TW200407434A - Tissue engineering material, production method thereof and application thereof - Google Patents
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200407434 A7 _B7_ 五、發明說明（丨） 發明背景 組織工程學近年來快速興起，現已成為組織或器官移 植的另一潛在選擇。最近，與細胞模組化、細胞外基質構 建和模倣身體結構的合成性聚合物相關的新技術迅速進 展，許多研究者開始思考藉由宿主組織自我再生來治療身 體缺陷。傳統生物材料科學已思及使用如金屬，陶瓷及合 成性聚合物等非生物性材料來修復受損組織。為了要克服 使用此類非活體材料的限制，已嘗試經由結合細胞外基質 雜合性人工合成聚合物與模組化細胞來建構模倣天然組織 的人工器官，產生許多具有生物功能之人工組織（Langer R 等人，5W⑼ce 1993，14;260:920·6)。 組織工程達成修復及再生的方式，係基於在支援加強 且，在某些情況下，重建再生組織的聚合物鷹架的使用。 鷹架可能用於以受調控速率釋放生物活性物質，或直接地 影響被納入細胞或内生細胞的行為。這些不同的功能通常 要求多孔鷹架微細構造與多孔性特質。鷹架化學中常希望 涵括或模倣與細胞外基質成分、生長因子或細胞表面受體 之間發生的專一***互作用。現已使用一些天然及合成性 聚合物作為組織鷹架（Murphy WL等人，J Μα仏r 2000;50:50-8 ; Thomson RC 等人， Biomaterial 1999;20:2007-18 ; Peter SJ 等人， 1998;43:422-7 ； Murphy WL 專 k，J Periodontal Res 1999;34:413-9)。這些系統的微細構造範圍自水凝膠，開孔 系統一直到纖維基質。自組織工程系統的潛在應用範圍變 ___3_ 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)200407434 A7 _B7_ V. Description of the Invention (丨) Background of the Invention Tissue engineering has risen rapidly in recent years and has become another potential choice for tissue or organ transplantation. Recently, new technologies related to the modularization of cells, the construction of extracellular matrix, and synthetic polymers that mimic the structure of the body have progressed rapidly, and many researchers have begun to think about treating body defects through self-regeneration of host tissues. Traditional biomaterial science has considered the use of non-biological materials such as metals, ceramics and synthetic polymers to repair damaged tissues. In order to overcome the limitation of using such non-living materials, attempts have been made to construct artificial organs that mimic natural tissues by combining extracellular matrix hybrid synthetic polymers with modular cells to produce many artificial tissues with biological functions (Langer R et al. 5Wcece 1993, 14; 260: 920 · 6). The way tissue engineering achieves restoration and regeneration is based on the use of polymer scaffolds that are enhanced in support and, in some cases, reconstructed regenerated tissue. The scaffold may be used to release bioactive substances at a regulated rate or directly affect the behavior of incorporated cells or endogenous cells. These different functions usually require the microstructure and porous properties of the porous scaffold. In scaffold chemistry, it is often desirable to include or mimic specific interactions with extracellular matrix components, growth factors, or cell surface receptors. Some natural and synthetic polymers have been used as tissue scaffolds (Murphy WL et al., J Μα 仏 r 2000; 50: 50-8; Thomson RC et al., Biomaterial 1999; 20: 2007-18; Peter SJ et al. 1998; 43: 422-7; Murphy WL Specialized, J Periodontal Res 1999; 34: 413-9). The microstructures of these systems range from hydrogels, open-cell systems to the fiber matrix. The potential application range of the self-organizing engineering system changes ___3_ Paper size applies to China National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling this page)
200407434 A7 __B7__ 五、發明說明（> ) 得寬廣以後，就一直有研究在著手找尋具有特別令人希望 的組織專一性，或可能有廣泛適用性而能用於數個組織系 統的材料。 聚乳酸（PLA)，聚羥基乙酸（PGA)和其共聚物如聚（乳酸 -共-羥基乙酸）(PLGA)已用於作為細胞移植的三度空間 (3D)聚合物鷹架，因為他們是生物可分解的及生物相容的 (Athanasiou KA 等人，1996;17:93-102)。直到 現在，在纖維間藉由PLA和PLGA的物理鍵結加強的非 編織性聚羥基乙酸（PGA)纖維網，由於其為高度多孔性及 交聯的結構，成為軟組織細胞移植中最普遍使用的鷹架 (Gao J 等人，α 1998;42:417-24)。在藥學， 醫學和生醫領域中生物可分解聚合物已用於發展有多種應 用的生物材料（Benicewicz Β. C.等人，《7 尸〇/少7刀 1999; 6:64-94; Holland S· J·等人，J 1986;4: 155-180 ； Kyriacos AA # A 9 Biomaterial 1993; 17: 93_102 ; Gilding D. K.，“Biodegradable polymers” in biocompatibility of Clinical Implant Materials, 1981;CRC Press，Inc·: pp. 209-232)。關於獲得允許表面修飾的生物可 分解聚合物之方法，亦有研究指出將聚合物鏈（例如聚乙二 醇，PEG)黏附到如PLA或PLGA之生物可分解聚合物鏈 (Suh JK 等人，价2000;21:2589_98)。然而整體觀 之，仍有需要去提昇這些材料的特性以達最佳化，提供醫 療上實際的應用。 聚合物系統可以被用於作為生長因素的補給，活體外 9¾ ---—.___f_ …_’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)200407434 A7 __B7__ 5. After the invention description was broad, research has been conducted to find materials that have particularly promising organizational specificity, or may have broad applicability and can be used in several organizational systems. Polylactic acid (PLA), polyglycolic acid (PGA) and its copolymers such as poly (lactic-co-glycolic acid) (PLGA) have been used as three-dimensional (3D) polymer scaffolds for cell transplantation because they are Biodegradable and biocompatible (Athanasiou KA et al., 1996; 17: 93-102). Until now, the non-woven polyglycolic acid (PGA) fiber network reinforced by the physical bonding of PLA and PLGA between fibers has become the most commonly used in soft tissue cell transplantation due to its highly porous and cross-linked structure. The scaffold (Gao J et al., Α 1998; 42: 417-24). In the fields of pharmacy, medicine and biomedicine, biodegradable polymers have been used to develop biomaterials with a variety of applications (Benicewicz Β. C. et al., "7 Corpse 0 / less 7 knives 1999; 6: 64-94; Holland SJ et al, J 1986; 4: 155-180; Kyriacos AA # A 9 Biomaterial 1993; 17: 93_102; Gilding DK, "Biodegradable polymers" in biocompatibility of Clinical Implant Materials, 1981; CRC Press, Inc. :: pp. 209-232). Regarding methods for obtaining biodegradable polymers that allow surface modification, there have also been studies that indicate that polymer chains (eg, polyethylene glycol, PEG) are adhered to biodegradable polymer chains such as PLA or PLGA (Suh JK et al., Price 2000; 21: 2589_98). However, as a whole, there is still a need to improve the characteristics of these materials to optimize them and provide practical medical applications. The polymer system can be used as a supplement for growth factors, in vitro 9¾ -----.___ f_… _ 'This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) (Please read the back (Please fill in this page again)
線 200407434 A7 B7 五、發明說明（）） 細胞培養以及改良活體内組織再生。當為了組織培養使用 生物可分解聚合物鷹架時’鷹架材料與細胞之間的交互作 用可能被聚合物性質所影響。舉例而言，可吸附的肽或蛋 白質常介入細胞的黏附（Myles JL等人，j Po—Ed 2000;1 1:69-86)。此外，在鷹架和細胞之間的交互 作用可以決定細胞功能··已顯示與聚合物表面之交互作用 不但景彡響增殖而且影響其他的過程，例如細胞分化和細胞 遷移（Bures P 等人 /(:⑽的/如/⑽“ 2001; l4:72:25-33;Park A 等人，Ed 1〇 ’· IkadaY， 价·〇漏阶ζ·α/ 1994;15:725-36) ° 對組織生長和組織功能控制的第一步在於抑制任何 非專性蛋白負或肽被吸附至用於細胞培養的聚合性裝 置，一旦聚合物表面被爲裝，其可藉由結合專一性蛋白質 或狀來達到功能化（Ratner BD，历㈣财方沁★价⑽ 1995;1():797-8()4·)。當抑制非專—性蛋白f的時候，藉由結 合專一性黏附因素（例如纖維凝集素），聚合物表面將能夠 促進專一性細胞黏附，並抑制非專一性蛋白質媒介之其他 種類細胞黏附。這在骨缺陷治療案例中很重要，將不欲细 胞種類從«位置排除成為主要的挑戰（MassiaSp等人^Line 200407434 A7 B7 V. Description of the invention () Cell culture and improvement of tissue regeneration in vivo. When a biodegradable polymer scaffold is used for tissue culture, the interaction between the 'scaffold material and the cell may be affected by the properties of the polymer. For example, adsorbable peptides or proteins often interfere with cell adhesion (Myles JL et al., J Po-Ed 2000; 1 1: 69-86). In addition, the interaction between the scaffold and the cell can determine the function of the cell ... The interaction with the polymer surface has been shown to affect not only proliferation but also other processes such as cell differentiation and cell migration (Bures P et al./ (: ⑽ 的 / 如 / ⑽ “2001; 14: 72: 25-33; Park A et al., Ed 10 ′ · IkadaY, Valence 〇 Leak Order ζ · α / 1994; 15: 725-36) The first step in tissue growth and tissue function control is to inhibit any non-specific protein negative or peptides from being adsorbed to the polymerizable device used for cell culture. Once the polymer surface is loaded, it can be combined with the specific protein or state. To achieve functionalization (Ratner BD, Li Fang Qin Fang Qin 1995; 1 (): 797-8 () 4 ·). When inhibiting non-specific protein f, by combining specific adhesion factors ( (Such as fibrectin), the polymer surface will be able to promote specific cell adhesion and inhibit non-specific protein mediators of other types of cell adhesion. This is important in the case of bone defect treatment, eliminating unwanted cell types from Main challenges (MassiaSp et al. ^
Biomed Mater Res 2001·，56··390_9) 〇 在很多外科手術案例中，有必要替換，補全或加強缺 少的或受傷的組織。目前並無理想的結締組織取代物。在 組織修復機制中，例如在傷害或外科手術之後，能夠使新 組織重建的細胞需要基質來支持細胞生長。一般已知膠原 本紙張尺度適用中關家標準（CNS)A4規格（21Q χ 2975公1 (請先閱讀背面之注意事項再填寫本頁) 訂.· .•線- 200407434 A7 五、發明說明（斗 蛋白可改變細胞的形態、遷移、黏附、以及在某些情況下 細胞的分化和生長。目前已有研究將不同種類的勝原蛋白 (第I ’ III和IV型），明膠，純化或重建的細胞外基質，醣 蛋白（例如纖維凝集素、laminin和vitr〇nectin)，纖維蛋白 原/纖維蛋白，包含葡萄糖胺聚糖之複合支持物，基底膜 (MatrigelT、，纖維素，去乙醯殼多醣和幾丁質衍生物等用 於促進細胞生長，以及可能單獨或合併使用於多孔基質。 以膠原蛋白做《的多1聚合物已作為基質來支援細胞 生長。然而讓細胞浸潤其間的膠原蛋白多孔聚合物之孔洞 易於毀壞。事實上這些孔洞無法維持一段足以讓一些細胞 (例纖維母細胞）建立群落的時間。 聚羥基乙酸在作為骨固定裝置之應用方面，已因為具 有較高機械穩定性之自我加強型聚羥基乙酸的發展而大為 增進（Muzzarelli RA 等人，五恐 1999;87:251_64)。然而，聚 羥基乙酸非編織性網不適合用於有組織_專一性的情況，因 為其不容易的進行修飾。 去乙醯殼多醣（聚1，4-D_葡萄糖胺）是幾丁質進行部份 去醯基化後的衍生物，幾丁質是節肢動物外骨骼主要結構 聚合物。商業化製備物之去醯基化程度從5〇%到9〇%。去 乙醯殼多醣為結晶狀多醣且通常不能溶解於pH值7以上 的水溶液中。