Reid J, Calderin L, Stott MJ, Yin X, MacKenie M, Smith TJN, Hendry JA, Langstaff SD. Structure and composition of silicon- stabilized tricalcium phosphate. Biomaterials 2003;24:369–382.
[32]Phong V. Phan, Mark Grzanna, James Chu, Anna Polotsky, Ahmed El-Ghannam, David Van Heerden, David S. Hungerford, Carmelita G. Frondoza. The effect of silica- containing calcium- phosphate particles on human osteoblasts in vitro. J Biomed Mater Res 2003;67A:1001–1008.
[33]Kokubo T. Mechanism of Apatite Formation on CaO-SiO2-P2O5 Glasses in a Simulated Body Fluid, J Non-Cryst Solids 1992;143:84–92.
[34]Broemer H, Deutscher K, Blencke B, Pfeil E, Strunz V. Properties of the bioactive implant material: Ceravital. Sci Ceramics 1977;9:219–225.
[35]Albee Y, Yamaguchi A, Fukui H. Calcium phosphate glass-ceramics. Japan Kokai 1976;76:24.
[36]Lin FH, Hon MH. A study on Bioglass ceramics in the Na2O-CaO-SiO2-P2O5 system. J Mater Sci 1988;23:4295–4299.
[37]Nakamura T, Yamamuro T, Higashi S, Kokubo T, Ito A. A new glass ceramic for bone replacement: Evaluation of its bonding to bone tissue. J Biomed Mater Res 1985;19:685–698.
[38]Lin FH, Yao CH, Huang CW, Liu HC, Sun JS, Wang CY. The bonding behavior of DP-Bioglass and bone tissue. Mater Chem and Phys 1996;46:36–42.
[39]Jarcho K, Kay JF, Gumaer KI, et al: Tissue, cellular and subcellular events at a bone-ceramic hydroxyapatite interface. JBio eng 1:79,1977.
[40]Block MS, Kent JN: Long term evaluation of hydroxylapatite augmentation of deficient mandibular alveolar ridges. J Oral Maxillofac Surg 1984;42:793–796.
[41]Kent JN, Zide MF, Kay JF, et al: Hydroxylapatite blocks and particles as bone graft substitutes in orthognathic and reconstructive surgery. J Oral Maxillofac Surg 1986;44:597–605.
[42]Zide MF, Kent JN, Machado L: Hydroxylapatite cranioplasty directly over dura. J Oral Maxillofac Surg 1987;45:481-486.
[43]Ono K. Yamamuro T. Nakamura T. Kakutani Y. Kitsugi T. Hyakuna K. Kokubo T. Oka M. Kotoura Y. Apatite-wollastonite containing glass ceramic-fibrin mixtures as a bone defect filler. J Biomed Mater Res 1988;22:869–885
[44]Smith L. Ceramic plastic materials as a bone structure. Arch Surg 1963; 87:653–661.
[45]Benum P, Lyng S, Alm T, Johannessen N. Porous ceramics as a bone substitute in the medical condyle of the tibia. An experimental study in sheep. Long-term observation. Acta Orthop Scand 1977;48:15.
[46]Gendler E. Perforated demineralized bone matrix: A new form of osteoinductive biomaterial. J Biomed Mater Res 1986;20:687–697.
[47]Ono K. Yamamuro T. Nakamura T. Kakutani Y. Kitsugi T. Hyakuna K. Kokubo T. Oka M. Kotoura Y. Apatite-wollastonite containing glass ceramic-fibrin mixtures as a bone defect filler. J Biomed Mater Res 1988;22:869–885.
[48]Yao CH, Chang YL, Lin FH. Preparation and evaluation of β-TCP powder and crosslinked gelatin composite as bone substitute. Chinese J Med Bioeng 1994;14:47–51.
[49]Tamura S, Kataoka H, Matsui Y, Shionoya Y, Ohno K, Michi KI, Takahashi K, Yamaguchi A. The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)/carrier complex on bone repair. Bone 2001;29:169–175.
[50]Saito N, Okada T, Horiuchi H, Ota H, Takahashi J, Murakami N,Nawata M, Kojima S, Nozaki K, Takaoka K. Local bone formation by injection of recombinant human bone morphogenetic protein–2 contained in polymer carriers. Bone 2003;32:381–386.
[51]Den Boer FC. Wippermann BW. Blokhuis TJ. Patka P. Bakker FC. Haarman HJ. Healing of segmental bone defects with granular porous hydroxyapatite augmented with recombinant human osteogenic protein-1 or autologous bone marrow. J Orthop Res 2003;21:521–528.
[52]Schmidt RJ, Chung LY, Andrews AM, Spyratou O, Turner TD: Biocompatibility of wound management products: a study of the effects of various polysaccharides on murine L929 fibroblast proliferation and macrophage respiratory burst. J Pharm Pharmacol 1993;6:508–513.
[53]Doyle JW, Roth TP, Smith RM, Li YQ, Dunn RM: Effects of calcium alginate on cellular wound healing processes modeled in vitro. J Biomed Mater Res 1996;4:561–568.
[54]Choi YS, Hong SR, Lee SB, Lee YM, Song KW, Park MH: Study on gelatin-based sponges. Part III: A comparative study of cross-linked gelatin/alginate, gelatin/hyaluronate and chitosan/hyaluronate sponges and their application as wound dressing in full-thickness skin defect of rat. J materials science 2001;1:67–73.
