201119693 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種治療股骨頭壞死之刺激血管新生性 注射式可降解骨水泥，尤指一種由可降解的材料聚富馬酸 二羥丙酯（p〇ly(propylene fumarate))及碟酸|弓水泥 (calcium phosphate cement)，添加人參息苦 (ginsenoside Rgl)製成強度足以支撐經核心減壓術處理 之股骨頭缺陷。 【先前技術】 股骨頭缺血性壞死是常見的骨疾之一，一般早期的治 療手術為核心減壓，但是病人在此種手術之後，股骨頭無 法立即得到機械強度上的支撐，必須避免讓股骨頭承受壓 力。有需要發展出生物可分解及促進血管新生的骨泥，於 病人核心減壓之後注入，初期提供機械強度及幫助血管新 生提供營養’以促進股組織復原，期望新的股組織能取代 分解掉的骨泥，使患部復原。 聚畐馬酸一每丙醋（PPF)作為生物可分解性組織工程 材料及藥物輸送材料係已知者（Lakshmi S et al. (2006) (Adv Biochera Engin/Biotechnol (2006) 102: 47 - 90: Polymers a,s Bioinaterials for Tissue Engineering and Con1:rolied Drug Delivery”）。聚富馬酸二羥丙酯 (PPF)與磷酸鹽水泥的複合材料已在林峰輝等人（2009)於 中華民國發明公開公報編號：200902469，名稱π具有生物 可分解性之骨水泥及其製備方法"中提及，其為一種具有 201119693 生物可分解性之骨水泥及’係在製備出高分子聚富馬酸二 羥丙酯（PPF)後，將其溶在N-乙烯基吡咯烷酮（Ν-VP)中 攪拌均勻’再將一磷酸四鈣（TTCP)/無水磷酸氫鈣（DCPA) >谷入N-VP/PPF溶液中’把一泡打粉 taking powder) (BP) 溶入溶液中，再將兩者混合均勻，在室溫下讓其固化完 全’即可得到該發明所述具有生物可分解性之骨水泥，具 有可注射性、生物可分解性、放射線不通透性、更接近骨 路強度的抗壓強度性、聚合溫度較低性，以及高生物相容 性等優點。 人蔘皂苷（Ginsenoside)是一種固醇類化合物，三萜 皂苷。其只在人蔘屬植物中發現到。其中，人蔘皂苷Rgl (Ginsenoside Rgl)為無色半结晶物（正丁醇—甲基乙基 酮），可溶於吡啶、甲醇及热丙酮，稍溶於醋酸乙酯及氣 仿。 人蔘皂苷Rgl經證實可以刺激微血管形成、及人類臍 靜脈内皮細胞（Human umbilical vein endothelial cel 1，HUVEC)的增生與移動（Lin al. Vascul. Pharmacol.，2008，49(1):37-43)。人蔘皂苷Rgl 可透過 基因途徑與非基因途徑刺激血管生成。於基因途徑中， Rgl可增加血官内皮生長因子的產生（Leung et al. J.201119693 VI. Description of the Invention: [Technical Field] The present invention relates to an angiogenic injectable degradable bone cement for treating femoral head necrosis, especially a polyhydroxyl fumarate from a degradable material. (p〇ly (propylene fumarate)) and calcium phosphate cement, added with ginsenoside Rgl to produce a femoral head defect that is strong enough to support core decompression. [Prior Art] Avascular necrosis of the femoral head is one of the common bone diseases. Generally, the early treatment is the core decompression, but after the operation, the femoral head cannot immediately obtain mechanical strength support, and must be avoided. The femoral head is under stress. There is a need to develop bio-decomposable and vascular-promoting bone mud, which is injected after the patient's core decompression, initially providing mechanical strength and helping angiogenesis to provide nutrition' to promote the recovery of the femoral tissue, expecting new tissue to replace the decomposed Bone mud, which restores the affected part. Polyglycerol mono- vinegar (PPF) is known as a biodegradable tissue engineering material and drug delivery material (Lakshmi S et al. (2006) (Adv Biochera Engin/Biotechnol (2006) 102: 47 - 90 : Polymers a, s Bioinaterials for Tissue Engineering and Con1:rolied Drug Delivery"). Composites of poly(hydroxypropyl) fumarate (PPF) and phosphate cement have been invented in Lin Fenghui et al. (2009) in the Republic of China. Bulletin No.: 200202469, the name π biodegradable bone cement and its preparation method, mentioned in the article, which is a bone cement with 201119693 biodegradability and 'system in the preparation of high molecular weight fumaric acid II After hydroxypropyl ester (PPF), it is dissolved in N-vinylpyrrolidone (Ν-VP) and stirred evenly. Then tetracalcium monophosphate (TTCP) / anhydrous calcium hydrogen phosphate (DCPA) > /PPF solution 'Plastic powder to take powder' (BP) is dissolved in the solution, and then the two are evenly mixed, and allowed to solidify at room temperature to obtain the biodegradable bone of the invention. Cement, injectable, biodegradable Radiation impermeability, compressive strength closer to the strength of the bone path, lower polymerization temperature, and high biocompatibility. Ginsenoside is a sterol compound, triterpenoid saponin. It is only found in the genus Amaranthus. Among them, ginsenoside Rgl (Ginsenoside Rgl) is a colorless semi-crystalline (n-butanol-methyl ethyl ketone), soluble in pyridine, methanol and hot acetone, slightly soluble Ethyl acetate and gas imitation. Glutathione Rgl has been shown to stimulate microvascular formation and proliferation and migration of human umbilical vein endothelial cel 1, HUVEC (Lin al. Vascul. Pharmacol., 2008, 49(1): 37-43). Human saponin Rgl stimulates angiogenesis through gene pathways and non-gene pathways. In the gene pathway, Rgl increases the production of blood-derived endothelial growth factor (Leung et al. J.
