1306767 玖、發明說明： 【發明所屬之技術領域】 本發明係關於一種生物醫學移植姑 材料、軟骨移植材料及皮膚移植材料的製備方法。方法’特別是骨移植 【先前技術】 已知傳統生姆學移撕料如轉植材·將人骨或動物骨經冷 凍乾燥去除其蛋自t抗雜，或聰處理財除其 去除所有有機•，或經二化 ===譬如已知有異種移植骨以雙氧水氧化反應 ^以月蛋自細化學個絲牛骨此蛋白抗原，舰 功以後（Mattz觸），技術㈣趨成熟。其製作過程也較為嚴謹，進 而商品化成鱗床可伽之⑽代品。但是在_骨_構下，化學 藥劑可能因絲内纽塞岭處理不全之慮，仍被f疑其蛋 白消除仍不夠徹底，其它類似商品_^Surgibc)ne%LubW&_^ 程則除化學方法外，再加上物理紐沖獅轉，所触之蛋白組成 約在20-29 %左右，目前已在歐洲國家做臨床使用。 軟骨的結構與骨頭的結構同樣是多孔性的結構，其成分則大部分是 軟骨質及第一型膠原蛋白。臨床上軟骨移植材料的用途不多，多為美 容醫學的用途，或是用來修復顎顳關節的關節盤。 另外，關於皮膚移植材料，臨床上是用來取代傷口上壞死皮膚的功 能。然而由於自體皮膚移植物的來源有限，因此必須採用異體的皮膚 移植物，或人造皮膚來進行移植。臨床上常使用的人造皮膚傷口敷料 的材料來源可分為兩大類：一為合成材料如polyurethane、p〇ly(vinyl alc〇h〇l)等；另一為天然材料如 collagen、chitosan、gelatin、biological tissue 等。使用天然材料的優點在於其具有良好的生物相容性，但是由於過 去所使用的材料修飾交聯劑如formaldehyde或glutaraldehyde等的細胞 毒性過高，若殘留在材料中使得材料在植入人體後，易導致過強且持 1306767 續性的發炎反應；使用合成材料的優點為量多且便宜，缺點則是其成 份多半不能吸收及其構造多半還不能提供良好的皮膚移植材料的功 能，故尋找-個能兼具良好生物相容性及近似人體皮膚的結構，便成 為在皮膚移植材料的發展上極為重要的—項課題。 【發明内容】 本發明係祕-麵的生师轉撕制製備紐，係將動物 組織樣本如骨塊、軟骨或皮膚’軸超臨界赫與縣交麵及特殊 修飾劑的處㈣做。雜界流_減作溫度及·壓力超過其臨 界溫度及臨界壓力時驗體。超臨界流_物雌f是介於氣、液相 之間的。例如，黏度接近於氣體，密度接近於液體，因密度高，可輸 送較氣體更彡的物質；職度低，輸送時賴的功軸較液體為低。 又如，擴散係數高於液體10至100倍，亦即質量傳遞阻力遠較液體為 小’因之在質量傳遞上較液體為快。此外，超臨界流體有如氣體幾無 表面張力，因此很容易滲入到多孔性組織中。除物理性質外，在化學 性質上亦與氣、液態時有所不同。例如，二氧化碳在氣體狀態下不具 萃取能力’赠進人細界狀驗’二氧化碳變舰錢性7因而具 有溶解有機物的能力，此溶解能力會隨溫度及壓力而有所不同。近年 來超臨界流體技術已被廣泛地應用在食品及製藥工業上。 目前常用的超臨界流體的種類有二氧化碳，其臨界溫度為311 I，臨界壓力為72.8 atm，還有氧化亞氮（36.4。(：，71.5 atm)，水(374.1 C，217.6 atm)等有數十種。其中二氧化碳沒有淨偶極矩，是一種非極性 物質，對於非極性及低極性物質有相當好的溶解度，但對於極性物質， 二氧化碳的溶解力就不盡理想。此時可藉由添加修飾劑（m〇difier)來 提高二氧化碳溶液的極性，如甲醇、乙醇都是常拿來使用的修飾劑； 另外也可以加入界面活性劑包覆溶質形成反微胞來降低溶質的極性。 氧化亞氮（凡0)雖然分子量、臨界壓力和溫度都和二氧化碳差不多， 卻具有較高的極性，對於極性物質萃取效果較佳，但是氧化亞氮本身 為一強氧化劑，使用上有安全上的顧慮。而超臨界水呈酸性具相當腐 银力與氧化力，多用於廢水處理與化武銷毀。 1306767 爰此’本發明係利用超臨界流體的高擴散性、1306767 发明, 发明发明发明: The present invention relates to a biomedical transplant material, a cartilage graft material, and a method for preparing a skin graft material. Method 'especially bone transplantation 【Prior Art】 It is known that traditional smear transfer materials such as transfer materials, freeze-dried human bones or animal bones to remove their eggs from t, or Cong treatment to remove all organic matter. , or by the second === 譬 譬 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异 异The production process is also more rigorous, and then commercialized into a scale bed can be gamma (10) generation. However, under the _ bone_structure, the chemical agent may still be incompletely resolved due to incomplete treatment of the Nesin in the silk, and other similar products _^Surgibc)ne%LubW&_^ In addition to the method, coupled with the physical New Zealand lion turn, the protein composition touched is about 20-29%, and has been used clinically in European countries. The structure of the cartilage is as porous as the structure of the bone, and most of its components are cartilage and type I collagen. There are not many clinically used cartilage graft materials, most of which are used for cosmetic medicine or for the repair of the ankle joint. In addition, regarding skin graft materials, it is clinically used to replace the necrotic