蠶絲蛋白微球體吸附生長因子於脂肪組織工程上的應用

Abstract

外傷、腫瘤移除或先天性缺陷所造成的軟組織缺陷，是起因於皮下脂肪組織的喪失，而脂肪組織工程現今面臨的重要課題為無法持久的維持移植後的脂肪體積，因此結合細胞、支架、生長因子組織工程三大要素於脂肪組織工程重建極為重要。脂肪前驅細胞(preadipocyte)為最常被應用於組織工程的細胞，因為當細胞分化為脂肪細胞後就將不會再增生，所以欲得到大量的成熟脂肪細胞，必需在脂肪前驅細胞階段促使增生。 脂肪再生需長時間的支架支撐，因此我們選用FDA認可的生醫材料:蠶絲蛋白(silk fibroin) ，其具有長期降解性的特色，並可用於長時間維持脂肪組織結構形成，利用組織工程之概念，並結合脂肪前驅細胞及適當的生長因子，將有助於脂肪組織的重建。此外，為了減少手術植入支架所造成的傷害，我們製備蠶絲蛋白微球體，以利於使用注射進注入體內，成為可注射式支架。我們使用W/O方式製備蠶絲微球體，水相為蠶絲蛋白水溶液，油相為油酸和甲醇混合液，並調控不同製備溫度和蠶絲水溶液濃度製備，來選擇最好的微球體條件，我們發現在25℃、8%蠶絲蛋白水溶液製備有最好的微球體外觀，因此我們選用此條件製備的微球體作為可注射式細胞支架。 生長因子bFGF已被廣泛應用於脂肪重建，因此我們選用bFGF吸附於我們製備成功的蠶絲微球體，並證實具有藥物釋放的效果，且發現控制bFGF釋放具有良好促使脂肪前驅細胞增生的能力；若與吸附bFGF明膠微球體比較，可發現吸附bFGF蠶絲微球體於後期有較好的脂肪前驅細胞增生能力。另外，在實驗過程中，我們也發現不含bFGF蠶絲微球體可能具有促使脂肪前驅細胞增生的功能。而動物實驗中，證實吸附bFGF微球體在活體內具有使脂肪再生的能力。 總結，我們成功製備長時間降解性蠶絲微球體，並於微球體上控制bFGF釋放促使脂肪前驅細胞增生，將此架構應用於軟組織再生具有其發展潛力存在。

The soft tissue defect caused by trauma, tumor removal, and congenital deformations that resulting from losing subcutaneous adipose tissue. Current challenge of adipose tissue engineering failed to maintain volume of adipose tissue after transplantation. Therefore, adipose regeneration requires combination of cells, scaffold, and growth factors. Most commonly used cells for tissue engineering are precursor cells, preadipocytes. While preadipocytes differentiate, cells are not capable of proliferation. For this reason, it is important to proliferate preadipocyte before differentiation. Because adipose regeneration requires long time support by scaffold for cell growth, we choose the material approving by FDA and is long-term biodegradation. It can use to maintain the formation of adipose tissue. The addition of combination with appreciate growth factor can promote adipogenesis. Moreover, we manufactured injectable silk microspheres as scaffold of transplantation for reducing damage from surgical operation. We prepared microspheres by water in oil method. Water phase is silk fibroin solution, and oil phase is the mix solution of oleic acid and methanol. We changed the temperature and the concentration of silk solution to prepare microspheres. The result showed that the condition of 25℃ and 8% silk solution for preparing had the better appearance of microspheres. Therefore, we used these microspheres as injectable scaffold for cells. Because bFGF commonly used to regenerate adipose tissue, we had chosen it absorbing on silk microspheres. The silk microspheres with bFGF can achieve controlled release and it had the proliferated ability of preadipocyte. The results that compare with gelatin microspheres with bFGF showed higher proliferation in the late period. It might cause by the delay of drug release in silk microspheres. Particularly, the silk microspheres without bFGF might promote preadipocyte proliferation. In animal study, it proved that microspheres with bFGF can promote adipose regeneration. In conclusion, we had accomplished manufacture of silk microspheres for a long-term degradation and controlled release bFGF upon silk microspheres. Application of the conception in soft tissue regeneration has the potential of development.