經濟部智慧財產局員工消費合作社印製 524699 A? ___ B7 五、發明說明（i ) 本發明係關於一種製備膠原蛋白(collagen)和生醫陶瓷粉 末(bioceramic)複合材料微粒的方法，特別是關於一種製備微 粒型式的複合材料的方法，此微粒內部及表面具有三維的膠 原蛋白纖維網狀結構。 人體骨組織中若因傷害或病變而缺損，需修補以儘量恢 復原組織之功能，如在缺損處加入骨駐植物作爲提供受力及 細胞生長的支架，將能有效輔助骨組織結構與功能上的再生 及修復。自體移植是骨駐植物最佳的選擇但其來源受限，且 需受二次手術之苦；異體或異種移植則有免疫及其他倂發症 的問題；人工合成的骨組織提供了另一個新選擇。以往所使 用的人工合成駐植體材料具惰性，以講求與生體組織共存。 但惰性物質反而會在植入體周圍產生纖維化，使組織與植入 物之間的界面性質形成嚴重落差，造成駐植體鬆動或周圍組 織纖維硬化，甚而壞死。習知技術已發現，具生物活性材料 若與組織之間有生物鍵結，便可表現出較好的生醫材料相容 性質。膠原蛋白是骨基質中含量最豐富的蛋白質，具有生物 相容性及低免疫性，適合作爲提供新生骨細胞貼附的基質。 再添加生醫陶瓷粉末（如tricalcium phosphate, TCP; hyd服yapatite，HAP，…等），形成複合物可提高其機械性質， 並能進一步模擬骨基質的成分。而以此生分性材料作爲載 體，攜入細胞和生長因子等物質，將具有誘導組織再生之功 能並能增快骨修補效果。 3 本紙張尺度適用中國國家標準（CNS)A4^格（210 x 297公釐） IL------黻-------—訂--------線丨·！. (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 524699 A7 B7 五、發明說明（2) 現有人工骨駐植物種類繁多，依形式可分爲塊狀、泥狀 和微粒等骨駐植物，但各有其缺點◦塊狀形式的骨駐植物， 機械強度較佳，但手術操作上不適合各種形狀的骨缺陷；泥 狀和微粒骨駐植物操作較方便，可依缺陷形狀不同而加以塑 形，且可以利用注射的方式放入人體骨缺陷處，減少動手術 之苦，缺點是材料不容易固定在缺陷處’且機械強度也較差。 習知技術利用經過燒結的多孔性陶瓷材料，以灌注或浸 泡的方式，將膠原蛋白填充在孔隙內。燒結後的陶瓷硬度 高，所以較能承受力量’但缺點是塑型上的限制，且此多孔 性陶瓷材料非生物可吸收，會防礙新生骨進一步生長。 習知技術直接將氫氧基磷灰石溶解於酸性溶液與重組 之膠原蛋白混合後調整溶液pH値至微鹼性使氫氧基憐灰石 在膠原蛋白上產生沉殿。習知技術亦將將磷灰石前驅物與膠 原蛋白溶液混合，利用酸鹼反應使前驅物轉變爲氫氧基磷灰 石沉殿在膠原蛋白纖維交互連結的基質中。1988、1989年Chu 等人(US Patent No. 4776890，4888366)，，將膠原蛋白纖維溶液 與氫氧基磷灰石顆粒或粉末直接混合，形成膠態溶液，具有 可注射式的優點，應用於軟組織的修復。上述〜般直接混合 的複合物，因膠原蛋白與陶瓷粉末密度相差較大，混合後經 過一段時間會因比重不同而漸漸分爲兩相，造成材料的不均 質◦所使用陶瓷粉末粒徑太大(約〇.5mm)，並不被生物體戶斤 吸收，且會在其周圍有纖維組織產生。另外從微觀結構的觀 察，此直接混合的複合材料，無法形成似骨組織中膠原蛋白 4 本紙張尺度適用中國國家標準（CNS)A4規格^川^^犯公釐) ' " -_ I I L ^1 I —^1 I I _ I ·11111--· I I I--I I * I ----— — — pill· — — — — — — — — — — ! — (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 524699 A7 B7 五、發明說明（3) 纖維網狀結構，以提供似骨組織中細胞所需的生長環境。 微粒型式的複合物中，膠原蛋白與陶瓷粉末分佈較均勻 且大量的微粒以各種不同方向隨意排列，所以在巨觀上能彌 補釋一顆微粒兩相的不均。另外它方便塑型，且因顆粒微 小，可製成注射形式使病人減少動手術之不便。習知技術以 矽油來製作膠原蛋白微粒，又進一步以氫氧基磷灰石粉末與 膠原蛋白溶液混合，滴入快速攪拌的ethyl-2-hexyl cocoate中， 使膠原蛋白成球，經離心及酒精去除ethyl-2-hexyl cocoate後 分離出膠原蛋白/氫氧基磷灰石微粒。2001年Wang等人(中華 民國專利公告號420604，證書號1273111)，將膠原蛋白與磷 酸鈣系陶瓷粉末混合成一混合溶液，逐滴滴入一油相(例如橄 欖油olive oil)中，使該混合溶液進行再重組而形成網狀纖維 結構顆粒;注入一交聯劑(例如戊二醛glutaraldehyde)於該油相 中使該顆粒進行交聯；及將該顆粒重從油相中分離出來。以 上述方法製備微粒過程中，有機溶劑的使用恐會破壞膠原蛋 白性質，且殘餘的有機油或交聯劑對組織有傷害的疑慮，另 外需較多的程序將油相分離移除。 基於習知複合材料及其製備方法、技術的缺點，本發明 的目的在於提供一種製備膠原蛋白和生醫陶瓷粉末複合材 料微粒的方法，以改善習知複合材料及其製備方法的缺點。 本發明的目的在於提供一種製備膠原蛋白和生醫陶瓷 粉末複合材料微粒的方法。使用膠原蛋白溶液及重組技術來 製備此複合材料微粒，此複合材料中膠原蛋白與生醫陶瓷粉 5 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -----1 —,-------------q I 訂：--------I (請先閱讀背面之注意事項再填寫本頁) 524699 A7 B7 五、發明說明（4：) 末能分佈均勻，微粒內外部具有三維的膠原蛋白纖維網狀結 構。本發明所提供方法之優點爲，在製備微粒過程中，無有 機溶劑、有機油或交聯劑殘餘對組織傷害的疑慮。本發明方 法製備的複合材料微粒，具足夠的機械強度能作爲載體攜入 細胞和包覆、固定各種不同的骨生長因子，誘導組織再生及 增快骨修補效果之功能，能以注射式或進一步依需求製作成 不同型式的基材模板，應用於骨組織創傷修復之用。 圖式之簡單說明 圖1:製備膠原蛋白和生醫陶瓷粉末複合材料微粒的方 法示意圖。 圖2A :係一 SEM圖，顯示9小時時，微粒切面10000X SEM 圖。 圖2B :係一 SEM圖，顯示9小時時，微粒表面10000X SEM 圖。 圖2C :係一^ SEM圖，顯不24小時時，微粒切面10000X SEM 圖。 圖2D :係一 SEM圖，顯示24小時時，微粒表面10000X SEM 圖。 圖3A :係一 SEM圖，顯示9小時時，微粒切面10000X SEM 圖。 圖3B :係一 SEM圖，顯示9小時時，微粒表面10000X SEM 圖。 6 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ---------% (請先閱讀背面之注意事項再填寫本頁) d丨訂/--------線丨 經濟部智慧財產局員工消費合作社印製 經濟部智慧財產局員工消費合作社印製 524699 A7 B7 五、發明說明（b ) 圖3C :係一 SEM圖，顯示24小時時，微粒切面10000X SEM 圖。 圖3D Μ系一 SEM圖，顯示24小時時，微粒表面10000X SEM 圖。 爲達上述之目的，本發明依下述方法來製備膠原蛋白和 生醫陶瓷粉末複合材料微粒： 將膠原蛋白溶液、褐藻酸(algimc acid)和生醫陶瓷粉末在 0-10°C下混合，以球滴形式滴入二價陽離子的水溶液中膠化 成微粒型。經由幾丁聚醣(chitosan)溶液批覆處理以鞏固微粒 的形狀並提供足夠的機械強度維持微粒的結構並能作爲載 體。再將微粒經液化處理以洗出內部的褐藻酸及表面的幾丁 聚醣，同時膠原蛋白在37°C左右下進行重組成三維纖維網狀 的結構。依不同操作條件可得到不同粒徑大小的微粒，將淸 洗後之含生醫陶瓷粉末的膠原蛋白微粒，經冷凍乾燥後置於 -20°C下保存。 本發明適用的膠原蛋白爲所有種類膠原蛋白，本發明實 施例使用經純化去免疫端(atelo)胺基酸的第一型、第二型動物 膠原蛋白來示範本發明。其乃利用酵素(例如胃蛋白酵素 pepsin)將膠原蛋白中會產生免疫反應的telopeptides切除，使 其無法聚集成膠原蛋白細纖維(collagen fibnlar)而形成膠原蛋 白溶液(soluble collagen)，經適當的條件可將其重組，而形成 膠原蛋白纖維的構造。由於將telopeptides切除，能使膠原蛋 7 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1_1 I H ϋ ·ϋ .^1 n ·_ϋ n · I n ϋ ϋ ϋ ^1 βϋ 一 口、I I aal I n ai_— I I I I ϋ ϋ ϋ · (請先閱讀背面之注意事項再填寫本頁) 524699 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（6) 白的免疫排斥降低◦本發明所使用的膠原蛋白含量爲微粒複 合材料重量的1-99 %，膠原蛋白溶液的濃度配成0.1-20 mg/ml 來使用。 骨組織之基質成分中礦物質的含量最大，其主要爲含 磷酸鈣和碳酸鈣所形成結晶程度較低之生物磷灰石 (bioapatite)。本發明所適用的生醫陶瓷粉末，主要有下列四 種··二銘磷酸鹽(a-or β-tricalcium phosphate)、氫氧基磷灰石 (hydroxyapatite)、硫酸鈣鹽(calcium sulfate hemi- hydrate)及碳 酸鈣鹽(calcium carbonate)，及上述這四種生醫陶瓷粉末依任 意不同組成或比例互相混合所構成。氫氧基磷灰石分子式爲 Ca1()(P04)6(0H)2，其成分和結晶構造與骨基質中之生物磷灰石 非常相似，其生物相容性極佳，可與骨組織產生直接的鍵 結，具有骨引導性(osteoconcuction)，能促進新生骨向其內部 生長。三鈣磷酸鹽分子式爲Ca3(P〇4)2，具有和氫氧基磷灰石 相似的生物相容性，能與骨組織直接鍵結，其生物可分解性 佳。硫酸鈣鹽的分子式爲CaS04· 1/2H20，與水混合變成Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 524699 A? ___ B7 V. Description of the invention (i) The present invention relates to a method for preparing collagen and bioceramic composite microparticles, especially about The invention relates to a method for preparing a composite material in the form of microparticles. The microparticles have a three-dimensional collagen fiber network structure inside and on the surface. If the human bone tissue is damaged due to injury or disease, it needs to be repaired to restore the function of the original tissue as much as possible. For example, if the bone plant is added to the defect as a scaffold to provide force and cell growth, it will effectively assist the bone tissue structure and function. Regeneration and repair. Autologous transplantation is the best choice for resident