476642 五、發明說明（1 登明背景 :原蛋白為一種具生物降解性的蛋白質，大部分以纖維 型悲存在於動物組織中’主要的功能為維持組織型態並提 供組織所需之張力。膠原蛋 虫曰刀于疋由二條多胜肽鏈彼此 互相纏繞而成之生物性高分+，立玫夕 同刀于，母條多胜肽鏈約由一千多 個胺基酸聚合而成，主要的胺篡醏古 戈们胺基^有甘胺酸、脯胺酸及羥 基脯胺酸。至目前為止，至少右+ 王乂百十九種不同類型之膠原蛋 白被發現。 一般膠原蛋白的應用係將其製成如海綿狀、膠體狀、管 狀及薄膜狀等各種不同型態之基質，可用於傷口敷料、止 血、組織修復、樂物載體、細胞培養基質以及各種人工臟 器等。為了使上述膠原蛋白基質具多孔狀結構以利細胞之 遷移、生長或藥物包埋及其釋放等，常使用冷凍乾燥法進 行基質孔洞的製備。一般而言，在冷凍乾燥過程中，常將 4樣置於-80 C冷凍或直接以液態氮急速冷凍後再進行冷凍 乾燥。由於急速冷凍時，所產生的冰晶顆粒較小，致使所 得之基質孔徑不大（小於30微米）。其次，若冷凍速率過 快’亦會造成基質中冰晶分佈不均而影響基質中孔洞的均 句度，甚至造成基質龜裂。 關於多孔狀膠原蛋白基質之製備程序，已揭示於許多先 前之專利中，例如美國專利第4，193,813號中，係先將膠原 蛋白溶於醋酸中，以戊二醛交聯後置於低溫長時間冷凍 (20小時），經解凍後以機械方式去水而成海绵狀基質，此 方法所得的基質孔徑約為8〇 - 1400微米。美國專利第 -4 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱） 裝 (請先閱讀背面之注意事項再填寫本頁) 訂--------- 經濟部智慧財產局員工消脅合作社印製 476642 A7 _____ B7 五、發明說明（2) 4,412,947號係將高純度的不可溶膠原蛋白顆粒懸浮於醋酸 溶液中，以約每分鐘0.3至0.4。(：之降溫速率進行冷;柬至-65 C ’經冷/東乾燥後製成多孔狀薄片基質。美國專利第 4,522,753號係將膠原蛋白與硫酸軟骨素（chondr〇ltm sulfate)混 合成共聚物，經戊二醛交聯並冷凍乾燥後得孔徑約2〇〜18〇 微米之多孔狀基質，其可作為人工皮膚之基材。美國專利 第4,970,298號則揭示將酸性膠原蛋白溶液或酸性膠原蛋白 與透明質酸、纖維連結蛋白（fibronectin)等混合之溶液，以 碳亞胺酸及高溫去水法交聯基質，經不同溫度之冷;東後進 行冷凍乾燥製成多孔狀基質，其使用之冷凍溫度為_30至 -50°C，所得之基質孔徑約為50〜250微米；而含透明質酸或 纖維連結蛋白之膠原蛋白基質則具有1〇〇〜15〇微米之孔 徑。美國專利第4,948,540號係將纖維樣及可溶性膠原蛋白 溶液混合並進行冷凍乾燥，在高壓（15,000〜30,000 psi )下將 其製成具高吸水性（15〜20倍重）薄片狀基質，再經高溫去 水法得最終產品。美國專利第5，116,552號將酸性膠原蛋白 溶液置於-40°C冷凍，經冷凍乾燥後成海綿狀基質，基質再 於105t：下靜置24小時後再以戊二醛交聯24小時後即得孔徑 為50 - 120微米之基質。將此基質以15%乙醇溶液浸潤，經 較低溫（-80°C或-135°C )冷凍後進行二次冷凍乾燥而得不易 瓦解之海綿狀基質。美國專利第5,869,080號係將膠原蛋白 分散於氫氧化鈉溶液中成纖維樣，加入適量乙醇溶液後於 低溫（約-5°C )預冷備用，將低溫（約-15°C )保存之適當大小 冰晶顆粒加入上述溶液中，並立即滴入二異氰酸六甲酯 -5 - 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) 裝 tr---------# 經濟部智慧財產局員工消費合作社印製 4/b642476642 V. Description of the invention (1) Background: The original protein is a biodegradable protein, most of which exist in animal tissues in the form of fiber type. The main function is to maintain the tissue type and provide the tension required by the tissue. The collagen egg worm is a biological high score + made of two multi-peptide chains intertwined with each other. Limeixi is the same as the multi-peptide chain. The parent multi-peptide chain is polymerized by about 1,000 amino acids. The main amines are Glycine amines, which include glycine, proline and hydroxyproline. So far, at least ninety-nine different types of collagens have been discovered. General collagen The application is to make it into various types of substrates such as sponge, colloid, tube and film, which can be used for wound dressing, hemostasis, tissue repair, music carrier, cell culture substrate and various artificial organs. In order to make the collagen matrix have a porous structure to facilitate cell migration, growth, drug embedding and release, etc., freeze-drying is often used to prepare matrix holes. Generally speaking, after freeze-drying In general, 4 samples are often frozen at -80 C or directly frozen with liquid nitrogen and then freeze-dried. Because of the small ice crystal particles produced during rapid freezing, the resulting pore size of the matrix is not large (less than 30 microns). Secondly, if the freezing rate is too fast, it will also cause uneven distribution of ice crystals in the matrix, affect the uniformity of the pores in the matrix, and even cause the matrix to crack. The preparation process of the porous collagen matrix has been disclosed in many previous In the patent, for example, U.S. Patent No. 4,193,813, the collagen is first dissolved in acetic acid, cross-linked with glutaraldehyde, and then frozen at low temperature for a long time (20 hours). After thawing, mechanically remove water and Into a sponge-like matrix, the pore diameter of the matrix obtained by this method is about 80-1400 microns. US patent No.