Biol· Chem. 2006; (281):36280-8)且增加與細胞骨重 塑（cytoskeleton remodeling)、細胞-細胞黏著和遷移 相關的基因之表現。於非基因途徑中，Rgl可提升氧化氮 的產生而誘導血管生成（Leung et al. FEBS Lett. 2006; 580(13):3211-6) 〇 ’ 201119693 本案發明人胁可_聚富馬酸二_自旨⑽)與猶 鹽骨水泥生物可降解的特性，釋放混合於骨水泥成分中之 促進血=新生因子Rg卜對促進上皮細胞型成血管具有統計 學上顯著的功效’預期應用於臨床可加速早射癒合，提 供骨缺損足夠的機械強度，促進股骨頭血f新生，且最後 被人體代謝動正常骨_取代，極研朗發，最後 經多年苦心孤錯虔心研究而開發出—種治療股骨頭壞死之 刺激血管新生餘射式可降水泥，因岐成本發明。 【發明内容】 本發明之目的為提供一種治療股骨頭壞死之刺激血管 新生性注射式可降解骨水泥，其可利用該骨水泥生物可降 解的特性，釋放齡於骨水泥成分t之促進血储生因子Biol Chem. 2006; (281): 36280-8) and increased the expression of genes associated with cytoskeleton remodeling, cell-cell adhesion and migration. In the non-gene pathway, Rgl can promote the production of nitric oxide and induce angiogenesis (Leung et al. FEBS Lett. 2006; 580(13):3211-6) 〇' 201119693 The inventor of this case threatens _ fumaric acid II _自(10)) and the biodegradable nature of the jelly cement, releasing the promoted blood = newborn factor Rg in the bone cement component has a statistically significant effect on promoting epithelial cell type angiogenesis 'expected for clinical use It can accelerate the healing of early shots, provide sufficient mechanical strength of bone defects, promote the regeneration of blood in the femoral head, and finally be metabolized by the human body to metabolize normal bones _ replaced, extremely research and development, and finally developed through years of painstaking research. The treatment of femoral head necrosis stimulates angiogenesis and can reduce cement, because of the cost of invention. SUMMARY OF THE INVENTION The object of the present invention is to provide an angiogenic injectable degradable bone cement for treating femoral head necrosis, which can utilize the biodegradable property of the bone cement to release the blood storage of the bone cement component t. Biofactor
Rgl，對促進上皮細胞型成血管具有統計學上顯著的功 效。 該刺激血管新生性注射式可降解骨水泥，包括： 1.純高分子聚富馬酸二羥丙酯（PPF)、磷酸四鈣 (ttcp)/無水磷酸氫鈣(DCPA)和人蔘皂苷Rgl，在^乙烯基吼 咯烷酮（Ν-VP)中的混合物，形成第一分劑； 2·提供泡打粉(Baking Pcwder)(BP)作為第二分劑； 3.提供N，N-二甲基-對-甲苯胺（N，N _ dimethyl 一 P ~ toluidine)(DMT)作為加速劑以加速交聯反應； 其中係於使用前刻，將泡打粉(BP)溶入第一分劑中， 混合均勻，形成刺激血管新生性注射式可降解骨水泥，加 入Ν’ N -二甲基—對-甲苯胺，混合均勻後即可注入。 201119693 人(二:酸二細(PPF)係根據林峰輝等 方法製備。簡述如下公開公報編號：200902469中所述 Λ驟’取莫耳比二甲基了稀二酸(_ :丙二醇 .氣化鋅（ZnCl2):對苯二酚（Hq) =1 _ 3: 士细丨·，進行均句授摔’將溫度升到i〇()°c，然後緩慢 U 15GC ’反應過程必須在隔絕空氣的狀態下進行，所 H氮氣。反應過程中，二甲基丁烯二酸⑽）跟丙二醇 反應會產生乙醇，利用冷凝管將其冷凝下來，當乙醇 不再冷凝出來時’表示第—步驟反應完成； 第二步驟’將溫度設定在loot：，將壓力抽至0.1托爾 t(T〇rr) ’在這個過程中，未反應的丙二醇（PG)將會被冷 凝出來，之後在兩小時内將溫度升至13〇_15(rc，此時開始 反，成尚分子PPF，在兩小時將溫度提高至·。c，並且在 20(TC恆溫12小時，之後冷卻至室溫，產生具有黏稠性的琥 珀色液體’就是所要的產物高分子ρρρ。 該高分子PPF尚須純化以移除裡面所含之催化劑、交 聯抑制劑，其純化步驟為： 將尚分子PPF以體積比一比一的比例溶解在二氯曱院 中’再加入氯化氫（HC1)去除催化劑； 以相同體積的二次蒸餾水、鹽水反覆萃取，移去有機 浴劑一氣甲烧，之後加入濃硫酸去除多餘的水分； 再將剩餘的高分子PPF和二氯曱烷溶液加入冷的乙 醚，以去除掉多餘的交聯抑制劑；以及 將產物利用真空乾燥去除多餘的有機溶劑。 本發明採用的是此種純高分子PPF。 201119693 而填酸四鈣（TTCP)/無水磷酸氫鈣(DCPA)雙相骨水苑 係依照下述步驟製備成： 1:1 第一步驟，依化學計量比取一莫耳焦磷酸辦（Ca2P2〇7) 粉末與兩莫耳碳酸鈣（CaC〇3)粉末，使其充分混合均句 後，將均勻之混合物平鋪在白金鉗鍋中，置於碳化矽 (SiC)高溫爐中進行高溫燒結； 第二步驟，將該粉末混合物以1(rc/分的加熱速率，加 熱升溫至燒結溫度144(TC，並在該溫度持溫三小時，之後 須迅速焊火至室溫，如此，即得到磷酸四鈣(TTCP);在本實 施例中，係以研砵研磨成細粉，再用細篩（篩網型號贴油 No· 106)過遽，其反應式如下：Rgl has a statistically significant effect on promoting epithelial cell type angiogenesis. The stimulating angiogenic injectable degradable bone cement comprises: 1. pure polymer poly(hydroxypropyl) fumarate (PPF), tetracalcium phosphate (ttcp) / anhydrous calcium hydrogen phosphate (DCPA) and saponin Rgl a mixture of ^ vinylpyrrolidone (Ν-VP) to form a first component; 2. a baking powder (Baking Pcwder) (BP) as a second component; 3. providing N, N-di Methyl-p-toluidine (DM, p-toluidine) (DMT) is used as an accelerator to accelerate the cross-linking reaction; in which the baking powder (BP) is dissolved in the first portion before use. , evenly mixed, form an angiogenic injection-type degradable bone cement, add Ν'N-dimethyl-p-toluidine, mix well and then inject. 201119693 Person (two: acid bismuth (PPF) is prepared according to the method of Lin Fenghui, etc. Briefly, as described in the publication number: 200002469, the following step is taken to take the molar ratio of dimethyl disulfide (_: propylene glycol. Zinc (ZnCl2): hydroquinone (Hq) =1 _ 3: 士细丨·, carry out the sentence of the sentence to 'fall the temperature to i 〇 () ° c, then slowly U 15GC 'the reaction process must be isolated Under the condition of air, H nitrogen. During the reaction, dimethyl butenedioic acid (10) reacts with propylene glycol to produce ethanol, which is condensed by a condenser, and when the ethanol is no longer condensed, it represents the first step. The reaction is completed; the second step 'set the temperature to loot:, pump the pressure to 0.1 torr t (T〇rr) 'In this process, unreacted propylene glycol (PG) will be condensed, followed by two hours The internal temperature was raised to 13 〇 _15 (rc, at this time, the reverse was started, the molecular PPF was increased, the temperature was raised to .c in two hours, and at 20 (TC constant temperature for 12 hours, then cooled to room temperature, resulting in The viscous amber liquid is the desired product ρρρ. The polymer PPF is still Purification to remove the catalyst and crosslinking inhibitor contained therein, the purification step is: dissolving the molecular PPF in a ratio of one to one by volume in the dichlorohydrazine yard and adding hydrogen chloride (HC1) to remove the catalyst; The same volume of double distilled water, brine is repeatedly extracted, the organic bath is removed, and then the concentrated water is added to remove excess water; then the remaining polymer PPF and dichlorosilane solution are added to the cold ether to remove Excess crosslinking inhibitor; and the product is vacuum dried to remove excess organic solvent. The pure polymer PPF is used in the present invention. 