1283569 九、發明說明： 【發明所屬之技術領域】 -^ 本發明係有關於一種生物可分解性載體，尤其係有關 於一種具有複數相之生物可分解性載體及其製造與使用方 法，其可被應用一生理系統中用以收容、誘導及支撐複數 種不同特性之組織細胞，藉以促進身體中不同組織交界處 的組織修復。 【先前技術】 習知組織工程上的技術開發重點，主要係著重於單一鲁 種組織的增殖培養上。不過由於身體係由許多經分化且具、 有不同特性的細胞所構成，因此於臨床上醫師所面臨需^ - 填補或修補的組織，往往為一具有兩種或兩種以上不同特 性的組織所構成之複合式組織。例如，人體的關節面即為 個相¥典型的兩相複合式組織(例如，軟骨組織與硬骨会且 織）。 關節軟骨組織是一種特殊的結締組織，能夠忍受長時 _ 間且局重量負载，其組成包含軟骨細胞及細胞外基質，例 如第二型膠原蛋白(type II collagen)與醣蛋白(pr〇te〇glycans) 等。早在西70 1743年前Hunter即指出，軟骨受到破壞後， 自行修復能力#常有限。即使到現在，也無法於缺損處重 建-塊與周邊正常組織具有相同生化、生物力學特性的新 生組織。 此外，由於關節軟骨組織中並無神經細胞的分布，所 1283569 以當關節軟骨組織發生損傷時，患者並無明顯的感覺。因 此，當患者開始感覺到關節不適或疼痛時，其受損的程度 大都已到達下方硬骨(subchondral bone)組織的海錦骨區。 此時需要修補的組織，除了軟骨組織外，尚包括硬骨組織。 為改善先前僅能用於修復單一種組織的增殖培養之缺 點’即有人提出對兩種不同的細胞同時進行培養，藉以形 成可進行修補的兩相複合組織。例如，Is〇gai等人(1999) 將聚乳酸(p〇ly_L_laCtide，PLLA)與聚甘醇酸(polyglyc〇lide， PGA)的共聚物製成一與中節指骨相似的形狀之支架，且此 支架包含一指關節，再將成骨細胞、軟骨細胞與肌腱細胞 分別種植於其上，之後植入裸鼠體内，藉以進行組織修補 (Formation of Phalanges and Small Joints by Tissue-Engineering. Journal of Bone and Joint Surgery^ 1999; 81* p.306-316)。廖俊仁(2002)提出了一種具有多層次的多孔隙 基材，此基材上方含有一空腔，空腔外圍為生物可降解性 高分子之多孔性薄膜，下方為聚乳酸甘醇酸共聚物 【poly(lactic-co_glycolic acid，PLGA)/ 氫氧基磷灰石 (Hydroxyapatite，HAP)的均勻性複合多孔隙結構之兩相支 架當載體，其係利用不同孔隙度支架的結構，以及組織塊 與細胞之體積差異，將不同的組織細胞予以分層，此雖可 使具有較大體積的細胞不會侵入具有較小體積細胞的組織 中，但其製造過程較為繁瑣，不易進行大規模生產(利用分 層次組織工程基材培養兩相關節組織，組織工程研討會， 2002 : ρ·107-108)。Schaefer等人(2000)將軟骨細胞與硬骨 1283569 細胞分別植入生物可降解性高分子支架，並在相同環境下 一起培養’ 4週後發現軟骨支架與硬骨支架之接觸面會結 合在一起’不過其高分子支架係藉由手術縫線將軟骨支架 與硬骨支架縫製在一起而成，此需要良好的縫合技巧才能 達成，且不利於商業化的實施。此外，由於軟骨細胞於生 長時會分泌出可避免軟骨細胞鈣化的葡萄糖胺聚醣 (glycosaminoglyCan，GAG)，這種將軟骨支架與硬骨支架直 接縫製在一起，會使得軟骨細胞所分泌之葡萄糖胺聚醣擴 散至硬骨支架中，而影響硬骨細胞的鈣化程度，致使此種 支架的修復效果不佳(In Vitro Generation of 〇ste〇ch〇ndral1283569 IX. Description of the invention: [Technical field to which the invention pertains] - The present invention relates to a biodegradable carrier, and more particularly to a biodegradable carrier having a complex phase and a method of making and using the same It is applied to a tissue system in a physiological system for accommodating, inducing and supporting a plurality of different characteristics, thereby promoting tissue repair at the junction of different tissues in the body. [Prior Art] The technical development focus of the conventional tissue engineering mainly focuses on the proliferation culture of a single tissue. However, because the body system is composed of many differentiated cells with different characteristics, the tissues that need to be filled or repaired by doctors in clinical practice are often organizations with two or more different characteristics. The composite organization that constitutes. For example, the articular surface of a human body is a phase of a typical two-phase composite tissue (for example, cartilage tissue and hard bone tissue). Articular cartilage tissue is a special connective tissue that can withstand long-term and local weight loading. Its composition consists of chondrocytes and extracellular matrices, such as type II collagen and glycoprotein (pr〇te〇). Glycans). As early as West 70 1743, Hunter pointed out that after cartilage is destroyed, the ability to repair itself is often limited. Even now, it is impossible to rebuild a new tissue with the same biochemical and biomechanical properties as the surrounding normal tissue. In addition, since there is no distribution of nerve cells in the articular cartilage tissue, 1283569 has no obvious sensation when the articular cartilage tissue is damaged. Therefore, when the patient begins to feel discomfort or pain in the joint, the degree of damage has mostly reached the area of the lower basal bone of the subchondral bone tissue. The tissue that needs to be repaired at this time, in addition to the cartilage tissue, also includes hard bone tissue. In order to improve the shortcomings of proliferation culture which was previously only used to repair a single tissue, it has been proposed to simultaneously culture two different cells to form a two-phase composite tissue that can be repaired. For example, Is〇gai et al. (1999) made a copolymer of polylactic acid (p〇ly_L_laCtide, PLLA) and polyglyc〇lide (PGA) into a stent of a shape similar to the middle phalanx, and this The scaffold contains a knuckle, and then osteoblasts, chondrocytes, and tendon cells are planted separately, and then implanted into nude mice for tissue repair (Formation of Phalanges and Small Joints by Tissue-Engineering. Journal of Bone) And Joint Surgery^ 1999; 81* p.306-316). Liao Junren (2002) proposed a multi-layered porous substrate with a cavity above the substrate, a porous film of biodegradable polymer on the periphery of the cavity, and a polylactic acid glycol copolymer underneath. Poly(lactic-co_glycolic acid, PLGA)/Hydroxyapatite (HAP) homogeneity Composite porous structure of two-phase scaffolds as carriers, which utilize structures of different porosity scaffolds, as well as tissue blocks and cells The difference in volume, stratification of different tissue cells, which can make cells with larger volume do not invade tissues with smaller volume cells, but the manufacturing process is cumbersome and difficult to mass production (utilization points) Hierarchical Tissue Engineering Substrate Culture Two Related Section Organizations, Tissue Engineering Seminar, 2002: ρ·107-108). Schaefer et al. (2000) implanted chondrocytes and hard bone 1283569 cells into biodegradable polymer scaffolds and cultured together under the same environment for 4 weeks. The contact surfaces of cartilage scaffolds and hard bone scaffolds were found to be combined. The polymer scaffold is made by sewing the cartilage scaffold and the hard bone scaffold by surgical suture, which requires good suturing techniques to achieve, and is not conducive to commercial implementation. In addition, since chondrocytes secrete glycosaminoglycan (GAG) which can prevent chondrocyte calcification during growth, this sewn together with the cartilage scaffold and the hard bone scaffold will cause the glycosaminoglycan secreted by the chondrocytes to aggregate. Sugar diffuses into the hard bone scaffold and affects the degree of calcification of the hard bone cells, resulting in poor repair of the scaffold (In Vitro Generation of 〇ste〇ch〇ndral