-4-this paper size applies to China National Standard (CNS) A4 specifications (210 X 297 public love) installed (please read the back first Please pay attention to this page before filling in this page) Order --------- Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs, Co-operative Society 476642 A7 _____ B7 V. Invention Description (2) No. 4,412,947 Collagen particles are suspended in an acetic acid solution and cooled at a rate of about 0.3 to 0.4 per minute. (: The cooling rate is cooled; Cambodia to -65 C 'after cold / east drying to make a porous sheet matrix. US Patent No. 4,522,753 Collagen is mixed with chondrolt sulfate to form a copolymer, which is crosslinked by glutaraldehyde and freeze-dried to obtain a porous matrix with a pore size of about 20 to 180 microns, which can be used as a substrate for artificial skin. U.S. Patent No. 4,970,298 discloses that an acidic collagen solution or a mixed solution of acidic collagen with hyaluronic acid, fibronectin, etc. is used to cross-link the substrate with carbimimic acid and high temperature dehydration method, and the temperature is different. It is cold; it is freeze-dried to make a porous matrix. Its freezing temperature is _30 to -50 ° C. The pore size of the matrix is about 50 ~ 250 microns; and collagen containing hyaluronic acid or fibronectin The protein matrix has a pore size of 100 to 150 microns. U.S. Patent No. 4,948,540 is a method of mixing fibrous and soluble collagen solutions and freeze-drying them, and forming them into a highly absorbent (15-20 times heavier) sheet-like matrix under high pressure (15,000 ~ 30,000 psi), and then The final product is obtained by high temperature dewatering. U.S. Patent No. 5,116,552 freezes the acidic collagen solution at -40 ° C and freeze-dries to form a sponge-like matrix. The matrix is left at 105t for 24 hours and then crosslinked with glutaraldehyde for 24 hours. A matrix with a pore size of 50-120 microns is obtained. This substrate was soaked with a 15% ethanol solution, frozen at a lower temperature (-80 ° C or -135 ° C), and then freeze-dried twice to obtain a sponge-like substrate that was not easily disintegrated. U.S. Patent No. 5,869,080 is a method of dispersing collagen in a sodium hydroxide solution to form a fibrous sample. After adding an appropriate amount of ethanol solution, it is pre-cooled at a low temperature (about -5 ° C) and stored at a low temperature (about -15 ° C). Size ice crystal particles were added to the above solution, and immediately dripped into the hexamethyl diisocyanate-5-This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before (Fill in this page) Install tr --------- # Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 4 / b642
五、發明說明（3 ) (hexamethylene diisocyanate，HMDI)交聯之，再經進一步冷凍 乾燥後製成海绵狀基質。由此法所得之基質雖具孔徑約 400微米之孔洞，但因孔徑分佈極廣（約5〇 _ 4〇〇微米），致 使其均勻度甚差。 上述專利所揭示製備膠原蛋白基質之方法，主要採用酸 性或鹼性膠原蛋白以高溫或化學交聯劑進行交聯，並採用 冷凍乾燥法進行孔洞之製造而得到多孔狀基質。由這些方 法所製得之基質，其孔洞均句性不佳，且孔徑之控制上十 分困難，更由於所使用之化學交聯劑大都具有毒性，於應 用上並不十分理想。 對於膠原蛋白基質之製備及應用，亟需有更精進之處理 技術以改良現有之膠原蛋白相關產品及突破現有產品之缺 點。 發明概述 本發明主要之目的係提供一種製備多孔狀膠原蛋白基質 之方法。 本發明又一目的係提供一種製備所需孔徑大小之多孔狀 膠原蛋白基質（方法，其係利用不同之操作條件控制多孔 狀膠原蛋白基質之孔徑大小。 本發明再一目的係提供一種由上述方法所製得之多孔狀 重組膠原蛋白基質。 邐式簡易說明 圖一係表7F落於醋酸中之膠原蛋白重組基質，於急逮降 溫冷凍後經冷凍乾燦所製成之多孔狀基質。（A)為基質經 _ 6 本紙張尺度適用中國國家標準（CN^^格（21〇 χ 297公楚） W-裝 (請先閱讀背面之注意事項再填寫本頁) 1T---------^9. 經濟部智慧財產局員工消費合作社印製 476642 經濟部智慧財產局員工消費合作社印製 A7 B7__ 五、發明說明（4 ) -20°C冷凍；（B)為基質經-40。(：冷凍；（C)為基質經_8(Γ(：冷 凍。 圖二為溶於醋酸中之膠原蛋白重組基質，於緩慢降溫冷 凍後經冷凍乾燥所製成之多孔狀基質。