plants, but its source is limited, and it needs to suffer from secondary surgery. Allogeneic or xenotransplantation has problems with immunity and other scurvy. Synthetic bone tissue provides another New choice. The artificial resident material used in the past was inert to coexist with the living tissue. However, the inert substance will produce fibrosis around the implant, which will cause a serious difference in the interface between the tissue and the implant, causing the implant to loosen or the surrounding tissue fibers to harden, and even necrosis. Conventional technology has found that bioactive materials with better biocompatibility can be exhibited if there is a biological bond with the tissue. Collagen is the most abundant protein in the bone matrix. It has biocompatibility and low immunity, and is suitable as a matrix for the attachment of new bone cells. Adding biomedical ceramic powder (such as tricalcium phosphate, TCP; hyd serving yapatite, HAP, ...) to form a complex can improve its mechanical properties and further simulate the components of the bone matrix. And using this biogenic material as a carrier, carrying cells and growth factors, etc., will have the function of inducing tissue regeneration and can accelerate the effect of bone repair. 3 This paper size applies to Chinese National Standard (CNS) A4 ^ (210 x 297 mm) IL ------ 黻 --------- Order -------- line 丨 ·! (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 524699 A7 B7 V. Description of the invention (2) There are many types of artificial bone resident plants, which can be divided into blocks according to the form, Clay-shaped and particulate bone-plants, but each has its own disadvantages. Bone-shaped bone-plants in block form have better mechanical strength, but are not suitable for bone defects of various shapes in surgical operation. Clay-shaped and particulate bone plants are more convenient to operate. It can be shaped according to the shape of the defect, and can be injected into the bone defect of the human body by injection to reduce the pain of surgery. The disadvantage is that the material is not easy to fix on the defect 'and the mechanical strength is also poor. Conventional techniques use sintered porous ceramic materials to fill the pores with collagen by infusion or impregnation. The sintered ceramic has high hardness, so it can withstand the force ’, but the disadvantage is the limitation of the shape, and the porous ceramic material is non-bioabsorbable, which will prevent the new bone from growing further. The conventional technique is to directly dissolve the hydroxyapatite in an acidic solution and mix it with the recombinant collagen, and then adjust the pH of the solution to a slightly alkaline state so that the hydroxyapatite produces a sink on the collagen. Conventional technology will also mix the apatite precursor with the colloidal protein solution, and use acid-base reaction to transform the precursor into a hydroxylapatite in the matrix of collagen fiber interlinkage. In 1988 and 1989, Chu et al. (US Patent No. 4776890, 4888366), directly mixed the collagen fiber solution with the hydroxide apatite particles or powder to form a colloidal solution, which has the advantages of injectability and is used in Repair of soft tissue. The above-mentioned direct-mixed composites have a large difference in the density of collagen and ceramic powders. After mixing for a period of time, they will gradually separate into two phases due to different specific gravity, resulting in heterogeneity of the material. The particle size of the ceramic powder used is too large. (Approximately 0.5mm), it is not absorbed by living organisms, and fibrous tissue is generated around it. In addition, from the observation of the microstructure, this directly mixed composite material cannot form collagen in bone-like tissue. 4 The paper size is applicable to the Chinese National Standard (CNS) A4 specification (Sichuan ^ ^ mm)) '" -_ IIL ^ 1 I — ^ 1 II _ I · 11111-- · II I--II * I ----— — — pill · — — — — — — — — — — Please read the precautions on the back first (Fill in this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 524699 A7 B7 V. Description of the invention (3) The fiber network structure provides the growth environment required for cells in bone-like tissues. In the composite of micro-particle type, collagen and ceramic powder are more uniformly distributed, and a large number of micro-particles are randomly arranged in various directions, so it can compensate for the unevenness of the two phases of a micro-particle. In addition, it is easy to shape, and because the particles are small, it can be made into an injection form to reduce the inconvenience of the patient. Conventional technology uses silicon oil to make collagen particles, and then further mixes the hydroxyl apatite powder with the collagen solution and drips into the rapidly stirring ethyl-2-hexyl cocoate to make the collagen into balls. After centrifugation and alcohol After removing ethyl-2-hexyl cocoate, collagen / hydroxyapatite particles were separated. In 2001, Wang et al. (Republic of China Patent Publication No. 420604, Certificate