200422064 五、發明說明（l) 發明所屬之技術領域 本發明係提供一種應用於引導組織再生（Guided Tissue Regeneration, GTR之複合膠原蛋白膜的製作方 法’尤指一種具有縫合能力（s u t u r a b 1 e )並可被緩慢吸收 之生物相容（biocompatible slow-resorbi n g)複合膠原 蛋白膜的製作方法。 先前技術 引導組織再生（Guided Tissue Regeneration, GTR) ·| 是目前被廣泛應用的一種外科手術程序，其目的是使因 為疾病或外傷而損害之組織或器官的型態（morph〇l〇gy) 以及功能以修復（r e s t 〇 r e )或再生（regeneration)。 在一個成功的組織再生過程中，再生的組織必須在 原本健康之該組織所在的位置和空間中重新分裂複製 (repopulate)。尤有甚者，為了使該區域中不同再生組 織之間的型態與功能關係亦能夠正常修復，該區域進行 之細胞分裂複製以及該再生細胞後續的分化 (differential: ion)都必須是一具有順序性（orderly)且 一致性地（concerted)程序。然而當一活組織中因為部分 組織損害而產生空洞（empty space)時，該空洞會被鄰近 且複製速度最快的細胞株（c e 1 1 1 i n e )所填滿。200422064 V. Description of the Invention (l) The technical field to which the invention belongs The present invention provides a method for producing guided collagen regeneration (Guided Tissue Regeneration, GTR), especially a method having suturab 1 e Preparation method of biocompatible slow-resorbing composite collagen film that can be slowly absorbed. Guidance Tissue Regeneration (GTR) is guided by the prior technology. It is a surgical procedure that is widely used at present, its purpose It is the morphology and function of tissues or organs that are damaged due to disease or trauma to rest or regenerate. In a successful tissue regeneration process, the regenerating tissue must Repopulate in the location and space where the tissue was originally healthy. In particular, in order to make the type and function relationships between different regenerating tissues in the area also be able to be repaired normally, the cells in the area proceed Both division and replication and subsequent differentiation: Must be an orderly and concerted procedure. However, when a void is created in a living tissue due to partial tissue damage, the void will be adjacent and the cell line with the fastest replication speed (Ce 1 1 1 ine).
200422064 五、發明說明（2) 以牙周病學（peri odontology)為例，在一個牙周病 灶裡，傷口的癒合有四種主要的來源細胞，分別為來自 牙齦的上皮細胞（e p i t h e 1 i u m )與結缔組織，以及來自骨 頭組織和牙周勒帶（periradicular ligament)的細胞， 其中唯有來自牙周勃帶的細胞有組織再生的能力。然而 由於上皮細胞的再生速度遠大於牙周韌帶的細胞，因此 使得損傷的牙周韌帶組織的修復十分困難。 而前述之引導組織再生（GTR)的技術即可使適當的組 織細胞得以進行一順序性且一致性地分裂複製以達到損 傷組織再生的目的。為達到此目的，現今大多是將—阻 障物（barrier)暫時於置於該再生組織以及可能會干擾該 再生組織之再生過程的組織之間，直到該組織損傷的部 分完全再生並且該再生的組織達到成熟（m a t u r i t y )為 止。以上述的牙周病灶為例，引導組織再生便是於置— 個生醫材料（B i omat er i a 1)膜以阻擋來自牙齦的表皮細胞 及結締組織和牙根相接觸，而僅允許牙周韌帶細胞的生 長。 目前有兩種主要的生醫材料被應用於引導組織再生 的技術上，第一種是膨体聚四氟乙烯（expanded polytetrafluoroethylene, EPTFE )膜以及高密度多孔聚 乙烯（MEDP0R)膜等不可被吸收高分子膜，由於該EPTFE膜200422064 V. Description of the invention (2) Take periodontology as an example. In a periodontal lesion, there are four main sources of wound healing cells, which are epithelial cells from the gums (epithe 1 ium). With connective tissue, as well as cells from bone tissue and periodontal ligament, only cells from periodontal ligament have the ability to tissue regeneration. However, because the regeneration rate of epithelial cells is much faster than that of periodontal ligament cells, it makes repair of damaged periodontal ligament tissue very difficult. The aforementioned technology of guided tissue regeneration (GTR) can enable appropriate tissue cells to perform a sequential and consistent division and replication to achieve the purpose of damaging tissue regeneration. To achieve this, barriers are currently placed between the regenerating tissue and the tissue that may interfere with the regenerating tissue until the damaged part of the tissue is completely regenerated and the regenerating Until the organization reaches maturity. Taking the above periodontal lesions as an example, guiding tissue regeneration is to place a biomedical material (Biomat er ia 1) membrane to block epidermal cells from the gums and connective tissue from contacting with the root of the teeth, while allowing only periodontal The growth of ligament cells. Currently, there are two main biomedical materials that are used to guide tissue regeneration. The first is expanded polytetrafluoroethylene (EPTFE) membrane and high-density porous polyethylene (MEDP0R) membrane. Polymer membrane due to the EPTFE membrane
