1275402 九、發明說明： 【發明所屬之技術領域】 本發明係關於一種用於進行組織拉 σ養的立體支架，特別是一 種生物可分解之多孔性立體支架及其製備方法。 』疋 【先前技術】 近幾年來，Pa者生物科技的進步而逐漸發展出組織工程 (W engm_g)，主要係藉由生醫材料提供立體結構 為組織細胞的培養支架，進而料_細胞生長成合適的 塊。未來，對於受損之組織或雜即可藉此類之姻^程再迭技 術，於體外修補或重建-新的組織或器官來替代受損部位，以恢 復病患的健康或延續病患的生命。 於組織工㈣技射，相當重要的_技術之―，係為發展 可为解性的多孔性支架’以作為組織細胞纟長的立體支架。由於 開發之多孔性支架需供細胞生長於其巾，故其需具備以下的特 性·可吸收分解性、南孔隙率、適當的孔徑、三度空間的多孔結 構，以及相互連通之孔隙結構。此外，於支架材料之種類中，目 前以生物可分解性的高分子材料最受研究者所重視。此類高分子 材料包括有·聚乙交酯酸（polyglycolicacid ; pGA)、聚丙交酯酸 (polylactic acid，PLA)、聚(乳酸-甘醇酸)(p〇iy (giyC〇iide_co_iactide) acid ’ PLGA)、聚己内醋（p〇iyCapr〇iaci〇ne ; pcL)、聚二戍烧g同 (polydioxanone)、聚原酸酯（poiyorthoester ; POE)、聚偶石粦氮 (polyphophazenes)、聚酯氨酯（p〇iyesterurethane)、聚酸酐亞胺 1275402 共聚物（poly(anhydride-co-imides))、聚經丁醋（polyhydroxyrate ; PHB)或其共聚物等材料’其於生物體内可分解微小分子鍵段， 以隨著人體内的新成代謝過程排出體外。 由於多孔性立體支架的架構乃係決定所要形成的組織的排 列及形態，目前，已提出用以製備生物可分解之高分子多孔性支 架的製備方法，只能製備單一孔隙結構或單一材料組成的多孔性 支架，然而人體組織係為連續性且多層次的結構，且其主要係由 #不同的組織間基質密度及不同材料所構成的多層次結構，引此開 發一可調控不同多孔隙架構及材料組合係為目前所需突破的關鍵 技術之一。 可歸納出現有之高分子多孔性支架的製備方法包括有：溶液 鍛造法（solution casting)、溶劑鍍造鹽洗法（s〇lvent_casting particulate leachmg)、膠體鍍造法（gel casting)、氣體發泡法（聊 foaming ) ^ ^ (phase separati〇n ). ^ ^ b〇nded fiber ^ 參及顆粒燒結法（particiesinter^ng)等。 夕於美國專利第5514378號中教示，利用溶劑鍍造鹽洗法製備 夕孔11越’再以二度空間的薄膜去重疊製備不同層次之立體支 木j此去因製備之單層支架厚度有限且其為不連續的製備方 法’因此需以人工進行加I堆疊，致使4繁複私乎成本效益， 法有效地進行量產。再者，其亦受限於鹽類與高分子溶液 =又目距太大而造成分布不均、鹽類顆粒可能被完全包覆以及支 糾部會殘留有機溶劑等_ 4外，於單層支架與單層支架間 1275402 _由溶劑將之連結在—起，如此將使不⑽界面部分的孔隙被 洛劑溶化而封死，因而造成不連通的孔隙結構。 此外，於相分離法中，雖然其利用凝結冰晶的型態來控制產 生之孔洞的形態，但目前於控制冰晶形態、顆粒大小、均勾度及 排列等技術上都尚未成熟，並無法直接麵於製備多層次多孔性 支架。 力再者，於中華民國專利第574273號中揭示多層次高分子支 製備方法，主要係將高分子材料和水溶性材料之混合物填入 -換具中，再將有機溶織經模制，贿高分子材料之表面溶 解並相互紐，最後再將水溶液流經模翻，以洗出水雜材料。 =此方法巾’所使用之模具係為可減壓過濾、之模具，並搭配使用 :減壓抽氣設備’藉贿錢關或水練驗模具内，使黏結 :刀子材料或洗出水溶性材料。因此，此方法雜可快速製備所 而之立體支架，但由於受限於使用模具，因而限制了其所能製備 # =支_狀及尺寸。再者，由於在黏結高分子材料上係藉由有機 ^劑溶解表面而使其相絲結，因此難以纽地控制製傷得之支 :尺寸和σσ貝’舉例來說，當過量之有機溶劑流麵具内時，易 ^成車乂丨尺寸之支架，反之，當流經模具内之有機溶劑不足時， 恐將因高分子材料未充分黏結而造成易鬆散之支架。此外，由於 =方去係藉由有機溶劑溶解表面而使高分子材料相互黏結，是故 貫際上於製備支架時’財機溶_溶解而有少許之高分子材料 被洗出’因而造成材料的浪費，並且許多高分子材料的價格都相 1275402 當昂貴，如此-來’勢必將造成成本的增加。 此外’此·it方法多需财機溶劑。然而有機溶劑大 多具有毒性，並且製備之支架最終都需應用於人體，因此有機容 _殘留與否-直係於使财機溶_製備方法中被受質疑的問 題點。 另外’於細胞的植入（seeding)技術上亦係目前多孔性立體 支架使用上不易克服的問題之_。由於此多孔性立體支架需提供 鲁細胞足夠發展成為組織的空間，因此其孔徑需大於細胞之尺徑: 以致細胞可深入支架内且貼附於支架上，進而生長於其上。然而， 當細胞與培養基的混合雜人多絲支料，纽的細胞往往會 因重力作用而於未貼附前即流出支架，不易流於支架的空隙内: 為克服此問題，目前多採用兩種做法：靜態植入法及動態植入法。 靜態植入法，主要係以高濃度之細就合液加於支架上，利 用多孔性支架本身的含水性將所植人之細胞含於支架内，待細胞 籲貼，於支架上後，再加入大量之培養基進行培養。此方法雖然可 確疋植入支架㈣細胞量，然而受到重力影響仍會造成上下層之 細胞分布不均的現象，此外為防止細胞溢出，能與細胞混合之培 養基相當有限，因此細胞貼附的效果及存活率亦需再考量。 動態植入法，目前-般係採用祕式反應器（spimierflask)， 以水拌的方式將細胞自培養基帶人支糾。此方法可得到較 佳之細胞貼附率及相對靜態之植入方式更均句的細胞分布，然 而所品使用之細胞數更多’且無法得知實際貼附於支架内之細 1275402 胞數·，此外，此方法的細胞植入仍是由外而内植入，故外圍的細 胞密度還是會高_部的細胞密度，於培養時，外__包生長 速率較為高，以致將形成-層阻礙，而阻相部細胞與培養基間 的養分交換，進而導致支架内外細胞生長明顯不均。 所以，對於改善多孔性支架的製備方法，仍是於組織工程的 技術中極重要且關鍵的研究課題之一。 【發明内容】 鐾於以上的問題，本發_主要目的在於提供—種生物可分 解，多孔性續該及其㈣方法，藉轉決先前技術所揭露的 問遞。於此’主要係藉由縣法而使基_結成型，以快速製備 多層次（包括：多孔徑、多孔隙率或不同材料組成）之立體支架， 並且可避免有機溶劑殘留於支架的問題。再者’搭配設置一凝膠 層，以提高細胞的留存率，並且還可提高支架強度。 、因此，本發明所揭露之生物可分解之多孔性立體支架的製備 方法’包括有下列步驟：依據一既定重量比將基材分別盘可水溶 轉料以關方式混合，以制至少—種_混合物；將固態混 ^物填入至模具巾；熱麵具巾之_混合物，以使基材黏結成 型；以及移除成型之_混合物内之可水溶性材料，以得到成型 之基材。 於此，可藉由模具的設計可使成型之基材具有一容置空間， 再將嘁膠，谷液填入至容置空間，待凝膠溶液凝結成固態膠後，可 知到複合支架。此外，於植入之細胞可於凝膠溶液填入至容置 1275402 ”先行與/旋膠溶液混合，再將混合液填入至容置空間内。 —2 ’可糊雜壓細機以-既定溫度鋪該目態混合物 既定日守間’藉以使模具中之基材黏結成型。 再者可將成型之固悲混合物置於水溶液中，以將内部之可 、六丨生材料溶解出來。於此，藉由定時更換水溶液，可確保並加 '欠’谷丨生材料的充分溶解。再者，亦可以僅以或辅以攪拌水溶 夜的方式/文泡成型之之固態混合物，來加速可水溶性材料溶解。 鲁其中，較佳之水溶液係為去離子水。 運用於本發明之基材可為高分子材料，或係為高分子材料混 合補強材料。其中，高分子材料可為聚乙交酯酸（p〇lyglyc〇lic acid ’ PGA)、聚丙交酯酸（p〇iyiactic acid ; PLA)、聚(乳酸_甘醇 酸）（poly (glyC〇lide-co_lactide) acid ; PLGA )、聚己内酯 (polycaprolactone ; PCL)、聚二戊：):完酮（polydioxanone)、聚原酸 酯（polyorthoester ; POE)、聚偶鱗氣（polyphophazenes)、聚酯氨 φ S旨（polyesterurethane)、聚酐（polyanhydrides)、聚酸酐亞胺共聚 物（poly(anhydride-co-imides))、聚羥丁酯（polyhydroxyrate ; PHB ) 或其共聚物。以及，可混合之補強材料包括有：無機材料，例如： 氫氧基填灰石（Hydroxyapatite ; HAP )、鱗酸鹽（calcium phosphates ; CP)、三I弓磷酸鹽（tricalciumphosphates ; TCP)、焦 石粦酸鹽（dicalcium phosphates )、四 #5 鱗酸鹽（tetracalcium phosphates ; TECP)、八妈鱗酸鹽（octacalciumphosphates)、非晶 質石粦酸鹽（amorphous calcium phosphates ; ACP)和貧|弓鱗酸鹽 11 1275402 ^Calcium deficient apatites ； CDA) # 〇 仏形狀係為顆粒狀，而較佳之粒徑尺寸係為於湖鋒以下。。 本發明之凝膠溶液可為水凝膠材料，例如：海 膠、膠原蛋白轉和纖維蛋白凝轉力，較此水凝膠材料 係為海藻膠。 本發明所揭露之生物可分解之複合支架，包括··—基材層， 具有複數觀隙；以及—導層，雜目絲該基材層上。 中基材層可具有一容置空間，並且凝膠層即位於此容置 空間内。 此基材層包括有至少一種高分子材料。適用之高分子材料可 為聚乙交酯酸（PGA)、聚丙交g旨酸（PLA)、聚(乳酸甘醇_ (PLGA)、己内酯（pCL)、聚二戊烷酮、聚原酸酯（p〇E)、 聚偶磷氮、聚酯氨酯、聚酐、聚酸酐亞胺共聚物、聚羥丁酯（pHB) 或其共聚物。 再者，此基材層更包括有至少一種無機材料，例如：氫氧基 磷灰石（HAP)、磷酸鹽（CP)、三鈣磷酸鹽（Tcp)、焦磷酸鹽、 四1¾磷酸鹽（TECP)、八舞磷酸鹽、非晶質磷酸鹽（ACp)和貧 鈣磷酸鹽（CDA)等。 此外，凝膠層包括有至少一種水凝膠材料。此水凝膠材料可 為海澡膠、瓊脂凝膠、膠原蛋白凝膠和纖維蛋白凝膠中之一。 