Composites·所omflimfl/，2000; 21: ρ·2599_2606)。 因此，本發明即致力於提供一種具有複數相之生物可 分解性載體，模擬軟骨組織與硬骨組織間所存在之不滲透 鈣化軟骨層及嗜鹼漲潮線的界面，以隔離來自骨髓的物 貝，以避免影響軟骨的生長與分化，並解決前述習知技術 上缺點。 【發明内容】 本發明之目的，係提供一種組織工程用載體，用以植 入一生理系統中（例如，動物體内）的不同類型組織連接處 (例如，軟骨組織與硬骨組織交界處、韌帶與肌腱附著處… 等），以使不同類型組織得以生長與再生，藉以進行組織細 胞的修復。 本發明之另一目的，係提供一種製作過程簡易之組織 1283569 工程:=L藉以簡化其製作程序及降低生產成本。 體的二體中== 出t載體可供組織向内生長進入該載 道(孔）而佔滿該==穿過設fr體本體中的生長通 、戰體的表面，這些生長通道能促進並收納 m且織。本發明的載體可被植入至一生理系統中，使 金·^至4雛質不相似的峨連接處，例如軟骨組織 ”石月組織、韌帶與肌腱附著處…等。 兩本發明載體包含一生物可分解的聚合物材料，當其暴 路於水溶液或生理純巾，㈣—段時間後會大致上或完 全被分解。於賴齡解的雜巾，尚未被分解的聚合物 材料可於組織的植人處提供—支撐，使組織得以進行再 生此外再生組織亦會進入被分解的載體所處的空間中， 供做為支架(scaff〇ld)”，以加速組織中受損或缺陷區域的 再生。因此，本發明載體係為一生物可分解性支架狀的多 孔狀結構，可用以促進、支撐與收納組織缺陷或受損處的 再生組織。由於本發明載體係為生物相容的，使用的患者 無須擔心身體内植入外來物可能造成的危險。另外，由於 本發明載體係為生物可分解的，故於植入後會慢慢的被分 解掉’原先所佔有的空間會由新生組織所取代，故無須進 行第二次手術將其取出。 為達成並符合本發明前述之目的，本發明在此提供一 種組織工程用載體，包含一由生物可分解之聚合物材料所 構成的第一支架與第二支架，該兩支架的聚合物材料可為 相同或不同，且可具有相似或不相似的機械性質；以及一 1283569 隔離薄膜，該第-支軸第二技齡職接於該隔離薄 膜的相反_，且該隔離賴於生㈣射的分解速率小 於該第-支架與該第二支架。本發日域體可被植入至一生 理系統中，使其位於兩種不囉類的组織交界處，原則上 載體的第-支㈣錄第-種_巾，第二支架會位於第 二種組織中，且隔離薄膜會位於第—種組織與第二種 的交界處。其中，該載體的第—支架可供容納第一種 的生長，該載體的第二支架可容納第二種組織的生長。 每-個支架中亦可進-步包含一酵素或其他習知的降 解劑，用峨進該聚合物材料的分解。本發日賊體中亦可 進-步包含-個或〇之可促進正常_分化及生長的生 長因子’或其他的_。酵素、生長因子或其他藥劑可根 據實際需要，依預設的比例分別添加至第一支架與第二支 架中，以產生不同的分解與組織修復速率。 ^發明亦提供-種具有上述結構之健的製造方法， 包含藉由將聚合物材料以—適當溶劑溶解成黏液狀，接著 將溶劑自此黏液狀聚合物中抽離，以分別製得具有預設形 狀之第一支架、第二支架與隔離薄膜。接著，使用濃 度的聚合物溶液塗抹於第一支架與隔離薄膜，以及第二支 架與隔離薄膜的連接處，使第一支架與第二支架分別黏合 於隔離薄膜的兩側。之後，進-步將殘餘溶劑絲後即可 獲得根據本發明所指出的載體。 本發明將藉由下述的詳細說明及實施例做進一步的說 明，這些實施例並不限制本發明前面所揭示之内容^熟習 1283569 ’但仍不脫離本 本發明之技藝者，可做鱗之改良與修飾 發明之範疇。 【實施方式】 參閱第一圖及第二圖，為根據本 數相的生物可分解性載體1〇 立X 才日出之/、有複 镇一*㈢繁士? 圖。該載體1〇包含- 第支架12、第二支架14，以及一將該第_支架η與該 第，支架14分隔開之隔離薄膜16，其中該第一支架u與 該第二支架14係分別連接於該隔離薄膜16的相反兩侧面 上。該第-支架U與該第二支架14可藉由任何f知且具 有生物相容性魅物可分娜之聚合崎料所製備，於本 發明中並沒有特別的限制。用於製備第一支架12與第二支 架14之聚合物麵可朗欲培養之組織選擇適合的聚合 物材料’ #父佳為聚g旨，所制之聚合物材料的麵可為相 同或不同，且兩者之機械性質可為相似或不相似。如欲使 用本發明載體培養軟骨細胞與硬骨細胞，做為本發明第一 支架12與第二支架14之聚合物材料的一實施例，較佳係 選自由聚L_乳酸(poly-L-lactide，以下簡稱pllA)、聚DL- 乳酉文、聚甘醇酸、聚己内g旨（P〇lyCapr〇lact〇ne，以下簡稱 PCL)與聚乳酸甘醇酸共聚物(p〇ly(iaetiC_eO_glyeQlie aeJd， 以下簡稱PLGA)所組成之族群所製備，但並不僅限於此。 若使用PLLA與PLGA進行製備時，pllA與PLGA之使 用比例較佳為1 : 1-6，更佳為1 : 3-6。其中，PLGA共聚 物較佳為藉由聚甘醇酸(polyglycolic acid，以下簡稱PLG) 1283569 與聚乳酸（polylactic acid，以下簡稱pLA)以濃度比 50/50〜85/15之比例所製備而得，例如當濃度比為5〇/5〇時 在此以PLGA50/50表示。 另外’隱薄膜16衬藉由任何f知具有生物相容性 與生物可分雜之聚合物·所製備，但胁生理系统中 被降解的速率需低於第-支架12與第二支架14，除此之 外於本發明巾並沒有其他_的_。做為本發明隔離薄 膜16之聚合物材料的一實施例，較佳 聚DL-乳酸'聚甘醇酸、聚己内臨與PLGA共聚 但並不僅限於此。其巾，PLGA共聚純佳為藉由pLG與 PLA以濃度比50/50〜85/15之比例所製備而得。若使用pCL 與PLGA 50/50進行製備時，PCL與pLGA 5〇/5〇之使用比 例較佳為1-9 : 1，更佳為3_9 : 1。 此外，為增加第-支架12與第二支架14的生物相容 性，可進-步對第-支架12與第二支架14以生物相容性 更佳之聚合物進行改質’可被應祕本發明巾用以進行改 質的聚合物材料’包含任何組駐程上習知用以進行表面 改質的聚合物材料’皆可被躺於本發財，於本發明中 並沒有特別制。做為本發明的—實施例，例如膠原蛋 白，但並不僅限於此m支架12較佳為藉由第二 型膠原蛋白進行改質，而第二支架14則較佳為藉由第一型 膠原蛋白進行改質。 *另一方面’為增強第一支架12、第二支架14及/或隔 離薄膜I6巾聚合物崎解速度’可進—步於其聚合物材料 11 1283569 中均勻散佈一可用以增強聚合物降解速率的酵素或降解 劑。可被應用於本發明中用以增強聚合物降解速率的酵 素，於本發明中並沒有特別的限制，例如習知具有酯鍵水 解能力的蛋白酶或水解酵素。做為這些酵素的例子，包含 蛋白酶K (proteinase K)、鳳梨蛋白酶(bromelain)、鏈黴蛋 白S# E (pronase E)、纖維素酶、聚葡萄糖酶(dextranase)、 彈性蛋白酶（elastase)、胞漿素鏈黴菌激酶（piasmin streptokinase)、騰蛋白酶、膜凝乳蛋白酶(chymotrypsifl)、 木瓜酶(papain)、木瓜凝乳蛋白酶(chymopapain)、膠原蛋白 酶(collagenase)、幾基胜肽酶 A(carboxypeptidase A)、氧化 酶等，但並不僅限於此。 再者，為促進新生細胞於本發明載體1〇上的生長，亦 可於第一支架12、第二支架14及/或隔離薄膜16中進一 步加入一生長因子（growth factor)，例如轉型生長因子 ^(transforming growth factor-beta)，或其他型式的治療藥 劑，例如用以增加組織細胞生長速率的類固醇或荷爾蒙。 而將這些治療藥劑及生長因子散佈於聚合物材料中，係為 熟習本技術領域者藉由先前之技藝可輕易達成之事，且可 藉由習知技術依所需之速率，使治療義自聚合物中釋放 出。 為使第支架12與第二支架14分別黏合於隔離薄膜 I6的相反兩側’於本發日种較鶴藉由—聚合物溶液 塗抹於第-支架12及第二支架14與隔離薄膜 16的連接區 上’此聚合物1st佳為具有生物相容性且具有生物可分解 12 1283569 性。做為本發明之一實施例，例如PCL以二氧陸圜 (1’4-dioxane)溶解後所形成之高濃度聚合物溶液，但並 限於此。 个m 參閱第三圖，為製備根據本發明所指出之具有複數相 的生物可分解性載體之流程圖。於製備本發明載體時，係 先將製備第-支架用之第一聚合物材料2〇與製備第二支 架用之第二聚合物材料22 ’分別以第—溶劑溶解Μ與以 第二溶劑溶解26形成第-聚合物溶液及第二聚合物溶 液，之後分縣除支架中溶劑28, 3G即可成形，做為本發# 明中去除支架中溶液的方法’例如冷束乾燥。之後將已成 形的第-支架與第二支架進-步分別以生物相容性更汽之 聚合物進行支架改質32, 34。另一方面，取隔離薄膜用聚 合物材料36以溶劑溶解38後，再去除溶劑4〇以形成所需 之隔離薄膜。將前述已預先製備好之第一支架與第二支架 分別黏合於隔離薄膜的兩侧42,其可藉由—聚合物溶液來 執行。最後，去除載體上的殘餘溶劑44 (例如，冷凍乾燥 法），藉此即可製得本發明之具有複數相的生物可分解性載# 體。 參閱第四圖及第五圖，係為本發明載體植入一生理環 境中之剖面示意圖。在此做為實例說明之生理環境，例如 軟骨與硬骨組織的交界區，但並不僅限於此。本發明載體 10於使用時，係將載體10植入欲進行修補的位置中，例 如設置並穿過軟骨組織46、硬骨組織48，以及軟骨組織 46與硬骨組織48交界之不渗透妈化軟骨層52的孔洞50 13 1283569 中。於植入後，本發明載體10中的第一支架12會位於軟 骨組織46中，第二支架14會位於硬骨組織48巾，且隔離 薄膜16會位於不滲透_軟骨層52中。於植人區域附近 的細胞會慢慢侵人第—支架12與第二支架14中，進而佔 據第支架12與第二支架14中原有存在的空隙。接著， 隧著第支架12與第二支架14於生理環境中慢慢的被分 解掉，留下來的空間則會為新生組織所取代。而這些新生 成的組織則會形成為新的支架系統，藉以使更多的新生組 織得於此一空間中生長，最後形成近似原有組織之型態， 以完成組織修補的工作。而位於不滲透鈣化軟骨層52中的 隔離薄膜16於第一支架12與第二支架14分解後，也會慢 慢的被分解掉。由於隔離薄膜16的分解速率低於第一支架 12與第二支架14，因此隔離薄膜16可有效隔離新生成的 軟骨細胞與硬骨細胞，並可阻止由軟骨細胞所分泌避免其 #5化的葡萄糖胺聚醣(glycosaminoglycan，GAG)滲入硬骨 細胞所處的位置中，而影響硬骨組織的生長。於待修復區 域之軟骨組織與硬骨組織修補完成後，隔離薄膜16隨後也 會被分解掉，由新生的組織所取代，形成近似於正常組織 中的不滲透鈣化軟骨層52。此外，由於第一支架12、第二 支架14與隔離薄膜16會於植入生理系統後，分別被分解 掉，因此進行組織修補之患者，無需再經第二次開刀將載 體取出。 熟習本發明所屬技術領域中具有通常知識者，依前述 之說明亦可瞭解，本發明載體也可於植入生理系統前，先 1283569 於載體的支架上植續欲進行修補處的細胞，先使其於一 生物反應1§巾進行培養後，再植人於生理纽巾的待修補 組織處，藉明強受損㈣修獅速度娜補效果。做為 本發明之-實關，例如於第—支架巾植人軟骨細胞，於 第二支架中植入骨髓基質幹細胞(Bone marr〇w str〇mai cells，BMSC)’接著將本發明載n置人_雙腔^生物反應器 54進行培養，如第六圖所示。雙腔式生物反應器54的第 一腔室56與第二腔室58，藉由一挾持裝置6〇連接，並以 本發明載體10的隔離薄膜16分隔開。於進行培養時，第 一腔室56與第二腔室58中分別先注入適合預先植入於第 一支条12與弟一支架14上之細胞生長的培養液，使植入 載體中的細胞得以生長。待培養一段時間後，即可將載體 10取出，將隔離薄膜16裁剪成適當小後即可植入待修補 組織處。 實施例一 製備具有複數相之生物可分解性載體 取PLLA與PLGA50/50以重量比丄：6之比例，溶於 適量的二氧陸圜(l，4-dioxane)溶劑中，配製成高分子溶液。 於麵模谷器中倒入不同體積之高分子溶液，先分別置 於冷藏溫度下(約4°C)預冷3分鐘，再放入液態氮中急速冷 凍30小時後，之後進行冷凍乾燥，以形成具有所需形狀的 第一支架與第二支架。 將上述支架分別以30%酒精潤濕5分鐘，使其完全濕 15 1283569 潤，之後再以二次水反覆清洗支架5次，每次3分鐘，以 將支架中的殘餘酒精沖洗掉。 將上述經潤濕後的第一支架與第二支架分別浸潰於 l%(w/v)的第二型與第一型膠原蛋白溶液中1小時，之後抽 真空5分鐘，再於冷藏溫度下(約4它)放置3〇分鐘，重複 上述動作三次。