[55]Choi YS, Hong SR, Lee YM, Song KW, Park MH, Nam YS: Study on gelatin-containing artificial skin: I. Preparation and characteristics of novel gelatin-alginate sponge. Biomaterials 1999;5:409–417.
[56]Cook W: Alginate dental impression materials: chemistry, structure, and properties. J Biomed Mater Res 1986;1:1–24.
[57]Sechoy O, Tissie G, Sebastian C, Maurin F, Driot JY, Trinquand C: A new long acting ophthalmic formulation of carteolol containing alginic acid. Int J Pharm 2000;2:109–116.
[58]Vandenberg GW, Drolet C, Scott SL, de la Noue J: Factors affecting protein release from alginate-chitosan coacervate microcapsules during production and gastric/intestinal simulation. J Control Release 2001;3:297–307.
[59]Fernandez H, Holgado MA, Fini A, Fell JT: In vitro evaluation of alginate beads of a diclofenac salt. int J Pharm 1998;163:23–34.
[60]Mi FL, Sung HW, Shyu SS. Drug release from chitosan-alginate complex beads reinforced by a naturally occurring cross-linking agent. Carbohydrate Polymers 2002;1:61–72.
[61]Kuo CK, Ma PX. Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. Structure, gelation rate and mechanical properties. Biomaterials 2001;6:511–521.
[62]Masuda K, Sah RL, Hejna MJ, Thonar EJ. A novel two-step method for the formation of tissue-engineered cartilage by mature bovine chondrocytes: the alginate-recovered-chondrocyte (ARC) method. J Orthop Res 2003;1:139–148.
[63]Marijnissen WJ, van Osch GJ, Aigner J, van der Veen SW, Hollander AP, Verwoerd-Verhoef HL, Verhaar JA. Alginate as a chondrocyte-delivery substance in combination with a non-woven scaffold for cartilage tissue engineering. Biomaterials 2002;6:1511–1517.
[64]Bigi A, Bracci B, Panzavolta S. Effect of added gelatin on the properties of calcium phosphate cement. Biomaterials 2004;25:2893–2899.
[65]Fujishiro Y, Takahashi K, Sato T. Preparation and compressive strength of a-tricalcium phosphate/gelatin gel composite cement. J Biomed Mater Res 2001;54:525–530.
[66]Fujishiro Y, Takahashi K, Sato T. Preparation and compressive strength of a-tricalcium phosphate/gelatin gel composite cement. J Biomed Mater Res 2001;54:525–530.
[67]Tetsu Katsumura, Tomihisa Koshino, Tomoyuki Saito. Viscous property and osteogenesis induction of hydroxyapatite thermal decomposition product mixed with gelatin implanted,into rabbit femurs. Biomaterials 1998;19:1839–1844.
[68]Kim HW, Kim HE, Salih V. Stimulation of osteoblast responses to biomimetic nanocomposites of gelatin–hydroxyapatite for tissue engineering scaffolds. Biomaterials 2005;26:5221–5230.
[69]Bigi A, Bracci B, Cojazz G, Panzavolta S, Roveri N. Drawn gelatin films with improved mechanical properties. Biomaterials 1998;19:2335–2340.
[70]Furthmayr H, Timpl R. Immunochemistry of collagens and procollagens. Int Rev coccect Tiss Res., 1976;7:61–70.
[71]Elisabetta E, Cortesi R, Nastruzzi C. Gelatin microspheres: Influence of preparation parameters and thermal treatment onchemico-physical and biopharmaceutical properties. Biomaterials 1996;17: 2009–2020.
[72]Link DP, Dolder J, Beucken J, Wolke JG, Mikos AG, Jansen JA. Bone response and mechanical strength of rabbit femoral defects filled with injectable CaP cements containing TGF-β1 loaded gelatin microparticles. Biomaterials 2008;29:675–682.
[73]Habraken WJEM, Jonge LT, Wolke JGC, Yubao L, Mikos AG, Jansen JA. Introduction of gelatin microspheres into an injectable calcium phosphate cement. J Biomed Mater Res Part A 2008;87:643–655.
[74]Huang Y, Onyeri S, Siewe M, Moshfeghian A, Madihally SV. In vitro characterization of chitosan–gelatin scaffolds for tissue engineering. Biomaterials 2005;26:7616–7627.
[75]Ogino M, Hench LL. Formation of calcium phosphate films onsilicat e glasses. J Noncryst Solids 1980;38:673–678.
[76]Koh HL, Woo SO. Chinese proprietary medicine in Singapore: Regulatory control of toxic heavy metals and undeclared drugs. Drug Saf 2000;23:351–362.
[77]Chan K. Progress in traditional Chinese medicine. Trends Pharmacol Sci 1995;16:182–187.
[78]Yuan R, Lin Y. Traditional Chinese medicine: An approach to scientific proof and clinical validation. Pharmacol Ther 2000; 86:191–198.
[79]Arnold MD, Thornbrough LM. Treatment of musculoskeletal pain with traditional Chinese herbal medicine. Phys Med Rehabil Clin N Am 1999;10:663–671.
[80]顏正華：中藥學，人民衛生出版社，1995。
[81]王輝武：中醫百家藥論匯粹，重慶出版社，1992。
[82]楊維傑：實用中醫方劑學，樂群文化事業，1988。
[83]本經疏證，清‧鄒潤安。
[84]張賢哲：本草備要解析，中國醫藥學院。
[85]Schnieders J, Gbureck U, Thull R, Kissel T. Controlled release of gentamicin from calcium phosphate—poly(lactic acid-co-glycolic acid) composite bone cement. Biomaterials 2006:27:4239-4249.