No. 1273111), mixed collagen and calcium phosphate-based ceramic powder into a mixed solution, and dropped it into an oil phase (such as olive oil) dropwise, so that the The mixed solution is recombined to form reticulated fibrous structure particles; a cross-linking agent (such as glutaraldehyde) is injected into the oil phase to cross-link the particles; and the particles are separated from the oil phase. In the process of preparing microparticles by the above method, the use of organic solvents may destroy the properties of collagen, and the residual organic oil or cross-linking agent may be harmful to the tissue. In addition, more procedures are required to separate and remove the oil phase. Based on the shortcomings of conventional composite materials, their preparation methods, and technologies, the object of the present invention is to provide a method for preparing collagen and biomedical ceramic powder composite material particles to improve the disadvantages of conventional composite materials and their preparation methods. The object of the present invention is to provide a method for preparing collagen and biomedical ceramic powder composite material particles. Collagen solution and recombination technology are used to prepare this composite material microparticles. Collagen and biomedical ceramic powder in this composite material. 5 The paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) ----- 1 —, ------------- q I Order: -------- I (Please read the notes on the back before filling this page) 524699 A7 B7 V. Description of the invention ( 4 :) The powder can be evenly distributed, and the particles have a three-dimensional collagen fiber network structure inside and outside. The advantage of the method provided by the present invention is that during the preparation of microparticles, there is no doubt about the damage to the tissue caused by residual organic solvents, organic oils or crosslinkers. The composite material particles prepared by the method of the invention have sufficient mechanical strength to carry cells as carriers and to coat and fix various bone growth factors, to induce tissue regeneration and to accelerate the effect of bone repair, and can be injected or further According to requirements, different types of substrate templates are made for bone tissue wound repair. Brief description of the figure Figure 1: Schematic diagram of the method for preparing collagen and biomedical ceramic powder composite particles. FIG. 2A is a SEM image showing a 10000X SEM image of a particle cross section at 9 hours. FIG. 2B is a SEM image showing a 10000X SEM image of the particle surface at 9 hours. FIG. 2C is a SEM image of 10000X SEM image of the particle cross section when it is not displayed for 24 hours. FIG. 2D is a SEM image showing a 10000X SEM image of the particle surface at 24 hours. FIG. 3A is a SEM image showing a 10000X SEM image of a particle cross section at 9 hours. FIG. 3B is a SEM image showing a 10000X SEM image of the particle surface at 9 hours. 6 This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) ---------% (Please read the precautions on the back before filling this page) d 丨 Order / --- ----- Line 丨 Printed by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs Printed by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs Printed by 524699 A7 B7 V. Description of the Invention (b) Figure 3C: It is a SEM image showing 24 hours, Particle cross section 10000X SEM image. FIG. 3D is a SEM image showing a 10000X SEM image of the particle surface at 24 hours. In order to achieve the above object, the present invention prepares collagen and biomedical ceramic powder composite material particles according to the following method: mixing a collagen solution, algimc acid and biomedical ceramic powder at 0-10 ° C, It is dropped into a divalent cation aqueous solution in the form of a ball drop and gelatinized into a particulate form. It is treated with a chitosan solution to consolidate the shape of the particles and provide sufficient mechanical strength to maintain the structure of the particles and to serve as a carrier. The microparticles were then liquefied to wash out the alginic acid inside and the chitosan on the surface. At the same time, the collagen was reorganized into a three-dimensional fibrous network structure at about 37 ° C. Microparticles with different particle sizes can be obtained according to different operating conditions. The collagen microparticles containing biomedical ceramic powder after washing are freeze-dried and stored at -20 ° C. The collagens applicable to the present invention are all kinds of collagens, and the embodiments of the present invention use purified first-type and second-type animal collagens that are immune to atelo amino acids to demonstrate the present invention. It uses enzymes (such as pepsin) to remove the telopeptides that produce immune response in collagen, making it unable to aggregate into collagen fibnlar to form a collagen solution. After appropriate conditions, It can be recombined to form a collagen fiber structure. Since the telopeptides are removed, the collagen paper can