再以超音波震盪器處理1〇分鐘後，再於冷 藏溫度下（約4。〇放置30分鐘，重複三次。藉以使膠原蛋 白完全滲入支架的孔洞中。Composites·omflimfl/, 2000; 21: ρ·2599_2606). Accordingly, the present invention is directed to providing a biodegradable carrier having a plurality of phases, simulating an interface between an impervious calcified cartilage layer and an alkaloid tide line existing between cartilage tissue and hard bone tissue to isolate the shellfish from the bone marrow. To avoid affecting the growth and differentiation of cartilage, and to solve the aforementioned technical disadvantages. SUMMARY OF THE INVENTION It is an object of the present invention to provide a tissue engineering carrier for implanting different types of tissue junctions in a physiological system (eg, in an animal) (eg, a junction of cartilage tissue and hard bone tissue, ligament Attachment to the tendon...etc.) to allow different types of tissue to grow and regenerate for tissue cell repair. Another object of the present invention is to provide an organization that is easy to make. 1283569 Engineering: =L to simplify its production process and reduce production costs. In the body of the body == the t-carrier is available for the tissue to grow inward into the channel (hole) and fills the == through the surface of the growth and warfare body in the body of the fr body, these growth channels can promote And store m and weave. The vector of the present invention can be implanted into a physiological system to make the sputum junctions of the gold to the 4 nectar dissimilar, such as cartilage tissue "stone structure, ligament and tendon attachment, etc." A biodegradable polymer material, when it is violently in an aqueous solution or a physiologically pure towel, (4) will be substantially or completely decomposed after a period of time. In the diarrhea disintegrated diaper, the polymer material that has not been decomposed can be The implanted tissue of the tissue provides support to enable the tissue to be regenerated and the regenerated tissue also enters the space in which the decomposed carrier is located, serving as a scaff〇ld to accelerate damaged or defective areas of the tissue. Regeneration. Thus, the carrier of the present invention is a biodegradable stent-like porous structure that can be used to promote, support and contain tissue regeneration or damaged tissue at the site of damage. Since the carrier of the present invention is biocompatible, the patient in use does not have to worry about the dangers that may be caused by the implantation of foreign objects in the body. In addition, since the carrier of the present invention is biodegradable, it will be slowly decomposed after implantation. The space originally occupied will be replaced by the new tissue, so that it is not required to be removed by a second operation. To achieve and meet the foregoing objectives of the present invention, the present invention provides a carrier for tissue engineering comprising a first stent and a second stent composed of a biodegradable polymer material, the polymer materials of the stents being Having the same or different, and may have similar or dissimilar mechanical properties; and a 1283569 isolating film, the first fulcrum of the second technical age is attached to the opposite of the isolating film, and the isolation depends on the raw (four) The decomposition rate is less than the first stent and the second stent. The hair field body can be implanted into a physiological system so that it is located at the junction of two kinds of tissues, in principle, the first branch of the carrier (four) records the first type - the towel, and the second stent is located at the first In both tissues, the barrier film will be located at the junction of the first tissue and the second. Wherein the first stent of the carrier is adapted to accommodate growth of the first species and the second stent of the carrier is capable of accommodating growth of the second tissue. Each of the scaffolds may further comprise an enzyme or other conventional degrading agent for breaking into the decomposition of the polymeric material. In this thief, the thief may also include - or a growth factor that promotes normal _ differentiation and growth or other _. Enzymes, growth factors, or other agents can be added to the first stent and the second stent, respectively, according to actual needs, to produce different rates of decomposition and tissue repair. The invention also provides a manufacturing method having the above structure, which comprises preparing a polymer material by dissolving it into a mucus in a suitable solvent, and then extracting the solvent from the viscous polymer to prepare a pre-prepared product. The first bracket, the second bracket and the isolating film are provided. Next, a concentration of the polymer solution is applied to the first holder and the separator, and the junction of the second holder and the separator, so that the first holder and the second holder are respectively adhered to both sides of the separator. Thereafter, the carrier as indicated in the present invention can be obtained by further feeding the residual solvent. The invention will be further illustrated by the following detailed description and examples, which are not intended to limit the disclosure of the present invention, but are not limited to those skilled in the art. And the scope of the invention. [Embodiment] Referring to the first figure and the second figure, it is a biodegradable carrier according to the number of phases, and the number of the bio-decomposable carrier is set to X. The carrier 1 includes a first bracket 12, a second bracket 14, and a separating film 16 separating the first bracket n from the first bracket 14. The first bracket u and the second