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不會被吸收’因此在植入（implanted)體内的這段時間内 可以維持其完整性，因而具有極佳的阻障效果。然而， 該EPTFE膜不可以無限期地置留於體内，因此在進行第一 次手術將該EPTFE膜植入的六至八星期後，需要再進行第 二次手術將之取出。 第一種則是由膠原蛋白（c〇llagen)或是其他聚合物 (polymer)，例如p〇lygiycolates，所構成的可吸收 (resorbable)膜’由於該可吸收膜最終會被吸收而於植 入體内消失，因此不需要額外進行一外科手術將之移 除。相對地，該可吸收膜可能無法完整地在體内存在一 段足夠的時間，以引導完成一成功的組織再生程序。 發明内容 因此，本發明之目的即在提供一種具縫合能力 (suturable)以及可被緩慢吸收（slow-resorbing)的生物 相容（biocompatible)複合膠原蛋白膜，以應用於引導組 織再生（GTR)的技術中並解決上述問題。 在本發明之最佳實施例中，本發明方法係先提供一膠原 蛋白膜，並將該膠原蛋白膜浸泡於一包含有膠原蛋白原 料以及GAGs(glycosaminoglycans)的混合溶液中，接著 依序進行一冷凍處理製程、一真空冷凍乾燥製程、一壓 縮製程清洗以及一乾燥製程，然後利用一交聯劑（cross-It will not be absorbed ’so it can maintain its integrity during the implanted period of time and therefore has an excellent barrier effect. However, the EPTFE membrane cannot be left in the body indefinitely. Therefore, six to eight weeks after the first operation of the EPTFE membrane, it is necessary to perform a second operation to remove the EPTFE membrane. The first is a resorbable membrane made of collagen or other polymers, such as poligiycolates, which is implanted because the resorbable membrane will eventually be absorbed. The body disappears, so no additional surgery is required to remove it. In contrast, the absorbable membrane may not remain intact in the body for a sufficient period of time to guide the completion of a successful tissue regeneration procedure. SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a biocompatible composite collagen film with suturable and slow-resorbing properties, which can be applied to guide tissue regeneration (GTR). Technology and solve the above problems. In a preferred embodiment of the present invention, the method of the present invention first provides a collagen film, and immerses the collagen film in a mixed solution containing collagen raw materials and GAGs (glycosaminoglycans), and then sequentially performs a Freezing process, a vacuum freeze-drying process, a compression process cleaning, and a drying process, and then a cross-linking agent (cross-
第7頁 200422064 五、發明說明（4) 1 inking agent)來使該膠原蛋白膜與該混合溶液令之膠 原蛋白原料以及GAGs發生交聯反應，以形成一膠原蛋白 複合膜。 由於本發明所提供之膠原蛋白膜具有複合結構，因 此可以減緩其被植入體吸收的速度，延長其在植入體内 置留的時間，進而增進其阻障的效果。另一方面，本發 明之複合膠原蛋白膜内另包含有至少一種抗生素（anti - microbial agent)、抗炎劑（anti-inflammatory agent) 或是誘導組織生長因子（growth factor)，以隨著時間而 釋放出不同療程所需之特定藥劑（specific agent)，進 而提昇傷口癒合（wound healing)以及組織再生的效果。 實施方式 在本發明之最佳實施例中，首先提供一膠原蛋白 膜。該膠原蛋白膜的製作方法是先將富含膠原蛋白之牛 皮（bovine skin)、肌腱（tendon)或是胎盤 placenta)以 胃液素（p e p s i η )進行一恆溫處理，接著將經過胃液素純 化後得到之膠原蛋白分子以（1-10 mg/ml)之濃度溶解於 0· 05M的醋酸溶液中，並置於4oC的環境下反應。 在經過一段適當的時間後，加入0 · 1Μ的氫氧化鈉溶 液進行酸鹼中和，並倒入一模型中在使之在20〜3 7oC的Page 7 200422064 V. Description of the invention (4) 1 inking agent) The collagen film and the mixed solution cause the collagen protein material and GAGs to undergo a cross-linking reaction to form a collagen composite film. Since the collagen film provided by the present invention has a composite structure, it can slow down the rate of absorption by the implant, prolong the time it stays in the implant, and thereby improve its barrier effect. On the other hand, the composite collagen film of the present invention further contains at least one antibiotic (anti-microbial agent), anti-inflammatory agent (anti-inflammatory agent), or growth factor (growth factor). It releases specific agents required for different courses of treatment, which in turn improves wound healing and tissue regeneration. Embodiments In a preferred embodiment of the present invention, a collagen film is first provided. The collagen film is prepared by first subjecting collagen-rich bovine skin, tendon or placenta to a constant temperature treatment with gastrin (pepsi η), and then purifying the gastrin to obtain The collagen molecules were dissolved in a 0.05 M acetic acid solution at a concentration of (1-10 mg / ml) and reacted at 4oC. After a suitable period of time, add 0.1 M sodium hydroxide solution for acid-base neutralization, and pour into a mold at 20 ~ 3 7oC.