be used for 7 paper sizes to comply with Chinese National Standard (CNS) A4 (210 X 297 mm) 1_1 IH ϋ · ϋ. ^ 1 n · _ϋ n · I n ϋ ϋ ϋ ^ 1 βϋ One mouth, II aal I n ai_— IIII ϋ ϋ ϋ (Please read the precautions on the back before filling out this page) 524699 A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (6) White immune rejection Decrease the content of collagen used in the present invention is 1-99% of the weight of the particulate composite material, and the concentration of the collagen solution is 0.1-20 mg / ml for use. Bone tissue has the largest mineral content in the matrix components. It is mainly bioapatite containing calcium phosphate and calcium carbonate with a low degree of crystallinity. The biomedical ceramic powders applicable to the present invention mainly include the following four types: a-or β-tricalcium phosphate, hydroxylapatite, and calcium sulfate hemi-hydrate ) And calcium carbonate (calcium carbonate), and the above four kinds of biomedical ceramic powder are mixed with each other according to any different composition or ratio. Hydroxyapatite has the molecular formula Ca1 () (P04) 6 (0H) 2. Its composition and crystal structure are very similar to those of biological apatite in bone matrix. It has excellent biocompatibility and can be produced with bone tissue. Direct bonding, with osteoconcuction, can promote the growth of new bone to its interior. The formula of tricalcium phosphate is Ca3 (P04) 2, which has biocompatibility similar to that of hydroxyapatite, can be directly bonded to bone tissue, and has good biodegradability. The molecular formula of calcium sulfate is CaS04 · 1 / 2H20, mixed with water to become
CaSCV 2H2〇，具有生物可吸收性。碳酸鈣鹽分子式爲 CaCCb，其生物可吸收性佳，植入體內數個月到一年可被組 織吸收◦生醫陶瓷粉末其顆粒大小會影響到組織與材料之間 的反應，顆粒太大不能被生物體所吸收且會在其周圍有纖維 組織產生；粒徑約5μιη以下可被巨噬細胞以包噬作用將顆粒 吸收。本發明所使用的生醫陶瓷粉末含量爲微粒複合材料重 量的1-99 %，粒徑大小約5μιη以下。 8 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） —--------m------- 訂--------線 |_! (請先閱讀背面之注意事項再填寫本頁) _ I ϋ ϋ ϋ ϋ -ϋ ϋ ϋ · 經濟部智慧財產局員工消費合作社印製 524699 A7 __— B7 五、發明說明（7 ) 褐藻酸爲無毒的天然的聚醣類高分子，於室溫下可溶於 水或鹼性溶液中形成黏稠溶液，但不溶於有機溶劑、酸性溶 液；在生理溶液的環境中爲帶負電的高分子，可與多種二價 陽離子反應形成J3寥體，應用於細胞或藥物的包覆。本發明所 使用的褐藻酸其濃度配成0.140 w/v %來使用。 本發明可使用的二價陽離子爲Cu2+，Pb2+，Ca' Ba2+，Cd2+， Co2+，Ζη2+，Μη2+，Nh2+，Sr2+等，而褐藻酸與二價陽離子所形成的 膠體其機械性質隨其之間親合力的增加而增加。由於錦離子 爲人體內所具有的離子，所以大部分都以鈣離子來進行膠 化。本發明所使用的二價陽離子水溶液配成0.1-10 w/v %使 用。 幾丁聚醣(chitosan，ploy(/5-l，4-D-glucosamine))是由幾丁 質(dntin)部份或完全去乙醯化所得之衍生物，分子結構與纖 維素相近，爲一種結晶狀的多醣類。幾丁聚醣在生物體內可 被溶菌酉_分解成N-D-glucosamine，具有抗菌性、可食用性 及良好的生物相容性，在生物醫學上應用廣泛。另外幾丁聚 醣在酸性環境下，其胺基上具有帶正電的特性。本發明乃利 用它帶正電的特性，使其批覆(coating)在帶負電的褐藻酸鹽微 粒上。當液化洗出微粒內部的褐藻酸及膠原蛋白進行重組期 間，用來鞏固微粒的形狀及提供足夠的機械強度維持微粒的 結構並能作爲載體。在褐藻酸液化洗出的期間，能同時將微 粒表面的幾丁聚醣漸漸一起帶離表面。本發明所使用的幾丁 聚醣係溶解於一酸性水溶液中以形成一幾丁聚醣水溶液，該 9 本紙張尺度適用中國國家標準（CNS)A4規格（21〇 x 297公釐) --------------- I I I---、訂 ^-----— II I . (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 524699 A7 B7 五、發明說明（8 ) 幾丁聚醣在該幾丁聚醣水溶液中的重量百分比含量爲 0.00001%〜10%，溶液的pH値在4.5-6.5之間。使用的酸係選 自甲酸(formic acid )、醋酸(acetic acid )、丙酸(propionic acid)、 乳酸(lactic acid)、蘋果酸(malic acid)、檸檬酸(citric acid)、抗壞 血酸(ascorbic acid)、草酸(oxalic acid)、丁二酸(succinic acid)、 丙二酸(malonic acid)、己二酸(adipic acid)、丙酮酸(pyruvic acid)、胺基戊二酸(glutaric acid)、酒石酸(tartaric acid)、丁氨二 酸(asparagic acid)、環氧丁二酸(epoxysuccinic acid)、一氯醋酸 (monochloroacetic acid)、水楊酸(salicylic acid)、亞甲基丁二酸 (itaconic acid)、D比略院酮羧酸(pyrrolidone carboxylic acid)、經 基醋酸(glycolic acid)、硝酸(nitric acid)、硫酸(sulfuric acid)、鹽 酸(hydrochloric acid)、磷酸(phosphoric acid)、及其混合物。 本發明使用磷酸系緩衝溶液，來液化洗出微粒內部的褐 藻酸及帶離表面的幾丁聚醣，同時微粒中的膠原蛋白在37°C 下，進行重組成三維的纖維網狀結構◦所使用磷酸系緩衝溶 液，爲磷酸緩衝溶液(PB，phosphate buffer )、檸檬酸鈉(sodium citrate)溶液等，來液化褐藻酸膠體。通常溶液濃度越高，其 液化洗出微粒內部的褐藻酸及帶離表面幾丁聚醣的速度越 快。可調節溶液濃度來控制洗出時間，讓膠原蛋白有足夠的 時間進行重組。本發明所使用的磷酸緩衝液配成0.0014M來 使用，pH値在7.2-7.4生理範圍値之間。 本發明利用一具針頭之容器以注射pump裝置(air jet-syringe pump extrusion)，將膠原蛋白、生醫陶瓷粉末和褐 10 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I — ϋ ϋ ϋ I I I I I I I I I ϋ I n ϋ ϋ 1 I ϋ I I ϋ ϋ n ϋ I I n I I ϋ I ϋ n I ϋ I ϋ ϋ ϋ n ϋ I I I I I - (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 524699 A7 B7 五、發明說明（9 ) 藻酸混合溶液擠壓成球滴形式，滴入二價陽離子的水溶液中 膠化成微粒型。調節氣體流量大小可得到不同粒徑大小的微 粒，氣體流量越大可得粒徑越小的微粒◦此氣體可爲氮氣、 氦氣、氬氣、氧氣、二氧化碳、空氣等氣體。 所製備出微粒型式的複合材料，以掃描式電子顯微鏡 (scanning electron microscope，SEM )，觀察其微粒內部及表面 的形態結構；以光學顯微鏡，觀察微粒中膠原蛋白與生醫陶 瓷粉末分佈情形及微粒粒徑大小。 以下列之實施例進一步說明本發明，但並不限制本發明 的精神和範圍。 【實施例1】 請參照圖1所示，依下列步驟進行製備膠原蛋白和生醫 陶瓷粉末複合材料微粒： 1 ·取經純化去免疫端(atelo)胺基酸的第一型膠原蛋白溶液6ml (濃度：6mg/ml)、β-三釣磷酸鹽(β-tricalcium phosphate)粉末 66.86 mg、褐藻酸溶液1.8ml (濃度：6%)和磷酸鹽緩衝液 (PBS，phosphate buffer saline) 1.2ml，將其充分混合。其中膠 原蛋白與β-三鈣磷酸鹽的重量百分比例爲35/65。 2.以air jet-syringe pump e血ision裝置，調節氮氣流量爲5 ml/min，將上述混合溶液以球滴形式滴入氯化錦(CaCK 1.5%)水溶液中膠化成微粒型。上述過程在4°C下進行以防 止膠原蛋白重組。 