bracket 14 are They are respectively connected to opposite side faces of the separator 16 . The first stent U and the second stent 14 can be prepared by any polymeric material having a biocompatible enchantment, which is not particularly limited in the present invention. The preparation of the polymer surface of the first stent 12 and the second stent 14 for selecting the suitable polymer material can be selected from the following aspects: And the mechanical properties of the two may be similar or dissimilar. If an embodiment of the polymer material of the first scaffold 12 and the second scaffold 14 of the present invention is used to culture chondrocytes and hard bone cells using the vector of the present invention, it is preferably selected from poly-L-lactide. , hereinafter referred to as pllA), poly DL- chylomicron, polyglycolic acid, polyglycolate (P〇lyCapr〇lact〇ne, hereinafter referred to as PCL) and polylactic acid glycolic acid copolymer (p〇ly(iaetiC_eO_glyeQlie) The preparation of aeJd, hereinafter referred to as PLGA) is not limited thereto. When using PLLA and PLGA for preparation, the ratio of pllA to PLGA is preferably 1: 1-6, more preferably 1: 3- 6. The PLGA copolymer is preferably prepared by polyglycolic acid (PLG) 1283569 and polylactic acid (hereinafter referred to as pLA) at a concentration ratio of 50/50 to 85/15. For example, when the concentration ratio is 5〇/5〇, it is represented by PLGA50/50. In addition, the 'hidden film 16 liner is prepared by any biocompatible and bio-separable polymer. However, the rate of degradation in the physiologic system needs to be lower than that of the first stent 12 and the second stent 14, in addition to this In addition to the invention, there is no other embodiment of the invention. As an embodiment of the polymer material of the release film 16 of the present invention, it is preferred that the poly-DL-lactic acid 'polyglycolic acid, polyhexanol and the PLGA are copolymerized but not only It is limited to this. The towel, PLGA copolymerization is preferably prepared by pLG and PLA at a concentration ratio of 50/50 to 85/15. When using pCL and PLGA 50/50, PCL and pLGA 5〇 The ratio of use of /5 较佳 is preferably from 1 to 9 : 1, more preferably from 3 to 9 : 1. Further, in order to increase the biocompatibility of the first holder 12 and the second holder 14, the first holder 12 can be further advanced. Modification with the second stent 14 with a more biocompatible polymer. The polymer material that can be modified by the invention towel comprises any group of polymers conventionally used for surface modification. The material 'is lie in the present invention, and is not specially prepared in the present invention. It is an embodiment of the present invention, such as collagen, but not limited thereto. The m stent 12 is preferably made of type II collagen. Modification is carried out, and the second stent 14 is preferably modified by the first type of collagen. 'To enhance the polymer scavenging speed of the first stent 12, the second stent 14 and/or the barrier film I6', it is possible to evenly distribute an enzyme which can be used to enhance the degradation rate of the polymer in the polymer material 11 1283569 or The degradation agent, which can be used in the present invention to enhance the rate of degradation of the polymer, is not particularly limited in the present invention, and is, for example, a protease or a hydrolyzing enzyme which is known to have an ester bond hydrolysis ability. Examples of such enzymes include proteinase K, bromelain, streptavidin S# E (pronase E), cellulase, dextranase, elastase, and cells. Piasmin streptokinase, TG, chymotrypsifl, papain, chymopapain, collagenase, carboxypeptidase A ), oxidase, etc., but not limited to this. Furthermore, in order to promote the growth of the nascent cells on the carrier 1 of the present invention, a growth factor such as a transforming growth factor may be further added to the first stent 12, the second stent 14 and/or the separator 16 . (transforming growth factor-beta), or other types of therapeutic agents, such as steroids or hormones used to increase the rate of tissue cell growth. Dispersing these therapeutic agents and growth factors in a polymer material is easily accomplished by those skilled in the art by the prior art, and can be treated at a desired rate by conventional techniques. Released from the polymer. In order to make the first bracket 12 and the second bracket 14 respectively adhere to the opposite sides of the isolating film I6, the first bracket 12 and the second bracket 14 and the isolating film 16 are applied on the present invention. On the junction area, this polymer 1st is preferably biocompatible and biodegradable 12 1283569. As an embodiment of the present invention, for example, a high concentration polymer solution formed by dissolving PCL in the presence of 1'4-dioxane is limited thereto. Referring to the third figure, a flow chart for preparing a biodegradable carrier having a complex phase as indicated in the present invention. In preparing the carrier of the present invention, the first polymer material 2〇 for preparing the first stent and the second polymer material 22' for preparing the second stent are first dissolved in the first solvent and dissolved in the second solvent. 