第8頁 200422064 五、發明說明（5) 溫度範圍内繼續作用24小時。之後利用一活塞（piston) 將溶液中之水分壓出，直到得到一預定厚度之膠原蛋白 膜。最後將該膠原蛋白膜浸入一含有體積濃度^ 5% diphenylphosphoryl azide (DPPA)之二甲基甲酿胺 (dimethylformamide, DMF)溶液中，以使該膝原蛋白膜 内之纖維（f i br i 1 s )彼此交聯，增強其對酵素分解作用 (enzyme degradat i on)的抵抗力（resistance)，進而得 到該膠原蛋白膜。 在得到該膠原蛋白膜之後，接著製備一由 glycosaminoglycansCGAGs)與第一型（Type 1}膠原蛋白 構成之混合溶液，其中GAGs相對於膠原蛋白所佔的比例 約為ίο%。該混合溶液的製備方法是先分別將GAGs以及膠 原蛋白製成重量濃度約為〇 · 5〜2 %之水溶液，該膝原蛋白 水溶液係將膠原蛋白膜纖維溶解於弱酸性水溶液' 0. 1M的醋酸水溶液）得之。最後將該二水溶液混合1並 利用一氫氧化鈉水溶液中和酸性，以調整該 酸鹼值至約為6. 5〜8。 狀 接下來將該膠原蛋白膜浸入該混合溶液中，並 液態氮以在溫度約-180〇C之環境下進行一冷 該冷來處理製程的目的在於使得膠蛋白策 傷之區域後，可以讓水分以及營養分+ 、、、且織扣 汉呂脣刀子通過微孔以供應Page 8 200422064 V. Description of the invention (5) Continue to operate for 24 hours in the temperature range. Then, a piston is used to squeeze out the water in the solution until a collagen film with a predetermined thickness is obtained. Finally, the collagen membrane was immersed in a dimethylformamide (DMF) solution containing a volume concentration of 5% diphenylphosphoryl azide (DPPA) to make the fibers (fi br i 1 s) in the knee protein membrane. ) Cross-links with each other to enhance their resistance to enzyme degradat i on, thereby obtaining the collagen film. After obtaining the collagen film, a mixed solution composed of glycosaminoglycansCGAGs and type 1 collagen is prepared, in which the proportion of GAGs relative to collagen is about ίο. The method for preparing the mixed solution Firstly, GAGs and collagen are respectively made into an aqueous solution with a weight concentration of about 0.5 ~ 2%, and the knee protein solution is obtained by dissolving collagen membrane fibers in a weakly acidic aqueous solution ('0.1M acetic acid aqueous solution). 5〜8。 Finally the two aqueous solutions were mixed 1 and neutralized acidic with an aqueous sodium hydroxide solution to adjust the pH value to about 6. 5 ~ 8. Next, the collagen film is immersed in the mixed solution, and the liquid nitrogen is cooled at a temperature of about -180 ° C. The purpose of the processing process is to make the gelatin damaged area. Moisture and nutrients + ,,, and woven buckle Han Lu lip knife is supplied through micro holes
200422064 五、發明說明（6) 組織再生過程程中所需之養分，同時又可以阻擋可能干 擾再生的纟田胞於該區域分裂複製。 隨後依序進行一需時約丨8小時之真空冷凍乾燥處理 製程以及一壓縮製程’以去除溶液中的水分並得到一預 疋之膜居。然後利用天然交聯劑-g e n i P i n或是戊二搭 (glutaraldehyde)作為交聯劑，以使該膠原蛋白膜與該 混合，谷液_之膠原蛋白以及glyC〇samin〇glyCans發生一 交聯（cross-linking)反應，而使該膠原蛋白膜吸收膠原 蛋白-GAGs混合液，以得到一複合結構之膠原蛋白膜。最 後清洗並乾燥該複合膠原蛋白膜，並可以視臨床所需再 重複上述步驟而製作一具有多重複合結構之複合膠原蛋 白膜。 本發明製作之複合膠原蛋白膜，除了具有可以被緩 慢吸收而增進其阻障的效果的優點之外，在進行複合膠 原蛋白膜之間的交聯反應時，更可以於交聯劑中摻入一 或數種的抗生素（anti_microbial agent)、抗炎劑 (anti- inflammatory agent)或是誘導組織生長因子 (growth factor)，進而包埋（immobilized)於該複合膠 原蛋白膜之中。 