去乙醯殼多醣及其複合物已開始用於生物醫 學的研究，包括藥物傳遞系統（Genta I等人，五尤^ 1999;87:305-13 ; Sabnis s 等人，加 ^ 咖施咖⑽ 2〇〇〇，13:27:181-6)及創傷敷料（L〇ke WK等人，J厶化所以 1 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) # · -線· 200407434 A7 五、發明說明（f )Biomed Mater Res 2001 ·, 56 · 390_9) 〇 In many surgical cases, it is necessary to replace, complement or strengthen missing or injured tissue. There is currently no ideal connective tissue substitute. In tissue repair mechanisms, such as after injury or surgery, cells capable of rebuilding new tissue require a matrix to support cell growth. It is generally known that the size of collagen paper is applicable to the Zhongguanjia Standard (CNS) A4 specification (21Q χ 2975 male 1 (please read the precautions on the back before filling this page). Order ... · Line-200407434 A7 V. Description of the invention ( It can change the cell's morphology, migration, adhesion, and cell differentiation and growth in some cases. At present, there are studies that have different types of vicinal proteins (types I'III and IV), gelatin, purified or reconstructed Extracellular matrix, glycoproteins (e.g., fibrin, laminin and vitroonectin), fibrinogen / fibrin, composite support containing glycosaminoglycan, basement membrane (MatrigelT, cellulose, deacetylated chitin And chitin derivatives are used to promote cell growth, and may be used alone or in combination with porous matrices. The poly-1 polymer made of collagen has been used as a matrix to support cell growth. However, the collagen infiltrated by cells is porous Polymer pores are easy to destroy. In fact, these pores cannot sustain a period of time sufficient for some cells (such as fibroblasts) to establish a colony. In terms of its application as a bone fixation device, it has been greatly enhanced by the development of self-reinforcing polyglycolic acid with higher mechanical stability (Muzzarelli RA et al., May 5 1999; 87: 251_64). However, polyglycolic acid Non-woven webs are not suitable for use in organized_specific situations, as they are not easily modified. Deacetylated chitin (poly 1,4-D_glucosamine) is a partial deacetylation of chitin. After the derivative, chitin is the main structural polymer of arthropod exoskeleton. The degree of dehybridization of commercial preparations ranges from 50% to 90%. Chitosan is a crystalline polysaccharide and usually cannot be dissolved. In an aqueous solution with a pH value of 7 or higher, desacetyl chitin and its complexes have begun to be used in biomedical research, including drug delivery systems (Genta I et al., Wuyou ^ 1999; 87: 305-13; Sabnis s Et al., Plus ^ Ka Shi Ka Ye 2000, 13: 27: 181-6) and wound dressing (Loke Ke WK et al., J Xiehua 1 X 297 mm) (Please read the note on the back first Please fill in this page for matters) # · -line · 200407434 A7 V. Description of Invention (f)
Maier 及以 2000;53:8-17)。 因此，組織工程相關技術領域中對於包含具穩定多孔 性而足以支援細胞建立群落的生物多孔性聚合物仍有明確 的需要。 另外，子宮内膜炎（子宮内膜異位）是描述子宮内膜空 間外的内膜組織内膜層細胞出現的情形。子宮内膜炎主要 發生在cul -de-sac (子宮後面），直腸***隔膜（這層組織位 在於直腸與***之間），直腸表層，輸卵管與卵巢，子宮薦 椎部韋刃帶，膀胱與骨盆腔壁。子宮内膜異位子宮内膜異位 子宮内膜異位子宮内膜異位推測罹患子宮内膜異位的婦女 人數是十分廣泛的。預估較保守的估計大約有9億的女性 被估算在這範圍中。但約只有1〇%的女性罹患，故使得子 呂内膜異位成為地球上最常見的一種疾病。 、μ目前對子宮内膜異位沒有治癒的方法，但在處理 上部有多種選擇。其目標可能包含：移除/減輕疼痛症狀， 減緩子宮内膜異位細胞生長，保存/回復生育力，與預防/ 延緩疾病的復發。Danazol，birth C〇ntrolpiu，。卯加，Maier and Iz. 2000; 53: 8-17). Therefore, there is still a clear need in the technical field related to tissue engineering to include a bioporous polymer having stable porosity sufficient to support the establishment of a cell colony. In addition, endometritis (endometriosis) describes the appearance of endometrial cells in the endometrial tissue outside the endometrial space. Endometritis mainly occurs in cul-de-sac (back of the uterus), the rectum-vaginal diaphragm (this layer of tissue is located between the rectum and the vagina), the surface of the rectum, the fallopian tubes and ovaries, the uterine sacral vertebrae, and the bladder and Pelvic wall. Endometriosis Endometriosis The number of women presumed to suffer from endometriosis is very wide. A more conservative estimate is that approximately 900 million women are estimated in this range. However, only about 10% of women suffer from it, which makes the endometriosis the most common disease on the planet. At present, there is no cure for endometriosis, but there are many options for treating the upper part. Its goals may include: removing / reducing pain symptoms, slowing the growth of endometriotic cells, preserving / restoring fertility, and preventing / delaying the recurrence of the disease. Danazol, birth Controlpiu ,. Add,
Synarel ’ Zoladex ’ dep〇t-Provera 與 N〇rplant 部論當主要 治劑或佐劑都未能證明其對子宮内膜異位相關的***症有 效治療（配合外科手術之治療）。當使用醫療處理可能可以 降低感染反應，使得外科矯正可以提早，並且降低因子宮 内膜異位所引起的疼痛，但在病人身上使用這些藥物時， 雖可以有最少的疾病但卻未能證明對***症有任何好處。 然而，選擇性之處理在這幾年間已證明可行，但經治療後 :：ί。^本紙張尺度剌巾關家標準（CNS)A4規格（210 X 297公爱）—-----------Synarel ’Zoladex’ depot- Provera and Norplant as main therapeutic agents or adjuvants have failed to prove that they are effective in treating infertility related to endometriosis (with surgical treatment). While the use of medical treatment may reduce the infection response, make surgical corrections earlier, and reduce the pain caused by the factor endometriosis, but when these drugs are used on patients, they have the least disease but have not proven to be effective. There are no benefits to infertility. However, selective treatment has proven feasible in these years, but after treatment :: ί. ^ The paper size of the paper towel family standard (CNS) A4 specification (210 X 297 public love) --------------
ί---f 訂： --線· -n n n n 1 (請先閱讀背面之注意事項再填寫本頁) n n n - i 200407434 A7 I------2Z____— 五、發明說明（& ) 的女性卻有非預期的骨盤疼痛，或需更進一步解決***症 的問題。 發明總％ 本發明相關一種生物可降解性材料，其包括由羥基乙 酸和去乙醯殼多醣聚合形成的多孔性雜合基質。本發明生 物可降解性材料之多孔性雜合基質中聚經基乙酸與去乙酿 殼多醣重量比通常自95%至5%，較佳為自8〇%至2〇%。 、本發明生物可降解性㈣之多孔性雜合基質係藉由熱 相分離方法合成。本發明生物可降解性材料之多孔性雜合 基質亦可藉由溶劑模造顆粒濾出方法合成，該溶劑模造顆 粒濾出方法較佳使用葡萄糖顆粒，其中所使用葡萄糖較佳 為佔整體重量的30%至60%重量。 本發明生物可降解性材料可用於作為細胞培養之支持 物，較佳作為哺乳動物細胞培養之支持物，更較佳作為上 皮細胞培養之支持物，上皮細胞較佳為纖維母細胞/ 树明生物可降解性材料亦可詩作為㈣工程廣架 I (scaffold) 〇 本發明亦相關一種製備生物可降解性材料的方法，其 包括經由熱相分離方式構建聚經基乙酸/去乙酿殼多醣多、 y生雜合基質。本發明生物可降解性材料之多孔性雜合基 質中聚羥基乙酸與去乙醯殼多醣重量比通常自燃至 5%，較佳為自80%至2〇0/〇。 本發明亦相關另一種製備生物可降解性材料的方法， 其包括經由經由溶劑模造顆粒遽出方式構建聚經基乙酸/ | - g 爾本紙張尺錢时關家鮮（CNS)A4絲（2W X 297公f ) — — — — — — — — — · I I (請先閱讀背面之注意事項再填寫本頁) ^訂 線丨丨 200407434 A7 ------------ -B7 _ 五、發明說明（^ ) ^酿殼Μ多孔性雜合基f。該_模造顆㈣出方法 $佳使用葡萄糖顆粒，其中所使用葡萄糖較佳為佔整體重 里的30〇/〇至6〇%重量。本發明生物可降解性材料之多孔性 雜合基質中聚羥基乙酸與去乙醯殼多醣重量比通常自95% 至5% ’較佳為自80%至20%。 本發明亦相關於一種生物性可降解性材料，其可於活 體内抑制不正常組織之增纟。較佳的I，本發明生物可降 解性材料可用於治療或預防子宮内膜炎。較佳的是，本發 明生物可降解性材料，可用於治療或預防子宮景内膜炎。 本發明生物可降解性材料較佳是經過誘發細胞凋亡的方 式’達成抑制細胞生長的功用。 凰^_簡單說明 圖1顯示鑑別式掃瞄熱量計掃瞄記錄。 圖2顯示鑑別式掃瞄熱量計掃瞄記錄。 圖3列出本發明雜合基質之多孔性。 圖4顯示本發明雜合基質水含量之膨脹率。 圖5顯示經由HPLC所決定的本發明雜合基質之活體 外降解型態。 圖6顯示培養24小時後黏附到本發明聚合物薄膜與 TCPS對照組受質的纖維母細胞數目。 圖7顯不經由_唑藍試驗估計之薄膜細胞毒性。 圖8顯示聚羥基乙酸/去乙醯殼多醣（p/c)雜合基質a 與小备基乙酸/去乙酸殼多醣雜合基質b對子宮内膜異位 …·'---------- ---9_ gg琢紙張尺度適用中國國家標準（CNS)A4規格（21Q χ 297公爱）- 一 一 (請先閱讀背面之注意事項再填寫本頁) . ;線_ 200407434 10 A7 五、發明說明（g ) 細胞的生長抑制圖表。 圖9顯示聚羥基乙酸/去乙醯殼多醣雜合基質a在500ί --- f Order: --line · -nnnn 1 (Please read the notes on the back before filling in this page) nnn-i 200407434 A7 I ------ 2Z ____— V. & Description of Invention Women, however, have unexpected pain in the pelvis, or need to further address infertility. Total invention% The present invention relates to a biodegradable material, which comprises a porous hybrid matrix formed by the polymerization of hydroxyacetic acid and chitosan. The weight ratio of polyacetic acid to chitosan in the porous hybrid matrix of the biodegradable material of the present invention is usually from 95% to 5%, preferably from 80% to 20%. 2. The porous hybrid matrix of the biodegradable rhenium of the present invention is synthesized by a thermal phase separation method. The porous hybrid matrix of the biodegradable material of the present invention can also be synthesized by a solvent-molded particle filtration method. The solvent-molded particle filtration method preferably uses glucose particles, wherein the glucose used is preferably 30% of the total weight. % To 60% by weight. The biodegradable material of the present invention can be used as a support for cell culture, preferably as a support for mammalian cell culture, more preferably as a support for epithelial cell culture, and the epithelial cell is preferably a fibroblast / shuming organism Degradable materials can also be used as a scaffold for engineering projects. The present invention also relates to a method for preparing biodegradable materials, which includes constructing polyacetic acid / deacetylated chitosan by thermal phase separation. , Y Health heterozygous matrix. The weight ratio of polyglycolic acid to chitosan in the porous hybrid matrix of the biodegradable material of the present invention usually spontaneously ignites to 5%, preferably from 80% to 200 / 〇. The present invention is also related to another method for preparing a biodegradable material, which comprises constructing polyacetic acid / |-g Erben paper ruler (CNS) A4 silk (2W) X 297 Male f) — — — — — — — — — II (Please read the precautions on the back before filling in this page) ^ Order line 丨 丨 200407434 A7 ------------ -B7 _ V. Description of the invention (^) ^ Fermentary shell M porous hybrid group f. The method of molding granules preferably uses glucose granules, in which the glucose used is preferably 30/60 to 60% by weight of the whole weight. The weight ratio of polyglycolic acid to chitosan in the porous hybrid matrix of the biodegradable material of the present invention is usually from 95% to 5% ', preferably from 80% to 20%. The invention also relates to a biodegradable material, which can inhibit the increase of abnormal tissues in vivo. Preferably, the biodegradable material of the present invention can be used for treating or preventing endometritis. Preferably, the biodegradable material of the present invention can be used for treating or preventing endometritis of uterine scene. The biodegradable material of the present invention preferably achieves the function of inhibiting cell growth through the manner of inducing apoptosis. Brief description ^ _ Figure 1 shows the scan log of a discriminating calorimeter. Figure 2 shows the scan log of a discriminating calorimeter. Figure 3 lists the porosity of the hybrid matrix of the present invention. Figure 4 shows the swelling rate of the water content of the hybrid matrix of the present invention. Figure 5 shows the in vitro degradation pattern of the hybrid matrix of the present invention determined by HPLC. Figure 6 shows the number of fibroblasts adhered to the polymer film of the invention and the TCPS control group after 24 hours of culture. Figure 7 shows the membrane cytotoxicity not estimated by the azole blue test. Figure 8 shows the endometriosis of polyglycolic acid / chitosan (p / c) hybrid matrix a and small acetic acid / chitosan hybrid matrix b on the endometrium ... · '------ ---- --- 9_ gg cut paper standards are applicable to Chinese National Standard (CNS) A4 specifications (21Q χ 297 public love)-one by one (please read the precautions on the back before filling this page).; Line_ 200407434 10 A7 5. Description of the invention (g) Graph of cell growth inhibition. Figure 9 shows the polyglycolic acid / desacetin chitosan hybrid matrix a at 500