26 forming a first polymer solution and a second polymer solution, and then dividing the solvent 28, 3G in the stent to form a method for removing the solution in the stent in the present invention, such as cold beam drying. The shaped first stent and the second stent are then further stepped into a scaffold modification 32, 34 with a biocompatible, more vaporous polymer. On the other hand, after the separation film is dissolved in a solvent 38 by a polymer material 36, the solvent is removed to form a desired separator. The previously prepared first stent and the second stent are respectively adhered to the two sides 42 of the separator, which can be performed by a polymer solution. Finally, the residual solvent 44 on the carrier (e.g., freeze-drying method) is removed, whereby the biodegradable carrier of the present invention having a complex phase can be obtained. Referring to the fourth and fifth figures, a schematic cross-sectional view of the carrier of the present invention implanted in a physiological environment is shown. Here, as an example, the physiological environment, such as the junction area between cartilage and hard bone tissue, is not limited thereto. When the carrier 10 of the present invention is used, the carrier 10 is implanted in a position to be repaired, for example, through the cartilage tissue 46, the hard bone tissue 48, and the impervious cartilage layer at the junction of the cartilage tissue 46 and the hard bone tissue 48. Hole 52 of 52 13 1283569. After implantation, the first stent 12 in the carrier 10 of the present invention will be in the soft bone tissue 46, the second stent 14 will be in the hard bone tissue 48, and the barrier film 16 will be in the impermeable-cartilage layer 52. The cells in the vicinity of the implanted area slowly invade the first and second stents 14, and thus occupy the voids existing in the first stent 12 and the second stent 14. Then, the tunneling bracket 12 and the second bracket 14 are slowly separated in the physiological environment, and the remaining space is replaced by the new tissue. These newly formed tissues will form a new scaffolding system, so that more new tissue will grow in this space, and finally form a pattern similar to the original tissue to complete the tissue repair work. The separator film 16 located in the impermeable calcified cartilage layer 52 is slowly decomposed after the first stent 12 and the second stent 14 are decomposed. Since the decomposition rate of the separation film 16 is lower than that of the first holder 12 and the second holder 14, the separation film 16 can effectively isolate the newly formed chondrocytes and hard bone cells, and can prevent the secretion of the glucose by the chondrocytes. Glycosaminoglycan (GAG) penetrates into the location of the hard bone cells and affects the growth of hard bone tissue. After the repair of the cartilage tissue and the hard bone tissue in the area to be repaired, the barrier film 16 is then decomposed and replaced by the nascent tissue to form an impermeable calcified cartilage layer 52 similar to that in the normal tissue. In addition, since the first stent 12, the second stent 14 and the barrier film 16 are separately decomposed after being implanted into the physiological system, the patient undergoing tissue repair does not need to remove the carrier by a second operation. Those skilled in the art to which the present invention pertains can also be understood from the foregoing description. The carrier of the present invention can also be used to implant the cells to be repaired on the carrier of the carrier before the implantation into the physiological system. After being cultured in a biological reaction 1 § towel, it is replanted in the tissue to be repaired of the physiological towel, and the damage is enhanced by Mingqiang (4). For the present invention, for example, in the first stent, the human chondrocytes are implanted, and the second stent is implanted with Bone marr〇w str〇mai cells (BMSC). The human_double chamber^ bioreactor 54 is cultured as shown in the sixth figure. The first chamber 56 and the second chamber 58 of the dual chamber bioreactor 54 are connected by a holding device 6 and are separated by a separator 16 of the carrier 10 of the present invention. During the culture, the first chamber 56 and the second chamber 58 are respectively filled with a culture solution suitable for the growth of cells pre-implanted on the first branch 12 and the first stent 14 to cause the cells implanted in the carrier. Can grow. After a period of time to be cultured, the carrier 10 can be taken out, and the separator film 16 can be cut into a small size to be implanted into the tissue to be repaired. Example 1 Preparation of a biodegradable carrier having a complex phase. The ratio of PLLA to PLGA 50/50 in a weight ratio of 丄:6 was dissolved in an appropriate amount of a solvent of 1,4-dioxane to prepare a high ratio. Molecular solution. Pour different volume of polymer solution into the surface mold, pre-cool for 3 minutes at refrigerated temperature (about 4 ° C), then freeze in liquid nitrogen for 30 hours, then freeze-dry. To form a first bracket and a second bracket having a desired shape. The above stents were each wetted with 30% alcohol for 5 minutes to completely wet 15 1283569, and then the stent was repeatedly washed with secondary water for 5 times for 3 minutes to rinse off the residual alcohol in the stent. The wetted first stent and the second stent were respectively immersed in 1% (w/v) of the second type and the first type collagen solution for 1 hour, followed by vacuuming for 5 minutes, and then refrigerating temperature. Place the next (about 4) for 3 minutes and repeat the above three times. After treatment with an ultrasonic oscillator for 1 minute, it was again placed at a temperature of about 4 minutes for 30 minutes, and the collagen was completely infiltrated into the pores of the stent.