（A)為基質經_2(rc 冷凍；（B)為基質經-80。(：冷凍。 圖三為溶於中性磷酸鹽緩衝液中之膠原蛋白重組基質， 於急速降溫冷東至_20°C後，經冷柬乾燥所製成之多孔狀膠 原蛋白基質’於掃瞄式電子顯微鏡下之照相圖。（A)放大 倍率為5〇倍；（B)放大倍率為400倍。 圖四為溶於中性磷酸鹽缓衝液中之膠原蛋白重組基質， 於急速降溫冷凍至-201：後，經冷凍乾燥所製成之多孔狀膠 原蛋白基質。⑷為缓衝液中含〇·5Μ氯化鈉；（B)為緩衝液 中含1.0M氯化鈉。 圖五為溶液對於基質表面孔洞之影響。（A)為基質浸潤於 戊二駿水溶液中；（B)為基質浸潤於100〇/〇乙醇溶液中。 發明詳細說明 為改進習知膠原蛋白基質製備技術之缺點，本發明採用 不文聯之中性膠原蛋白溶液作為基材，經重組、冷束及冷 滚減壓乾燥程序而得多孔狀膠原蛋白基質。過程中以不同 之操作條件控制所需基質之孔徑大小。由本發明之方法， 可獲得高孔隙均勻度，及不同孔徑大小之多孔狀膠原蛋白 基質。 本發明提供一種製備多孔狀膠原蛋白基質之方法，該方 法包括將中性膠原蛋白溶液靜置於3〇至45C下一段時間， 本紙張尺度適用中國國家標準(cns〉A4規格（ χ 297公爱） (請先閱讀背面之注意事項再填寫本頁) 訂---------#. 476642V. Description of the invention (3) (hexamethylene diisocyanate (HMDI)) is crosslinked, and then further freeze-dried to make a sponge-like matrix. Although the matrix obtained by this method has pores with a pore size of about 400 microns, its uniformity is very poor due to the extremely wide pore size distribution (about 50-400 microns). The method for preparing a collagen matrix disclosed in the above patents mainly uses acidic or basic collagen to crosslink at a high temperature or a chemical crosslinking agent, and freeze-drying to produce pores to obtain a porous matrix. The pores of the matrix prepared by these methods are poor in sentence structure, and the control of the pore diameter is very difficult. Moreover, most of the chemical crosslinkers used are toxic, which is not very ideal in application. For the preparation and application of collagen matrix, more sophisticated processing technology is urgently needed to improve existing collagen related products and break through the shortcomings of existing products. SUMMARY OF THE INVENTION The main object of the present invention is to provide a method for preparing a porous collagen matrix. Another object of the present invention is to provide a porous collagen matrix with a desired pore size (method, which uses different operating conditions to control the pore size of the porous collagen matrix. Another object of the present invention is to provide a method by the above method. The obtained porous reconstituted collagen matrix. Brief description of the formula: Figure 1 shows the collagen reconstituted matrix falling in acetic acid as shown in Table 7F. The porous matrix was made by freeze-drying after freezing and cooling. (A ) Is the matrix warp_ 6 This paper size applies to the Chinese national standard (CN ^^ format (21〇χ297297) W-pack (please read the precautions on the back before filling this page) 1T ------- -^ 9. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 476642 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7__ V. Description of the invention (4) Freezing at -20 ° C; (B) The matrix is -40. (: Frozen; (C) is a porous matrix made by the matrix through _8 (Γ (: frozen. Figure 2 is a collagen reconstituted matrix dissolved in acetic acid, freeze-dried after slow cooling and freezing. (A) The substrate is frozen by _2 (rc; (B) The matrix is -80. (: Frozen. Figure 3 shows the collagen reconstituted matrix dissolved in neutral phosphate buffer solution. After rapid cooling and cooling to -20 ° C, the porous collagen is dried by cold cooling. Photograph of the protein matrix 'under a scanning electron microscope. (A) A magnification of 50 times; (B) A magnification of 400 times. Figure 4 shows a collagen reconstituted matrix dissolved in a neutral phosphate buffer solution. Frozen to -201 after rapid cooling: Porous collagen matrix made by freeze-drying. ⑷ is 0.5M sodium chloride in buffer solution; (B) 1.0M sodium chloride in buffer solution Figure 5 shows the effect of the solution on the pores