201119693 The tetrahydrate (TTCP) / anhydrous calcium hydrogen phosphate (DCPA) duplex The skeletal water system is prepared according to the following steps: 1:1 The first step is to take a Mohr pyrophosphate (Ca2P2〇7) powder and a two-mole calcium carbonate (CaC〇3) powder according to the stoichiometric ratio. After thoroughly mixing the homogenous sentences, the uniform mixture is laid flat in a white gold clamp pot and placed in a high temperature furnace of silicon carbide (SiC) for high temperature sintering; in the second step, the powder mixture is heated at a rate of 1 (rc/min, Heating up To the sintering temperature of 144 (TC, and holding the temperature at this temperature for three hours, after which it must be quickly welded to room temperature, thus, to obtain tetracalcium phosphate (TTCP); in this embodiment, it is ground into a fine powder by a mortar. Then, using a fine sieve (screen type oil No. 106), the reaction formula is as follows:
Ca2P2〇7 + 2CaC〇3 — Ca4(P〇4)2〇 + 2C〇2 ; 第三步驟，將上述得到之磷酸四鈣(TTCP)粉末與一無水 填酸氫鈣（DCPA)以一莫耳比一莫耳的比例混合均勻後，即得 到磷酸四鈣(TTCP)/無水磷酸氫鈣(DCPA)雙相骨水泥。 於本發明中係將高分子聚富馬酸二經丙酯（ρρρ)溶在N一 乙烯基吼咯烧酮（N-VP)中攪拌均勻。 接著將磷酸四鈣(TTCP)/無水磷酸氫鈣(DCPA)溶入該N-VP/PPF溶液中，形成一混合物。 然後將人蔘皂苷Rgl (Ginsenoside Rgl)溶於該混合物 中，形成第一分劑。 之後，提供泡打粉(Baking Powder)作為第二分劑。 最後，於使用前刻，將泡打粉(BP)溶入該第一分劑 中，混合均勻，形成刺激血管新生性注射式可降解骨水 泥。 其中，純高分子PPF的分子量經膠透層析術（gei 201119693 permeation chromatography)測定分別具有 1060 Da之數 平均分子量與1839之重量平均分子量。其多分散指數 (Polydispersity index) (PDI)為 1.729502。 其中，泡打粉為含澱粉、小蘇打、磷酸鹽、燒明凡、 琥麟之食品添加劑。於本發日种时固化本發明刺激血管 新生性注射式可降解骨水泥的固化劑。該固化可在室 完成。 本發明要訂列概雛實_予崎_步閣明。 【實施方式】 實施例1 純高分子聚富馬酸二羥丙酯（ppF)之製備 ^取莫耳比二甲基丁烯二酸（DEF):丙二醇（pG): 氣)匕鋅（ZnCIO :對苯二酚（Hq) =1 · 3 : 〇 〇1 : 〇 〇〇2 進行均勻攪拌，將溫度升到靴，然後緩慢加熱到 fOC，反應過程必須在隔絕空氣的狀g下進行，所以通 氮氣。反應過程中，二甲基丁烤二酸（DEF)跟丙二醇 (PG)反應會產生乙醇，利用冷凝管將其冷凝下來，當乙 醇不再冷凝出來時，表示第_步驟反應完成。 將溫度設定在lOOt ’將壓力抽至〇1托爾 _(T〇rr)，在這個過程中，未反應的丙二醇將會被 冷凝出來，之後在兩小時内將溫度升至13〇_15〇它，此 開始反應成高分子PPF，在兩小時將溫度提高至2⑼。C， 並且在200 C恆溫12小時，之後冷卻至室溫，產生且 黏稠性的號騎液體，就是所要的產物高分子PPF。 將該高分子PPF以體積比一比一的比例溶解在二氣Μ 宁，再加入氯化氫（HC〗）去除催化劑； 201119693 以相同體積的二次蒸顧水、鹽水反覆萃取，移去 溶劑二氯甲烷，之後加入濃硫酸去除多餘的水分； 再將剩餘的高分子PPF和二氣甲烧溶液加入冷的乙 醚，以去除掉多餘的交聯抑制劑；以及 將產物利用真空乾燥去除多餘的有機溶劑。 該純高分子PPF的分子量經膠透層析術制奶；515 HPLC栗，717自動樣品注射器，剛折射率侧器）測 定分別具有1060 Da之數平均分子量與1839之重量平均 分子量。其多分散指數(PDI)為1,729502。 實施例2 磷酸四鈣(TTCP)/無水磷酸氫鈣(DCPA)雙相骨水泥之製備 第一步驟，依化學計量比取一莫耳焦磷酸辦（Ca2P2〇7) 粉末與兩莫耳碳酸鈣（cac03)粉末，使其充分混合均勻 後，將均勻之混合物平鋪在白金鉗銷中，置於碳化矽 (SiC)高溫爐中進行高溫燒結； 第二步驟’將該粉末混合物以1〇。〇/分的加熱速率，加 熱升溫至燒結溫度144(TC，並在該溫度持溫三小時，之後 須迅速淬火至室溫，如此，即得到磷酸四鈣(TTCP);在本 實施例中，係以研缽研磨成細粉，再用細篩（篩網型號 mesh No. 1〇6)過濾。 】 第三步驟，將上面所得磷酸四鈣（TTCP)粉末與一無 水磷酸氫鈣（DCPA)以一莫耳比一莫耳的比例混合均勻 後’即得到磷酸四鈣（TTCP)/無水磷酸氫鈣（DCPA)雙相骨 水泥。 實施例3 /σ療知骨頭壞死之刺激血管新生性注射式可降解骨水泥 201119693 配方之最優化 該配方的設計如下表所示。首先，將PPF和BP溶解在 N-乙烯基吡咯炫_(N-VP)中。然後，將TTCP/DCPA摻加到 該PPF/N-VP糊内。最後，快速混合入dmt以加速交聯反 應。將該骨泥複合材料混合物注射到圓柱型模子内。在 室溫下交聯之後，從模子取出樣品。 含量 PPF N-VP CPC BP DMT C/P=0 1克 0.7克 0克 0. 002 克 2微升（eL) C/P=l 1克 0.7克 1克 0. 002 克 2微升 C/P=2 1克 0.7克 2克 0. 002 克 2微升 PPF .聚富馬酸一經丙S旨；N-VP :卜乙稀基0比嘻烧網； CPC :磷酸鈣水泥；BP :泡打粉；DMT : N，N-二甲基-對-甲苯胺 實施例4 機械檢驗 根據ASTM D695-02a ’將該骨泥複合材料液注射到直 徑6毫米與高度12毫米的模子内。於室溫下交聯之後， 從模子取出樣品。使用機械檢驗機（MTS Bionix 858 Test System)測量樣品的機械性質。試驗樣品係在1.3 201119693 毫米/分的十字頭速度下壓縮。使用所記錄到的負載和變 形數據來標繪應力-應變曲線。應力係以負載除以初始橫 截面面積而測定而應變係以位移除以初始圓柱長度而定 出。壓縮強度係以應力-應變曲線的初始線型部份之最大 應力來測定。楊氏模數（Young，s m〇dulus)係以應力—應 變曲線的初始線型部份之之斜率來分析。衰竭能量 (Failure energy)為材料吸收能量至破斷的能力之測量。 應力-應變曲線下至破斷的面積決定該衰竭能量。結果標 • 繪於圖1至圖3中。圖1顯示出在c/P=〇與C/P=2之間 有明顯差異。其趨勢為增加TTCP/DCPA比例可提高壓縮 強度之值。人類海綿骨（human cancellous bone)的壓縮 強度為約4-12 Mpa。於本實施例f所有配方的壓縮強度 都高於此範圍。於圖2中，所有組別之間都有統計學意 義。人類海綿骨的楊氏模數取於骨密度絕為約50_1000 Mpa。配方C/P=l與C/P=2的楊氏模數都位於此範圍之 春 内。於 2003 中，Giesen et al. (Calcif Tissue Int. 2003; 73(3) :225-31)報導防腐處理過的人類屍體之下顎 髁（mand i bu 1 ar condy 1 e )所得海綿骨之衰竭能量為約 0. 041MJ/m3 (19女性，5男性平均年齡± SD: 74. 8 士 11. 7歲）。所有配方的衰竭能量都高於此值（圖3)。此 外’在C/P=0組與C/P=2組之間有明顯差異。 實施例5 胞毒性-LDH檢定 乳酸脫氫酶(Lactate dehydrogenase) (LDH)為細胞 201119693 裂解後釋放出的穩定性細胞溶質酵素。在菸酰胺腺嘌呤二 核苷酸(NAD+)與乳酸等反應物之下，LDH可產生丙酮酸與 菸醯胺腺嘌呤二核苷酸（NADH)。再配合黃遞酶 (diaphorase)之下，所形成的NADH與四唑鏽鹽（INT)反應 將INT轉化為紅色的formazan產物。紅色formazan產物 的量係以標準96-洞盤讀取器予以偵測。溶解細胞的數目 正比於紅色formazan產物的量。於此實施例中，胞毒性 係經由萃取液體來檢驗。將1〇4細胞/洞接種於96_洞盤 中。一天之後，用萃取液體取代培養基。將各盤溫置一籲 天。用市售檢定套組（CytoTox 96® Assay, Promega)檢 驗各檢驗洞。 經由分析對3T3細胞系的LDH檢定結果（圖4)，發現 在空白組與實驗組之間沒有明顯差異。對人類臍靜脈内 皮細胞（HUVEC )的胞毒性檢定結果顯示添加CPC具有明顯 的影響（圖5)。在用萃取液取代培養基後的第三天，於空 白組與C/P = 〇組之間有明顯差異。此外，在第三天，空 白組與C/P = 1組之間也有明顯差異。我們推斷較高的鲁 TTCP/DCPA含量可減低胞毒性。 