Ag/ml對子宮内膜異位細胞的生長抑制圖表。 圖1 〇顯示經不同濃度的聚羥基乙酸/去乙醯殼多醣雜 合基質A處理4天後的子宮内膜異位細胞型態分析之照相 圖（150X)。 圖Π顯不經不同濃度的聚羥基乙酸/去乙醯殻多醣雜 合基質A處理4天後的子宮内膜異位細胞型態分析之照相 圖（100X)〇 圖12顯示經聚羥基乙酸/去乙醯殼多醣雜合基質八誘 發細胞凋亡之照相圖。 圖13顯示經聚羥基乙酸/去乙醯殼多醣雜合基質a與 經聚羥基乙酸/去乙醯殼多醣雜合基質B之細胞其流式細 胞分析表。 * 圖14顯示經不同濃度之聚羥基乙酸/去乙醯殼多醣雜 合基質處理之細胞其caspase_3之活性圖表。 發明詳細說明 本發明相關新穎生物可降解性材料，其主要特徵在於 f備過程中藉由將混合去乙酿殼多聽與聚經基乙酸而製 得，成功將從前單獨使用聚窥基乙酸或去乙酿殼多酶作為 生物材料的優點結合。本發明生物可降解性材料呈有非主 性，生物相容性及生物可分解性等優異特質，故^實驗= 臨床上極有利之生物材料。 只欢 989^紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）Graph of Ag / ml inhibition of growth of endometriotic cells. Fig. 10 shows a photograph (150X) of morphological analysis of endometriotic cells after 4 days of treatment with different concentrations of polyglycolic acid / chitosan hybrid matrix A. Figure Π shows a photograph of the analysis of endometriotic cell type after treatment with polyglycolic acid / chitosan hybrid matrix A for 4 days without different concentrations (100X). Figure 12 shows the polyglycolic acid / Photograph of apoptosis induced by deacetylated chitin hybrid matrix VIII. Fig. 13 shows a flow cytometric analysis table of cells treated with polyglycolic acid / chitosan hybrid matrix a and cells treated with polyglycolic acid / chitosan hybrid matrix B. * Figure 14 shows a graph of caspase_3 activity of cells treated with different concentrations of polyglycolic acid / chitosan hybrid matrix. Detailed description of the invention The novel novel biodegradable material related to the present invention is mainly characterized in that it is prepared by mixing and removing polyethyl acetate and polyacetic acid during the preparation process, and it has been successfully used alone before. Combine the advantages of deacetylated polyenzyme as a biomaterial. The biodegradable material of the present invention exhibits excellent characteristics such as non-subjectivity, biocompatibility, and biodegradability, so ^ experiment = clinically extremely advantageous biomaterial. Only Huan 989 ^ Paper size applies to China National Standard (CNS) A4 (210 X 297 mm)
.丨—I —丨—丨—111 — · — I (請先閱讀背面之注意事項再填寫本頁) -線- -I I I n n n 200407434 五 A7 B7 、發明說明（^ ) “本發明湘不同的構建方法來混合聚録乙酸和去乙 w多酶而製得生物可降解性材料。本發明並針對該生物 可降解性材料特性（多孔性，機械性質和親水性等）及_ 工程應用性作進-步探究，使該生物可降解性材料可有效 作為、.且4工私學鷹架或其他支持細胞生長的用途。 ,本發明生物可降解性材料可以用目前已知的用於構建 、且、哉材料的方法進行構建，例如熱相分離方式伸 phase separation)或溶劑模造顆粒濾出（s〇i_^sting paniculate leaehing)方式。在這些方法中，熟習該項技術者 可以藉由改變製備條件而控制該生物可降解性多孔材料的 孔徑大小。例如，在溶劑模造顆粒濾出方法中，可以經由 調整所使用粒子（例葡萄糖）的大小來製備具有希望孔徑大 小之基質。 在本备明一些具體事實中，亦發現氫鍵對於本發明材 料的許多特性均有很大影響，例如親水性，活體外降解和 細胞黏附。氫鍵對於本發明材料親水性的影響視未鍵結的 殘餘羥基乙酸單體份量而定。未鍵結的殘餘羥基乙酸單體 份置增加時，本發明材料具較大的親水性。 此情形亦同樣發生於本發明材料之降解特性上。在構 建期間發生降解是由於暴露於空氣及水之故。在本發明的 具體事實中，大量殘餘的經基乙酸與水分子迅速形成氫鍵 而會造成較大的降解率。 此外，此氫鍵模型亦能解釋本發明材料之生物相容 性。在本發明的具體事實中，纖維母細胞培養於本發明材. 丨 —I — 丨 — 丨 —111 — · — I (Please read the notes on the back before filling out this page) -line- -III nnn 200407434 Five A7 B7, Invention Description (^) "The different constructions of this invention Method for mixing polyacetic acid and deacetylase to obtain biodegradable materials. The present invention also aims at the characteristics of the biodegradable materials (porosity, mechanical properties, hydrophilicity, etc.) and _ engineering applicability -Step-by-step investigation, so that the biodegradable material can be effectively used as a scaffold or other support for cell growth. The biodegradable material of the present invention can be used for construction, and And materials, such as thermal phase separation or solvent-molded particle filtration (soi_sting paniculate leaehing). In these methods, those skilled in the art can change the preparation conditions by And control the pore size of the biodegradable porous material. For example, in the solvent-molded particle filtration method, the size of the particles (such as glucose) can be adjusted to prepare Matrix of pore size. In some specific facts of this note, it has also been found that hydrogen bonding has a great impact on many characteristics of the material of the present invention, such as hydrophilicity, degradation in vitro and cell adhesion. Hydrogen bonding is hydrophilic to the material of the present invention. The effect depends on the amount of unbonded residual glycolic acid monomer. When the amount of unbonded residual glycolic acid monomer is increased, the material of the present invention is more hydrophilic. This situation also occurs in the material of the present invention. Degradation characteristics. Degradation occurs during construction due to exposure to air and water. In the specific facts of the present invention, a large amount of residual acetic acid quickly forms hydrogen bonds with water molecules, resulting in a large degradation rate. This hydrogen bond model can also explain the biocompatibility of the material of the present invention. In the specific facts of the present invention, fibroblasts are cultured in the material of the present invention
----.---Γ 訂. (請先閱讀背面之注意事項再填寫本頁) 線. -n n n n · ZUUH-U /H-JH- A7 B7----.--- Order. (Please read the precautions on the back before filling this page). -N n n n · ZUUH-U / H-JH- A7 B7
五、發明說明（P 料上可存活且維持細長形態。 乙醯殼多酿之間的氫鍵形成較少聚經基乙酸和去 較高的密度在本發明材料上生長夺’、義維母細胞細胞可以 去乙酿设多酶是N-乙酿其片# 連鲈彳組s •土匍甸糖胺和葡萄糖胺（1—4 、口、、、一兀，、聚糖，結構類似於葡萄糖胺聚糖。葡萄 手在㈣々t 形成蛋白多酶。蛋白多醣分 胞形態，分化和功能方面松演主要角色。因此， 一 =4去乙醯殼多_與蛋白多㈣成分相似可能有益於 、，、田胞黏附和功能促進。 ，乙酸是眾所週知的生物材料，其優良的生物相 谷師生物可降解性導致其在醫學上的廣大應用。聚經基 乙酉夂在生物體内降解成為可被代謝者或被排出體外。 本^月首先心及並構建結合去乙醯殼多釀及聚經基乙 酉夂兩者優點的雜合材料，並成功製得高度多孔性的魔架而 使細胞合易黏附和生長。因此，本發明製得的乙酿殼多聽 及水經基乙酸雜合材料提供組織工程學之新生物材料，特 別是有利於創傷癒合或組織再生。 另一方面，本發明亦首先將乙醯殼多醣及聚羥基乙酸 雜曰材料應用在子宮妹膜異位症。通常子宮内膜異位多發 生在較深層的構造。在月經來潮時期，可發現子宮内膜異 位細胞位在子宮之後的區域。最廣泛被採用的理論，直腸 ***的月經週期，是指子宮内膜異位發生的階段，即當子 呂内膜異位片段與鄰近骨盆構造連結時之生長狀況。其他 理論包含組織移植，在腹膜腔内襯細胞引入改變，分離子 12 •紙張尺度適用甲國國家標準（CNS)A4規格（21ΰ X 297公爱）V. Description of the invention (P material can survive and maintain a slender form. Hydrogen bond formation between acetonitrile shells is less polyacetic acid and a higher density is grown on the material of the present invention. Cells can be deacetylated. The multi-enzyme is N-ethyl stuffed. # 彳 彳 彳 group • Toxidian glucosamine and glucosamine (1-4, mouth ,,,,,,,,, and glycan, the structure is similar to Glycosaminoglycans. Grape hands form protein polyenzymes in proteoglycans. Proteoglycans play a major role in cell morphology, differentiation, and function. Therefore, a = 4 deacetylated polysaccharides can be beneficial and similar to polyproteins The adhesion and function of field cells are promoted. Acetic acid is a well-known biological material, and its excellent biodegradability leads to its wide application in medicine. Polyethylene glycol is degraded into It can be excreted by the metabolizer or excreted from the body. This month, we first focused on the construction of a hybrid material that combines the advantages of acetonitrile dehulling and polyacrylamide, and successfully produced a highly porous magic frame. Cells bind and grow easily. Therefore, this The invented acetonitrile shell and water-based acetic acid hybrid materials provide new biomaterials for tissue engineering, which are particularly beneficial to wound healing or tissue regeneration. On the other hand, the present invention also firstly uses acetochitin and Polyglycolic acid materials are used in endometriosis. Endometriosis usually occurs in deeper structures. During menstrual period, endometriosis cells can be found in the area behind the uterus. Most The widely adopted theory is that the menstrual cycle of the rectum and vagina refers to the stage of endometriosis, that is, the growth condition when the ectopic endometrial segment is connected to the adjacent pelvic structure. Other theories include tissue transplantation in the peritoneum Introduced changes in cavity lining cells, isolate 12 • Paper size applies to National Standard A (CNS) A4 (21ΰ X 297 public love)
----.----- 訂. (請先閱讀背面之注意事項再填寫本頁) —線· -— I I · 200407434 A7 B7 五、發明說明（ 呂月？脈，與直接延伸淋巴系統。但沒有一個理論可以用 解釋所有的狀況，當一個子宮Λ 七丄、 们于呂内膜異位細胞經常出現在所 有女性生理期間時的腹膜區域，因此有人推測子宮内膜里 位可旎在每一個人身上都會生長。顯然地，這與事實不符， 仁不幸的是沒有人知道為什麼。然而可能有某些免疫上的 發現可以與子宮内膜異位或因子宮内臈異位而發炎的的情 形符合。----.----- Order. (Please read the notes on the back before filling in this page) —Line · -— II · 200407434 A7 B7 V. Description of the invention (Lu Yue? Vein and direct extension lymphatic system But there is no one theory that can explain all the conditions. When a uterus of the uterus, the endometriotic cells often appear in the peritoneal area of all women during the physiological period, so some people speculate that the endometrium can be trapped in the endometrium. Everyone grows. Obviously, this is not consistent with the fact. Ren unfortunately no one knows why. However, there may be some immune findings that can be inflamed with endometriosis or factor ectopic uterus. meets the.