on the surface of the substrate. (A) The substrate is infiltrated in the aqueous solution of glutarium; (B) The substrate is infiltrated in a 100/0 ethanol solution. Detailed description of the invention is to improve the conventional collagen matrix. Disadvantages of the preparation technique: The present invention uses a non-textually-linked neutral collagen solution as a substrate, and obtains a porous collagen matrix through recombination, cold beam, and cold rolling and reduced pressure drying procedures. During the process, different operating conditions are required to control the need. The pore size of the matrix. According to the method of the present invention, a porous collagen matrix with high pore uniformity and different pore sizes can be obtained. The present invention provides a method for preparing a porous collagen matrix, which comprises placing a neutral collagen solution at 30 ° C. Until the next 45C, this paper size applies to the Chinese national standard (cns> A4 size (χ 297 public love) (Please read the precautions on the back before filling this page) Order --------- #. 476642
以進行膠原蛋白之纖維重組得到膠原蛋白膠狀基質 孩膠原蛋白膠狀基質進行冷東減壓乾燥後回收 原蛋白基質。 修 由於動物的結缔I織富含膠原蛋白，因此用於本發明之 膠原蛋白材料’可由動物結缔組織中抽取製得，或以生物 科技方式製備基因重組之膠原蛋白而得。 抽取純化膠原蛋白的方式，可由任何熟習此項技術者所 習知之方式進行。例如，豬皮中膠原蛋白之抽取純化，可 以去毛及油脂之豬皮碎片浸於0 5M醋酸溶液中均質之，並 經胃蛋白酶去除膠原蛋白分子的末端抗原後，調至中性使 f蛋白酶去活化，經反覆的鹽析、酸溶及透析等步驟進行 抽取及純化，以得到膠原蛋白溶液。 上述得到：C膠原蛋白，可以任何熟習此項技術者所習知 之方法‘備成中性之膠原蛋白溶液。本發明中將膠原蛋白 製成中性膠原蛋白溶液之方法，可將溶於酸性溶液之膠原 蛋白以鹼性溶液，例如氫氧化鈉溶液，將此膠原蛋白溶液 調整成中性，此酸性溶液包括但非限於醋酸、檸檬酸、草 故、鹽酸或硫fe:等溶:液，較佳為錯酸溶液。亦可將膠原蛋 白透析至已調整成中性之鹽類緩衝液，此鹽類缓衝液包括 但非限於磷故緩衝食鹽水溶液、磷酸鹽、醋酸鹽、碳酸 鹽、滷鹽（例如氯化鈉）等之緩衝液。 本發明進行膠原蛋白重組之方式，係將所製得之中性膠 原蛋白溶液，靜置於30至45°c之恆溫恆濕箱中，較佳為37 °C，靜置時間為0.5小時以上，較佳為靜置24小時，使膠原 -8 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注音？事項再填寫本頁) 裝 ----訂------ 經濟部智慧財產局員工消脅合作社印製 A7 B7 五、發明說明（6 忠白分子重組成纖維，形成膠原蛋白膠狀基質。 (請先閱讀背面之注意事項再填寫本頁) 中性膠原蛋白溶液’可再透析或添加金屬鹽類至溶液後 進行膠原蛋白重組成膠原蛋白膠狀基質。藉由調整中性膠 原蛋白溶液之組成，再經適當冷凍過程及冷凍乾燥後可得 不同孔徑大小之多孔狀膠原蛋白基質。適用於本發明之金 屬鹽’包括但非限於氯化鈉、氯化鉀、氟化鈉、醋酸鈉、 碳酸鈉等鹽類。例如，使用的起始原料為溶於含氯化鈉濃 度為0.13M之中性磷酸鹽緩衝液，則最後所形成之多孔狀 膠原蛋白基質孔徑可達100微米以上。 經濟部智慧財產局員工消f合作社印製 冷凍的方式，可以任何熟習此項技術者所習知之方式操 作。其中’冷凍的降溫速率與冷凍的溫度與孔徑形成的大 d及均句度’關。冷;東的溫度與基質形成的孔徑關係，就 透析至醋酸並調至令性之重組膠原蛋白而言，在·20它冷凍 時，其基質所形成的孔徑較大，約為50微米以上；在_40它 冷柬時’基質所形成的孔徑約為30微米以上；在·8<rc冷束 時，基質所形成的孔徑則約為15微米以上。冷凍溫度為 -20及-40 °C時，各基質的纖維有融合現象，冷凍溫度為_8〇 °C時，基質中則具有明顯的纖維結構。當冷凍的降溫速率 較缓和時（降溫速率為每小時1(rc至每小時仞^，所得的 基負孔徑範圍較大，但均勻性較差，其中，以緩慢降溫冷 凍至-20 C之基質，其孔徑約為5〇至3〇〇微米，以緩慢降溫 冷凍至_80 C之基質，則具有約為15至15〇微米之孔徑。當 冷凍的降溫速率較急速時，所得膠原蛋白基質的孔徑範圍 較乍亦即均勻性較佳，例如降溫速率為每分鐘i 〇°c以 -9 - 本紙張尺度適用_國國豕標準（CNS)A4規格（21〇 X 297公楚） 476642 五、發明說明（ 上’則基質的孔徑差異範圍可以控制在30微米以内。 其次’就透析至中性磷酸鹽緩衝液之膠原蛋白溶液而 言’在急速降溫冷凍之條件下，冷凍溫度為-20t:時，其基 處孔徑約為100至125微米；而冷凍溫度為-8〇。〇時，其基質 孔徑約為30至50微米。而當於中性磷酸鹽之膠原蛋白溶液 中的氯化鈉濃度提咼至0.5至丨〇M時，冷凍溫度為·2〇ι 時’所形成之膠原蛋白基質孔徑約為3〇至4〇微米。 為維持膠原蛋白基質孔徑的大小以利將來之應用，本發 明之製備方法可進一步將冷凍乾燥後的多孔狀膠原蛋白基 質以不同之有機溶劑處理。適用於本發明之有機溶劑為醇 類及酮類，其包括但非限於甲醇、乙醇、丙醇、異丙醇、 丁醇、異丁醇、丙酮等，較佳為乙醇，更佳為無水乙醇， 其可元全維持原有的膠原蛋白基質之孔洞型態。 根據上述不同之透析溶液、冷凍降溫速率、冷凍溫度及 金屬鹽濃度等條件之改變，可控制所需膠原蛋白基質之孔 徑大小，其舉例如表一所示。 (請先閱讀背面之注意事項再填寫本頁) 裝 丁 n an fl— ϋ J i ·ϋ ϋ 1ϋ ·ϋ H «ϋ ϋ I · 經濟部智慧財產局員工消费合作社印製 表一、不同製備程序所得多孔狀膠原蛋白基質之孔徑分佈 及均勻度 透析溶液 冷凍溫度 冷凍降溫速 率* 膠原蛋白基質 孔徑(微米） 均勻度 醋酸溶液 -20°C 緩慢 50-300 差 醋酸溶液 -80°C 緩慢 15-150 差 錯酸溶液 -20°C 急速 50-75 佳 酷酸溶液 -40°C 急速 30-50 佳 476642 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（8 ) 醋酸溶液 -80°C 急速 15-40 佳 醋酸溶液(膠原蛋 白未經重組） -20°C 急速 100-300 甚差 中性磷酸鹽溶液 -20°C 急速 100-125 佳 中性磷酸鹽溶液 -80°C 急速 30-50 佳 中性磷酸鹽溶液 /0.5M氯化納 -20°C 急速 30-40 佳 中性磷酸鹽溶液 /1.0M氯化納 -20°C L— ----- 急速 30-40 佳 *表中冷凍降溫速率有急速及缓慢兩者。急速冷凍表示降 溫速率為每分鐘5°C以上者；而緩慢冷凍表示降溫速率為 每小時lOt：至每小時60°c者。 下列之實施例係用於對本發明作進一步之例示說明，並 非用以限制本發明，任何熟習此項技術者根據本發明說明 書之教示所達成之修飾及應用，皆屬本發明之範疇。 