實施例6 細胞存活性-WST-1檢定 為了知道釋放出的化學品是否會刺激内皮細胞增生， 乃使用水溶性四唑鏽鹽-l(WST-l)細胞增生檢定套組來檢 驗細胞活性。存在於粒線體内且只在活細胞中有活性的 丁二酸-四 u坐鏽鹽還原酶（Succinate-tetrazolium 12 201119693 reductase)(粒線體脫氫酶（mitochondrial dehy dr ogenase))會將四唑鏽鹽（WST-1)切斷而形成可溶 性formazan。活細胞的增加導致丁二酸-四唑鏽鹽還原酶 總活性的提高。酵素活性的增加造成formazan形成的增 加，因而formazan的量正比培養基内代謝活性細胞之數 目。活細胞產生的formazan可用ELISA讀取器測量該染料 溶液在440 nm的吸光度予以定量且較高的formazan染料 溶液吸光度值係由更加的細胞活性所造成。於本實施例 中，係以萃取液檢驗細胞增生。將1〇4細胞/洞接種於 96-洞盤中。一天之後，以萃取液取代培養基。將各盤溫 置一天與三天。用用市售檢定套組（Cyt〇T〇x 96® Assay， Promega)檢驗各檢驗洞。WST-1試劑處理每一檢驗洞。 更高的光學密度值（optic density (0D))代表較高 的在活細胞内的粒線體脫氫酶之活性且經認為正比於活 細胞的數目。根據對3T3細胞系的WST-1檢定之結果（圖 6)在空白組與實驗組之間沒有明顯差異。對即VEC的 WST-1檢定結果顯示在空白組與c/p = 〇址之間有明顯差 異（圖7)。整體而言，添加TTCP/DCPA可減低PPF系統的 負面影響。 實施例7 藥物釋放 進行藥物釋放實驗時，係將直徑3. 4毫米與高度6. 8 毫米的圓柱形骨水泥複合材料樣品浸沒在小管瓶所裝2 毫升的磷酸鹽緩衝食鹽水（PBS)中。將該管瓶溫浸於 13 201119693 37°C下。於特定時間間隔（6小時、1天、2天、3天、5 天、7天、其後每週）收集PBS且用2毫升新鮮PBS補充。 在固相萃取之後，使用超性能液體層析術-三段四極質譜 儀（ultraperformance liquid chromatography)(Waters Acquity UPLC) - triple quadrupole mass spectrometer)(Quattro Premier XE MS) (UPLC-MS/MS) 定量所收集的PBS内之人參皂苷Rg卜使用得自Waters的 有加VanGuard Pre-column 之Acquity UPLC BEH C18 管 柱（100毫米x2. 1毫米直徑，1· 7微米）。分析時管柱溫 度係調定在45°C。樣品係使用由甲醇（69%)和水（31%)組 成的移動相予以濃縮。移動相的流速為〇· 35毫升/[分且 注射體積為30微升（Guan J et al.，J Pharm Ri隱Η Anal. 2007; 44(4):996-1000)。所收集到的樣品係用電 噴霧正電離法（electrospray ionization positive method (ESI+))喷灑到級聯四極質譜儀内。使用多重反 應監測模式（MRM)來測定樣品内的Rgi之量。 從前面諸實施例可以找出最佳配方。c/p = 2組不 具胞毒性。C/P = 2組的機械性質比其他組都強。與其他 組相比，C/P = 2組的交聯溫度較低且吸收較少的水分。 所以選擇C/P = 2組來混合Rgi以進行藥物分析。其組成 如下表所示。 201119693Ca2P2〇7 + 2CaC〇3 — Ca4(P〇4)2〇+ 2C〇2; In the third step, the tetracalcium phosphate (TTCP) powder obtained above and one anhydrous calcium hydrogenate (DCPA) are one molar. After mixing evenly than the molar ratio, a tetracalcium phosphate (TTCP) / anhydrous calcium hydrogen phosphate (DCPA) duplex cement is obtained. In the present invention, the polymer polyfumaric acid dipropyl propyl ester (ρρρ) is dissolved in N-vinylpyrrolidone (N-VP) and stirred uniformly. Tetracalcium phosphate (TTCP) / anhydrous calcium hydrogen phosphate (DCPA) is then dissolved in the N-VP/PPF solution to form a mixture. The saponin Rgl (Ginsenoside Rgl) is then dissolved in the mixture to form a first sub-agent. Thereafter, Baking Powder was provided as a second sub-agent. Finally, prior to use, the baking powder (BP) is dissolved in the first portion and uniformly mixed to form an inflammatory neoplastic injectable degradable bone cement. Among them, the molecular weight of the pure polymer PPF was determined by gel permeation chromatography (gei 201119693 permeation chromatography) to have an average molecular weight of 1060 Da and a weight average molecular weight of 1839, respectively. Its Polydispersity Index (PDI) is 1.729502. Among them, the baking powder is a food additive containing starch, baking soda, phosphate, burning Mingfan, and Hulin. The curing agent for stimulating angiogenic neoplastic injectable bone cement is cured in the present invention. This curing can be done in the chamber. The present invention is to be classified as a _ _ _ _ _ _ ge [Examples] Example 1 Preparation of pure polymer poly(dihydroxypropyl) fumarate (ppF) ^Merbi dimethyl phthalate (DEF): propylene glycol (pG): gas) bismuth zinc (ZnCIO) : hydroquinone (Hq) =1 · 3 : 〇〇1 : 〇〇〇2 Stir well, raise the temperature to the boot, and then slowly heat to fOC, the reaction must be carried out in the form of air isolation, so Nitrogen gas. During the reaction, dimethyl butadiene dicarboxylic acid (DEF) reacts with propylene glycol (PG) to produce ethanol, which is condensed by a condenser tube. When the ethanol is no longer condensed, it indicates that the first step reaction is completed. Set the temperature at 100 t' and pump the pressure to 〇1 Torr (T〇rr). During this process, the unreacted propylene glycol will be condensed, and then the temperature will rise to 13 〇 _15 within two hours. It starts to react into a polymer PPF, raises the temperature to 2 (9) C in two hours, and is kept at 200 C for 12 hours, then cooled to room temperature, resulting in a viscous ride on the liquid, which is the desired product polymer. PPF. Dissolve the high molecular weight PPF in a ratio of one to one by volume in the two gas cylinders. Then add hydrogen chloride (HC) to remove the catalyst; 201119693 Repeat the extraction with the same volume of secondary steam, brine, remove the solvent dichloromethane, then add concentrated sulfuric acid to remove excess water; then the remaining polymer PPF and The second gas-burning solution is added with cold diethyl ether to remove excess crosslinking inhibitor; and the product is vacuum dried to remove excess organic solvent. The molecular weight of the pure polymer PPF is made by gel chromatography; 515 HPLC Chestnut, 717 automatic sample injector, rigid-refractive-index