以具有多重複合結構之複合膠原蛋白膜為例，吾人 可利用摻有不同藥劑之交聯劑，使膠原蛋白膜進行至少200422064 V. Description of the invention (6) Nutrients required during the tissue regeneration process, and at the same time can prevent the Putian cells that may interfere with regeneration from dividing and replicating in this area. Subsequently, a vacuum freeze-drying process and a compression process', which take about 8 hours, are sequentially performed to remove moisture from the solution and obtain a pre-filmed membrane. Then use the natural cross-linking agent-geni Pin or glutaraldehyde as the cross-linking agent to make the collagen film mix with the collagen, and the cross-linking of the collagen and glycans of the gluten_ cross-linking) reaction, so that the collagen film absorbs the collagen-GAGs mixed solution to obtain a collagen film with a composite structure. Finally, the composite collagen film is washed and dried, and the above steps can be repeated as needed to make a composite collagen protein film with multiple composite structures. In addition to the advantages that the composite collagen film produced by the present invention can be slowly absorbed to enhance its barrier effect, it can be incorporated into the cross-linking agent during the cross-linking reaction between the composite collagen films. One or more kinds of antibiotics (anti-microbial agent), anti-inflammatory agent (anti-inflammatory agent) or growth factor (growth factor), and then immobilized in the composite collagen membrane. Taking a composite collagen film with multiple composite structures as an example, we can use a cross-linking agent mixed with different agents to make the collagen film at least
第10頁 200422064 五、發明說明（7) 一次以上的複合過程，以形成一於 同藥劑之多重複合膠原蛋白膜。所 素、抗炎劑或是誘導組織生長因子 之膠原蛋白膜逐漸吸收而分解的過 釋放出這些不同功能的特定藥齊丨， 及組織再生的效果。 相較於習知之膠原蛋白膜，本勒 蛋白膜可以減緩其被吸收的速度，契 時間，進而增進其阻障的效果，並巧 不同療程所需之特定藥劑（specif ic 傷口癒合（wound heal ing)以及組織 以上所述僅為本發明之較佳實施 請專利範圍所做之均等變化與修飾， 之涵蓋範圍。 同結構區域含有不 的藥劑可能是抗生 因此在該多重複合 中，便可隨著時間 而提昇傷口癒合以 卜明提供之複合膠原 L長其在體内置留的 -以隨著時間釋放出 agent),進而提昇 再生的效果。 i例，凡依本發明申 皆應屬本發明專利 200422064 圖式簡單說明 « 第12頁Page 10 200422064 V. Description of the invention (7) Compounding process more than once to form a multiple composite collagen film with the same agent. Sodium hormones, anti-inflammatory agents, or collagen membranes that induce tissue growth factors are gradually absorbed and decomposed to release these specific drugs with different functions, and the effect of tissue regeneration. Compared with the conventional collagen film, the Benler protein film can slow its absorption rate, increase the time, and thereby improve its barrier effect. It also coincides with specific medications required for different treatments (specif ic wound healing). ) And the organization described above are only equivalent changes and modifications made by the scope of the patent for the preferred implementation of the present invention. The scope of coverage is covered by the same scope. Drugs containing non-identical structures may be antibiotics, so in this multiple compound, it can follow Over time, wound healing is improved. The complex collagen L provided by Bu Ming is long in the body-to release the agent over time), thereby improving the regeneration effect. Example i. Any application according to the present invention shall belong to the patent of the present invention. 200422064 Schematic illustration «Page 12