將膠原蛋白透析至0.5M醋酸溶液中，形成酸性之膠原蛋 白溶液。以氫氧化鈉調整此膠原蛋白溶液成中性（pH = 7.2 ) 後’置於底面積約2平方公分之破璃容器中，於37C之恨 溫f亙濕箱中靜置24小時，形成重組之膠原蛋白基質。隨後 將此膠原蛋白基質進行急速降溫冷凍（降溫速率每分鐘2〇它） 分別至溫度為-20 C、-40 C及*80 C後，於壓^力⑴毫托耳下 進行冷凍乾燥2天，即得高孔隙均勻度之多孔狀膠原蛋白 -11 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 裝--------訂--- (請先閱讀背面之注意事項再填寫本頁) 476642 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（9) 基質’結果如圖一所示，其中基質孔徑分別約為5〇 _ 75、 30 - 50 及 15-40微米。 實例2 將純化後之膠原蛋白透析至0 5M醋酸溶液中，形成酸性 之膠原蛋白溶液。以氫氧化鈉調整此膠原蛋白溶液成中性 (pH = 7.2 )後’置於底面積約2平方公分之玻璃容器中，於 37°C之恆溫恆濕箱中靜置24小時，形成重組之膠原蛋白基 質。隨後將此膠原蛋白基質進行缓慢降溫冷凍（降溫速率 為母小時20 C )至溫度為-20°C〜-80°C後，於壓力1〇毫托耳下 進行冷凍乾燥2天，即得多孔狀膠原蛋白基質。此種基質 可具有大於200微米之孔徑，但其孔徑範圍分佈大，亦即 孔隙均勾度較差，如圖二所示。 實例3 將純化後之膠原蛋白透析至中性磷酸鹽緩衝液（含〇· 135M 氯化鈉之0.02M磷酸鹽緩衝液，pH = 7.2)中，隨後將此膠原 蛋白溶液置於底面積約2平方公分之玻璃容器中，於37c 之恒溫怪濕箱中靜置24小時，形成重組之膠原蛋白基質。 再將此膠原蛋白基質進行急速降溫冷凍（降溫速率為每分 鐘20 C )’冷〉東至溫度為-20 C後’於壓力1〇毫托耳下進行 冷凍乾燥2天，即得到孔隙均勻度佳，孔徑範圍為1〇〇至 125微米之多孔狀膠原蛋白基質，結果如圖三所示。 實例4_ 根據實例3所述之方法製備膠原蛋白基質，但於中性麟 故鹽緩衝液中添加氯化鈉的濃度為0.5與i.OM。最後製得孔 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） n n 1 I ϋ n n n n · flu lap ^^1 n ϋ —ϋ ϋ ^ ^ I ϋ i <ϋ ϋ i·— I n I ' (請先閲讀背面之注意事項再填寫本頁) A7 五、發明說明（10 ) 徑為30至40微米之多孔狀膠原蛋白基質，如圖四所示。 實例5 · (請先閱讀背面之注意事項再填寫本頁) 將冷凍乾燥後之膠原蛋白基質，分別浸於純水、中性磷 fe鹽緩衝落液、2%戊二醛水溶液、5〇%乙醇、75〇/〇乙醇及 典水乙畴中。圖五為溶液成份對於基質表面孔洞影響的情 形’分別測量基質收縮比（shrinkage rati〇)及缴密表層厚度 比’其結果如下列表二所示。 表二、以不同有機溶劑處理多孔狀膠原蛋白基質之收縮比 及緻密表層厚度比 無水乙醇 75%乙醇 50%乙醇 2%戊二醛 水溶液 PBS水溶液 水 直徑收縮 比(％) 0_39 土 0.23 2.99 士 1.88 4.48±1.80 2.49 士 1.42 20.77土 11.19 29.93士 1.1S 厚度收縮 比(％) 1.75 士 0.10 22.96士 0.39 33.10 士 1.56 17· 86 士 0.15 38.51 士 2.90 56.75土 5.18 緻密表層 厚度比(％) 一 3.3 5.0 2.5 39.3 45.8 經濟部智慧財產局員工消費合作社印製 由表二的結果顯示，浸潤於無水乙醇的膠原蛋白基質， 其收縮程度最小，且無緻密表層形成，孔徑的穩定性輕 佳，其可幾乎維持膠原蛋白基質原有的孔徑大小及結構。 -13 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）The collagen gelatinous matrix was obtained by reorganizing collagen fibers, and the collagen gelatinous matrix was dried under cold pressure and dried to recover the original protein matrix. Since the connective tissue of animals is rich in collagen, the collagen material used in the present invention can be obtained by extracting from connective tissues of animals, or it can be obtained by preparing biorecombinant collagen in a biotechnology manner. The method of extracting and purifying collagen can be performed by any method known to those skilled in the art. For example, the extraction and purification of collagen from pig skin can be done by immersing pig skin fragments of depilation and oil in 0.5 M acetic acid solution and homogenizing it, and then removing the terminal antigen of collagen molecules by pepsin, then adjusting to neutral to make f proteinase Deactivation, extraction and purification through repeated steps of salting out, acid dissolution and dialysis to obtain a collagen solution. The above-mentioned: C collagen can be prepared by any method known to those skilled in the art ′ to prepare a neutral collagen solution. The method for making collagen into a neutral collagen solution in the present invention can adjust the collagen solution dissolved in an acidic solution to a neutral solution, such as a sodium hydroxide solution, to adjust the collagen solution to be neutral. The acidic solution includes However, it is not limited to acetic acid, citric acid, forage, hydrochloric acid, or sulfur, and other solvents: liquids, and is preferably a mixed acid solution. Collagen can also be dialyzed to a neutral salt buffer that has been adjusted to neutral. This salt buffer includes, but is not limited to, phosphate buffered saline solution, phosphate, acetate, carbonate, halide (such as sodium chloride). Wait for the buffer. The method of collagen recombination in the present invention is to place the prepared neutral collagen solution in a constant temperature and humidity box at 30 to 45 ° C, preferably 37 ° C, and the standing time is more than 0.5 hours. It is better to let it stand for 24 hours to make collagen-8.