side device) measured the average molecular weight of 1060 Da and the weight average molecular weight of 1839, respectively. Its polydispersity index (PDI) is 1,729,502. Example 2 Preparation of Tetracalcium Phosphate (TTCP)/Anhydrous Calcium Phosphate (DCPA) Duplex Bone Cement The first step is to take a Mohr Pyrophosphate (Ca2P2〇7) powder and two molar calcium carbonate according to stoichiometric ratio. (cac03) powder, after it is thoroughly mixed uniformly, the homogeneous mixture is laid flat in a platinum clamp, and placed in a high-temperature furnace of cerium carbide (SiC) for high-temperature sintering; the second step 'the powder mixture is 1 Torr. The heating rate of 〇/min is heated to a sintering temperature of 144 (TC, and held at this temperature for three hours, after which it must be rapidly quenched to room temperature, thus obtaining tetracalcium phosphate (TTCP); in this embodiment, It is ground into a fine powder by a mortar and then filtered with a fine sieve (mesh type mesh No. 1〇6).] The third step is to obtain the above-mentioned tetracalcium phosphate (TTCP) powder and an anhydrous calcium hydrogen phosphate (DCPA). After mixing uniformly in a molar ratio of one molar to one molar, a tetracalcium phosphate (TTCP)/anhydrous calcium hydrogen phosphate (DCPA) duplex cement was obtained. Example 3 / σ treatment of bone necrosis stimulated angiogenesis injection Degradable Bone Cement 201119693 Optimization of Formulation The design of this formulation is shown in the following table. First, PPF and BP are dissolved in N-vinylpyrrole_(N-VP). Then, TTCP/DCPA is added. The PPF/N-VP paste was finally mixed. Finally, the dmt was quickly mixed to accelerate the cross-linking reaction. The bone mud composite mixture was injected into a cylindrical mold. After cross-linking at room temperature, the sample was taken out from the mold. N-VP CPC BP DMT C/P=0 1 g 0.7 g 0 g 0. 002 g 2 μl (eL) C/P=l 1 g 0.7 g 1 g 0. 002 g 2 μl C/P=2 1 g 0.7 g 2 g 0. 002 g 2 μl PPF. Polyfumaric acid once C; N-VP : 乙乙稀基0比嘻烧网; CPC: calcium phosphate cement; BP: baking powder; DMT: N,N-dimethyl-p-toluidine Example 4 Mechanical inspection according to ASTM D695-02a 'The bone The mud composite liquid was injected into a mold having a diameter of 6 mm and a height of 12 mm. After cross-linking at room temperature, the sample was taken out from the mold. The mechanical properties of the sample were measured using a mechanical inspection machine (MTS Bionix 858 Test System). Compression at a crosshead speed of 1.3 201119693 mm/min. The stress-strain curve is plotted using the recorded load and deformation data. The stress is determined by dividing the load by the initial cross-sectional area and the strain is removed in bits. The initial cylinder length is determined. The compressive strength is determined by the maximum stress of the initial linear portion of the stress-strain curve. The Young's modulus (Young, sm〇dulus) is the initial linear portion of the stress-strain curve. The slope is analyzed. The failure energy is the energy absorbed by the material. Measurement of the ability to break. The area under the stress-strain curve to the breaking area determines the energy of the failure. The results are shown in Figure 1 to Figure 3. Figure 1 shows the difference between c/P = 〇 and C/P = 2. There is a significant difference between them. The trend is to increase the TTCP/DCPA ratio to increase the value of compressive strength. The human cancellous bone has a compressive strength of about 4-12 Mpa. The compressive strength of all formulations in this example f was above this range. In Figure 2, there is statistical significance between all groups. The Young's modulus of the human sponge bone is taken from the bone density of about 50_1000 Mpa. The Young's modulus of the formula C/P = 1 and C/P = 2 is in the spring of this range. In 2003, Giesen et al. (Calcif Tissue Int. 2003; 73(3): 225-31) reported the energy of the sponge bone failure obtained from the preserved human corpse (mand i bu 1 ar condy 1 e ). It is about 0. 041 MJ/m3 (19 female, 5 male mean age ± SD: 74. 