在本發明說明中所引述之文獻均以參考資料的方式併 入本案。 本發明其他的特徵及優點將可明顯見於下列較佳具體 事實及申請專利範圍。〃 實例 下列實施例用於示範說明本發明。這些實施例不以任 何方式意欲限制本發明之範圍，但用於指示如何實施本發 明的材料及方法。 $ 線 31MJ」聚羥基乙醢/去乙醯殼吝醣薄膜的製備 1·1·經由熱相分離方式構建聚羥基乙酸/去乙醯殼多醣雜合 基質發泡物 σ 在DMSO及乙酸混合物中，製備聚羥基乙酸/去乙醯殼 多醣（卩/〇)(81§11^，81：1〇1^，^10，1；8八)重量比分別為7〇%， 50%及30%的溶液。將透明的聚合物溶液倒入直徑1〇公分 的玻璃皿中，置於-20°C冷凍24個小時後，浸入1Ν氫氧化 鈉溶液中30分鐘以便去除溶劑，最後進行真空乾燥5 99未紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公t ) 200407434 A7 ___ B7 " - ' ' ----—— 五、發明說明（丨>) 時。 1 ·2·經由溶劑模造顆粒濾出方式構建聚羥基乙酸/去乙醯殼 多醣雜合基質發泡物 ' 在DMSO及乙酸混合物中，製備聚羥基乙酸/去乙醯殼 多醣（Sigma ’ St· Louis ’ MO，USA)重量比分別為 70%，50% 及3 0°/。的溶液。將透明的聚合物溶液倒入直徑} 〇公分的玻 璃jni中，置於-20°C冷凍24個小時後，浸入i Ν氫氧化鈉 溶液中3 0分鐘以去除溶劑，最後進行真空乾燥5小時以去 除殘餘溶劑。 1.3.薄膜的製備 聚羥基乙酸/去乙醯殼多醣薄膜使用真空乾燥法製 得。將聚羥基乙酸聚合物溶液（8%wt)與同體積的去乙醯殼 多醣溶液（2%wt)混合。將溶液倒入内徑3公分的盤中，然 後在真空乾燥24小時形成薄膜。那些薄膜在經蒸餾的去離 子水中清洗表面。 實例2 :掃瞄式電子顯微鏡（SEN/Π掄杏 膜鷹架以2.5%戊二醛固定。檢體使用自5〇%至1〇〇% 的乙醇逐步脫水，每步驟約5分鐘。最後將檢體以臨界點 乾燥機（Hitachi HCP-2臨界點乾燥機，Hitachi，東京，曰 本）乾燥，在不同的平面切片，以金進行喷濺塗覆（Hitachi HUS_5GB高度真空揮發器，Hitachi，東京，曰本），以掃 瞄式電子顯微鏡（SEM，Hitachi S_2400，Hitachi，東京，曰 qcn-—---1--- 14 •〜本紙張尺度適用中國國家標準（CNS)A4規袼（210 X 297公t ) (請先閱讀背面之注意事項再填寫本頁) 訂： 線 200407434 A7 五、發明說明（Θ) 本)觀察。 使用SEM去定性_基乙叫乙《多ϋ雜合基f ^田構造’藉此檢視藉不同製造方法和不同聚經基乙酸/ 去乙醯殼多醣重量濃度所形成基質的内部。針對根據實例 M.所述方法（經由熱相分離方式構建聚羥基乙酸/去乙醯 殼多酶雜合基質發泡物）製得的鹰架。橫切面圖顯示雖然 50父互連接的微孔出現在内部，但是在鹰架底面有幾乎 緻也的皮膚層。這些結果示範，以熱相分離方式製造的聚 經基乙酸/去乙醯殼多膽雜合基質，不同的成分卻展現類似 的開放***互連接的孔結構，只有在表面和底面的孔尺寸 不同。 、針對根據貝合J 1.2.所述方法（經由溶劑模造顆粒滅出 方式構建聚經基乙酸/去乙酿殼多醣雜合基質發泡物）製得 的鹰架。肖經由熱相分離方式構建方法製備的檢體相反， 該對稱性膜具較小的孔尺寸。孔徑自^至5()_，與葡 萄糖粒子的散佈大小間有相關性。葡萄糖種類對於多孔性 及孔徑沒有影響。發泡物多孔性質與所使用的粒子無關， 僅為聚羥基乙酸/去乙醯殼多醣相對量和粒子尺寸的函 數。 熱性質之定性 使用鑑別式掃瞄熱量計（DSC821 ， mettlertoledo，瑞士）檢查所製得鷹架和薄膜的熱性 質。將5至8毫克之聚合物檢體置於密封DSC鋁檢體平盤 99秦紙張尺度適用中_冢標準（CNS)A4規格巧1() χ 297公|^ (請先閲讀背面之注意事項再填寫本頁)The documents cited in the description of the present invention are incorporated into the present case by reference. Other features and advantages of the present invention will be apparent from the following preferred specific facts and the scope of patent applications. 〃 Examples The following examples are used to illustrate the present invention. These examples are not intended to limit the scope of the invention in any way, but are used to indicate how to implement the materials and methods of the invention. $ 线 31MJ ″ Preparation of polyhydroxyacetic acid / deacetylated chitosan thin film 1.1 · Construction of polyglycolic acid / deacetylated chitosan hybrid matrix foam σ by thermal phase separation in DMSO and acetic acid mixture , Preparation of polyglycolic acid / deacetylamidine chitin (多糖 / 〇) (81§11 ^, 81: 101, ^ 10,1; 88) The weight ratios are 70%, 50% and 30%, respectively. The solution. Pour the transparent polymer solution into a 10 cm diameter glass dish, freeze at -20 ° C for 24 hours, then immerse it in 1N sodium hydroxide solution for 30 minutes to remove the solvent, and finally vacuum dry 5 99 paper The standard is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 metric t) 200407434 A7 ___ B7 "-'' -------- V. Description of the invention (丨 >). 1 · 2 · Construction of polyglycolic acid / chitosan hybrid matrix foam through solvent-molded particle filtration method 'In the mixture of DMSO and acetic acid, polyglycolic acid / chitosan (Sigma' St · Louis' MO, USA) weight ratios are 70%, 50% and 30 ° /. The solution. The transparent polymer solution was poured into glass jni with a diameter of 〇 cm, frozen at -20 ° C for 24 hours, immersed in a sodium hydroxide solution for 30 minutes to remove the solvent, and finally vacuum-dried for 5 hours. To remove residual solvents. 1.3. Preparation of the film Polyglycolic acid / chitosan film was prepared by vacuum drying method. The polyglycolic acid polymer solution (8% wt) was mixed with the same volume of the deacetylated chitosan solution (2% wt). The solution was poured into a 3 cm inner diameter dish and then dried under vacuum for 24 hours to form a film. Those films were washed in distilled deionized water. Example 2: Scanning electron microscope (SEN / Π apricot membrane scaffold is fixed with 2.5% glutaraldehyde. The specimen is gradually dehydrated with 50% to 100% ethanol, each step is about 5 minutes. Finally, Specimens were dried with a critical point dryer (Hitachi HCP-2 critical point dryer, Hitachi, Tokyo, Japan), sliced on different planes, and spray-coated with gold (Hitachi HUS_5GB high vacuum volatilizer, Hitachi, Tokyo Scanning electron microscope (SEM, Hitachi S_2400, Hitachi, Tokyo, qcn ---- 1 --- 14) • This paper size applies the Chinese National Standard (CNS) A4 Regulation (210 X 297 male t) (Please read the notes on the back before filling in this page) Order: Line 200407434 A7 V. Description of the invention (Θ) This is observed. Use SEM to characterize the _____ ^ Tian Tectonics' to view the inside of the matrix formed by different manufacturing methods and different polyacetic acid / chitosan weight concentrations. For the method according to Example M. (Construction of polyglycolic acid via thermal phase separation / Deacetylated shell multi-enzyme hybrid matrix foam) The cross-sectional view shows that although 50 interconnected micropores appear in the interior, there is almost a skin layer on the bottom of the scaffold. These results demonstrate that the polyacetic acid / deacetamidine produced by thermal phase separation Shell-bile hybrid matrix, but different components exhibit similar open and interconnected pore structure, only the pore size on the surface and the bottom surface are different. For the method according to Behe J 1.2. The scaffold made of polyacetic acid / deacetylated chitin hybrid matrix foam was constructed in the same way. In contrast, the specimen prepared by the thermal phase separation method has a smaller pore size. The pore size ranges from ^ to 5 () _, and has a correlation with the size of the glucose particles. The type of glucose has no effect on the porosity and pore size. The porous nature of the foam is independent of the particles used, only polyglycolic acid / deacetylated As a function of the relative amount of chitin and particle size. Qualitative thermal properties The thermal properties of the scaffolds and films produced were examined using a discriminating scanning calorimeter (DSC821, mettlertoledo, Switzerland). 5 to 8 mg of polymer sample is placed on a sealed DSC aluminum sample flat plate 99 Qin paper standard is applicable _ Tsukazumi (CNS) A4 size 1 () 297 297 | (Please read the precautions on the back first (Fill in this page)
200407434 A7 五、發明說明（丨(/〇 裡，在電子天平型號HF_3000(AND，日本）上被精確稱重。 檢體平盤使用檢體包膜壓製法密封，並將空的密封平盤作 為對照組。首先將檢體和對照組在3〇〇c平衡，隨後將檢體 以5.0C/分鐘的加熱速率自3〇。〇加熱到25(rc。DSC可用 於聚合物的熱轉換的定量，例如熔融和玻璃態轉換；亦用 於決定在鷹架製造期間是否發生聚合物結構的任何改變。 圖1顯示DSC掃瞄，包括純聚羥基乙酸和去乙醯殼多 醣的記錄。聚羥基乙酸的一個熔融波峰在2〇〇-22〇。〇之間， 其與文獻數據（223-233。〇—致。去乙醯殼多醣的熔融波峰 為80 C。本發明發現，經由熱相分離方式與經由溶劑模造 顆粒濾出方式在混合過程中有相對氫鍵之現象。從聚羥基 乙酸，去乙醯殼多醣和葡萄糖構成，指出在聚合物之間形 成氫鍵的數目。依去乙醯殼多醣的含量，在去乙醯殼多醣 的結構中有較多的立體障礙會影響分子間或分子内氫鍵。 氫鍵形式結果與DSC掃描結果一致。圖2顯示，由Dsc 掃描來看，不同的構建方法及不同的聚羥基乙酸/去乙醯殼 多醣（P/C )重量具有影響力。70% p/c基質有三個波峰約 在125 C，15(TC和200。〇。50% P/C基質有三個波峰約在 120°C，148。(：和 20(TC。30。/〇 P/C 基質只有在 132。(：有一個 波峰。熔融波峰信號與聚羥基乙酸/去乙醯殼多醣相對量有 關。在200°C的信號來自聚羥基乙酸。只有3〇% p/c基質 有一個波峰，指出在聚羥基乙酸和去乙醯殼多醣份量間存 在相對比例關係。 ί請先閱讀背面之注意事項再填寫本頁) · •線 經由熱相分離方式與經由溶劑模造顆粒濾出方式所構200407434 A7 V. Description of the invention (丨 (/ 〇, is accurately weighed on the electronic balance model HF_3000 (AND, Japan). The specimen flat plate is sealed with the specimen envelope compression method, and the empty sealed flat plate is used as the Control group. The specimen and control group were first equilibrated at 300 ° C, and then the specimen was heated from 5.0 ° C to 25 ° C at a heating rate of 5.0C / min. DSC can be used to quantify the thermal conversion of polymers. , Such as melting and glass transition; it is also used to determine whether any changes in the polymer structure occurred during the fabrication of the scaffold. Figure 1 shows a DSC scan including records of pure polyglycolic acid and deacetylated chitin. Polyglycolic acid One melting peak is between 200 and 220.0, which is consistent with the literature data (223-233.00). The melting peak of desacetin chitosan is 80 C. The present invention finds that it can be separated by thermal phase. There is a phenomenon of relative hydrogen bonding in the mixing process with the way of filtering out the particles by solvent molding. From polyglycolic acid, deacetylated chitosan and glucose, indicate the number of hydrogen bonds formed between the polymers. Polysaccharide content in There are more steric obstacles in the structure of acetamidine chitin, which will affect intermolecular or intramolecular hydrogen bonding. The results of hydrogen bonding are consistent with the results of DSC scanning. Figure 2 shows that from the Dsc scanning, different construction methods and different Polyglycolic acid / chitosan (P / C) weight has an influence. 70% p / c matrix has three peaks at about 125 C, 15 (TC and 200. 50% P / C matrix has three peaks At about 120 ° C, 148. (: and 20 (TC. 30. / 〇P / C matrix only at 132. (: There is a peak. The melting peak signal is related to the relative amount of polyglycolic acid / desacetyl chitosan. The signal at 200 ° C comes from polyglycolic acid. Only 30% p / c matrix has a peak, indicating that there is a relative proportional relationship between the polyglycolic acid and the amount of chitosan. Ί Please read the precautions on the back first (Fill in this page) • • The wire is constructed by thermal phase separation and filtration by solvent-molded particles