11 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱y (請先閱讀背面之注意事項再填寫本頁)CaSCV 2H2O is bioabsorbable. The molecular formula of calcium carbonate is CaCCb, which has good bioabsorbability. It can be absorbed by tissues for several months to one year after implantation. The particle size of biomedical ceramic powder will affect the reaction between tissues and materials. It is absorbed by the organism and there will be fibrous tissue around it; the particle size of about 5 μm or less can be absorbed by the macrophages by encapsulation. The content of the biomedical ceramic powder used in the present invention is 1-99% by weight of the particulate composite material, and the particle size is about 5 μm or less. 8 This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) —-------- m ------- Order -------- line | _! (Please read the notes on the back before filling out this page) _ I ϋ ϋ ϋ ϋ -ϋ ϋ ϋ · Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 524699 A7 __ — B7 V. Description of the invention (7) Alginic acid is non-toxic It is a natural polysaccharide polymer that can be dissolved in water or alkaline solution to form a viscous solution at room temperature, but is insoluble in organic solvents and acidic solutions. It is a negatively charged polymer in the environment of physiological solutions and can be used with A variety of divalent cations react to form J3 oligosomes, which are used to coat cells or drugs. The alginic acid used in the present invention is used at a concentration of 0.140 w / v%. The divalent cations usable in the present invention are Cu2 +, Pb2 +, Ca 'Ba2 +, Cd2 +, Co2 +, Zη2 +, Mη2 +, Nh2 +, Sr2 +, and the like, and the mechanical properties of the colloid formed by alginic acid and the divalent cation depend on the affinity between Increase. Since bromide ions are ions in the human body, most are gelled with calcium ions. The divalent cation aqueous solution used in the present invention is used in an amount of 0.1-10 w / v%. Chitosan (ploto (/ 5-1, 4-D-glucosamine)) is a derivative obtained by partial or complete deacetylation of chitin (dntin). Its molecular structure is similar to that of cellulose. A crystalline polysaccharide. Chitosan can be broken down into N-D-glucosamine by lysobacteria in vivo, which has antibacterial, edible and good biocompatibility, and is widely used in biomedicine. In addition, chitosan has a positively charged property on its amine group in an acidic environment. The present invention takes advantage of its positively charged properties to allow it to be coated on negatively charged alginate particles. When the alginic acid and collagen inside the microparticles are liquefied and washed out for reorganization, it is used to consolidate the microparticle shape and provide sufficient mechanical strength to maintain the microparticle structure and can be used as a carrier. During the liquefaction and washing out of alginic acid, the chitosan on the microparticle surface can be gradually taken away from the surface at the same time. The chitosan used in the present invention is dissolved in an acidic aqueous solution to form a chitosan aqueous solution. The 9 paper sizes are applicable to the Chinese National Standard (CNS) A4 specification (21 × 297 mm) --- ------------ II I ---, order ^ -----— II I. (Please read the precautions on the back before filling out this page) Employees ’Cooperatives, Intellectual Property Bureau, Ministry of Economic Affairs Printing 524699 A7 B7 V. Description of the invention (8) The weight percentage content of chitosan in the chitosan aqueous solution is 0.00001% to 10%, and the pH of the solution is between 4.5 and 6.5. The acid used is selected from formic acid, acetic acid, propionic acid, lactic acid, malic acid, citric acid, ascorbic acid , Oxalic acid, succinic acid, malonic acid, adipic acid, pyruvic acid, glutaric acid, tartaric acid ( tartaric acid, asparagic acid, epoxysuccinic acid, monochloroacetic acid, salicylic acid, itaconic acid, D. Compared with pyrrolidone carboxylic acid, glycolic acid, nitric acid, sulfuric acid, hydrochloric acid, phosphoric acid, and mixtures thereof. The present invention uses a phosphate buffer solution to liquefy and wash out alginic acid and chitosan from the surface of the microparticles. At the same time, the collagen in the microparticles is reconstituted into a three-dimensional fibrous network structure at 37 ° C. The alginic acid colloid is liquefied using a phosphoric acid-based buffer solution, such as a phosphate buffer solution (PB), a sodium citrate solution, or the like. Generally, the higher the concentration of the solution, the faster the liquefaction and washing out of the alginic acid and the chitosan off the surface of the particles. The concentration of the solution can be adjusted to control the washout time, allowing enough time for collagen to reconstitute. The phosphate buffer solution used in the present invention is formulated at 0.0014M for use, and the pH is between 7.2 and 7.4 physiological range. The present invention uses a container with a needle to inject a pump (air jet-syringe pump extrusion), collagen, biomedical ceramic powder and brown 10 paper size applicable to Chinese National Standard (CNS) A4 (210 X 297 mm) ） I — ϋ ϋ ϋ IIIIIIIII ϋ I n ϋ ϋ 1 I ϋ II ϋ ϋ n ϋ II n II ϋ I ϋ n I ϋ I ϋ ϋ ϋ n ϋ IIIII-(Please read the precautions on the back before filling this page) Printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 524699 A7 B7 V. Description of the invention (9) The alginic acid mixed solution is extruded into a ball drop form, and is dripped into an aqueous solution of divalent cations to be gelled into a particulate form. The particle size can be adjusted by adjusting the gas flow