8 ± 11.7 years old). The depletion energy of all formulations is above this value (Figure 3). In addition, there is a significant difference between the C/P=0 group and the C/P=2 group. Example 5 Cytotoxicity-LDH assay Lactate dehydrogenase (LDH) is a stable cytosolic enzyme released after cell lysis in 201119693. Under the reaction of nicotinamide adenine dinucleotide (NAD+) with lactic acid, LDH produces pyruvate and nicotinamide adenine dinucleotide (NADH). In combination with diaphorase, the formed NADH reacts with the tetrazolium rust salt (INT) to convert INT to a red formazan product. The amount of red formazan product was detected using a standard 96-well disk reader. The number of lysed cells is proportional to the amount of red formazan product. In this example, cytotoxicity was tested by extracting the liquid. Inoculate 1〇4 cells/hole in a 96-well plate. After one day, the medium was replaced with an extraction liquid. Set each plate to a standstill. Each test hole was tested with a commercially available test kit (CytoTox 96® Assay, Promega). By analyzing the LDH assay results for the 3T3 cell line (Fig. 4), it was found that there was no significant difference between the blank group and the experimental group. Cytotoxicity assays on human umbilical vein endothelial cells (HUVEC) showed a significant effect on the addition of CPC (Figure 5). On the third day after the medium was replaced with the extract, there was a significant difference between the blank group and the C/P = 〇 group. In addition, on the third day, there was also a significant difference between the empty group and the C/P = 1 group. We conclude that higher levels of TTCP/DCPA can reduce cytotoxicity. Example 6 Cell viability - WST-1 assay In order to know whether the released chemical would stimulate endothelial cell proliferation, the water-soluble tetrazolium rust-l (WST-1) cell proliferation assay kit was used to test cell viability. Succinate-tetrazolium 12 201119693 reductase (mitochondrial dehy dr ogenase), which is present in the mitochondria and active only in living cells, will The tetrazolium rust salt (WST-1) is cleaved to form a soluble formazan. An increase in viable cells results in an increase in the total activity of the succinate-tetrazole rust reductase. An increase in the activity of the enzyme causes an increase in the formation of formazan, and thus the amount of formazan is proportional to the number of metabolically active cells in the medium. The formazan produced by living cells can be quantified by measuring the absorbance of the dye solution at 440 nm using an ELISA reader and the higher formazan dye solution absorbance value is caused by more cell activity. In this example, cell proliferation was examined with an extract. Inoculate 1〇4 cells/hole in a 96-well dish. After one day, the medium was replaced with an extract. Set each plate to one or three days. Each test hole was tested using a commercially available assay kit (Cyt〇T〇x 96® Assay, Promega). WST-1 reagent treats each test well. A higher optical density (0D) represents a higher activity of mitochondrial dehydrogenase in living cells and is considered to be proportional to the number of viable cells. There was no significant difference between the blank group and the experimental group based on the results of the WST-1 assay on the 3T3 cell line (Fig. 6). The WST-1 test results for the VEC show a significant difference between the blank group and the c/p = 〇 address (Figure 7). Overall, adding TTCP/DCPA reduces the negative impact of the PPF system. Example 7 Drug Release In the drug release experiment, a sample of a cylindrical bone cement composite having a diameter of 3.4 mm and a height of 6.8 mm was immersed in a 2 ml phosphate buffered saline (PBS) in a vial. . The vial was immersed in 13 201119693 at 37 °C. PBS was collected at specific time intervals (6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, and thereafter every week) and supplemented with 2 ml of fresh PBS. After solid phase extraction, ultra performance liquid chromatography (Waters Acquity UPLC) - triple quadrupole mass spectrometer (Quattro Premier XE MS) (UPLC-MS/MS) The ginsenoside Rg in the collected PBS was obtained from an Acquity UPLC BEH C18 column (100 mm x 2.1 mm diameter, 1.7 μm) with a VanGuard Pre-column from Waters. The column temperature was set at 45 °C during the analysis. The sample was concentrated using a mobile phase consisting of methanol (69%) and water (31%). The flow rate of the mobile phase was 〇·35 ml/[minute and the injection volume was 30 microliters (Guan J et al., J Pharm Ri, Conceal Anal. 2007; 44(4): 996-1000). The collected samples were sprayed into a cascade quadrupole mass spectrometer by electrospray ionization positive method (ESI+). The multiple reaction monitoring mode (MRM) was used to determine the amount of Rgi within the sample. The best formulation can be found from the previous examples. c/p = 2 groups were not cytotoxic. The mechanical properties of the C/P = 2 group were stronger than those of the other groups. Compared with the other groups, the C/P = 2 group has a lower cross-linking temperature and absorbs less moisture. So choose C/P = 2 to mix Rgi for drug analysis. Its composition is shown in the table below. 201119693