skin on the wound. However, due to the limited source of autologous skin grafts, it is necessary to use allogeneic skin grafts or artificial skin for transplantation. The material sources of artificial skin wound dressings commonly used in clinical practice can be divided into two categories: one is synthetic materials such as polyurethane, p〇ly (vinyl alc〇h〇l), and the other is natural materials such as collagen, chitosan, gelatin, Biological tissue, etc. The advantage of using natural materials is that they have good biocompatibility, but the cytotoxicity of the material-modified cross-linking agent such as formaldehyde or glutaraldehyde used in the past is too high, and if it remains in the material, the material is implanted in the human body. It is easy to cause too strong and has an inflammatory reaction of 1306767. The advantage of using synthetic materials is that it is more and cheaper. The disadvantage is that most of its components can not be absorbed and its structure can not provide good skin grafting materials, so look for - A structure that combines good biocompatibility and approximate human skin has become an extremely important topic in the development of skin graft materials. SUMMARY OF THE INVENTION The invention is a secret-face-to-face preparation for tearing a tissue, such as a bone tissue, a cartilage or a skin axis, a supercritical hegemony junction with a county and a special modifier (4). Miscellaneous flow _ is reduced to temperature and pressure when the temperature exceeds its critical temperature and critical pressure. The supercritical flow_object female f is between the gas and the liquid phase. For example, the viscosity is close to that of gas, the density is close to that of liquid, and because of its high density, it can transport substances that are more sturdy than gas; the job is low, and the work axis is lower than that of liquid. For another example, the diffusion coefficient is 10 to 100 times higher than that of the liquid, that is, the mass transfer resistance is much smaller than that of the liquid, which is faster than the liquid in mass transfer. In addition, supercritical fluids, like gas, have little surface tension and therefore easily penetrate into porous tissue. In addition to physical properties, it is also chemically different from gas and liquid. For example, carbon dioxide does not have the ability to extract in a gaseous state, and it has the ability to dissolve organic matter. This solubility varies with temperature and pressure. Supercritical fluid technology has been widely used in the food and pharmaceutical industries in recent years. At present, the commonly used supercritical fluids are carbon dioxide with a critical temperature of 311 I, a critical pressure of 72.8 atm, and nitrous oxide (36.4. (:, 71.5 atm), water (374.1 C, 217.6 atm), etc. Ten kinds of carbon dioxide, which has no net dipole moment, is a non-polar substance with good solubility for non-polar and low-polar