rate. The larger the gas flow rate, the smaller the particle size can be. ◦ This gas can be nitrogen, helium, argon, oxygen, carbon dioxide, air and other gases. Scanning electron microscope (SEM) was used to observe the morphology and structure of microparticles in the prepared composite material. The distribution of collagen and biomedical ceramic powder in microparticles and microparticles were observed with an optical microscope. Particle size. The following examples further illustrate the present invention, but do not limit the spirit and scope of the present invention. [Example 1] Please refer to FIG. 1 to prepare collagen and biomedical ceramic powder composite material particles according to the following steps: 1 Take 6ml of a type 1 collagen solution that has been purified to remove immunogenic (atelo) amino acids ( Concentration: 6mg / ml), β-tricalcium phosphate powder, 66.86 mg, alginic acid solution, 1.8ml (concentration: 6%), and phosphate buffer saline (PBS, phosphate buffer saline), 1.2ml. It's well mixed. An example of the weight percentage of colloidal protein and β-tricalcium phosphate is 35/65. 2. Using an air jet-syringe pump e blood ision device, adjust the nitrogen flow rate to 5 ml / min, and drop the above mixed solution in the form of a ball drop into an aqueous solution of chlorinated bromide (CaCK 1.5%) to gel into a particulate form. The above process was performed at 4 ° C to prevent collagen reorganization. 11 This paper size applies to China National Standard (CNS) A4 specifications (210 X 297 public love y (Please read the precautions on the back before filling this page)
524699 A7 B7 五、發明說明（in) 3. 以 Ca-CHES(2-N-cyclohexylaminoethane-sulfuric acid )置換氯 化鈣水溶液，並使上述微粒在其中時效(aging) 5分鐘進行 淸洗。再置換至0.05w/v%的幾丁聚醣溶液似0.05%的乳酸 配製，pH値=5.5)中披覆(coating)處理5分鐘。以0.5M磷 酸緩衝液先進行液化處理5分鐘，部分洗出褐藻酸及表面 的幾丁聚醣。上述過程在室溫下進行。 4. 接著將上述微粒以0.02M磷酸緩衝液，於37°C下進行液化 處理，並在第1，3, 6, 9, 12, 24小時時，更換新鮮的磷酸緩 衝液，以達到完全洗出褐藻酸及表面的幾丁聚醣的目的， 同時膠原蛋白能進行重組成具有三維纖維網狀結構。 5. 以磷酸鹽緩衝液(PBS)淸洗，將淸洗後之含生醫陶瓷粉末的 膠原蛋白微粒，經冷凍乾燥後置於-20°C下保存，或置於磷 酸鹽緩衝液中於4°C下保存。 利用光學顯微鏡圖觀察微粒，由實驗結果顯示，所製備 的微粒型式複合材料，其β-三鈣磷酸鹽與膠原蛋白能均勻分 佈在其中◦上述微粒，依不同時間以磷酸緩衝液洗出褐藻 酸，利用偵測磷酸緩衝液中褐藻酸的含量以了解褐藻酸是否 完全被洗出。由實驗結果發現，在9小時後褐藻酸已有90 % 以上洗出，而在12小時以後褐藻酸已幾乎完全被洗出。另外 微粒以掃描式電子顯微鏡，觀察不同時間微粒切面及表面結 構，如圖2Α至2D所示。圖2Α與2Β係9小時時，圖2C與 2D係24小時時，以10000倍觀察微粒切面及表面結構之SEM 照片◦實驗結果顯示，在9小時後微粒內部及表面具有立體 12 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 I « — ϋ I I ϋ a I I I ϋ ϋ ϋ ϋ I I ϋ ϋ II ϋ I -I ϋ H -ϋ —.1 ϋ H ϋ ^1 ϋ I H - 524699 A7 B7 五、發明說明（fl) 膠原蛋白纖維結構，而表面則有些微局部的幾丁聚醣殘留； 在24小時後微粒內部及表面都具有三維膠原蛋白纖維網狀 結構，表面的幾丁聚醣大部分都隨著褐藻酸一起被洗離微粒 表面。微粒的平均粒徑（average diameter)爲1560 土 70 μπι。 【實施例2】 裝置、步驟如貫施例1 ^除在步驟3中以0.005w/v %的 丁聚醣溶液似0.005%的乳酸配製，pH値=5.5)中披覆5分鐘 處理，其它同實施例1中的步驟。 由本實驗結果顯示，在9小時時褐藻酸已有95 %以上洗 出，而在12小時以後褐藻酸已幾乎完全洗出。微粒以掃描式 電子顯微鏡觀察結構，圖3A與3B係9小時時，圖3C與3D 係24小時時，以10000倍觀察微粒切面及表面結構之SEM 照片。實驗結果發現，在9小時後微粒內部及表面具有立體 膠原蛋白纖維結構，表面有微量的幾丁聚醣殘留；在24小時 後微粒內部及表面都具有三維膠原蛋白纖維結構，表面的幾 丁聚醣幾乎全跟著褐藻酸一起被洗出。 【實施例3】 裝置、步驟如實施例1，除在步驟1中將膠原蛋白與β-三銘磷酸鹽的比例改爲25/75，其它同實施例1。實驗結果(掃 描式電子顯微鏡圖顯示類似實施例1，2所示的結構形態）顯 示微粒內部及表面都具有三維的膠原蛋白纖維網狀結構。在 13 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐) -ϋ ϋ ϋ n I ·ϋ I ϋ I I ·1 1 i a··— -1· in ϋ J 、I ϋ n (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 線 iiilli------------ 524699 A7 ______ B7 五、發明說明（12) 複合材料微粒中，膠原蛋白的比例越高越具有三維網狀纖維 結構，但微粒強度較弱且成本較高；如其比例越低則強度較 強且成本較低，但三維網狀纖維結構較差，無法有效提供類 似骨組織中膠原纖維網狀結構，以讓細胞生長的環境。通常 膠原蛋白與生醫陶瓷粉末的比例在10/90-90/10之間較佳，而 以35/65的比例爲最佳，因爲此比例與骨基質內的兩成分比 例相似。 【實施例4】 裝置、步驟如實施例1，除在步驟2中，氮氣的流量調 爲7.5 ml/mm其它同實施例1。實驗結果顯示如同實施例1， 微粒內部及表面都具有三維膠原蛋白纖維網狀結構，微粒的 平均粒徑爲835 ± 36 μιη。 。 【實施例5】 裝置、步驟如實施例1，除在步驟2中，氮氣的流量調 爲10 ml/min其它同實施例1。實驗結果顯示如同實施例！， 其微粒的平均粒徑爲210 ± 12 μιη。調節氣體流量大小可得到 不同粒徑大小的微粒，氣體流量越大可得粒徑越小的微粒。 【實施例6】 裝置、步驟如貫施例1，除在步驟1中以氯氧基憐灰石 取代β-三鈣磷酸鹽粉末，其它同實施例1的中步驟。實驗結 14 (請先閱讀背面之注意事項再填寫本頁) 着m-------:丨訂··1 經濟部智慧財產局員工消費合作社印製 線—瘳！ --------I*------------ 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） A7 524699 五、發明說明（13) 果顯示’微粒內部及表面都具有三維的膠原蛋白纖維網狀結 構(掃描式電子顯微鏡圖及光學顯微鏡圖顯示，其結果類同實 施例1的結構形態)。微粒中的生醫陶瓷粉末可依本發明所述 四種生醫陶瓷材料，以任意不同組成或比例互相混合所構 成。 【實施例7】 裝置、步驟如實施例1，除在步驟1中取經純化去免疫 端(atelo)胺基酸的第二型膠原蛋白溶液―（濃度：6mg/ml)取 代第一型膠原蛋白溶液，其它同實施例1的中步驟。實驗結 果顯示’微粒內部及表面都具有三維的膠原蛋白纖維網狀結 構(掃描式電子顯微鏡圖及光學顯微鏡圖顯示，其結果類同實 施例1的結構形態)。微粒中的生醫陶瓷粉末可依本發明所述 四種生醫陶瓷材料，以任意不同組成或比例互相混合所構 成。 以本發明的方法，所製備出的微粒型式複合材料，其微 粒的粒徑大小範圍可爲5〇 ，具有下列優點：使用 經純化去免疫端胺基酸的膠原蛋白，能降低其免疫排斥性； 製備方法爲水相系統，能避免有機溶劑破壞膠原蛋白性質及 殘餘的有機油對組織傷害的疑慮；微粒已具有足夠的機械強 度’不需再經交聯劑的作用，能避免交聯劑對組織的傷害； 使用膠原蛋白重組技術，微粒內部及表面都具有三維的膠原 蛋白纖維網狀結構· ，生醫陶瓷粉末能均勻分佈在膠原蛋白纖 維網狀結構中；可依不同操作條件得到不同粒徑大小的微 15 (請先閱讀背面之注意事項再填寫本頁) 11 訂·：----- 線蜂- 經濟部智慧財產局員工消費合作社印製 氏張尺度適用中國國家標準（CNS)A4 ϋ210 X 公釐) 524699 Α7 Β7 五、發明說明（Μ) 粒；具足夠的機械強度，因此可用來包覆、固定各種不同的 骨生長因子，有誘導組織再生及增快骨修補效果之功能；並 因在製備過程完全使用溫和、且不會危害細胞的條件，因此 也適合包覆細胞或組織等生物活體，藉由組織工程的槪念’ 以進行人體組織修補；而其施用的方式，能以注射式或進一 步依需求製作成不同型式的基材模板等優點。另外所使用的 材料如膠原蛋白、生醫陶瓷粉末、褐藻酸及幾丁聚醣等皆爲 美國食品及藥物管理局(FDA)所核准用於人體。此微粒複合 材料具有類似骨組織的成份及膠原蛋白纖維網狀結構，可提 供似骨組織中細胞生長的環境，應用於骨組織創傷修復之 用。 雖然本發明已以上述之較佳實施例揭露如上，然其並 非用以限定本發明，任何熟習此技藝者，在不脫離本發明之 精神和範圍內，當可做些許之更動與潤飾，因此本發明之保 護範圍當視後附之申請專利範圍所界定者爲準。 16 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製524699 A7 B7 V. Description of the invention (in) 3. The calcium chloride aqueous solution was replaced with Ca-CHES (2-N-cyclohexylaminoethane-sulfuric acid), and the above-mentioned microparticles were aged for 5 minutes for rinsing. The chitosan solution replaced with 0.05 w / v% was prepared by coating with 0.05% lactic acid, pH 値 = 5.5) for 5 minutes. 