-This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) (Please read the note on the back? Matters before filling out this page.) Packing- --Order ------ Printed by the staff of the Intellectual Property Bureau of the Ministry of Economic Affairs, A7, B7. V. Description of the invention (6 Loyal molecules recombine fibers to form a collagen gel matrix. (Please read the precautions on the back first) (Fill in this page again) Neutral collagen solution 'can be re-dialyzed or added with metal salts to the solution, and then collagen can be reconstituted into collagen gelatinous matrix. By adjusting the composition of the neutral collagen solution, and then through appropriate freezing process and After freeze-drying, porous collagen matrices with different pore sizes can be obtained. Metal salts suitable for the present invention include, but are not limited to, sodium chloride, potassium chloride, sodium fluoride, sodium acetate, sodium carbonate and other salts. For example, Used starting materials Soluble in a neutral phosphate buffer solution containing sodium chloride at a concentration of 0.13M, the pore size of the porous collagen matrix formed at the end can reach more than 100 microns. The method of printing and freezing by the cooperative of the staff of the Intellectual Property Bureau of the Ministry of Economic Affairs, It can be operated in any way familiar to those skilled in the art. Among them, 'the cooling rate of freezing and the temperature of freezing and the large d and average degree of pore diameter formation' are related. Cold; the relationship between the temperature of the east and the pore diameter of the substrate, dialysis For acetic acid and adjusted to orderly recombinant collagen, when it is frozen at 20, the pores formed by its matrix are larger, about 50 microns or more; at _40 it is cold, the pores formed by the matrix are about It is more than 30 microns; in the cold beam of 8 rc, the pore diameter formed by the matrix is about 15 microns or more. When the freezing temperature is -20 and -40 ° C, the fibers of each matrix have a fusion phenomenon, and the freezing temperature is _ At 80 ° C, the matrix has obvious fibrous structure. When the cooling rate is slower (the cooling rate is 1 hr per hour to 仞 ^ per hour, the range of the basic negative pore diameter is larger, but the uniformity Poor, of which Substrates frozen to -20 C with slow cooling have a pore size of about 50 to 300 microns, and substrates frozen to _80 C with slow cooling have a pore size of about 15 to 150 μm. When frozen, the temperature decreases. When the speed is faster, the pore size range of the obtained collagen matrix is better than that at first glance, for example, the cooling rate is i 0 ° c per minute to -9-this paper size applies _ National Standards (CNS) A4 specifications ( 21〇X 297) 476642 V. Description of the invention (above), the pore size difference of the matrix can be controlled within 30 microns. Secondly, 'for collagen solution dialyzed to neutral phosphate buffer solution', it is frozen at rapid cooling Under these conditions, when the freezing temperature is -20t :, the pore diameter at the base is about 100 to 125 microns; and the