於此，較佳之孔隙尺寸係為在以下。 有關本發明的特徵與實作，茲配合圖示作最佳實施例詳細說 12 1275402 明如下。 【實施方式】 以下舉出具體實施例以詳細說明本發明之内容，並以圖示作 •為辅助說明。說明中提及之符號係參照圖式符號。 於此實施例中，所選用之材料為：（1)基材（即，支架的原 料）：為了方便，在此以聚(乳酸_甘醇酸）（PLGA)之粉碎的顆粒 作S兄明；及（2)可水溶性材料：為了方便，在此以氯化納（Na⑴ 魯顆粒作說明。 芩照第1圖’首先，依據-既定重量比將上述各材料顆粒以 固態形式混合均勻（步驟110)，混合後，將混合之顆粒倒入一模 具中（步驟120)，熱壓倒入模具中之混合顆粒，藉以使顆 粒黏結成型，以得到所需之支架（步驟13〇)，取出成型之支架， 並將支架内之氯化鈉顆粒水洗出來（步驟丨4〇)。 於此’可直接將模具置入一熱麗成型機中，進行熱壓，以得 馨到所需之支架◦是故，僅需根據所需之立體支架的形狀和尺寸， 設計適當的模具（即，具有相應所需立體支架的形狀和尺寸的容 • 置空間之模具），即可快速製得所需之立體支架。於此，可利用熱 壓成型機以約95 C之溫度進行約2小時的熱壓，以得到所需之支 ' 架。 其中，於「步驟140」中，可將成型之支架置於裝有水溶液 的燒杯中，以將内部之氯化鈉顆粒洗出。於此，可定時更換燒杯 中之水溶液以充分洗出氯化執顆粒，並且亦可加速氯化納顆粒的 13 1275402 洗出。此外，亦可以的方式浸泡水洗，以加速氯化納顆粒的 洗出。較佳之水溶液係為去離子水。 水洗後之支架可藉由真空供箱加熱乾燥或靜置風乾。於採用 真空烘箱加熱乾燥時，可以約坑之溫度進行加熱乾燥。 於此’欲控健備之支架的品質及孔徑大小，可於混合前， 先將氯化鈉顆粒鱗（步驟搬）’如第2圖所示。舉例來說，將 氯化納顆粒通過60-80孔目的篩網，過筛後可得到粒徑範圍約為 • 88"m至250//m的氯化鈉顆粒。 再者’就「步驟110」來說，可於混合前，先依據所需之支 架尺寸及PLGA #氯化鈉的既定重量比分別計算所需之pLGA顆 粒和氯化麵㈣重量（步驟112)，再依據所計算的重量混合 PLGA顆粒和氯化鈉顆粒（步驟ι14)，如第3圖所示。 因此，可藉由量測乾燥後之支架重量，再將量測值與初始祥 取之PLGA顆粒的重量相比較，即可得知是否已完全洗出氯化納 0顆粒。 其中，PLGA和氯化納的既定重量比可介於至ι··9 $之 間（PLGA比氯化鈉）。較佳之plga和氯化鈉的既定重量比約為 1:9 (PLGA比氯化納）。 第4A、4B圖係顯示利用掃描式電子顯微鏡，分別於15〇〇〇 倍和35000倍的放大倍率下，所觀察得之根據本發明一實施例的 製備方法所製得之立體支架的結構。於圖中，可知已熱壓方式所 得到之立體支架係呈現多孔隙狀態，且PLGA顆粒間亦能充分黏 14 1275402 結。 於此，立體支架之孔隙粒徑可藉由添加的可水溶性材料 °.氣化#0的選定粒躲調控，社體支架的厚度亦可 (例如· PLGA)的添加量來調控。 再者，於製備多層次立體支架時，只需取得多種粒押之 納顆粒，依據既定重量比將其分別與PLGA雛混合㈣（步驟 210)，混合後，再依據粒徑大小將混合之顆粒倒入模具中（=驟 220) ’熱壓倒人模具中之混合顆粒，藉以使pLGA .黏結成型， 以得到所需之支架（步驟13〇)，取出成型之支架，並將支°架内之 氣化鈉顆粒水洗出來（步驟14〇)，如第5圖所示。 於此，雖僅描述多層次立體支架之製備方式，然事實上亦可 依據所需之立體支架的層次形態，變化PLGA顆粒和/或氯化鈉顆 粒，以及/或者變化混合之顆粒倒入模具的順序以得到各種型態之 多層次立體支架。 再者，本發明之基材可為高分子材料，或係為高分子材料混 合無機材料。其中，高分子材料可為聚乙交酯酸（p〇lyglyc〇lic acid ; PGA)、聚丙交酯酸（polylactic acid ; PLA)、聚(乳酸-甘醇 酸）（poly (glycolide-co_lactide) acid ; PLGA )、聚己内酯 (polycaprolactone ; PCL)、聚二戊烷酮（polydioxanone)、聚原酸 酯（polyorthoester ; POE)、聚偶鱗氮（polyphophazenes)、聚酯氨 酯（polyesterurethane)、聚酐（polyanhydrides)、聚酸酐亞胺共聚 物（poly(anhydride-co-imides))、聚經丁酯（polyhydroxyrate，· PHB ) 15 1275402 或其共聚物。以及，可混合之無機材料，例如··氫氧基磷灰石 (Hydroxyapatite ; HAP)、磷酸鹽（calciumphosphates，· CP)、三 鈣磷酸鹽（tricaldum phosphates ; TCP)、焦磷酸鹽（dica】dum phosphates)、四鈣鱗酸鹽（tetracalciumphosphates ; TECP)、八舞 磷酸鹽（octacalcium phosphates )、非晶質磷酸鹽（am〇rph〇us calcium phosphates ; ACP)或貧鈣磷酸鹽（Caldum defident apatites ; CDA)等。 • 並且，基材的形狀和尺寸並無特別限制，然較佳之形狀係為 顆粒狀或纖維狀。 此外，本發明之可水溶性材料包括無毒之水溶性材料，諸如 鹽類（例如：氯化鈉、氯化鉀（NaK)、氯化鉀（κα)、溴化鉀（ΚβΓ)、 氯化鈣（CaCl2)、氯化鎂（MgCl2 )、御細、偏、鱗酸卸、 矽酸_、碳酸納、碳酸氫氨 ' 硫酸納、硫酸钟、硫酸鎮、聚乙二 户等）A白貝（例如·明膠及洋菜膠等）、生物可相容之單或 _ 、又醣（例如·糊精、葡萄糖、嚴糖、果糖、左旋糖、麥芽糖、乳 糖等）、及多_聚合物（例如：殿粉、紐鹽、幾丁聚糖等）中之 ’或者係為以上之混合物。其中，可水溶性材料之較佳形狀係 :、顆粒狀’喻佳之粒徑尺寸係為於5GG//m以下。 <、再^，為增加植入支架内之細胞的留存性，可藉由上述方法 二成如第6圖所不之具有容置空間312的支架·，再填入凝膠 一夜3—2〇 (步驟15〇)，待凝膠溶液320固化後可得到如第7圖所 、支木300a (步驟16〇)，如第8圖所示。並且，藉由此製 16 1275402 備方法可提高支架的強度。 於此，可於將凝膠溶液填入支架的容置空間前，先將欲植入 之細胞與凝膠溶液混合均勻（步驟ls2)，混合後再將細胞盘凝膠 溶，的混合液填入至容置空間中（步驟154)，待凝膠溶液凝結成 固態膠後（步驟160)，如第9圖所*，即可得到已植入細胞之複 合支架，隨後即可進行後續之細胞培養或支架植入等程序。 適用於本發明之凝膠溶液可為水凝膠材料，例如：海藻膠、 •瓊脂凝膠、膠原蛋白凝膠和纖維蛋白凝膠等。其中，較佳之水凑 膠材料係為海藻膠。 免 其中，於「步驟丨60」中，可將填入凝膠溶液32〇後之支架 置於裝有溶液410的燒杯400巾，待凝膠溶液32〇凝結成膠狀: 待凝結後即可得到—複合支架·a，如第ω圖所示。於此，此 溶液可為—水溶液，其中較佳之水溶液係為具有_子之水、容 液^就是說，可將填人凝膠溶液後之支架浸泡於具有_子之 癱水溶液-既定時間’以使凝膠溶液凝結成勝狀。此既定時間可 !里。於此’製得之複合支架遍包括有孔Μ結構_ 和/母澡膠結構L320，如第π圖所示。 根據本發明之製備方法所製得之支架/複合支架可用以植入 =動物之組織“官的缺陷部位或異常部位，則丨導組織細胞生 =成合適的組織塊，進而達到修補或重建受損組織或器宫的目 時的:_機係於需將支架7複合支架植入哺乳動物 ^根據本發明之鶴方紗騎需之支架/複合支架。 17 1275402 再者，於支架/複合支架製備之後，可於支架/複合支架植入 哺乳動物體内之前或當時，將組織細胞植入於支架/複合支架中。 於此，此組織細胞可視預期之應用或待治療之疾病而定。其中， 細胞的培養可以於此領域中熟悉此項技術者所熟悉的標準及方法 的條件下進行。因此，欲植入支架/複合支架中之細胞可於植入前 先進行培養，以增加細胞數和/或誘導分化至所欲之表現型。再 者，細胞於植入支架/複合支架後，可於植入活體前，先於活體外 _進行培養，以促進適當生物基質的增加和積存。此外，細胞於植 入支架/複合支架後，亦可不經過體外培養即將支架/複合支架植入 活體内。 雖然本發咖前狀較佳實_揭露如上，财鱗用以限 定本發明，任何熟習娜技藝者，在不麟本發明之精神和範圍 内’當可作些狀更動無飾’因此本發明之專娜護範圍須視 本說明書所附之申請專利範圍所界定者為準。 【圖式間早說明】 第1圖係為說明根據本發明第一實施例之多紐立體支 製備方法的流程圖； 第2圖係為說明根據本發明第二實施例之多孔性立體支苹的 製備方法的流程圖； 第3圖係為說明根據本發明第三實施例之多孔性立體支苹的 製備方法的流程圖； μ 弟4A、4B圖係為顯示利用掃描式電子顯微鏡觀察得之根據 18 1275402 本發明一貫施例之多孔性立體支架的結構； 第5圖係為說明根據本發明帛四實施例之多孔性立體支架的 製備方法的流程圖； 第6圖係為說明根據本發明第一實施例之多孔性立體支架的 不意圖， 第7圖係為說明根據本發明第二實施例之多孔性立體支架的 示意圖； 第8圖係為說明根據本發明第五實施例之多孔性立體支架的 製備方法的流程圖； 第9圖係為說明於第δ圖中「步驟15〇」之一實施例的細部 流程圖； 第1〇圖係為說明於第8圖中「步驟150」之另-實施例的示 意圖；以及1275402 IX. Description of the Invention: [Technical Field] The present invention relates to a stereoscopic stent for performing tissue pulling, in particular a biodegradable porous stereo stent and a preparation method thereof. 