200407434 A7 ___ B7 _ 五、發明說明（[Γ) 建的產物有不同的結果。70% P/C/G基質只有一個在約120 °(：的波峰。50%P/C/G基質在約130°C和140°C分別有波 峰。30%P/C/G基質在約86°C，113°C和143°C分別有三個 波峰。經由熱相分離方式所得產物中，30% P/C/G之混合 程度比50% P/C/G和70% P/C/G好；經由溶劑模造顆粒濾 出方式所構建的產物中，70% P/C/G之混合程度比30% P/C/G和50% P/C/G好。可想而知，在這個過程中加入的 葡萄糖會與去乙醯殼多醣競爭而與聚羥基乙酸形成氫鍵。 實例4 :雜合基質多孔性的測定 · 鷹架的多孔性依下列公式計算：200407434 A7 ___ B7 _ 5. Description of the invention ([Γ) The products produced have different results. 70% P / C / G matrix has only one peak at about 120 ° (:. 50% P / C / G matrix has peaks at about 130 ° C and 140 ° C, respectively. 30% P / C / G matrix has peaks at about 130 ° C and 140 ° C, respectively. There are three peaks at 86 ° C, 113 ° C and 143 ° C. Among the products obtained by thermal phase separation, the mixing ratio of 30% P / C / G is 50% P / C / G and 70% P / C / G is good; 70% P / C / G is better than 30% P / C / G and 50% P / C / G in the product constructed by the solvent-molded particle filtration method. It is conceivable that in The glucose added in this process will compete with deacetylammonium chitin and form hydrogen bonds with polyglycolic acid. Example 4: Determination of the porosity of the hybrid matrix · The porosity of the scaffold is calculated according to the following formula:
Dx A-M/ 1 · 多孔性（％)= = —Β~χ/Ρρ χ100〇/ο vm和νρ分別為基質體積和被聚合物占領的體積。ρ ρ 是基質的密度。在測量區Α之後，可以估算質量Wm和基 質厚度D以求得總多孔性。去乙醯殼多醣的密度為0.942 公克/公分3,且純聚羥基乙酸的密度是1.723公克/公分3。 經計算後70% P/C的密度是1.609，且50% P/C及30% P/C 的密度分別為是1.532與1.456。 將實例1所得不同的基質依上列等式計算出多孔性， 結果見於圖3。大體上，所有三種重量比以及不論熱相分 離方式或溶劑模造顆粒濾出方式所構建的產物，鷹架的多 孔性都大於40%。多孔性隨聚羥基乙酸/去乙醯殼多醣的混 合重量比增加而增加，而且在兩種不同的製造方法中均可 L—------17 __ 9珠纸張尺度適用中國國家標準（CNS)A4規格（21〇 X 297公釐） (請先閱讀背面之注意事項再填寫本頁)Dx A-M / 1 · Porosity (%) = = —β ~ χ / ρρ χ100〇 / ο vm and νρ are the volume of the matrix and the volume occupied by the polymer, respectively. ρ ρ is the density of the matrix. After the measurement area A, the mass Wm and the matrix thickness D can be estimated to obtain the total porosity. The density of deacetylated chitin was 0.942 g / cm3, and the density of pure polyglycolic acid was 1.723 g / cm3. After calculation, the density of 70% P / C is 1.609, and the density of 50% P / C and 30% P / C are 1.532 and 1.456, respectively. The porosity of the different substrates obtained in Example 1 was calculated according to the above equation. The results are shown in FIG. 3. In general, the porosity of the scaffold is greater than 40% for all three weight ratios and the products constructed regardless of the thermal phase separation method or the solvent-molded particle filtration method. The porosity increases with the increase in the weight ratio of polyglycolic acid / desacetin chitosan, and it can be used in two different manufacturing methods. L —------ 17 __ The size of 9-bead paper is applicable to Chinese national standards (CNS) A4 specification (21〇X 297 mm) (Please read the precautions on the back before filling this page)
200407434 A7 _B7__ 五、發明說明（lb ) 高到90%附近。 實例5 :雜合基質的機械性質測定 在乾燥態的機械性質測定之前，將基質儲存於室溫。 為了研究雜合基質的機械性質，將基質個別切割為4公分 XI公分小塊。將樣本以5公釐/分鐘的速度放置在二個架 設盤（拉力儀器：LRX型，LLOYD儀器，美國）之間的薄膜 承接器上。 針對相異方法構建及聚羥基乙酸和去乙醯殼多醣相異 重量比所製得的雜合基質測量Young’s模數，最大負載和 硬度。這些結果顯示雜合基質之機械性質隨著聚羥基乙酸 從30%增到70%而增加。最大的負載值從70% P/C之近3 N 增加到30% P/C之13 N。此結果指出在混合時使用聚羥基 乙酸可改善機械硬度。 實例6 :雜合基質水含量之測量 基質的水含量被定義為在水飽和的基質裡的水重量百 分比。基質的水含量測量如下··將每個基質在室溫下浸濕， 並重複地稱重直到得到脹大基質的恆定重量。隨後將基質 在真空下乾燥至怪定重量。解釋基質水含量之待測定的二 個重要參數，分別為多孔性及在水分子和基質之間形成氫 鍵。 如圖4所示，基質水含量之膨脹率約在200到500% 的範圍。70% P/C/G的膨脹率比50% P/C，30% P/C和70% __18__ 9衡紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ----I--— II----· I I (請先閱讀背面之注意事項再填寫本頁) · --線- 200407434 A7 _____ B7 五、發明說明（) P/C要快且高。 t例7:雜合某質的活體外降解 將聚羥基乙酸/去乙醯殼多醣雜合基質被放在15毫升 瓶中，每瓶包含0·2Μ，pH 7.4的磷酸鹽生理緩衝液（PBS)。 將體被儲放在搖盛機上，搖盘條件為5〇轉/分及37。〇下 進行隶長到到3 5天的不同時間。將瓶密封以避免在長時間 試驗當中液體部份揮發。 為了要正確地定義雜合基質的降解模式，因此發展快 速HPLC方法而可以同時測定降解終產物降解（也就是羥基 乙酸）。在此研究中，經由直接同時測定雜合基質水解當中 被釋放在培育培養基中經基乙酸單體來決定降解特質。數 天後’取出少量（〇·5毫升）緩衝液並離心（5〇〇〇轉/分）;將 所得上清液的一部份直接注射於HPLC管柱上。在培育緩 衝液中所釋放的經基乙酸使用jasc〇儀器（型號uv一 970/975 ’不同的波長偵測器和型號ρυ_98〇泵，&⑶，東 京日本）和HPLC管柱（十八烷基（C18)，4.6X250毫米，j. T.Baker’美國）。流動相是2%(仏)在磷酸鹽緩衝劑中的甲 醇’其包含0.002M蛾代四丁基鈹。色層分析法以〇·6毫升 /分鐘的流速進行，且洗提物的吸光度在21〇毫微米檢測。 L基乙&L峰巔的石隹認係精由色層分析法使用經認證的標 準。經基乙酸的定量值藉由使用外在標準化方法將波峰區 積分而得。降解情況係以溶液中羥基乙酸重量百分比而加 以定義。 ^本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公董) " ---- 1 I I I---· I 1 (請先閱讀背面之注意事項再填寫本頁) 訂： i線· 200407434 A7 ----------B7 "---------- 五、發明說明（/g ) • I----— — — — — — — — --- (請先閱讀背面之注意事項再填寫本頁) 以上述hplc戶斤決定的雜合基質之活體外降_能， 見於圖5。、經35天暴露後，70% P/C冑架雜合基失 近8 0 %的經基乙酸。經熱相分離方式或溶劑模造顆粒渡出 方式構建的|物所釋放的經&乙酸具有類似曲線和降解速 率。所有的雜合基質的大體外觀在降解期間隨時間改變。。 :雜合基質的接觸角度測詈 聚羥基乙酸/去乙醯殼多醣雜合基質和組織培養級聚 笨乙烯（tissue culture polystyrene，TCPS)使用影像接觸角系 統（型號SZ-ST，Olympus，日本）測量水接觸角度。水接觸 角度在乾燥受質上測量。 _線. P/C和那P/C/G基質的水接觸角度變化從50% P/C之 近20°到30% P/C/G之70。。水接觸角是受質親水性 (hydrophilicity)的測量，較大親水性受質有比較小的水接觸 角。經熱相分離方式或溶劑模造顆粒濾出方式所構建的雜 合基質之接觸角度均低於TCPS(83。），即具較大的親水 性。 實例9 :雜合基質的細胞黏附研究 對於細胞黏附的動力學的評估，將5x105細胞/平方公 分密度的纖維母細胞細胞植於TCPS和聚經基乙酸/去乙醯 殼多醣雜合基質上。雜合基質溫和地以漢克生理緩衝液溶 液（HBSS，Life Technologies)沖洗，且黏附細胞數目藉由使 用錐藍染色細胞而予以定量 L·____20____ Γ "本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -- 200407434 A7 -—- _B7__ 五、發明說明（1 ) 與其他的雜合基質相比，植於70% P/C上的纖維母細 胞細胞生存能力被發現較高（對照組為植於TCPS上的細 胞）。排列順序為：70% P/C > 50% P/C > 70% P/C/G > 30% P/C > 50% P/C/G > 30% P/C/G。 宽i列10 :細胞相容性試驗 將滅菌膜片2x2公分放入6-孔培養盤（IWAKI，日本） 中’並以TCPS作為對照組。將纖維母細胞細胞以ΐχΐ〇5 細胞/平方公分密度植於基質和對照組孔中。檢測細胞形態 學和黏附，其在被連接到倒置相位差顯微鏡（1X70， Olympus，日本）的〇τ影像分析系統上進行。以CCD影像 照相機（彩色照相機型號VK-C370, Hitachi，曰本）捕捉影 像並得到影像（Optimas 5.2，Optimas，美國）。 受質對於在第24小時培養後對於細胞形態造成影 響。細胞形態學顯微相片顯示P/C和P/C/G雜合基質上分 佈延伸紡錘體形式的細胞。如圖6所示，在24小時培養後 黏附到所有聚合物薄膜的纖維母細胞數目與黏附到TCPS 對照組受質者相仿。在所有雜合基質中，70% P/C基質在 培養48小時後出現最大程度的纖維母細胞細胞黏附。令人 驚訝地，70% P/C基質不只親水性較大，且較多細胞黏附 其上。 實例11 :使用nt唑i_(Mrn試驗測試細胞毒性 將膜濾出產品加入DMEM中並經由0.22微米過濾器 _________21____ 0雜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 I - -線. 200407434 A7 ___Β7_ 五、發明說明) (Millipore，USA)過濾。以不同濃度使用來試驗其對細胞生 存能力的效應。將纖維母細胞細胞植入在24-孔碟中，且 於37t及5% C02、95%空氣下培育24小時。隨後將細胞 曝於不同濃度（10%-50%)的膜濾出產品達24小時。_唑藍 (MTT)試驗依照標準程序進行來評估細胞生存能力。膜經 由MTT估計之細胞毒性示於圖7。 實例12 :聚經基乙酸/去乙醯殼多醣雜合基質對生長 抑制之影響 12.1細胞、培養液與培養技術 初級子宮内膜異位細胞由具有生育力的女性病人之子 宮内膜異位症的組織切片中分離出來。子宮内膜異位症組 織的使用是經由在VGHTPE的婦科醫學部門所允許使用。 組織樣本（1-2公克）轉移至實驗室的DMEM-H培養液中， 其内含有高量的葡萄糖（Life Techconologies，Inc.，U.S.A)與 5%FBS(Life Techconologies，Inc·，U.S.A)，並加入 2X 抗生 素與抗黴劑，使最終的濃度為200單位的盤尼西林 (penicillin)，0.2克的鏈黴素，與0.5//g的二性黴素B/ml (Antibiotic/antimycotic solution ; Sigma，St.Louis，MO) 0 組織以Hanhs BSS溶液沖洗，以去除血液和。經溫和 的離心（600X g)移除上清液，將組織放置在一 300-mm的塑 膠組織培養皿上（Corning-Costar，Cambridge，MA)。最後的 產物在無菌操作台上操作。組織以滅菌的手術刀切成1 -mm2 的小塊，而後以膠原酶（2mg/ml; Sigma，St· Louis，MO) 紙張尺度適用中國國家標準（CNS)A4規格（210 χ 297公釐） (請先閲讀背面之注意事項再填寫本頁)200407434 A7 _B7__ 5. The description of the invention (lb) is as high as 90%. Example 5: Determination of mechanical properties of hybrid substrates Prior to determination of mechanical properties in the dry state, the substrates were stored at room temperature. In order to study the mechanical properties of the hybrid matrix, the matrix was individually cut into 4 cm XI cm pieces. The sample was placed at a speed of 5 mm / min on a film holder between two mounting plates (pulling instrument: LRX model, LLOYD instrument, USA). The Young's modulus, maximum load, and hardness were measured for heterogeneous matrices prepared by dissimilar methods and heterogeneous weight ratios of polyglycolic acid and deacetylated chitosan. These results show that the mechanical properties of the hybrid matrix increase as the polyglycolic acid increases from 30% to 70%. The maximum load value increased from nearly 3 N at 70% P / C to 13 N at 30% P / C. This result indicates that the use of polyglycolic acid during mixing improves mechanical hardness. Example 6: Measurement of water content of hybrid substrate The water content of a substrate is defined as the weight percentage of water in a water-saturated substrate. The water content of the substrate was measured as follows: Each substrate was soaked at room temperature and repeatedly weighed until a constant weight of the expanded substrate was obtained. The substrate was then dried to a strange weight under vacuum. Two important parameters to be determined to explain the water content of the matrix are porosity and the formation of hydrogen bonds between water molecules and the matrix. As shown in Fig. 4, the expansion rate of the water content of the substrate is in the range of about 200 to 500%. The expansion ratio of 70% P / C / G is 50% P / C, 30% P / C and 70%. __18__ 9 scale paper size is applicable to Chinese National Standard (CNS) A4 (210 X 297 mm) ---- I --- II ---- · II (Please read the notes on the back before filling this page) · --- 200407434 A7 _____ B7 V. Description of the invention () P / C should be fast and high. Example 7: Degradation of a hybrid substance in vitro The polyglycolic acid / chitosan hybrid matrix was placed in a 15 ml bottle, each bottle containing 0.2 M phosphate buffer (PBS, pH 7.4 ). The body was stored on a shaker, and the shaker conditions were 50 rpm and 37. 