freezing temperature is -80. At 0, the pore size of the matrix is about 30 to 50 microns. When the concentration of sodium chloride in the collagen solution of the neutral phosphate is raised to 0.5 to 0, and the freezing temperature is · 200m, the pore size of the collagen matrix formed is about 30 to 40. Microns. In order to maintain the pore size of the collagen matrix for future applications, the preparation method of the present invention can further treat the porous collagen matrix after freeze-drying with different organic solvents. Organic solvents suitable for the present invention are alcohols and ketones, which include, but are not limited to, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, acetone, etc., preferably ethanol, more preferably anhydrous ethanol It can maintain the pore shape of the original collagen matrix. The pore size of the required collagen matrix can be controlled according to the above-mentioned changes in conditions such as the dialysis solution, the freezing cooling rate, the freezing temperature, and the concentration of the metal salt. Examples are shown in Table 1. (Please read the precautions on the back before filling this page) Decoration n an fl— ϋ J i · ϋ ϋ 1ϋ · ϋ H «ϋ ϋ I · Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs I. Different preparation procedures Pore size distribution and uniformity of the obtained porous collagen matrix Dialysis solution Freezing temperature Freezing cooling rate * Collagen matrix pore size (microns) Uniformity Acetic acid solution -20 ° C Slow 50-300 Poor Acetic acid solution -80 ° C Slow 15-150 Error acid solution -20 ° C rapid 50-75 Jiaku acid solution -40 ° C rapid 30-50 good 476642 A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (8) Acetic acid solution -80 ° C Rapid 15-40 best acetic acid solution (collagen is not reconstituted) -20 ° C rapid 100-300 very poor neutral phosphate solution -20 ° C rapid 100-125 good neutral phosphate solution -80 ° C rapid 30- 50 best neutral phosphate solution / 0.5M sodium chloride -20 ° C rapid 30-40 best neutral phosphate solution / 1.0M sodium chloride -20 ° CL — ----- rapid 30-40 best * table There are both rapid and slow cooling rates. Rapid freezing means that the cooling rate is above 5 ° C per minute; and slow freezing means that the cooling rate is from 10t per hour: to 60 ° c per hour. The following examples are intended to further illustrate the present invention, and are not intended to limit the present invention. Any modification and application made by those skilled in the art according to the teachings of the description of the present invention belong to the scope of the present invention. The collagen was dialyzed into a 0.5 M acetic acid solution to form an acidic collagen protein solution. Adjust this collagen solution to neutrality (pH = 7.2) with sodium hydroxide, and then place it in a broken glass container with a bottom area of about 2 square centimeters, and leave it in a humid box at 37 ° C for 24 hours to form a reorganization. Collagen matrix. Subsequently, this collagen matrix was rapidly cooled and cooled (the cooling rate was 20 ° per minute) to a temperature of -20 C, -40 C, and * 80 C, respectively, and then freeze-dried under a pressure of 2 mTorr for 2 days. ， Porous collagen with high pore uniformity-11-This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm). -------- Order --- (please first Read the notes on the back and fill in this page) 476642 A7 B7 printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (9) The results of the matrix