自上述膠原蛋白溶液中取出支架，將支架以冷凍乾燥 機處理12-24小時使其乾燥。將乾燥後之支架置入1%二曱 胺基丙基碳化二亞胺鹽酸鹽【C2H5N=C=N(CH2)3NMe2_HCl， EDC】溶液(交聯劑）中，於4。〇下進行交聯反應牝小時。’ 之後取出支架再以二次水將支架上的交聯劑清洗掉，最 後以冷凍乾燥機使支架乾燥。The stent was taken out from the above collagen solution, and the stent was dried in a freeze dryer for 12 to 24 hours. The dried scaffold was placed in a solution of 1% diammonium propyl carbodiimide hydrochloride [C2H5N=C=N(CH2)3NMe2_HCl, EDC] (crosslinking agent) at 4. The cross-linking reaction was carried out for a few hours. After that, the stent was taken out and the cross-linking agent on the stent was washed away with secondary water, and finally the stent was dried by a freeze dryer.
另外，取聚酯類高分子(例如，pCL及/或pLGA5〇/5〇) 溶於二氧陸圜溶劑中，配製成2%(w/v)的高分子溶液，之 後倒=禱難巾(例如，賴触），使高分子溶液平均分 ^於鑄膜盤巾。接著，以加熱法及域冷;東乾燥法，使其乾 燥並去除隔離薄膜中的溶劑，藉此即可製得隔離薄膜。 古、最，’取PCL以二氧麵關配製成高;農度之pcL 二刀=喊(例如’ 5G%(W/W))。將依前述方法所製得之第 二Ϊ架二支架分別以高濃度之PCL高分子溶液做為黏 :劑抽二:隔離薄膜的相反兩面，再於真空下將殘餘的 乾°#此即可製得根據本發明所指出之具有複數相 16 1283569 之生物可分解性載體。 實施例二 依實施例-中隔離薄膜的製造方法In addition, a polyester polymer (for example, pCL and/or pLGA5〇/5〇) is dissolved in a dioxane solvent to prepare a 2% (w/v) polymer solution, and then it is difficult to pray. The towel (for example, the touch) allows the polymer solution to be divided evenly over the cast film. Next, a barrier film can be obtained by heating and domain cooling; east drying method, drying and removing the solvent in the separator film. Ancient, most, 'take PCL with a dioxic surface to make a high; agronomic pcL two knives = shout (for example '5G% (W / W)). The second truss two scaffolds prepared according to the above method are respectively made of a high concentration of PCL polymer solution as a viscous agent: two opposite sides of the separator film, and then the residual dryness under vacuum ## A biodegradable carrier having a plurality of phases 16 1283569 as indicated in accordance with the present invention is prepared. Embodiment 2 According to the embodiment - the method for manufacturing the isolating film
，取 PCL 與 PLGA 50/50以3: 1、5: 1、7: i與9: i之重量比製成不同成分 比例的隔離薄膜’將其切成小片狀，秤得重量w。，之後以 95%酒精滅菌’再以磷酸鹽緩衝溶液(沖〇叩血忪buffered yaline’ PBS)^洗二次後，浸泡於磷酸鹽缓衝溶液，於37 C、5% C〇2培養箱中進行降解測試，並於每一預設時間取 出樣品’以二次水去除樣品中的鹽類。之後，抽真空使其 麟後’㈣ίΐ %。最後，藉由下列公式計算其經降解 後之剩餘重量比，所得結果如表一所示。 經降解後之剩餘重量比(％)=Wi/w〇x 1〇〇% 參閱表一，可以看出經13週的測試，PCL:PLGA50/50 3·1、5·1、7·1與9:1的隔離薄膜降解比率分別約為 25% 15%、15%與12% ’且不同組成比例之隔離薄膜皆會 隨著時間的增加而增加降解程度。另外，從結果中亦可看 =Ik著隔離薄膜巾PCL所佔的的賴愈高，隔離薄膜被 降解的比例愈高，且於測試初期隔離薄膜的降解速率較 決，^後則趨於緩慢，其係由於較易被降解的 PLGA 50/50 :皮大里的降解’使得隔離薄膜中剩下主要以較不易降解之 =為主的成分。因此’由上述職結果可知，熟習本技 ^員域者可藉_整不同降解速度的聚合物組成比製得具 有所需降解速率的隔離薄膜。 1283569 $—隔離薄膜於體外進行降解測試之結果 間(週） PCL ： PLGA^-^ 0 ，Ϊ ，鏽 | CXNJ V， 2 一^一-............ 5 9 13 3:1 100 98.33 土 1.81* 87.23 ± 3.74 78.6 + 0.92 75.49 士 0.74 5:1 100 97.67 ± 2.71 96.85 ± 0.07 85.2 ± 1.35 84.03 ± 3.52 7:1 100 99.37 ± 1.18 92.80 士 2.21 86.8 ± 0.4 85.82 士 1.07 9:1 100 100.98 ± 3.68 96.04 ± 5.26 91.33 ± 2.63 88.08 士 5.08 *，經降解後之剩餘比率(％) 實施例三 取年齡2年6個月、30-35公斤重的蘭峻豬，自其膝 蓋無受力區取出膝蓋軟骨組織，經以習知方式處理後，懸 浮於無企清的 DMEM (Dulbecco，s Modified Eagle Media)培 養基中。另外’自豬隻的腸骨脊(niac crest)中取出骨髓， 經以習知方式去除紅血球及其他雜物後，自骨髓中取得骨 髓基質幹細胞並將其懸浮kl_dmem培養基中。 取上述預先製備細胞濃度為lxlO6 cells/ml的軟骨細 胞與2xl06cells/ml的骨髓基質幹細胞之細胞懸浮液，利用 支架固定化動態植入方式分別植入實施例一中所製得之載 體中的第一支架與第二支架。並藉由一雙腔式生物反應器 (例如，如第六圖所示）中進行靜態培養，其中第一支架與 第二支架係分別位於雙腔式生物反應器的第一腔室與第二 18 1283569 腔室"ϋ使兩腔室_由本翻載體雜離薄膜分隔。 於進仃培養時，先於第—腔室與第二腔室中分別注入 DMEM培養基與l，DMEM培養基，之後放人37°C、5% C02 的培養箱中進行培養，1週更換兩次培養基。於培養-週 後’第一腔室中的培養基更換成含有5_/Ιη1Ι^抗壞金酸 的8〇μεμ培養基，而第二腔室中的培養基則更換為含有 10 8Μ曱基脫氫皮質固醇(dexamethas〇ne)、1〇mM ρ甘油磷 酸(β-glycerophate)與 50 pg/mi L_抗壞血酸的 L-DMEM 培養 基’以促使骨髓基質幹細胞分化成硬骨。經培養後分析各 支架的測試結果分述於表二及表三。 多二軟骨細胞植么多一★架德的生化分拚結果 天） 測試項 1 14 28 49 支架内細胞數 8.84 ± 11.6 ± 12.29 ± 37.23 ± (lxlO4 cell) 1.45 0·13 1.13 1.99 支架内葡萄糖胺 17.66 ± 18·71 土 27.10 土 49·13 士 聚醣的含量^g) 0.74 1.05 2.77 2.97 支架内膠原蛋白 15.33 ± 21·98 ± 39.87 土 56.55 土 的含量(Kg) 2.86 2.96 14.76 23.32 19 1283569 硬骨細胞^植入蔓三支架後的生化分析結l 事ι(天 Γ[ — Ί ~PCL and PLGA 50/50 were prepared in a ratio of 3:1, 5:1, 7:i and 9:i to form a separator of different composition ratios, which were cut into small pieces and weighed by weight w. , then sterilized with 95% alcohol and then washed twice with phosphate buffer solution (buffered yaline' PBS), soaked in phosphate buffer solution at 37 C, 5% C 〇 2 incubator The degradation test is carried out, and the sample is taken out at each preset time to remove the salt in the sample with secondary water. After that, evacuate it to make it ’((四)ίΐ %. Finally, the residual weight ratio after degradation was calculated by the following formula, and the results are shown in Table 1. Residual weight ratio after degradation (%) = Wi/w〇x 1〇〇% Referring to Table 1, it can be seen that after 13 weeks of testing, PCL: PLGA50/50 3·1, 5·1, 7·1 and The 9:1 barrier film degradation ratio is about 25%, 15%, 15%, and 12%, respectively, and the separators of different composition ratios will increase the degree of degradation with time. In addition, from the results, it can be seen that the IK with the isolating film towel PCL is higher, the higher the ratio of the barrier film is degraded, and the degradation rate of the barrier film is determined at the initial stage of the test. It is due to the degradation of PLGA 50/50, which is more easily degraded: the degradation of the skin is mainly caused by the component which is mainly less susceptible to degradation. Therefore, it can be seen from the above-mentioned results that a person skilled in the art can obtain a separator having a desired degradation rate by using a polymer composition ratio of different degradation rates. 1283569 $—The result of the degradation test of the release film in vitro (week) PCL : PLGA^-^ 0 , Ϊ , rust | CXNJ V, 2 一一一-......... 