〇2 Under different periods from 3 to 5 days. Seal the bottle to avoid volatilization of liquids during prolonged testing. In order to correctly define the degradation mode of the hybrid matrix, a rapid HPLC method was developed to simultaneously measure degradation of degradation end products (ie, glycolic acid). In this study, the degradation characteristics were determined by the simultaneous simultaneous determination of the glycoacetic acid monomer released in the incubation medium during the hydrolysis of the hybrid matrix. After a few days, a small amount (0.5 ml) of the buffer was removed and centrifuged (5,000 rpm); a portion of the resulting supernatant was injected directly onto the HPLC column. Glycolic acid released in the incubation buffer was using a JASCO instrument (model UV-970 / 975 'different wavelength detector and model ρ_98 pump, & ⑶, Tokyo Japan) and HPLC column (octadecane (C18), 4.6 x 250 mm, j. T. Baker 'USA). The mobile phase was 2% (仏) methanol 'in phosphate buffer, which contained 0.002M moth tetrabutyl beryllium. Chromatographic analysis was performed at a flow rate of 0.6 ml / min, and the absorbance of the eluate was detected at 210 nm. The L-Bei & L Peak's Recognition System uses certified standards using chromatographic analysis. The quantitative value of glyoxylic acid was obtained by integrating the peak region using an externally standardized method. Degradation is defined as the weight percent of glycolic acid in the solution. ^ This paper size applies to China National Standard (CNS) A4 (210 X 297 public directors) " ---- 1 II I --- · I 1 (Please read the precautions on the back before filling this page) Order: i-line · 200407434 A7 ---------- B7 " ---------- V. Description of the invention (/ g) • I ----— — — — — — — — --- (Please read the precautions on the back before filling out this page) The in vitro energy reduction of the hybrid matrix determined by the above hplc households is shown in Figure 5. After 35 days of exposure, 70% of the P / C stilbene hybrids lost nearly 80% of the acetic acid. The & acetic acid released by the thermal phase separation method or the solvent-molded particle escape method has a similar curve and degradation rate. The general appearance of all hybrid matrices changes over time during degradation. . : Measurement of contact angle of hybrid matrix 基质 Polyglycolic acid / chitosan hybrid matrix and tissue culture polystyrene (TCPS) using imaging contact angle system (model SZ-ST, Olympus, Japan) Measure the water contact angle. The water contact angle is measured on a dry substrate. _Line. The water contact angle of the P / C and that of the P / C / G substrate varies from approximately 20 ° at 50% P / C to 70 at 30% P / C / G. . Water contact angle is a measure of hydrophilicity. Larger hydrophilic substrates have smaller water contact angles. The contact angle of the hybrid matrix constructed by the thermal phase separation method or the solvent-molded particle filtration method is lower than TCPS (83.), that is, it has greater hydrophilicity. Example 9: Cell Adhesion Study of Hybrid Matrix For the assessment of the kinetics of cell adhesion, 5x105 cells / cm² density fibroblast cells were implanted on TCPS and polyacetic acid / deacetylamidine chitin hybrid matrices. The hybrid matrix was gently washed with Hank's physiological buffer solution (HBSS, Life Technologies), and the number of adherent cells was quantified by staining the cells with cone blue. L · ____ 20____ Γ " This paper size applies Chinese National Standard (CNS) A4 size (210 X 297 mm)-200407434 A7 ------ _B7__ V. Description of the invention (1) Compared with other hybrid matrices, the viability of fibroblast cells planted on 70% P / C was found Higher (control group was cells implanted on TCPS). Sort order: 70% P / C > 50% P / C > 70% P / C / G > 30% P / C > 50% P / C / G > 30% P / C / G . Column i 10: Cytocompatibility test Put a sterilized membrane 2x2 cm into a 6-well culture plate (IWAKI, Japan) 'and use TCPS as a control group. Fibroblast cells were implanted in the matrix and control wells at a density of χ × 05 cells / cm 2. Cell morphology and adhesion were measured on a ττ image analysis system connected to an inverted phase contrast microscope (1X70, Olympus, Japan). A CCD image camera (color camera model VK-C370, Hitachi, Japanese) was used to capture and obtain images (Optimas 5.2, Optimas, USA). The substrate affected the cell morphology after 24 hours of incubation. Cell morphology photomicrographs show cells in the form of extended spindles distributed on P / C and P / C / G hybrid matrices. As shown in Figure 6, the number of fibroblasts adhered to all polymer films after 24 hours of culture was similar to that of TCPS control subjects. Among all hybrid matrices, 70% P / C matrix exhibited the greatest degree of fibroblast cell adhesion after 48 hours in culture. Surprisingly, the 70% P / C matrix is not only more hydrophilic, but also more cells adhere to it. Example 11: Use ntazole i_ (Mrn test to test cytotoxicity. Add membrane filtration products to DMEM and pass through a 0.22 micron filter _________21____ 0 Miscellaneous paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) Please read the notes on the back before filling in this page) Install I--line. 200407434 A7 ___ Β7_ V. Description of the invention) (Millipore, USA) filter. Use at different concentrations to test its effect on cell viability. Fibroblast cells were implanted in 24-well dishes and incubated for 24 hours at 37t, 5% CO2, and 95% air. The cells were then exposed to membrane filtration products of different concentrations (10% -50%) for 24 hours. The azole blue (MTT) test is performed according to standard procedures to assess cell viability. The cytotoxicity estimated by the membrane via MTT is shown in Figure 7. Example 12: Effect of polyacetic acid / desacetin chitosan hybrid matrix on growth inhibition 12.1 Cells, culture fluids and culture techniques Primary endometriosis cells Endometriosis from a female patient with fertility Tissue sections were isolated. Endometriosis tissue is used with permission from the Gynecological Department of VGHTPE. Tissue samples (1-2 g) were transferred to the laboratory's DMEM-H broth, which contained high amounts of glucose (Life Techconologies, Inc., USA) and 5% FBS (Life Techconologies, Inc., USA), 2X antibiotics and antimycotics were added to make the final concentration of 200 units of penicillin, 0.2 grams of streptomycin, and 0.5 // g of amphotericin B / ml (Antibiotic / antimycotic solution; Sigma, St. Louis, MO). Tissues were rinsed with Hanhs BSS solution to remove blood and. The supernatant was removed by gentle centrifugation (600X g), and the tissue was placed on a 300-mm plastic tissue culture dish (Corning-Costar, Cambridge, MA). The final product is handled on a sterile operating table. The tissue was cut into 1-mm2 small pieces with a sterilized scalpel, and then collagenase (2mg / ml; Sigma, St. Louis, MO) was used as the paper standard of China National Standard (CNS) A4 (210 x 297 mm). (Please read the notes on the back before filling this page)