0.5M phosphoric acid buffer solution was firstly subjected to liquefaction treatment for 5 minutes to partially wash out alginic acid and chitosan on the surface. The above process is performed at room temperature. 4. Next, the above microparticles were liquefied with 0.02M phosphate buffer solution at 37 ° C, and the fresh phosphate buffer solution was replaced at 1, 3, 6, 9, 12, and 24 hours to achieve complete washing. The purpose of alginic acid and chitosan on the surface, and collagen can be reconstituted with a three-dimensional fibrous network structure. 5. Rinse in phosphate buffered saline (PBS), and freeze the collagen particles containing biomedical ceramic powder after lyophilization, and store them at -20 ° C after freeze drying, or put them in phosphate buffered saline. Store at 4 ° C. Observe the microparticles with an optical microscope image. The experimental results show that the β-tricalcium phosphate and collagen can be uniformly distributed in the prepared microparticle composites. The above microparticles are washed out with alginic acid in a phosphate buffer solution at different times. To detect whether the alginic acid was completely washed out by detecting the content of alginic acid in the phosphate buffer solution. From the experimental results, it was found that after 9 hours, alginic acid was washed out more than 90%, and after 12 hours, the alginic acid was almost completely washed out. In addition, the scanning electron microscope was used to observe the cross-section and surface structure of the particles at different times, as shown in Figs. 2A to 2D. Figures 2A and 2B are at 9 hours, and Figures 2C and 2D are at 24 hours. The SEM pictures of the cross section and surface structure of the particles are observed at 10000 times. The experimental results show that after 9 hours, the inside and surface of the particles have three dimensions. China National Standard (CNS) A4 (210 X 297 mm) (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs I «— ϋ II ϋ a III ϋ ϋ ϋ ϋ II ϋ ϋ II ϋ I -I ϋ H -ϋ —.1 ϋ H ϋ ^ 1 ϋ IH-524699 A7 B7 V. Description of the invention (fl) Collagen fiber structure, but the surface has some localized chitosan residue ; After 24 hours, the microparticles have a three-dimensional collagen fiber network structure inside and on the surface, and most of the chitosan on the surface is washed away from the microparticle surface with alginic acid. The average diameter of the particles was 1560 to 70 μm. [Example 2] The device and steps are as in Example 1. ^ Except that in step 3, 0.005w / v% butan solution is prepared like 0.005% lactic acid, pH 値 = 5.5), and coated for 5 minutes, and other Same steps as in Example 1. According to the results of this experiment, alginic acid was eluted more than 95% at 9 hours, and alginic acid was almost completely eluted after 12 hours. The microstructure of the microparticles was observed with a scanning electron microscope. Figures 3A and 3B show the SEM images of the cross-section and surface structure of the microparticles at 10,000 times at 9 hours and Figures 3C and 3D at 24 hours. The experimental results found that after 9 hours, the microparticles had a three-dimensional collagen fiber structure inside and on the surface, and a small amount of chitosan remained on the surface; after 24 hours, the microparticles had a three-dimensional collagen fiber structure on the surface and on the surface. Almost all the sugar was washed out with alginic acid. [Example 3] The device and steps are the same as those in Example 1, except that the ratio of collagen to β-triammonium phosphate was changed to 25/75 in step 1, and the others were the same as in Example 1. The experimental results (scanning electron microscopy images showing structures similar to those shown in Examples 1 and 2) showed that the inside and surface of the microparticles had a three-dimensional collagen fiber network structure. In 13 paper sizes, the Chinese National Standard (CNS) A4 specification (210 X 297 mm) is applied -ϋ ϋ ϋ n I · ϋ I ϋ II · 1 1 ia ·· — -1 · in ϋ J, I ϋ n ( Please read the precautions on the back before filling out this page) Printed line for consumer cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs iiilli ------------ 524699 A7 ______ B7 V. Description of the invention (12) Composite material particles The higher the proportion of collagen, the more it has a three-dimensional network fiber structure, but the strength of the particles is weaker and the cost is higher; if the ratio is lower, the strength is stronger and the cost is lower, but the three-dimensional network fiber structure is poor and cannot be effectively provided An environment similar to the network of collagen fibers in bone tissue to allow cells to grow. Generally, the ratio of collagen to biomedical ceramic powder is between 10 / 90-90 / 10, and the ratio of 35/65 is the best, because this ratio is similar to the ratio of the two components in the bone matrix. [Embodiment 4] The apparatus and procedures