substances, but for polar substances, the solubility of carbon dioxide is not ideal. A modifier (m〇difier) is used to increase the polarity of the carbon dioxide solution. For example, methanol and ethanol are often used as modifiers. Alternatively, a surfactant may be added to coat the solute to form an anti-microcell to reduce the polarity of the solute. Although nitrogen (Wan 0) is similar to carbon dioxide in terms of molecular weight, critical pressure and temperature, it has a higher polarity and better extraction effect on polar substances, but nitrous oxide itself is a strong oxidant, which has safety concerns. The supercritical water is acidic with considerable rosin strength and oxidizing power, and is mostly used for wastewater treatment and chemical weapons destruction. 1306767 爰This invention uses supercritical High diffusivity body,
原性，並保留無機的礦物或有機的膠原蛋白的多 利用超臨界流體可去除病原體的滅菌特性，作為 學移槿材料的製程，可解決前述德统晋弒始从土.丨 孔性的骨架成分，並The originality, and retaining the inorganic mineral or organic collagen, the use of supercritical fluid can remove the sterilization characteristics of the pathogen, as a process of learning the transfer material, can solve the above-mentioned skeleton of the Tudor Ingredients, and
【實施方式】 本發明之實施方法示於圖i。其詳細實施步驟如下： 1. 準備一動物組織，如骨塊，清除其上肉屑、脂肪、骨膜、皮質骨 及多餘組織，將其中的海綿狀骨裁成若干大小之骨塊，或磨成顆 粒狀；如軟骨，清除其上肉屑、脂肪、骨膜及多餘組織，裁成若 干大小之軟骨塊；或如皮膚，去除其上的脂肪、毛髮及多餘組織， 裁成若干大小，並以支持器固定之。 2. 將别述組織樣本置於超臨界流體設備之樣本處理籃7中，放入處 理槽5中並關緊處理槽5閘門。 3. 開啟流體來源高壓儲存鋼瓶如液態二氧化碳高壓儲存鋼瓶丨閥門 使之經由傳輸管線2注入超臨界流體處理槽5，此時槽内壓力約 為50至60kgw/cm2 ’然後開啟流體加壓幫浦3，並設定控溫設備 6使操作溫度不高於45 °C，而使液態二氧化碳逐漸加壓加溫而達 到超臨界態，此時槽内壓力約為8〇到7〇〇 kgw/cm2。 4. 關閉二氧化碳流入及流出處理槽5的閥門’並將5〇0/。乙醇置入交 鏈劑修飾劑儲存槽4，以幫浦4使之加入處理槽5中，作為修飾 1306767 劑，調整其操作濃度為0.5 %，處理15分鐘。 5.没定限流器8使二氧化碳的交換流速設定為每小時2Kg，處理30 分鐘。 6·關閉二氧化碳流入及流出處理槽5的閥門，將戊二醛水溶液置入 交鏈劑修飾劑儲存槽4 ’以幫浦5使之加入處理槽5中，作為交 鏈劑，調整其操作濃度為〇_5 %，或使用genjpin或 (3-dimethyl aminopropyl) -carbodiimide (EDC)，於處理槽 5 中 以超臨界流體處理30分鐘。 7. 待洩壓冷卻後，將50 %乙醇置入交鏈劑修飾劑儲存槽4，以幫浦 4使之加人處理槽5中，作為修飾劑，調整其操作濃度為〇 5 %， 按步驟3操作15分鐘。 8. 待舰冷，後’按步驟3以純超臨界二氧化碳流體處理之，並開 啟-氧化碰人及流丨處賴關門，並蚊雜胃8使流速設 定為每小時2 Kg，處理30分鐘。 9. 待顏冷卻後，開啟處理槽5，取出並樣本處理藍7，並以無 菌之钳子將製備完成的生物醫學移植材料取出，置於密封無菌之 玻璃瓶或培養皿中，並保存於冷凍庫中。 … 10_處理槽5排出的氣體，得排於大氣中，或經由傳輸管線2 ίΐϊ 9後’再經傳輸管線2送至流體回收設備μ以回收 再刺用。 【圖式簡單說明】 圖1為本發明處理方法設備之示意圖。 【主要部分代表符號說明】 高壓儲存鋼瓶 1流體來源高壓儲存鋼瓶如液態二氧化碳 2傳輸管線 3液態氣體加壓幫浦 4交鏈劑及修飾劑加壓幫浦 1306767 5超臨界流體處理鋼槽 6控溫設備 7超臨界流體樣本處理槽 8限流器 9收集設備 10流體回收設備[Embodiment] The method of the present invention is shown in Fig. i. The detailed implementation steps are as follows: 1. Prepare an animal tissue, such as a bone block, to remove the meat, fat, periosteum, cortical bone and excess tissue, and cut the sponge bone into several sizes of bone, or grind it. Granular; such as cartilage, removes upper meat, fat, periosteum and excess tissue, cut into several sizes of cartilage blocks; or as skin, removes fat, hair and excess tissue, cut into several sizes, and supports The device is fixed. 2. Place the tissue sample in the sample processing basket 7 of the supercritical fluid device, place it in the treatment tank 5 and close the gate of the treatment tank 5. 