200407434 A7 _ - __B7___ 五、發明說明) 在37°C的DMEM-H培養液中消化2.5小時，並將其放置在 震盪器上。經消化後的組織塊，以吸量管劇烈的上下反覆 吸取，而後放置到一滅菌篩孔其為# 100篩孔，另外用一 個# 400篩孔（37//m)。將已消化的子宮内膜異位細胞放置 篩孔的表層，當過濾基質細胞時，上皮組織的腺體將保留 在#100與# 400篩孔。 培養初級細胞所用的培養液内含有Ml99及F12 (1:1) 並在其中加入孕酮（Sigma，St· Louis, MO)，ITS+[内含胰 蛋白酿（0.62//忌/1111)，運鐵蛋白（〇_62//运/1111)，及石西（〇.62 //2/1111)，牛血清白蛋白（125//2/1111)與亞麻油酸（52.6// g/ml)] ’並加入100單位的盤尼西林，〇· ig的鏈黴素與〇. 25 // g — 性黴酗 /ml(antibiotic/ antimycotic solution，200407434 A7 _-__B7___ V. Description of the invention) Digest in DMEM-H medium at 37 ° C for 2.5 hours and place it on a shaker. The digested tissue pieces were sucked up and down vigorously with a pipette, and then placed in a sterilizing sieve with a # 100 sieve and another with a # 400 sieve (37 // m). Place the digested endometriotic cells on the surface of the sieve hole. When filtering the stromal cells, the glands of the epithelial tissue will remain in the # 100 and # 400 sieve holes. The medium used to culture the primary cells contains Ml99 and F12 (1: 1), and progesterone (Sigma, St. Louis, MO) is added to it. ITS + [contains tryptin (0.62 // bogey / 1111). Ferritin (〇_62 // 运 / 1111), and Shixi (〇.62 // 2/1111), bovine serum albumin (125 // 2/1111) and linoleic acid (52.6 // g / ml )] 'And add 100 units of penicillin, ig streptomycin and 0.25 // g — antifungal / ml (antibiotic / antimycotic solution,
Sigma)。培養液每 3-4 天更換一次（Aronld JT，Kaufman DG，Sigma). The culture medium is changed every 3-4 days (Aronld JT, Kaufman DG,
Seppala M, Lessey BA. Hum Reprod 2001 May ; 16(5):836-45)。 人類皮膚纖維母細胞以含有l〇%FCS的DMEM培養液培 養，並將其放置在含有5%二氧化碳潮濕空氣中的37。〇培養 箱。 12.2化學物質處理 在本發明中，發明者使用兩種型式的聚羥基乙酸/去乙 醯殼多醣（P/C)雜合基質。一種為P/c雜合基質a其為pGA 溶液（24%重量百分比）與聚甲殼糖溶液混合（2%重量百分 比）。另一種型式為P/C雜合基質B，其為聚羧乙酸/聚甲 叙糖的緊密薄膜，於PBS溶液中可溶且可以冊％溶液調整 --—--- 2^_ tW.5本紙張尺度適用中國國家標準（CNS)A4規格（210 x 297公爱）--—----- (請先閱讀背面之注意事項再填寫本頁)Seppala M, Lessey BA. Hum Reprod 2001 May; 16 (5): 836-45). Human skin fibroblasts were cultured in DMEM medium containing 10% FCS and placed in 37% humidified air containing 5% carbon dioxide. 〇 Incubator. 12.2 Chemical Substance Treatment In the present invention, the inventors used two types of polyglycolic acid / desacetylacetin (P / C) hybrid matrix. One is a P / c hybrid matrix a which is a pGA solution (24% by weight) mixed with a polychitosan solution (2% by weight). The other type is P / C hybrid matrix B, which is a compact film of polyacetic acid / polymethylose, which is soluble in PBS solution and can be adjusted by% solution. ------- 2 ^ _ tW.5 This paper size applies to China National Standard (CNS) A4 specification (210 x 297 public love) ----------- (Please read the precautions on the back before filling this page)
200407434 A7 ---------B7___ _ 五、發明說明 PH值。當用來處理子宮内膜異位細胞時，p/c雜合基質△ 則以培養液作連續的稀釋濃度。當使用P/C雜合基質A時， 八為使用培養液連績稀釋產生不同的濃度，而後在使用於 子宮内膜異位細胞。將以不同濃度之p/c雜合基質A處理 之子s内膜異位細胞，以1χ1〇6細胞數/毫升培養在6孔的 培養盤中。在實施各類分析前，細胞之培養維持2-了天。 12. 3生長抑制分析 經由WST-1方法分析因P/c雜合基質Α對生長細胞之 衫響（cell proliferation assay kit, Chemicon International Inc· CA)。簡而言之，將5xl04細胞數培養 在96孔的培養盤内24小時後，將培養液置換成一含有雜 合基質的培養液，細胞再經4天培養，而後將1〇 #丨的WST—i 溶液加入，共同再培養2小時。存活的細胞數經微盤讀值 機（microplate re ader)( Molecular Devices, Spectra MAX) 以450nm的波長測量。過去一般常使用之波長為600nm。 NA0(Nonyl acridine orange)與 PI(pr〇pidium iodiume)的 結合物’可以用來作細胞多樣性分期的刺激性流式細胞儀 分析。 12. 4結果 在WST-1的分析中，約有15%的細胞經96小時以P/C 雜合基質處理並與控制組比較下，其存活情形呈劑量-依賴 性降低，其最高濃度為500 //2/]111(圖8)。在p/c雜合基質 A濃度約250 /zg/ml沉澱雜合基質，以WST-1色度計分析。 而後發明者結合細胞存活率的流式細胞儀與螢光影像分 _______ ___ 24____ 00钵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) 訂· -線· 200407434 A7 _ B7_ 五、發明說明（>3 ) --------------· I I (請先閱讀背面之注意事項再填寫本頁) 析。P/C雜合基質A(500 //g/ml)對子宮内膜異位細胞的影 響與其時間間隔表示在圖9。經4天處理後期抑制速率近 乎20%。經4天於雜合基質A架雜合基質B培養後，存活 之細胞百分比約近於90%(圖8)，顯示經雜合基質A加入雜 合基質B後，細胞生長受到抑制。 實例13 :聚羥基乙酸/去乙醯殼多醣雜合基質誘發細 胞凋亡 13. 1經P / C雜合基質處理之細胞的型態分析 線· 細胞型態的改變可以由將早期計畫性細胞死亡得知， 經ΝΑ0染色具活性的粒腺體的步驟來分析（Ferlini C，Di Cesare S, Rainaldi G, Malorni W, Samoggis P, Biselli R， Fattorossi A. Cytometry 1996 Jun 1 ; 24(2):106-15。Vermes I， Haanen C, Steffens-Nakken H, Reutelingsperger C. J. Immunol Methods 1995 Jul 17 ; 184(1):39-51)，並經由一 遞增的膜滲透性的方式收集較晚期的細胞凋亡之細胞，其 可允許 PI 進入細胞（Koopman G，Reutelingsperger CP， KuijtenGA, Keehnen RM, Pals ST, van Oers MH. Blood 1994 Sep 1 ; 84(5):1415-20)。而ΝΑ0與PI的結合體可以用來做 細胞凋亡與細胞週期分布階段的流式細胞儀與螢光影像分 析。Flourescein(FITC) annexin V是一種綠色螢光嵌合 物，其可以延伸使用在實驗室中，用來偵測凋亡細胞的數 量。Flourescein annexin V常與PI結合用來偵測壞死細 胞，例如使用細胞凋亡彳貞測試劑組（Apoptosis Detection Kit)(R&D system, U.S.A)。將 2.5 x 105 細胞/ 毫升，經 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 200407434 A7 __—__B7______ 五、發明說明)200407434 A7 --------- B7___ _ 5. Description of the invention PH value. When used to treat endometriotic cells, the p / c hybrid matrix △ is continuously diluted with culture medium. When the P / C hybrid matrix A is used, eight different concentrations are used for serial dilution of the culture medium, and then used in endometriotic cells. The ectopic endometrial cells of the child s treated with the p / c hybrid matrix A of different concentrations were cultured in a 6-well culture plate at 1 x 106 cells / ml. Cell culture was maintained for 2-days before performing various analyses. 12. 3 Growth inhibition analysis The cell proliferation assay kit (Chemicon International Inc. CA) for P / c hybrid matrix A was analyzed by the WST-1 method. In short, 5xl04 cells were cultured in a 96-well culture plate for 24 hours, and the culture medium was replaced with a culture medium containing a hybrid matrix. The cells were cultured for another 4 days, and then the 10 # 丨 WST— Add the solution and incubate for another 2 hours. The number of surviving cells was measured at a wavelength of 450 nm using a microplate reader (Molecular Devices, Spectra MAX). The wavelength commonly used in the past is 600nm. The combination of NA0 (Nonyl acridine orange) and PI (propiadium iodiume) 'can be used for stimulating flow cytometry analysis of cell diversity staging. 12.4 Results In the analysis of WST-1, about 15% of the cells were treated with a P / C hybrid matrix for 96 hours and compared with the control group, their survival showed a dose-dependent decrease, and the highest concentration was 500 // 2 /] 111 (Figure 8). The hybrid matrix was precipitated at a p / c hybrid matrix A concentration of about 250 / zg / ml and analyzed by a WST-1 colorimeter. Then the inventor combined the flow cytometry of the cell survival rate with the fluorescence image analysis. _______ ___ 24____ The paper size of the 00 bowl is applicable to the Chinese National Standard (CNS) A4 (210 X 297 mm). (Please read the precautions on the back first. Fill in this page) Order · -line · 200407434 A7 _ B7_ V. Description of the invention (> 3) -------------- · II (Please read the precautions on the back before filling this page ) Analysis. The effect of P / C hybrid matrix A (500 // g / ml) on endometriotic cells and their time intervals are shown in Fig. 9. After 4 days of treatment, the inhibition rate was nearly 20%. After 4 days of culture on hybrid matrix A and hybrid matrix B, the percentage of viable cells was approximately 90% (Fig. 8), showing that after hybrid matrix A was added to hybrid matrix B, cell growth was inhibited. Example 13: Polyglycolic acid / deacetylamidine chitosan hybrid matrix induces apoptosis 13.1 Type analysis line of cells treated with P / C hybrid matrix · Changes in cell type can be determined by early planning Cell death was learned, and analysis was performed by ΑΝ0 staining of active granulocytes (Ferlini C, Di Cesare S, Rainaldi G, Malorni W, Samoggis P, Biselli R, Fattorossi A. Cytometry 1996 Jun 1; 24 (2) : 106-15. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger CJ Immunol Methods 1995 Jul 17; 184 (1): 39-51), and collect the later stage cells through an increasing membrane permeability. Dead cells that allow PI to enter the cells (Koopman G, Reutelingsperger CP, KuijtenGA, Keehnen RM, Pals ST, van Oers MH. Blood 1994 Sep 1; 84 (5): 1415-20). The combination of NA0 and PI can be used for flow cytometry and fluorescence image analysis at the stage of apoptosis and cell cycle distribution. Flourescein (FITC) annexin V is a green fluorescent chimera, which can be extended in the laboratory to detect the number of apoptotic cells. Flourescein annexin V is often used in combination with PI to detect necrotic cells, such as using the Apoptosis Detection Kit (R & D system, U.S.A.). Apply 2.5 x 105 cells / ml to the Chinese standard (CNS) A4 (210 X 297 mm) according to the paper size. 200407434 A7 __—__ B7______ 5. Description of the invention)