藥物釋放分布曲線係用UPLC串聯MS/MS取得。分析 所收集到的PBS樣品。結果顯示於圖8中。Rgi在PBS 中的每日釋放量係表成所釋放出的Rgl濃度（卯m)除以相 應的以天計的釋放時間因而表為ppm/天。圖形顯示在樣 品内有較高的Rgl含量者會釋放出相對較高的Rgl到周 圍的PBS内。如從圖9明顯可看出者，三個實驗組具有 幾乎相同的累積釋放分布曲線。 實施例8 HUVEC管形成The drug release profile was obtained by UPLC tandem MS/MS. The collected PBS samples were analyzed. The results are shown in Figure 8. The daily release of Rgi in PBS is expressed as the released Rgl concentration (卯m) divided by the corresponding daily release time and thus expressed as ppm/day. The graph shows that a higher Rgl content in the sample will release a relatively high Rgl into the surrounding PBS. As is apparent from Fig. 9, the three experimental groups have almost the same cumulative release profile. Example 8 Formation of HUVEC tube
HUVEC經研究在接種於Matrigel基質（Becton Dickinson Biosciences)上之後，具有貼附、遷移和形成 管狀結構之能力（Huang YC，et al. Pharm Res. 2005; 22(4):636-46; Yu LC, et al., 2007; 32 Int J Pharm. 8(2):168-76)。HUVEC 管形成檢定係在每洞塗覆著20微升Matrigel的96-洞盤抗 試驗。將其量為7xl04細胞/洞的HUVEC分別接種到塗覆盤 15 201119693 上的空白組萃取液以及有掺加與沒有摻加Rgl的水泥之萃 取液内。將諸盤至於37°C保溫箱内12小時。以附帶 Nikon數位攝影機的光學顯微鏡用4倍放大倍率攝取顯 微域内的五張照片。血管新生活性係以照片域中管子網 絡所覆蓋的面積和直徑測定。面積分析係用ImageJ軟體 予以量化。管結構的直徑係從HUVECs所圈住的空位面積 及圓圈面積方程式導出。 吆1的試管内（in vitro)血管生成效應業經探討過。 Sengupta et al. ( 2004; 110(10):1 Circulation. 219 25)報導過相對最佳濃度為1250 nM (lppm)。在此 濃度之下，Rgl會比空白組明顯地刺激HUVEC的增生。於 此檢定中’使用培養基中R/N=0.01組的萃取液來處理 HUVEC。培養基内的藥物釋放樣式經假設與在PBS内者相 同方式。收集第二天到第三天R/N=〇. 01組在2毫升培養 基内的萃取液。根據先前的藥物釋放分布曲線，假設 岐1在所收集的培養基内之濃度為8. 19 ppm如同在PBS 内的釋玫量。將所收集的培養基内8. 19 ppm的假設濃度 稀釋到1 ppm。於接種12小時之後，HUVECs形成如圖 10中所示的蜂巢狀結構之網絡。與空白組和無Rgl組的 X/尼相異者，有組（1 ppm)的水泥形成相當完全，壓 縮且狹窄的管狀結構。所觀察到的管子形成進一步經由 分析官子網絡的面積和直徑在量上評估。管形成活性經 定為相對於空白組的面積和直徑百分比。圖U和12顯 不出從水泥釋放出的Rgl會刺激HUVEC管形成，且此刺 201119693 激相比於空白組和無Rgl組的水泥具有統計顯著性。經 由Rgl的刺激，HUVEC會聚集形成較密的結構導致每攝影 域更咼的面積覆蓋率。計算出的直徑也小於其他組因而 更肯定Rgl的刺激作用。 綜上所述，本發明成功地製造出可治療股骨頭壞死 之刺激血管新生性注射式生物可降解骨水泥。經由LDH和 WST-1檢定，隨著CPC對PPF的比例增加，本發明骨水泥 更具生物相容性。機械檢驗也顯示，增加TTCp/j)cPA比例 可提升壓縮強度、楊氏模數和衰竭能量之值。簡言之， C/P=2組的配方為所有實驗配方中相對較優者。因此，本 發明含聚畐馬酸一經丙酯（PPF)、填酸四飼（TTCP)/無水 磷酸氫鈣（DCPA)和人蔘皂苷Rgi的生物可降解骨水泥係新 穎者。 藥物釋放分析證明樣品内有較高Rgl含量者可釋放 相對較高量的Rgl到周圍PBS中。根據釋放樣式，使用萃 取液來試驗釋放出的Rgl之血管新生功能。其結果證明釋 放出的Rgl之刺激作用相對於空白組與不Rgl的骨泥組別 都具有統計上的明顯性。釋放出的R g丨仍然能夠明顯地刺 激血管新生。整體而言，本發明骨水泥複合材料有希望 用來治療股骨頭的缺血性壞死。因此本發明含聚富馬酸 二羥丙酯（PPF)、磷酸四鈣（TTCP)/無水磷酸氫鈣（DcpA) 和人蔘皂苷Rgl的生物可降解骨水泥具有進步性與產業應 用性。 總而言之，本發明刺激血管新生性注射式可降解骨 17 水泥具有可專雜，㈣啸ίϋ專师ΐ請，懇請准予 專利權為禱。 【圖式簡單說明】 圖1顯示出本發明刺激血管新生性注射式可降解骨 水泥的壓縮強度表在ρ<0.05層次的統計顯著性。數據 係表為平均值±標準偏差）。 圖2顯示出本發明刺激血管新生性注射4可降解骨 水泥的楊氏模數(Young，s m〇duU) 表在p<〇 〇5層次 的統計顯著性。數據係表為平均值±標準偏差）。 圖3顯示出本發明刺激血管新生性注射式可降解骨 水泥的衰竭能量（Failure energy) 表在p<〇 〇5層次 的統計顯著性。數據係表為平均值±標準偏差）。曰 圖4顯示出本發明刺激血管新生性注射式可降解骨 水泥對3T3細胞糸的胞毒性。 圖5顯示出本發明刺激血管新生性注射式可降解骨 水泥對_毒性㈣知〈請層㈣統計顯著 性。數據係表為平均值±標準偏差）。 圖6顯示出本發明刺激血管新生性注射式可降解骨 水泥對3T3的WST-1檢定結果。 圖7顯示出本發明刺激血管新生性注射式可降解骨 水泥對HUVEC的WST-1檢定結果（*表在p<〇 〇5層次的統 計顯著性。數據係表為平均值±標準偏差）。 、 圖8顯示出本發明刺激血管新生性注射式可降解骨 水泥中Rgl在PBS中的每日釋放曲線。 201119693 圖9齡iti本發_激血管新生处料可降解骨 水泥中Rgl在PBS中的累積釋放量。 圖10顯示出本發明刺激血管新生性注射式 水泥中Rgl的HUVEC管形成。 圖11顯示出相對於空白組的管形成面積百分比（*表 差f。0· 〇5層次的統計顯著性。數據係表為平均冊票準偏HUVEC has been studied to have the ability to attach, migrate and form tubular structures after inoculation on Matrigel matrix (Becton Dickinson Biosciences) (Huang YC, et al. Pharm Res. 2005; 22(4): 636-46; Yu LC , et al., 2007; 32 Int J Pharm. 8(2): 168-76). The HUVEC tube formation assay was applied to a 96-well disk test with 20 microliters of Matrigel per well. HUVEC in an amount of 7 x 10 4 cells/well was inoculated separately into a blank group extract on a coating plate 15 201119693 and in an extract having a cement mixed with and without Rgl. The plates were placed in an incubator at 37 ° C for 12 hours. Five photographs in the microdomain were taken at 4 times magnification with an optical microscope with a Nikon digital camera. Angiogenesis activity is determined by the area and diameter covered by the tube subnetwork in the photo field. Area analysis was quantified using ImageJ software. The diameter of the tube structure is derived from the vacancy area and the circle area equation enclosed by the HUVECs. The in vitro angiogenesis effect of 吆1 has been explored. Sengupta et al. (2004; 110(10):1 Circulation. 219 25) reported a relative optimal concentration of 1250 nM (lppm). Below this concentration, Rgl significantly stimulated HUVEC proliferation compared to the blank group. In this assay, HUVEC was treated with an extract of R/N = 0.01 in the medium. The drug release pattern in the medium was assumed to be the same as in PBS. Collect the extract from the next day to the third day R/N = 〇. 01 in 2 ml of medium. Based on the previous drug release profile, the concentration of 岐1 in the collected medium was 8.19 ppm as in the PBS. Dilute the assumed concentration of 8.19 ppm in the collected medium to 1 ppm. After 12 hours of inoculation, HUVECs formed a network of honeycomb structures as shown in FIG. In the case of X/N, which differs from the blank