were the same as those in Embodiment 1, except that in Step 2, the flow rate of nitrogen was adjusted to 7.5 ml / mm, and the same procedures as in Embodiment 1. The experimental results show that, as in Example 1, the microparticles have a three-dimensional collagen fiber network structure inside and on the surface, and the average particle diameter of the microparticles is 835 ± 36 μm. . [Embodiment 5] The apparatus and steps are the same as those in embodiment 1, except that in step 2, the flow rate of nitrogen is adjusted to 10 ml / min. The experimental results show the same as the examples! The average particle size of the particles is 210 ± 12 μm. By adjusting the gas flow rate, particles with different particle sizes can be obtained. The larger the gas flow rate, the smaller particles can be obtained. [Example 6] The device and steps are the same as those in Example 1, except that the β-tricalcium phosphate powder is replaced with chlorooxylimite in step 1, and the other steps are the same as those in Example 1. Lab 14 (Please read the precautions on the back before filling this page) With m -------: 丨 Order · 1 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs— 瘳! -------- I * ------------ This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) A7 524699 V. Description of the invention (13) The results show that both the inside and the surface of the microparticles have a three-dimensional network structure of collagen fibers (scanning electron microscope images and light microscope images show that the results are similar to the structural form of Example 1). The biomedical ceramic powder in the microparticles can be formed by mixing the four biomedical ceramic materials according to the present invention with any different composition or ratio. [Example 7] The device and steps are the same as those in Example 1, except that in step 1, the purified type II collagen solution without immune ate (atelo) amino acid is taken-(concentration: 6mg / ml) instead of type I collagen The solution and other steps are the same as those in Example 1. The experimental results show that the three-dimensional collagen fiber network structure is present inside and on the surface of the microparticles (scanning electron microscope image and optical microscope image, the results are similar to the structural form of Example 1). The biomedical ceramic powder in the microparticles can be formed by mixing the four biomedical ceramic materials according to the present invention with any different composition or ratio. According to the method of the present invention, the particle type composite material prepared can have a particle size range of 50, and has the following advantages: the use of purified collagen deimmunized amino acid can reduce its immune rejection ; The preparation method is an aqueous phase system, which can avoid the doubt that the organic solvent destroys the properties of collagen and the residual organic oil will damage the tissue; the microparticles have sufficient mechanical strength, and the crosslinking agent can be avoided without the action of the crosslinking agent Damage to tissues; Using collagen recombination technology, there is a three-dimensional collagen fiber network structure inside and on the surface of the particles. The biomedical ceramic powder can be evenly distributed in the collagen fiber network structure; it can be obtained according to different operating conditions. Particle size of micro 15 (Please read the precautions on the back before filling out this page) 11 Orders: ----- Line bee-Printed scales for employees' cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs apply Chinese national standards (CNS ) A4 ϋ210 X mm) 524699 Α7 Β7 V. Description of the invention (M) grain; with sufficient mechanical strength, it can be used to cover and fix various Bone growth factor has the function of inducing tissue regeneration and accelerating the effect of bone repair; and because it uses mild conditions that do not harm cells during the preparation process, it is also suitable for covering biological living cells such as cells or tissues. Engineering's thoughts' are used to repair human tissues; and its application method can be injected or further made into different types of substrate templates as required. In addition, materials such as collagen, biomedical ceramic powder, alginic acid, and chitosan are approved by the US Food and Drug Administration (FDA) for use in humans. This particulate composite material has a bone-like composition and a collagen fiber network structure, which can provide an environment for the growth of cells in bone tissue, and is used for bone tissue wound repair. Although the present invention has been disclosed as above with the above-mentioned preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications and retouches without departing from the spirit and scope of the present invention. The protection scope of the present invention shall be determined by the scope of the attached patent application. 16 This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) (Please read the notes on the back before filling this page)