處理 500 // g/ml，250 // g/nU，125 // g/ml 與 25 // g/ml 的 P/C 雜合基質A後’培養在維持之條件下24小時。經四天的培 養，單層細胞以培養液清洗，而後將細胞培養再含有螢光 引子的培養液中，37°C含5%的二氧化碳濃度，而後以螢光 顯微鏡分析（AX-70, Olympus, Tokyo)。 13. 2 FITC-Annexin V和PI雙重染色之流式細胞儀分 析 以FITC-Annexin V和PI雙重染色作流式細胞儀分析。 經以PBS清洗兩次後，經處理與未經處理的1〇6細胞數於 室溫下離心收集。溫和的在Annexin V培養試劑下將細胞 打散，並將其培養在室溫下避光環境15分鐘。混合物以 FACS 伏特式流式細胞儀（FACS Vantage flow cytometer)和 Cel lQuest 軟體分析（Becton Dickinson)。 13. 3 DNA片段分析 細胞以2·5χ105細胞數/毫升的濃度培養在完全培養液 中，並經由維持條件24小時，在將培養液更新成含有連續 稀釋濃度之P/C雜合基質A或B，並培養72小時，經過此 步驟後，將細胞刮下並進行分析。以suicide-trackTMDNA ladder kit (Oncogene，ΜΑ)分析 DNA 片段。 13.4結果 在經4天培養之後，子宮内膜異位細胞經營光染劑 (ΝΑΟ與PI)後被染上明顯的綠色，與控制組比較下大於95% 的細胞可存活。85%以上的細胞被染成紅色，顯示在雜合基 質Α濃度500 # g/ml條件下培養4天後，大部分的細胞會 ______26__ β0參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) . --線_ 200407434After treatment of 500 // g / ml, 250 // g / nU, 125 // g / ml and 25 // g / ml of P / C hybrid matrix A, the culture was maintained for 24 hours. After four days of culture, the monolayer cells were washed with the culture medium, and then the cells were cultured with a fluorescent primer-containing medium at 37 ° C containing 5% carbon dioxide, and then analyzed with a fluorescence microscope (AX-70, Olympus , Tokyo). 13. 2 Flow cytometry analysis with FITC-Annexin V and PI dual staining Flow cytometry analysis was performed with FITC-Annexin V and PI dual staining. After washing twice with PBS, the number of treated and untreated 106 cells was collected by centrifugation at room temperature. Gently disperse the cells in Annexin V culture reagent and incubate at room temperature for 15 minutes in the dark. The mixture was analyzed with a FACS Vantage flow cytometer and CelQuest software (Becton Dickinson). 13.3 DNA Fragment Analysis Cells were cultured in complete culture at a concentration of 2.5 × 105 cells / ml, and after maintaining for 24 hours, the culture was renewed to contain a serially diluted concentration of P / C hybrid matrix A or B, and cultured for 72 hours. After this step, the cells were scraped off and analyzed. DNA fragments were analyzed with suicide-trackTM DNA ladder kit (Oncogene, MA). 13.4 Results After 4 days of culture, endometriotic cells were stained with a distinct green color after operating light stains (NAO and PI). Compared with the control group, more than 95% of the cells were viable. More than 85% of the cells were stained red, showing that after culturing for 4 days at a concentration of 500 # g / ml of hybrid matrix A, most of the cells will be ______26__ β0. The paper scale applies the Chinese National Standard (CNS) A4 specification ( 210 X 297 mm) (Please read the notes on the back before filling out this page). --Line_ 200407434
五、發明說明（>y) 死亡（圖10)。為了證明哪些種類的細胞為雜合基質a誘發 死亡的，發明者以annexin V與PI雙重染色研究細胞的外 型改變。經由螢光annexinV與ρι結合物染色的細胞，可 偵測出非細胞凋亡之存活細胞（annexin V正，pi負），早期 細胞凋亡細胞（annexinV正，PI負）與晚期細胞凋亡細胞 (annexin V正）。圖η表示在經雜合基質a處理後的細胞 核片段化與染色質濃縮化。這些為細胞凋亡的特徵。經4 天於雜合基質A在濃度500 培養後，大部分的細胞 被染成紅色，顯示子宮内膜異位細胞屬於晚期細胞凋亡。 為了確定經由P/C雜合基質A處理可誘發細胞凋亡， 發明者研究在子宮内膜異位細胞的片段化的圖式，經 暴露在雜合基質A4天後（圖l2)〇，，DNA片段化（DNA-ladder)，， 經72小時培養後觀察到。在第4天時經瓊脂醣膠電泳的定 量分析，其可發現經P/C雜合基質A濃度5〇〇//g/ml處理 後’細胞走向細胞凋亡的路徑（圖12)。為證明在經p/c雜 合基質A處理後的細胞期細胞凋亡的頻度，發明者以經 AnnexinV技術染色進行流式細胞儀分析（圖13)，推測p/c 雜合基質A可誘發已黏著的細胞產生細胞凋亡。發明者過 去就已經證明將近80%的細胞經p/c雜合基質a處理4天 後，會產生細胞凋亡。 實例Π : Caspase-3酵素法怡 17. 1沉澱細胞萃取物與酵素分析 以’’EnzChek”分析試劑組測量在細胞溶解物中的 Caspase-3酵素之活性。收集經處理的細胞，以冰冷的pBs ------27 0游紙張尺度適用中國國家標準(CNS)A4規格(210 x 297公愛）--- -------------裝--- (請先閲讀背面之注意事項再填寫本頁) 訂· · 200407434 A7 ______B7_ 五、發明說明（) 清洗兩次，並將細胞懸浮在細胞溶解緩衝液中，其為試劑 組所附之溶液，而後將細胞溶解物注入至一冷凍-解凍循環 中。依據操作手冊之指導，細胞溶解物於微量離心機中以 5000rpm五分鐘離心。所得之上清液用來分析Caspase-3 酵素之活性。螢光物質則以微盤讀值機（Molecular Devices, Spectra MAX)於激發波長為355nm與放射波長為460nm。蛋 白質濃度經蛋白質分析染料試劑組（Bio-Rad protein assay dye reagent )決定 0 17.2 Caspase-3 酵素活性 為分析經P/C雜合基質處理的誘發事實，發明者試驗 在子宮内膜異位細胞中caspase-3酵素的活性。經4天的 培養，經P/C雜合基質A處理的細胞其caspase-3酵素活 性與正常細胞相較之下，約降低2倍（圖14)。如圖14所 表示，P/C雜合基質B並不改變caspase-3酵素活性。所有 試驗中的螢光強度以溶解狀態的蛋白質量作標準化。 Caspase-3酵素活性的斜率，以經處理的細胞與未經處理 的細胞作計算。 17.3 Caspase-3酵素抑制劑對經P/C雜合基質A處理 的細胞之影響 根據caspase_3酵素的活性，以Ac-DEVD-CH0抑制劑 確定被誘發與正常細胞族群中所觀察之螢光信號。根據圖 14所示，推測caspase-3酵素的活性在P/C雜合基質A所 處理的細胞中，牽涉誘發細胞凋亡的路徑。 28 紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公一 ----I----I----· I I (請先閱讀背面之注意事項再填寫本頁) · 200407434 A7 B7 五、發明說明 者而可作之不同修正及變化對於熟習該項技術 ==不會偏離本發明的範圍與精神。雖然本發明 不當地限制於該等特定具體事實上瞭=:树明不應被 明之已述模式方面’對於熟習該項技術二本發 不同修正亦被涵蓋於下列申請專利範圍之/為易知之 -II I — — — — — 1 — — —— · I I (請先閱讀背面之注意事項再填寫本頁) · 29 y表紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）V. Explanation of the invention (> y) Death (Figure 10). In order to prove which types of cells are induced by heterozygous matrix a, the inventors studied the changes in cell appearance by double staining with annexin V and PI. Non-apoptotic viable cells (annexin V positive, pi negative), early apoptotic cells (annexinV positive, PI negative) and late apoptotic cells can be detected by cells stained with fluorescent annexinV and ρ conjugate (annexin V positive). Figure η shows fragmentation of nucleus and chromatin concentration after treatment with hybrid matrix a. These are characteristics of apoptosis. After 4 days of incubation with hybrid matrix A at a concentration of 500, most of the cells were stained red, indicating that endometriotic cells belonged to advanced apoptosis. In order to determine whether apoptosis was induced by treatment with P / C hybrid matrix A, the inventors studied the fragmentation pattern of endometriotic cells, and exposed to hybrid matrix A for 4 days (Figure 12). DNA-ladder was observed after 72 hours of culture. Quantitative analysis by agarose gel electrophoresis on the 4th day revealed that the cells' path to apoptosis after treatment with P / C hybrid matrix A concentration of 500 // g / ml (Fig. 12). In order to prove the frequency of apoptosis in the cell phase after treatment with p / c hybrid matrix A, the inventors performed flow cytometry analysis with AnnexinV staining (Figure 13), and speculated that p / c hybrid matrix A can induce Adhered cells produce apoptosis. The inventors have shown in the past that nearly 80% of cells will undergo apoptosis after treatment with p / c hybrid matrix a for 4 days. Example Π: Caspase-3 Enzyme Method 17.1 Analysis of Precipitated Cell Extracts and Enzymes The activity of Caspase-3 enzymes in cell lysates was measured using the "EnzChek" analysis reagent group. The treated cells were collected and subjected to ice-cold pBs ------ 27 0 travel paper size applicable to China National Standard (CNS) A4 specifications (210 x 297 public love) --- ----------- install --- (Please Read the precautions on the back before filling this page) Order · · 200407434 A7 ______B7_ V. Description of the invention () Wash twice and suspend the cells in the cell lysis buffer, which is the solution attached to the reagent set, and then place the cells The lysate was injected into a freeze-thaw cycle. According to the instructions of the operation manual, the cell lysate was centrifuged in a microcentrifuge at 5000 rpm for five minutes. The resulting supernatant was used to analyze the activity of the Caspase-3 enzyme. The fluorescent substance was A micro-disk reader (Molecular Devices, Spectra MAX) was used at the excitation wavelength of 355 nm and the emission wavelength of 460 nm. The protein concentration was determined by the Bio-Rad protein assay dye reagent 0 17.2 The activity of the Caspase-3 enzyme was Analysis of P / C hybrid matrices Induced fact of treatment, the inventors tested the activity of caspase-3 enzyme in endometriotic cells. After 4 days of culture, cells treated with P / C hybrid matrix A had caspase-3 enzyme activity similar to normal cells In contrast, it was reduced about 2 times (Figure 14). As shown in Figure 14, the P / C hybrid matrix B did not change the caspase-3 enzyme activity. The fluorescence intensity in all experiments was normalized to the protein mass in the dissolved state. The slope of Caspase-3 enzyme activity is calculated from treated cells and untreated cells. 17.3 Effects of Caspase-3 enzyme inhibitors on cells treated with P / C hybrid matrix A. According to the activity of caspase_3 enzyme, Ac-DEVD-CH0 inhibitors were used to determine the fluorescence signals observed in the induced and normal cell populations. As shown in Figure 14, the activity of the caspase-3 enzyme was speculated in cells treated with the P / C hybrid matrix A, Involved in the path of inducing apoptosis. 28 Paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 public one ---- I ---- I ---- · II (Please read the precautions on the back first) (Fill in this page again) 200407434 A7 B7 The different amendments and changes are familiar with the technology == will not deviate from the scope and spirit of the present invention. Although the present invention is improperly limited to these specific specific facts =: the tree model should not be clearly described in terms of the model Familiar with this technology. The different amendments of this issue are also covered by the scope of the following patent applications / for easy understanding -II I — — — — — 1 — — — II (Please read the precautions on the back before filling out this page) · 29 y sheet paper size applicable to China National Standard (CNS) A4 (210 X 297 mm)
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