and Rgl-free groups, a group (1 ppm) of cement forms a fairly complete, compressed and narrow tubular structure. The observed tube formation is further quantitatively evaluated via the area and diameter of the analysis subnetwork. The tube formation activity was determined as the area and diameter percentage relative to the blank group. Figures U and 12 show that Rgl released from the cement stimulates the formation of HUVEC tubes, and this thorn 201119693 is statistically significant compared to the blank and non-Rgl groups. Stimulated by Rgl, HUVECs will aggregate to form a denser structure resulting in a more ambiguous area coverage per photographic field. The calculated diameter is also smaller than the other groups and thus more positive for the stimulation of Rgl. In summary, the present invention successfully produces an angiogenic injectable biodegradable bone cement that can treat femoral head necrosis. The bone cement of the present invention is more biocompatible as the ratio of CPC to PPF is increased via the LDH and WST-1 assays. Mechanical testing has also shown that increasing the TTCp/j)cPA ratio increases the values of compressive strength, Young's modulus, and depletion energy. In short, the C/P=2 formula is the relatively best of all experimental formulations. Accordingly, the present invention is a novel biodegradable bone cement system containing poly(p-Methylate)-propyl ester (PPF), acid-filled four-feed (TTCP)/anhydrous calcium hydrogen phosphate (DCPA) and saponin Rgi. Drug release analysis demonstrated that a higher Rgl content in the sample could release a relatively higher amount of Rgl into the surrounding PBS. The extract was used to test the angiogenic function of the released Rgl according to the release pattern. The results demonstrate that the stimulatory effect of the released Rgl is statistically significant relative to the blank group and the non-Rgl bone mud group. The released R g丨 is still able to significantly stimulate angiogenesis. Overall, the bone cement composite of the present invention is promising for the treatment of ischemic necrosis of the femoral head. Therefore, the biodegradable bone cement containing polyhydroxylfusate (PPF), tetracalcium phosphate (TTCP) / anhydrous calcium hydrogen phosphate (DcpA) and saponin Rgl of the present invention has advancement and industrial applicability. In summary, the present invention stimulates angiogenic injection-type degradable bones. 17 Cement has special effects, and (4) 啸 ϋ ϋ ϋ ϋ ϋ 恳 恳 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the statistical significance of the compression strength table of the angiogenic injectable degradable bone cement of the present invention at the ρ < 0.05 level. The data is tabulated as mean ± standard deviation). Fig. 2 is a graph showing the statistical significance of the Young's modulus (Young, s m〇duU) table of the present invention for stimulating angiogenic injection of 4 degradable bone cement at the p < 〇 〇 5 level. The data is tabulated as mean ± standard deviation). Fig. 3 is a graph showing the statistical significance of the failure energy table of the angiogenic injectable degradable bone cement of the present invention at the p<〇5 level. The data is tabulated as mean ± standard deviation). Figure 4 shows the cytotoxicity of the angiogenic injectable degradable bone cement of the present invention against 3T3 cell rafts. Fig. 5 shows the statistical significance of the stimulating angiogenic injectable degradable bone cement of the present invention for _toxicity (four). The data is tabulated as mean ± standard deviation). Fig. 6 shows the WST-1 assay results of the angiogenic injectable degradable bone cement of the present invention for 3T3. Fig. 7 shows the results of WST-1 assay of HUVEC stimulating angiogenic injectable degradable bone cement of the present invention (* the statistical significance of the table at p< 〇 〇 5 level. The data is expressed as mean ± standard deviation). Figure 8 shows the daily release profile of Rgl in PBS in the angiogenic injectable degradable bone cement of the present invention. 201119693 Figure 9 age iti hair _ angiogenesis new material degradable bone The cumulative release of Rgl in PBS. Fig. 10 shows the formation of HUVEC tubes of Rgl in the angiogenic injectable cement of the present invention. Figure 11 shows the percentage of tube formation area relative to the blank group (* statistical significance of the difference of the scale f. 0· 〇 5 level. The data sheet is the average ticket quasi-bias
在形成直徑百分⑽表 差）。 生。數據係表為平均值士標準偏 【主要元件符號說明】In the formation of the percentage of diameter (10) difference). Health. The data system is the average standard deviation [main component symbol description]