』疋[Prior Art] In recent years, the progress of biotechnology in Pa has gradually developed tissue engineering (W engm_g), mainly by providing biomechanical materials to provide a three-dimensional structure for the culture of tissue cells, and then the cells grow into The right block. In the future, for damaged tissue or miscellaneous, this method can be used to repair or reconstruct a new tissue or organ to replace the damaged part in order to restore the health of the patient or to continue the patient's disease. life. In the organization of workers (four) technology, it is quite important that the technology is to develop a dissipative porous stent as a stereoscopic stent for tissue cell length. Since the developed porous scaffold is required to grow cells in its tissues, it is required to have the following characteristics: absorbable decomposition, south porosity, appropriate pore size, three-dimensional porous structure, and interconnected pore structures. In addition, among the types of scaffold materials, biodegradable polymer materials are currently highly valued by researchers. Such polymer materials include polyglycolic acid (pGA), polylactic acid (PLA), poly(lactic-glycolic acid) (p〇iy (giyC〇iide_co_iactide) acid 'PLGA ), polycaprolactone (p〇iyCapr〇iaci〇ne; pcL), polydioxanone, polyisic acid ester (poiyorthoester; POE), polyphophazenes, polyester ammonia A material such as poly(anhydride-co-imides), polyhydroxylate (PHB) or a copolymer thereof, which is capable of decomposing micromolecules in vivo. The key segment is excreted in the body with a new metabolic process. Since the structure of the porous stereo scaffold determines the arrangement and morphology of the tissue to be formed, a preparation method for preparing a biodegradable polymer porous scaffold has been proposed, and only a single pore structure or a single material can be prepared. Porous stent, however, the human tissue is a continuous and multi-layered structure, and it is mainly composed of different matrix density between different tissues and a multi-layer structure composed of different materials, which leads to the development of a different porous structure and The material combination is one of the key technologies for the current breakthrough. The preparation methods of the polymer porous stent which can be summarized include: solution casting, solvent plating, galvanic coating, gel casting, gas foaming. Method (talking foaming) ^ ^ (phase separati〇n ). ^ ^ b〇nded fiber ^ Participate in the particle sintering method (particiesinter^ng). According to the teachings of U.S. Patent No. 5,514,378, the solvent-plated salt-washing method is used to prepare the outer layer 11 and then the two-dimensional film is used to overlap and form different layers of the three-dimensional branch. Moreover, it is a discontinuous preparation method. Therefore, it is necessary to manually add I to stack, which makes the 4 complex and cost-effective, and the method is effective for mass production. Furthermore, it is also limited by the salt and polymer solution = the eye distance is too large to cause uneven distribution, the salt particles may be completely coated, and the organic solvent may remain in the support portion, etc., in a single layer. 16474002 between the stent and the single-layer stent _ is connected by a solvent, so that the pores of the (10) interface portion are melted by the agent and sealed, thereby causing a disconnected pore structure. In addition, in the phase separation method, although the shape of the formed pores is controlled by the type of condensed ice crystals, the techniques for controlling ice crystal morphology, particle size, uniformity and arrangement are not yet mature, and it is not directly For the preparation of multi-layer porous scaffolds. Further, in the Republic of China Patent No. 574273, a multi-layer polymer branch preparation method is disclosed, which mainly comprises filling a mixture of a polymer material and a water-soluble material into a-transformer, and then molding the organic solvent-dyed, bribe The surface of the polymer material is dissolved and mutually added, and finally the aqueous solution is flowed through the mold to wash out the water-containing material. = The mold used in this method is a mold that can be decompressed and filtered, and used in combination: decompression pumping equipment 'bring money or water to test the mold to make the glue: knife material or wash out water-soluble materials. Therefore, this method can quickly prepare a stereo stent, but it is limited by the use of the mold, thereby limiting its ability to prepare #=支___ and size. Furthermore, since the surface of the bonded polymer material is melted by the organic solvent to dissolve the surface, it is difficult to control the wounded branch: size and σσ贝', for example, when the excess organic solvent When the flow mask is inside, it is easy to form a bracket of the size of the rut. On the contrary, when the organic solvent flowing through the mold is insufficient, it is feared that the polymer material is not sufficiently bonded to cause a loose bracket. In addition, since the polymer is bonded to the surface by the organic solvent, the polymer material is bonded to each other, so that the material is dissolved and a small amount of the polymer material is washed out when the stent is prepared. The waste, and the price of many polymer materials are all 1275402 when it is expensive, so - come 'will inevitably lead to an increase in costs. In addition, this method requires more solvent. However, organic solvents are mostly toxic, and the prepared scaffolds are ultimately required to be applied to the human body, so the organic content _ residual or not - is directly related to the problem that has been questioned in the preparation method. In addition, the "seeding" technique of cells is also a problem that is difficult to overcome in the current use of porous stereo stents. Since the porous stereo scaffold needs to provide a space for the Lu cells to develop into a tissue, the pore size needs to be larger than the size of the cell: so that the cells can penetrate into the scaffold and attach to the scaffold, thereby growing thereon. However, when the cells are mixed with the medium and the multifilaments are mixed, the cells of the New Zealand tend to flow out of the stent before being attached due to gravity, and are not easily flowed into the gap of the stent: To overcome this problem, two Methods: static implantation and dynamic implantation. The static implantation method is mainly applied to the stent with a high concentration of fine fluid, and the cells of the implanted human body are contained in the stent by using the water content of the porous stent itself, and the cells are attached to the stent, and then the stent is placed on the stent. A large amount of medium is added for cultivation. Although this method can confirm the amount of cells implanted in the scaffold (4), the influence of gravity still causes uneven distribution of cells in the upper and lower layers. In addition, in order to prevent cell overflow, the medium that can be mixed with the cells is rather limited, so the cells are attached. The effect and survival rate also need to be considered. The dynamic implantation method, currently using the secret reactor (spimierflask), uses water to mix the cells from the medium. This method can obtain a better cell attachment rate and a more uniform cell placement than a static implant method. However, the number of cells used is more 'and the number of fine 1275402 cells actually attached to the stent is not known. In addition, the cell implantation of this method is still implanted from the outside, so the peripheral cell density will still be high _ part of the cell density, in the culture, the outer __ packet growth rate is relatively high, so that will form a layer Obstruction, and the exchange of nutrients between the cells in the phase-blocking phase and the culture medium leads to significant uneven growth of the cells inside and outside the stent. Therefore, the improvement of the preparation method of the porous stent is still one of the most important and key research topics in the technology of tissue engineering. SUMMARY OF THE INVENTION In view of the above problems, the main purpose of the present invention is to provide a biodegradable, porous, and (4) method, which is based on the prior art. Here, the base is formed by the county method to rapidly prepare a multi-layered (including multi-aperture, multi-porosity or different material composition) stereo stent, and the problem of residual organic solvent remaining on the stent can be avoided. Furthermore, a gel layer is provided in combination to increase the retention rate of the cells and also to increase the strength of the stent. Therefore, the method for preparing a biodegradable porous stereo stent disclosed in the present invention includes the following steps: mixing the substrate with a water-soluble transester in a closed manner according to a predetermined weight ratio to make at least one type _ a mixture; a solid mixture is filled into the mold towel; a mixture of heat mask towels to bond the substrate; and the water-soluble material in the formed mixture is removed to obtain a molded substrate. Herein, the molded substrate can be designed to have a receiving space, and then the silicone and the valley liquid are filled into the accommodating space. After the gel solution is condensed into a solid gel, the composite stent can be known. In addition, the implanted cells can be filled in the gel solution to accommodate the 1275402" prior to mixing with the / gel solution, and then the mixture is filled into the housing space. - 2 ' can be mixed with the fine machine to - The predetermined temperature is used to lay the mixture of the target, so that the substrate in the mold is bonded and formed. In addition, the solid mixture of the molding can be placed in an aqueous solution to dissolve the internal hexavalent material. Therefore, by periodically replacing the aqueous solution, it is possible to ensure sufficient dissolution of the 'undeum' glutinous material. Further, it is also possible to accelerate the water solubility by using only a solid mixture of a stirred water solution or a foamed molding. The material is dissolved. Among them, the preferred aqueous solution is deionized water. The substrate used in the present invention may be a polymer material or a mixed material of a polymer material, wherein the polymer material may be polyglycolide. (p〇lyglyc〇lic acid 'PGA), polylactide (P〇iyiactic acid; PLA), poly(glyC〇lide-co_lactide); PLGA, polycaprolactone (polycaprolactone; PCL), poly E:: dioxin (polydioxanone), polyorthoester (POE), polyphophazenes, polyester ammonia s S (polyester urethane), polyanhydrides (polyanhydrides), polyanhydride copolymers (poly(anhydride-co-imides)), polyhydroxyrate (PHB) or its copolymers. Also, the reinforcing materials include: inorganic materials such as: Hydroxylpatite (Hydroxyapatite; HAP) ), calcium phosphates (CP), tricalcium phosphates (TCP), dicalcium phosphates, tetracalcium phosphates (TECP), eight amygic acid Octacalcium phosphates, amorphous calcium phosphates (ACP) and lean | oleate 11 1275402 ^Calcium deficient apatites ; CDA) # 〇仏 shape is granular, and preferred particle size The gel solution of the present invention may be a hydrogel material, such as: jelly, collagen, and fibrin cohesion, and the hydrogel material is seaweed. The biodegradable composite stent disclosed in the present invention comprises: a substrate layer having a plurality of gaps; and a guiding layer on the substrate layer. The middle substrate layer may have an accommodating space, and the gel layer is located in the accommodating space. The substrate layer includes at least one polymer material. Suitable polymer materials may be polyglycolide (PGA), polyacrylic acid (PLA), poly(lactic acid glycol (PLGA), caprolactone (pCL), polydipentanone, polyphosphate An acid ester (p〇E), a polyphosphoryl nitrogen, a polyester urethane, a polyanhydride, a polyanhydride copolymer, a polyhydroxybutyl ester (pHB) or a copolymer thereof. Further, the substrate layer further includes At least one inorganic material, such as: hydroxyapatite (HAP), phosphate (CP), tricalcium phosphate (Tcp), pyrophosphate, tetraphosphonate (TECP), baho phosphate, amorphous Phosphate (ACp) and calcium-depleted phosphate (CDA), etc. In addition, the gel layer comprises at least one hydrogel material. The hydrogel material can be sea bath glue, agar gel, collagen gel and One of the fibrin gels. Here, the preferred pore size is as follows. Regarding the features and implementations of the present invention, the preferred embodiment will be described in detail with reference to 12 1275402. The specific embodiments are described in detail to explain the details of the present invention, and are illustrated by the accompanying drawings. Schematic symbols. In this embodiment, the materials selected are: (1) substrate (ie, the material of the stent): for convenience, the granulated particles of poly(lactic-glycolic acid) (PLGA) are used here. (S) Water-soluble material: For convenience, sodium chloride (Na(1) Lu particles are used for explanation. Referring to Figure 1 first, the above materials are solid in accordance with the established weight ratio. The form is uniformly mixed (step 110). After mixing, the mixed particles are poured into a mold (step 120), and the mixed particles in the mold are hot pressed, whereby the particles are bonded and molded to obtain a desired stent (step 13). 〇), take out the formed bracket, and wash the sodium chloride particles in the holder (step 丨4〇). Here, the mold can be placed directly into a hot forming machine for hot pressing to obtain a sweet The required bracket is the only one. It is only necessary to design a suitable mold according to the shape and size of the required three-dimensional bracket (that is, a mold having a space for the shape and size of the corresponding three-dimensional bracket). Make the required three-dimensional bracket. Here, The hot press is performed at a temperature of about 95 C for about 2 hours by a hot press to obtain a desired support. In the "Step 140", the formed stent can be placed in a beaker containing an aqueous solution. In order to wash out the internal sodium chloride particles. Here, the aqueous solution in the beaker can be periodically replaced to fully wash out the chlorinated particles, and the 13 1275402 of the sodium chloride particles can be accelerated to be washed out. The method is soaked in water to accelerate the elution of the sodium chloride particles. The preferred aqueous solution is deionized water. The water-washed stent can be dried by vacuum or statically dried. When heated by a vacuum oven, it can be about The temperature of the pit is heated and dried. Here, the quality and pore size of the stent to be controlled can be adjusted as shown in Fig. 2 before the mixing. For example, the sodium chloride particles are passed through a 60-80 mesh screen and sieved to obtain sodium chloride particles having a particle size ranging from about 88 "m to 250//m. Furthermore, in the case of "Step 110", the required pLGA particles and the chlorinated surface (IV) weight can be separately calculated according to the required stent size and the predetermined weight ratio of PLGA #Sodium chloride before mixing (Step 112). Then, PLGA particles and sodium chloride particles are mixed according to the calculated weight (step ι14), as shown in Fig. 3. Therefore, by measuring the weight of the stent after drying, and comparing the measured value with the weight of the initially taken PLGA pellet, it can be known whether or not the sodium chloride particles have been completely washed out. Among them, the established weight ratio of PLGA and sodium chloride can be between ι··9 $ (PLGA vs. sodium chloride). Preferably, the established weight ratio of plga to sodium chloride is about 1:9 (PLGA vs. sodium chloride). 4A and 4B are views showing the structure of a stereoscopic stent obtained by a production method according to an embodiment of the present invention, which was observed at a magnification of 15 和 and 35,000 times by a scanning electron microscope. In the figure, it can be seen that the stereo stent obtained by the hot pressing method exhibits a porous state, and the PLGA particles can also sufficiently adhere to the 14 1275402 junction. Here, the pore size of the stereo scaffold can be controlled by the selected particle of the added water-soluble material °, gasification #0, and the thickness of the social scaffold can also be controlled by the addition amount of (for example, PLGA). Furthermore, in the preparation of the multi-layered stereo scaffold, it is only necessary to obtain a plurality of granules of the granules, and mix them with the PLGA chicks according to the predetermined weight ratio (IV) (step 210), and then mix the granules according to the particle size. Pour into the mold (= step 220) 'Hot pressure to pour the mixed particles in the mold, so that the pLGA. is bonded to form the desired bracket (step 13〇), take out the formed bracket, and The vaporized sodium granules are washed out (step 14), as shown in Figure 5. Here, although only the preparation method of the multi-layer stereo stent is described, in fact, depending on the morphological form of the desired stereo stent, the PLGA particles and/or the sodium chloride particles may be changed, and/or the mixed particles may be poured into the mold. The order is to obtain a multi-layered stereo bracket of various types. Further, the substrate of the present invention may be a polymer material or a polymer material mixed with an inorganic material. Among them, the polymer material may be poly(glycolidec〇lic acid; PGA), polylactic acid (PL), poly(glycolide-co-lactide) ; PLGA ), polycaprolactone (PCL), polydioxanone, polyorthoester (POE), polyphophazenes, polyester urethane, poly Polyanhydrides, poly(anhydride-co-imides), polyhydroxyrate (PHB) 15 1275402 or copolymers thereof. And, inorganic materials that can be mixed, such as hydrogen hydroxyapatite (HAP), phosphate (calciumphosphates, CP), tricalcium phosphates (TCP), pyrophosphate (dica) dum Phosphates), tetracalcium phosphates (TECP), octacalcium phosphates, amorphous phosphates (ACP) or calcium-depleted phosphates (Caldum defident apatites; CDA) )Wait. Further, the shape and size of the substrate are not particularly limited, but the preferred shape is granular or fibrous. Further, the water-soluble material of the present invention includes non-toxic water-soluble materials such as salts (for example, sodium chloride, potassium chloride (NaK), potassium chloride (κα), potassium bromide (ΚβΓ), calcium chloride. (CaCl2), magnesium chloride (MgCl2), fine, partial, scaly acid, citric acid _, sodium carbonate, ammonium hydrogencarbonate 'sodium sulphate, sulfuric acid clock, sulphuric acid, polyethylene, etc.) A white shell (for example Gelatin and acacia gum, etc., biocompatible single or _, and sugar (eg, dextrin, glucose, Yan sugar, fructose, levulose, maltose, lactose, etc.), and poly-polymer (for example: temple 'In powder, neo-salt, chitosan, etc.) or a mixture of the above. Among them, the preferred shape of the water-soluble material is: granules, the size of the particle size is less than 5 GG / / m. <, again, in order to increase the retention of the cells implanted in the stent, the above method can be used to form a stent having the accommodating space 312 as shown in Fig. 6, and then filled in the gel overnight 2-3. 〇 (Step 15〇), after the gel solution 320 is solidified, the branch 300a (step 16〇) as shown in Fig. 7 can be obtained, as shown in Fig. 8. Moreover, the strength of the stent can be improved by the method of preparing 16 1275402. In this case, before the gel solution is filled into the accommodating space of the stent, the cells to be implanted are uniformly mixed with the gel solution (step ls2), and after mixing, the cell plate is melted and the mixture is filled. After entering the accommodating space (step 154), after the gel solution is condensed into a solid glue (step 160), as shown in Fig. 9, a composite scaffold of the implanted cells can be obtained, and then the subsequent cells can be obtained. Procedures such as culture or stent implantation. The gel solution suitable for use in the present invention may be a hydrogel material such as: seaweed gum, agar gel, collagen gel, and fibrin gel. Among them, a preferred water-repellent material is seaweed gum. In addition, in "Step 丨 60", the stent filled with the gel solution 32 置于 can be placed in a beaker 400 containing the solution 410, and the gel solution 32 condensed into a gel: after being condensed Obtained - composite scaffold · a, as shown in the figure ω. Herein, the solution may be an aqueous solution, wherein the preferred aqueous solution is a water having a water content and a liquid content. That is to say, the stent after the filling of the gel solution may be immersed in an aqueous solution having an aqueous solution of _ _ _ _ _ In order to make the gel solution condense into a win. This time is set! The composite stent produced herein comprises an apertured structure _ and / a mother bath structure L320, as shown in Fig. π. The stent/composite stent prepared according to the preparation method of the present invention can be used to implant the defect site or the abnormal site of the animal tissue, and then the tissue cell is converted into a suitable tissue block to achieve repair or reconstruction. At the time of the damage of the tissue or the uterus: _ the machine is required to implant the stent 7 composite stent into the mammal. The stent/composite stent for the crane yarn according to the present invention. 