3. Open the fluid source high-pressure storage cylinder such as liquid carbon dioxide high-pressure storage cylinder 丨 valve to inject into the supercritical fluid processing tank 5 via the transfer line 2, at this time the pressure in the tank is about 50 to 60 kgw / cm 2 ' then open the fluid pressure pump 3, and set the temperature control device 6 so that the operating temperature is not higher than 45 ° C, and the liquid carbon dioxide is gradually pressurized and heated to reach the supercritical state, at this time the pressure in the tank is about 8 〇 to 7 〇〇 kgw / cm 2 . 4. Turn off the flow of carbon dioxide into and out of treatment tank 5 and set 5〇0/. Ethanol was placed in the crosslinker modifier storage tank 4, and the pump 4 was added to the treatment tank 5 as a modification 1306767, and the operation concentration was adjusted to 0.5% for 15 minutes. 5. The current limiter 8 is not set to set the exchange rate of carbon dioxide to 2 kg per hour for 30 minutes. 6. Close the valve of the carbon dioxide flowing into and out of the treatment tank 5, and place the aqueous glutaraldehyde solution into the cross-linking agent modifier storage tank 4' to be added to the treatment tank 5 by the pump 5, and adjust the operating concentration as a crosslinking agent. For 〇5 %, or using genjpin or (3-dimethyl aminopropyl) -carbodiimide (EDC), treat in a treatment tank 5 with a supercritical fluid for 30 minutes. 7. After the pressure is released, 50% ethanol is placed in the cross-linker modifier storage tank 4, and the pump 4 is added to the treatment tank 5 as a modifier, and the operating concentration is adjusted to 〇5 %. Step 3 is operated for 15 minutes. 8. Wait for the ship to cool, then follow step 3 with a pure supercritical carbon dioxide fluid, and open-oxidize the person and the hooligan to close the door, and set the flow rate to 2 Kg per hour for 30 minutes. 9. After the face is cooled, open the treatment tank 5, take out the sample and process the blue 7 and take out the prepared biomedical transplant material with sterile pliers, place it in a sealed sterile glass bottle or petri dish, and store it in the freezer. in. 10_ The gas discharged from the treatment tank 5 may be discharged to the atmosphere or sent to the fluid recovery device μ via the transfer line 2 via the transfer line 2 to be recovered for re-spinning. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view of a processing method apparatus of the present invention. [Main part representative symbol description] High-pressure storage cylinder 1 fluid source high-pressure storage cylinder such as liquid carbon dioxide 2 transmission line 3 liquid gas pressurized pump 4 cross-linking agent and modifier pressurized pump 1306767 5 supercritical fluid processing steel tank 6 control Temperature equipment 7 supercritical fluid sample processing tank 8 restrictor 9 collection equipment 10 fluid recovery equipment