5 9 13 3:1 100 98.33 Soil 1.81* 87.23 ± 3.74 78.6 + 0.92 75.49 ± 0.74 5:1 100 97.67 ± 2.71 96.85 ± 0.07 85.2 ± 1.35 84.03 ± 3.52 7:1 100 99.37 ± 1.18 92.80 ± 2.21 86.8 ± 0.4 85.82 ± 1.07 9:1 100 100.98 ± 3.68 96.04 ± 5.26 91.33 ± 2.63 88.08 ± 5.08 *, residual ratio after degradation (%) Example 3 takes the age of 2 years and 6 months, 30-35 kg of Lang Jun pig, from The knee cartilage tissue was taken out in the unstressed area of the knee, and after being treated in a conventional manner, it was suspended in DMEM (Dulbecco, s Modified Eagle Media) medium. Further, bone marrow was taken out from the intestine ridge (niac crest) of the pig, and after removing red blood cells and other debris in a conventional manner, bone marrow stromal stem cells were obtained from the bone marrow and suspended in kl_dmem medium. Taking the above-mentioned cell suspension of chondrocytes having a cell concentration of lxlO6 cells/ml and bone marrow stromal cells of 2×10 6 cells/ml, and implanting the cells prepared in the first embodiment by stent-immobilized dynamic implantation. A bracket and a second bracket. And performing static culture in a double chamber bioreactor (for example, as shown in Figure 6), wherein the first stent and the second stent are respectively located in the first chamber and the second chamber of the dual chamber bioreactor 18 1283569 The chamber "ϋ makes the two chambers_ separate from the negative carrier film. In the culture of the sputum, the DMEM medium and the DMEM medium were injected into the first chamber and the second chamber, respectively, and then cultured in a 37 ° C, 5% C02 incubator, and replaced twice a week. Medium. After the culture-week, the medium in the first chamber was changed to 8〇μεμ medium containing 5_/Ιη1Ι^corrosic acid, and the medium in the second chamber was replaced with 108% dehydrocorticotrope. Alcohol (dexamethas〇ne), 1 mM ρ glycerol phosphate (β-glycerophate) and 50 pg/mi L_ascorbic acid in L-DMEM medium to promote differentiation of bone marrow stromal stem cells into hard bone. The test results of analyzing each scaffold after culture are described in Tables 2 and 3. More than two chondrocytes planted a lot of ★ ★ Deer biochemical spelling results days) Test item 1 14 28 49 Number of cells in the stent 8.84 ± 11.6 ± 12.29 ± 37.23 ± (lxlO4 cell) 1.45 0·13 1.13 1.99 Glucosamine in the stent 17.66 ± 18·71 Soil 27.10 Soil 49·13 Content of sulphate^g) 0.74 1.05 2.77 2.97 Collagen in the scaffold 15.33 ± 21·98 ± 39.87 Soil 56.55 Soil content (Kg) 2.86 2.96 14.76 23.32 19 1283569 Hard bone cells ^ Biochemical analysis after implantation of the vine three brackets l Things ι (天Γ[— Ί ~
由表二中可以看出，隨著培養時間的增加，軟骨細胞 生長有逐漸向上攀升的趨勢，於㈣天時軟f細胞生長速 率較為緩慢’ Μ 2849天時，軟骨細胞生錢率上升幅 ，較大。而支架内由軟骨細胞所分泌之㈣糖胺聚釀的含 量’於培養初期(1-14天），僅有少量的葡萄糖胺聚膽生成， 而至培養中後期（14_49天）則有大量的葡萄糖胺聚畴生 成，其分泌速率約騎養初_ 5倍。至於軟骨細胞之膠 原蛋白的分泌量嗜培養初期軟骨細胞合成膠原蛋白 率較為緩慢，僅有少量的膠縣白合成。而在培養中 膠原蛋㈣合成速相顯的增加，較培養初期增加約工件。 ，外’骨赌質幹細胞在培養過程中，係先進行“ 1週後’接著再進行骨分析培養6週。 ' 骨聽基質幹細胞於培養初射，已進行骨分化旦 有稍微的下降，至培養中後期後 里 的趨勢。此外，妙養油胞數里又有開始增加 °養_ ’由骨難質幹細胞骨分化過 20 1283569 程中所分泌之葡驗性磷酸酶(alkaline phosphatase，ALP)的 含量可以看出’於培養初期有較高之葡驗性麟酸酶的分泌 量，之後葡鹼性磷酸酶的分泌量則有下降之趨勢。顯示骨 髓基質幹細胞於更換培養基進行骨分化的初期，即已大量 分化成硬骨細胞。骨髓基質幹細胞於骨分化過程的初期， 膠原蛋白的分泌速度較為緩慢，而至後期則有較為明顯的 膠原蛋白合成速率。 綜上所述，本發明載體於分別植入軟骨細胞與骨髓基 質幹細胞後，以培養4週以上後植入生理系統中較為適宜。 另參閱第七A圖’為第一支架經培養49天以後之組 織切片圖（以Hematoxylin-Eosin組織染色）。從第七a圖中 可以觀察到支架外圍已形成一層薄薄的軟骨細胞，且支架 内部已可見大量細胞的生長。參閱第七B圖，為第二支架 經培養49天以後之組織切片圖（以Hemat〇xylin E〇sin組織 染色）。從第七B圖中可以明顯看出支架内外有均勻的鈣化 現象，愈靠近支架外圍鈣化程度愈明顯，且亦可觀察到支 架内外均有大量細胞聚集的現象。另外，由組織切片中可 以觀察到，細胞會先於支架外圍形成組織結構，之後再逐 漸往支架内部遷移，並於支架内部繼續生長。 顯示本發明載體確實可供軟骨細胞生長，以及供骨髓 基質幹細胞生長分化成硬骨細胞，且載體中之分隔薄膜可 、文將位於同-載體上生長之軟骨細胞與硬骨細胞分隔 ^使於軟骨細胞生長過程中所分泌用以抑觸化之葡萄 胺聚酿不會錢至第二支針，*干制硬骨細胞的生 21 1283569 長。 實施例四 將年齡2年6個月、3G_35公斤重之蘭铺麻醉後， 打開其股骨與脛骨_膝錢，職徑7mm環刀於不受力 區(patellar groove)製造之傷口。接著’將實施例三中已預 先於生物反應器中培養四週之載體，依第四圖中所示方式 植入受損部位中後縫合傷口。之後，於6则後打開傷口， 觀察支架值人後之修復狀況，並將支架取出後做進一步的 分析。 麥閲第八A圖 1 ^7、、、两見又永植入猪隻内經六個月修;j 後的關節組織外觀。從第人A圖中可以看出植人本發明、 體所修復的關節處與其周圍的關節面非常的平整光%骨(毫 圈處）’顯示受損處的修復狀況十分良好。另參^第s 圖，為本發明載體於植入豬隻體内經修復3個月後關節名 織中軟骨修復處的組織切片圖。由第八B圖中可以觀客】 所修復軟骨有許多新生軟骨_，並與周_原生組^ 結合性非常良好’完全看;^到本發明紐朗岐織的; 面，且支架完全降解。參閱第八C圖為本發明载體於植^ 豬隻體内經修復3则後關節組射硬骨修復處的組^ 片圖。由第八C圖中可以觀察到所修復的硬骨已新、味 管與新生骨的產生。 4生』 【圖式簡單說明】 22 1283569 第一圖為根據本發明所指出之具有複數 性载體之立體示意圖； 圖為根據本發明所指出之具有複數 性載體之剖面示意圖； 圖備根據本發明所指出之具有複數相的生物可 为解性載體之流程圖； =2發明載體植入一生理環境中之剖面示意圖； ®二發明載體植入一生理環境中之另一剖面示意 第 第三 圖 •相的生物可分解 ‘相的生物可分解 第六圖4將本發日輯體置人―雙腔式生物反應器進行培 養之示意圖； f A圖為第—支架經培養49天以後之組織切片圖； ^ 圖為第一支架經培養49天以後之組織切片圖； 第八AH為細胞支架植人豬隻内經六個月修復後的關節 組織外觀； 第八：8圖為本發明健於植讀隻體⑽修復3個月後 關節組織中軟骨修復處的組織切片圖；以及 第/VC®為本發8賴植人豬隻體⑽修復3個月後 關節組織中硬骨修復處的組織切片圖。 【主要元件符號說明】 10載體 12第一支架 14第二支架 16 隔離薄膜 23 聚合物溶液 第一聚合物材料 第二聚合物材料 以第一溶劑溶解 以第二溶劑溶解 去除支架中溶劑 去除支架中溶劑 支架改質 支架改質 隔離薄膜用聚合物材料 以溶劑溶解 去除溶劑 將第一支架與第二支架分別黏合於隔離薄膜的兩側 去除載體上的殘餘溶劑 軟骨組織 硬骨組織 孔洞 不滲透#5化軟骨層 雙腔式生物反應器 第一腔室 第二腔室 挾持裝置 24It can be seen from Table 2 that with the increase of culture time, the growth of chondrocytes gradually increases upwards. At (4) days, the growth rate of soft f cells is slower. Μ 2849 days, the rate of chondrocyte production increases. Larger. In the scaffold, the amount of glycosaminoglycan secreted by chondrocytes is 'in the early stage of culture (1-14 days), only a small amount of glucosamine is produced, and in the middle and late culture (14_49 days) there are a large number of The glucosamine polydomain is formed, and its secretion rate is about 5% times that of the first riding. As for the secretion of collagen protein in chondrocytes, the rate of collagen synthesis in chondrocytes in the early stage of culture is relatively slow, and only a small amount of rubber is synthesized in white. In the culture, the increase in the speed of collagen (4) synthesis is increased, and the workpiece is increased compared with the initial stage of culture. In the process of culturing, the skeletal stem cells were first cultured for 1 week after the "one week later" and then subjected to bone analysis for 6 weeks. 