聚乳酸微球體結合膠原蛋白/玻尿酸膠體於脂肪組織工程之應用

Abstract

由於先天上的疾病，或是後天意外、手術導致皮下脂肪層發生的組織缺失，就是所謂的軟組織缺陷。針對這些問題，利用組織工程的概念所建立的替代物來重建軟組織，是目前被看好的解決方法之一。在組織工程裡，除了細胞及棚架，還需要生長訊息來幫助組織修復，為了持續提供這些生長訊息，常利用藥物釋放系統來保護並釋放這些生長訊息，以乳化法製備成的微球體就是其中一種。本研究即著重於加入含有誘導分化物質之聚乳酸微球體至膠原蛋白與玻尿酸膠體形成的棚架中，誘導脂肪前驅細胞分化。 在實驗中，我們將取自Wistar公鼠的脂肪前驅細胞培養在膠原蛋白與玻尿酸混合的膠體，再加入含有胰島素及dexamethasone的聚乳酸微球體，目的是希望能利用微球體控制釋放這些物質，使脂肪前驅細胞能有效分化成脂肪細胞，達到軟組織重建的目的。首先，我們利用乳化法將胰島素及dexamethasone包覆於聚乳酸微球體中，並進行控制釋放試驗。經過高效能液相層析儀的分析得到胰島素在70天的累積釋放量為29.62 μg/ml，dexamethasone為2.4 μg/ml，並配合穩定性試驗證明微球體能控制釋放這兩種藥物。之後將此微球體加入細胞中一起培養，利用Oil Red O染色定量脂肪細胞分化的程度，發現微球體的加入，的確能誘導細胞分化。而在膠體中的實驗，從MTS assay的實驗結果顯示，適量的微球體加入有助於脂肪前驅細胞的增生。另外，我們使用非線性光學顯微鏡觀察脂肪前驅細胞在膠體內分化的情形，得知玻尿酸的加入，使膠原蛋白膠體產生的網狀結構可能增加其機械強度而減少膠體收縮；而且在膠體中，也觀察到了脂肪細胞的生成，再次證明微球體的加入，確實有幫助脂肪分化的效果。 研究結果證實，利用乳化法製備的微球體，的確能達到控制釋放的效果，幫助脂肪前驅細胞分化，雖然分化的程度仍需進一步的確認，但是結合脂肪前驅細胞、微球體及水膠，做為幫助作為軟組織重建的替代物是有潛力的。

Soft tissue defects and tissue deficits resulted from congenital defects and acquired diseases are often occured in deep subcutaneous fat layer. A promising solution for these defects is tissue engineered adipose tissue. In addition to cells and scaffolds, signals are required to promote tissue regeneration. Drug controlled release systems are often used to protect and release these signals and microspheres fabricated by emulsion technique is a kind of drug carriers. In this study, we focused on the differentiation induced by microspheres which contained inducing factors in the collagen/ hyaluronic acid (HA) hydrogel scaffold. In order to reconstruct soft tissue, we combine preadipocytes isolated from male Wistar rats cultured in collagen/ HA gels blended with microspheres containing insulin and dexamethasone. PDLLA microspheres containing insulin and dexamethasone were fabricated through emulsion technique and controlled release studies were conducted. These samples were analyzed using high performance liquid chromatography (HPLC) method and the cumulative release in a 70 days controlled-release experiment: the concentration of insulin and dexamethasone are 29.62 μg/ml and 2.4 μg/ml, respectively. According to the release studies and stability tests, we found that microspheres can controll release these two drugs. Then, preadipocytes were cultured with microspheres containing inducing factors, and adipogenesis were evaluated by Oil Red O staining. The adipogenesis induced by the microspheres containing insulin and dexamethasone was observed. We also found that incorporating adequate PDLLA microspheres into gel can promote cell proliferation through MTS assay. Moreover, the differentiation of preadipocytes in the gel was observed by nonlinear optical microscopy. We found that the formation of network structure in collagen gel caused by adding HA, and it may increase the mechanical properties and limit contraction of the gel. The formation of adipocytes in the gel was observed and this result can proof that adipogenesis can be induced by microspheres containing insulin and dexamethasone in the gel. These results supported that insulin and dexamethasone can be controlled released from microspheres although the adipogenesis was still evaluated further. The combination of collagen/ HA gel with microspheres and preadipocytes is a promising tissue engineered adipose tissue.