are shown in Figure 1, where the pore sizes of the matrix are about 50-75, 30-50 and 15-40 microns. Example 2 The purified collagen was dialyzed into a 0.5 M acetic acid solution to form an acidic collagen solution. Adjust this collagen solution to neutrality (pH = 7.2) with sodium hydroxide, and then place it in a glass container with a bottom area of about 2 square centimeters, and leave it in a constant temperature and humidity box at 37 ° C for 24 hours to form a reconstituted product. Collagen matrix. Subsequently, the collagen matrix is slowly cooled and cooled (the temperature reduction rate is 20 C for mother hours) to a temperature of -20 ° C ~ -80 ° C, and then freeze-dried under a pressure of 10 millitorr for 2 days to obtain a porous structure. Collagenous matrix. This matrix can have a pore size greater than 200 microns, but its pore size range is large, meaning that the average porosity of the pores is poor, as shown in Figure 2. Example 3 The purified collagen was dialyzed into a neutral phosphate buffer solution (0.02M phosphate buffer solution containing 0.135M sodium chloride, pH = 7.2), and then the collagen solution was placed on a bottom area of about 2 Reconstituted collagen matrix was formed in a glass container of 37 cm in a constant temperature and humidity box at 37c for 24 hours. This collagen matrix is then rapidly cooled and cooled (the cooling rate is 20 C per minute) 'cold> east to a temperature of -20 C' and freeze-dried under a pressure of 10 millitorr for 2 days to obtain the pore uniformity The porous collagen matrix with a pore size ranging from 100 to 125 microns is shown in Figure 3. Example 4_ A collagen matrix was prepared according to the method described in Example 3, but sodium chloride was added to the neutral sodium salt buffer solution at a concentration of 0.5 and i.OM. Finally, the size of the hole paper is applicable to the Chinese National Standard (CNS) A4 (210 X 297 mm). Nn 1 I ϋ nnnn · flu lap ^^ 1 n ϋ —ϋ ϋ ^ ^ I ϋ i < ϋ ϋ i · — I n I '(Please read the precautions on the back before filling this page) A7 V. Description of the invention (10) A porous collagen matrix with a diameter of 30 to 40 microns, as shown in Figure 4. Example 5 · (Please read the precautions on the back before filling this page) The lyophilized collagen matrix was immersed in pure water, neutral phosphorous fe salt buffer solution, 2% glutaraldehyde aqueous solution, 50% Ethanol, 7500/0 ethanol and ethyl acetate. Figure 5 shows the effect of the solution components on the pores on the surface of the substrate. The results are shown in Table 2 below, which measures the shrinkage ratio of the matrix and the thickness ratio of the dense surface layer. Table 2. Shrinkage ratio and dense surface thickness of porous collagen matrix treated with different organic solvents than anhydrous ethanol 75% ethanol 50% ethanol 2% glutaraldehyde aqueous solution PBS aqueous solution water diameter shrinkage ratio (%) 0_39 soil 0.23 2.99 ± 1.88 4.48 ± 1.80 2.49 ± 1.42 20.77 ± 11.19 29.93 ± 1.1 Thickness shrinkage ratio (%) 1.75 ± 0.10 22.96 ± 0.39 33.10 ± 1.56 17 · 86 ± 0.15 38.51 ± 2.90 56.75 ± 5.18 Thickness ratio of dense surface layer (%)-3.3 5.0 2.5 39.3 45.8 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. The results shown in Table 2 show that the collagen matrix immersed in anhydrous ethanol has the smallest degree of shrinkage and no dense surface layer formation. The original pore size and structure of the collagen matrix. -13-This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm)