聚乙二醇-聚乳酸聚丁內醯胺共聚物溫度敏感型水膠製備及其於軟骨組織工程之應用

Injectable thermosensitive copolymer mPEG-poly(pyrrolidione-co-lactide) hydrogel: Synthesis and application for cartilage tissue engineering

鄭博元

指導教授 ： 朱一民

國立清華大學/工學院/化學工程學系/碩士(2013年)

摘要

本研究主要探討可注射溫度敏感型poly(ester-amide)水膠methoxy poly(ethylene glycol)-b-poly(pyrrolidone-co-lactide)(簡稱mPDLA)之性質以及其作為軟骨組織工程支架之潛力。利用D,L-lactide (LA)、2-pyrrolidone (PD)單體與mPEG550開環聚合反應，在固定單體比例([PD]/[LA]=30/70)下合成一系列不同親/疏水分子量比例之mPDLA(命名為P3L7)，找出具有最佳成膠特性之親/疏水比例，並且以未含PD單體的mPEG-PLA作為對照組(命名為P0L10)。結果顯示P3L7與P0L10之共聚物水溶液皆可形成奈米微胞，臨界微胞濃度介於0.1~0.3 mg/ml之間。由DLS測定結果發現P3L7共聚物之奈米微胞均隨溫度上升而有聚集的現象。藉由倒置試管法初步測試其成膠性質，兩團聯共聚物中最佳的親/疏水比例為550/1405。由流變儀可以證實P3L7兩團聯共聚物的黏度以及機械性質會隨著溫度而變化，確實具有溫度敏感性，並且可以在生理溫度下快速成膠。此外，P3L7之吸水膨潤程度較P0L10高，可以提供細胞較佳的生長環境。體外降解實驗結果則顯示P3L7降解產物造成的酸性於初期較P0L10低。在水膠包埋軟骨細胞的實驗部分可以發現軟骨細胞可以在P3L7水膠中生長並且具有正常軟骨化之表現。動物實驗結果則顯示以15 wt% P3L7水膠修復軟骨缺陷可以得到良好的結果。綜合上述結果，我們可以推論P3L7具有可注射、原位成膠以及生物相容性等優點，並且比聚酯類水膠具有較低的降解酸性、較高的含水率，因此適宜做為軟骨組織工程之水膠支架。

關鍵字

聚酯醯胺 ； 溫度敏感型水膠 ； 軟骨組織工程

並列摘要

The objective of this study was to discuss the thermoresponsive properties of a novel poly(ester-amide) polymer, i.e. methoxy poly(ethylene glycol)-poly(pyrrolidone-co-lactide)(mPDLA) diblock copolymers and evaluate its capability of chondrocyte encapsulation for cartilage tissue engineering. A series of amphiphilic diblock copolymers were synthesized by ring-opening polymerization of mPEG550, D,L-lactide and 2-pyrrolidone. The initial ratio of monomers in mPDLA was [PD]/[LA]=30/70 and the target molecular weight of hydrophobic segment were 1105, 1405, 1705 respectively. The copolymers were characterized via1H-NMR,FT-IR spectroscopy, and GPC. The results indicated that the diblock copolymers formed nano-micelles at low concentrations in aqueous phase. The micelle properties were also measured. The critical micelle concentration(CMC) was ranged from 0.1 to 0.3 mg/ml.As the temperature increased, micelles aggregation was observed by DLS. The diblock copolymer P3L7-1405 solution underwent a sol-to-gel phase transition, which was confirmed by test tube inverting method. Rheology results showed that viscoelastic properties of the copolymer solution varied with temperature, indicative of the formation of a gel. The mPDLA diblock copolymer solutions exhibited sol-gel transition behavior as a function of temperature. In vitro degradation test showed that the acidity of degradation was effectively reduced by introducing the monomer PD into polyester hydrogel. mPDLA alsoexhibited higher water content from swelling ratio. It would provide an environment that is preferred by cells; therefore, mPDLA diblock copolymer exhibited better biocompatibility in vitro.
As an injectable scaffold, the viability, cell proliferation and chondrogenesis of chondrocytes encapsulated in mPDLA hydrogel were investigated. MTT and DNA quantification showed proliferation of cells within 2 weeks. By Live/Dead stain we can confirm that the morphology of cells in hydrogel was typical sphere which mature chondrocytes supposed to be. Also, ECM content was significant increased within 2 weeks compared with initial amount. In vivo test showed the repair of cartilage defect treated with 15% mPDLA hydrogel. From above results, we deduced that this thermosensitive hydrogel was sutible as an injectable scaffold for cartilage tissue engineering.

並列關鍵字

poly(ester-amide) ； thermosensitive hydrogel ； cartilage tissue engineering

參考文獻

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