'Bone stromal stem cells were initially injected in the culture, and the bone differentiation has been slightly decreased. The trend of cultivating in the middle and late stages. In addition, there is an increase in the number of cells in the cultivating oil. _ 'Akaline phosphatase (ALP) secreted by the skeletal stem cell bone differentiation in 20 1283569 The content can be seen as 'the secretion of higher phytase in the early stage of culture, and then the secretion of alkaline phosphatase is decreasing. It shows the initial stage of bone differentiation of bone marrow stromal cells in the medium. That is, it has been largely differentiated into hard bone cells. In the early stage of bone differentiation, bone marrow stromal cells have a slower rate of collagen secretion, and at the later stage, there is a more obvious rate of collagen synthesis. In summary, the vectors of the present invention are respectively After implantation of chondrocytes and bone marrow stromal stem cells, it is more suitable to be implanted into the physiological system after culture for more than 4 weeks. See also Figure 7A for the first stent. The tissue section of the tissue after 49 days (stained with Hematoxylin-Eosin tissue). It can be observed from the seventh a picture that a thin layer of chondrocytes has been formed on the periphery of the stent, and a large number of cells have been seen inside the stent. Figure B shows the tissue section of the second scaffold after 49 days of culture (stained with Hemat〇xylin E〇sin tissue). It can be seen from Figure 7B that there is uniform calcification inside and outside the scaffold, and the closer to the periphery of the scaffold The more obvious the degree of calcification, the large amount of cells aggregated inside and outside the stent can be observed. In addition, it can be observed from the tissue section that the cells will form a tissue structure before the stent, and then gradually migrate to the inside of the stent. The inside of the stent continues to grow. It is shown that the carrier of the present invention can be used for the growth of chondrocytes, and for the differentiation and differentiation of bone marrow stromal stem cells into hard bone cells, and the separation membrane in the carrier can separate the chondrocytes grown on the same carrier from the hard bone cells. ^Let the cobamine produced during the growth of chondrocytes to inhibit the touch of the grape will not cost money to the second needle * The life of dried hard bone cells is 21 1283569 long. Example 4 After anesthesia for 2 years and 6 months, 3G_35 kg of orchids, open the femur and tibia _ knee money, the diameter of 7mm ring knife in the unstressed area a wound made by (patellar groove). Then, the carrier which has been cultured for four weeks in the bioreactor in the third embodiment is inserted into the damaged portion in the manner shown in the fourth figure, and then the wound is sutured. Thereafter, at 6 After opening the wound, observe the repair status of the scaffold value, and take out the scaffold for further analysis. The first reading of Fig. 8A, Fig. 1 ^7, and the two are also implanted in the pig for six months; The appearance of the joint tissue after j. It can be seen from the figure A of the human being that the implanted body, the joint repaired by the body and the surrounding joint surface are very flat and light (% of the circle) The situation is very good. In addition, the figure s is a histogram of the cartilage repair site in the joint name of the carrier in the pig body after being repaired for 3 months. From the eighth B picture, you can see that the cartilage has many new cartilage _, and it has a very good combination with the Zhou _ original group. 'Completely looking at it; ^ to the New Zealand woven fabric of the present invention; . Referring to the eighth C diagram, the carrier of the present invention is repaired in the body of the pig, and the posterior joint group is subjected to the hard bone repair. The repair of the bony, the taste tube and the new bone can be observed from Figure 8C. BRIEF DESCRIPTION OF THE DRAWINGS The first figure is a perspective view of a multiplicity carrier according to the present invention; the figure is a schematic cross-sectional view of a multiplicity carrier according to the present invention; The biofilm having the complex phase indicated by the invention can be a flow chart of the decomposable carrier; = 2 is a schematic cross-sectional view of the invention implanted in a physiological environment; the second invention is implanted into a physiological environment, and the other cross-section is shown as the third Figure 2: Biodegradable 'biodegradable phase' Figure 6 is a schematic diagram of the culture of the two-chamber bioreactor in the present day; f A is the first stent after 49 days of culture. Tissue section map; ^ The figure shows the tissue section of the first scaffold after 49 days of culture; the eighth AH is the appearance of the joint tissue after repairing the cell in the cell scaffold for six months; eighth: 8 is the invention The tissue section of the cartilage repair in the joint tissue after 3 months of repairing the body (10); and the /VC® is the repair of the bone in the joint tissue after 3 months of repair Organization Slice graph. [Main component symbol description] 10 carrier 12 first support 14 second support 16 isolation film 23 polymer solution first polymer material second polymer material is dissolved in the first solvent and dissolved in the second solvent to remove the solvent removal stent in the stent The solvent scaffold modified stent is modified with a polymer material to dissolve the solvent by solvent. The first scaffold and the second scaffold are respectively adhered to the two sides of the separator to remove the residual solvent on the carrier. The cartilage tissue is not penetrated. Cartilage layer double chamber bioreactor first chamber second chamber holding device 24