17 1275402 Furthermore, the stent/composite stent After preparation, the tissue cells can be implanted into the stent/composite stent before or at the time the stent/composite stent is implanted into the mammal. Here, the tissue cells can be determined depending on the intended application or the disease to be treated. The culture of the cells can be carried out under the conditions of the standards and methods familiar to those skilled in the art. Therefore, the cells to be implanted in the scaffold/composite scaffold can be cultured before implantation to increase the number of cells. And/or induce differentiation to the desired phenotype. Furthermore, after implantation of the stent/composite stent, the cells can be cultured prior to implantation into the living body to promote appropriate biobased In addition, after the implanted stent/composite stent is implanted, the stent/composite stent can be implanted into the living body without in vitro culture. Although the pre-cafe shape is better than the above, the scale is used to limit In the present invention, any skilled person skilled in the art will be able to make a change in the spirit and scope of the present invention. Therefore, the scope of the present invention is defined by the scope of the patent application attached to the present specification. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart illustrating a method for preparing a multi-dimensional stereoscopic branch according to a first embodiment of the present invention; and Fig. 2 is a view showing porosity according to a second embodiment of the present invention A flow chart of a method for preparing a stereoscopic branch; Fig. 3 is a flow chart for explaining a method for preparing a porous three-dimensional branch according to a third embodiment of the present invention; μD 4A, 4B is a display using a scanning electron microscope The structure of the porous stereo scaffold according to the consistent embodiment of the present invention is observed according to 18 1275402; FIG. 5 is a flow chart for explaining the preparation method of the porous stereo scaffold according to the fourth embodiment of the present invention. Fig. 6 is a schematic view showing a porous stereoscopic stent according to a first embodiment of the present invention, and Fig. 7 is a schematic view showing a porous stereoscopic stent according to a second embodiment of the present invention; A flowchart of a method for preparing a porous stent according to a fifth embodiment of the present invention; FIG. 9 is a detailed flow chart for explaining an embodiment of "Step 15" in the δ diagram; A schematic diagram of another embodiment of "Step 150" in Figure 8;
第11圖係為說明根據本發明第二實施例之多孔性立體支架 的截面圖。 【主要元件符號說明】 步驟102.........將氯化鈉顆粒過篩 ν驟0..........將聚(乳酸-甘醇酸)顆粒和氯化納顆粒混合均勻 步驟112 分別計算所需之聚(乳酸甘醇酸)顆粒和氯化納 顆粒的重量 ν驟14..........均勾混合聚(乳酸_甘醇酸)顆粒和氯化納顆粒 步驟120.........將混合顆粒填入至模具中 19 1275402 步驟130…· .....熱壓成型 步驟140…· .....取出模具中成型之支架，並將其内之氯化鈉顆 粒水洗出來 .步驟 150 ····, .....將凝膠溶液填入至支架中 步驟152 ••… .....將細胞與凝膠溶液混合均勻 步驟154 ••… .....將細胞與凝膠溶液的混合液填入至容置空間中 步驟160… .....待凝膠溶液凝結成固態膠 ^ 步驟 210 ····. .....將不同粒徑之氯化鈉顆粒分別與聚(乳酸-甘醇 酸)顆粒混合均勻 步驟220 •.… .....依據粒徑大小倒入模具中 300............... .....支架 300a............. .....複合支架 312............... .....容置空間 320............... .....凝膠溶液 • 400............... 410............... .....燒杯 .....溶液 L310............ .....聚(乳酸甘醇酸)結構 ‘ L320............ .....海藻膠結構 20Figure 11 is a cross-sectional view showing a porous stereo stent according to a second embodiment of the present invention. [Explanation of main component symbols] Step 102.........Sintering sodium chloride particles to ν. 0. Poly(lactic-glycolic acid) particles and chlorination The nanoparticle mixing uniformity step 112 respectively calculates the weights of the desired poly(lactic acid glycol) particles and the sodium chloride particles, respectively, and then the mixture is mixed with poly(lactic acid-glycolic acid). Granules and sodium chloride particles step 120...filling the mixed particles into the mold 19 1275402 Step 130...·.. hot press forming step 140...·.. removing the mold The formed stent is washed out of the sodium chloride particles therein. Step 150 ····, ..... Fill the gel solution into the holder Step 152 ••... Mixing with the gel solution step 154 ••.........filling the mixture of cells and gel solution into the accommodating space. Step 160... The gel solution is condensed into a solid gel. 210 ····...... Mixing different sizes of sodium chloride particles with poly(lactic-glycolic acid) particles. Step 220 •.....Pour the mold into the mold according to the particle size. Medium 300....................... bracket 300a................... Coupling 312........................... accommodating space 320.......................... gel solution • 400............... 410............... ..... beaker.....solution L310.... ........ ..... Poly (lactic acid glycolic acid) structure 'L320................... Seaweed structure 20