軟組織填充物之無細胞皮膚基質的製程開發
Process Development of Acellular Dermal Matrix (ADM) for Soft
Tissue Augmentation
中文摘要 軟組織填充物在臨床上已成功的被應用在臉部的輪廓修復或是治療尿失禁。理想 的軟組織填充物在發展時必須以提高堅持度為目標而又不影響其注射力，採用注 射的方式，是因為這樣比較方便使用，而且和注射部位的組織能夠有比較緊密的 結合。而本研究的目的是希望建立一套方法，能將豬皮製備成可供注射用的無細 胞皮膚基質來當做軟組織的填充物。在我們的實驗中，發現將豬皮利用 0.25% trypsin 在 25℃下處理 24 小時，再加上 560 unit/L dispase 處理 12 小時，可以徹 底的將豬皮內的細胞去除乾淨，得到無細胞皮膚基質。藉由組織切片的觀察可以 清楚的觀察到酵素清除細胞的效果，而且從切片中也可看到還保有膠原蛋白的網 狀纖維型態。另外，利用穿透式電子顯微鏡，SDS-PAGE 和 size-exclusion HPLC 的觀察，可確定無細胞皮膚基質含有第一型膠原蛋白，而且分子量分布的主要波 峰也分為多體和單體兩部份。將無細胞皮膚基質均質成較小纖維使其可當作注射 用的無細胞皮膚基質，同時利用 0.9 %的氯化鈉溶液來配製不同濃度的可供注射 用的無細胞皮膚基質。 在本研究中將利用動態機械分析儀來測試這些可供注射用的無細胞皮膚基質的 流變特性。測試的項目包括，潛變曲線，應力應變曲線和儲存模數，我們藉由這 些測試的結果來討論堅持度的問題。除了測試不同濃度的可供注射用無細胞皮膚 基質的流變特性，還測試α-hydroxy acids 和其他不同添加物以及溫度（25℃和 37℃）對於流變特性的影響。實驗之後發現，所有的潛變曲線經過迴歸之後都可 以用 Kelvin-Voigt 的模型來描述，受力後形變的方程式可用 表示（r：形變，σ： 受力，E：楊氏係數，τ：延滯時間，t：時間）而其中以添加 2 %的甘醇酸其延 滯時間（τ，retardation time）最長，所以添加 2 %的甘醇酸後，可供注射用的 無細胞皮膚基質會呈現最強的剛性和最好的堅持度。因為物質的 值越高，會 呈現越固結的特性，相反的，τ值越低，會呈現越偏流體的特性。而堅持度是影 響臨床效果的一個重要指標。另一方面，溫度也會影響流變學的實驗結果，在 25℃下堅持度的表現要比 37℃來得好。 生體相容性試驗藉由與纖維母細胞（3T3）共同培養的體外細胞毒性測試來確 定，由觀察細胞的生長狀況和速度來當作細胞毒性的定性判斷。結果發現在實驗 期間並沒有發現細胞型態的改變，因此無細胞皮膚基質具有良好的生體相容性， 可當作軟組織填充物的發展材料。 英文摘要contour defects or urinary incontinence. Ideal soft tissue filler should possess the persistence as high as possible without deteriorating the injectability because it would be easy to introduce by injection and that could match the tissue as closely as possible. The object of this study was to develop a method of processing porcine skin to
produce an injectable acellular dermal matrix (ADM) as a soft tissue replacement material. The results demonstrated that lengthy incubation of pieces of porcine skin with 0.25% trypsin (24 hr) and 560 unit/L dispase (12 hr) at 25℃ removed cells and cellular components in the skin efficiently. Histological examinations revealed that the epidermis, dermal fibroblasts, and epidermal appendages were completely absent after treatments, and the basic dermal architecture of collagen bundles were in a loose meshwork. Analysis by TEM, SDS-PAGE, and size-exclusion HPLC revealed that the ADM contains type I collagen mostly and showed two typical component peaks identified as oligomers and monomers, resepctively. After then, ADM was
homogenized to a particulate form as an injectable soft tissue replacement material and 0.9 % sodium chloride was used as the medium to prepare various concentration of injectable ADM.
The persistence of injectable ADM was discussed based on the rheological
characteristics of injectable ADM with the analysis of the stress-strain curve, creep compliance, and storage modulus, which measured by using Dynamic Mechanical Analyzer (DMA). In addition to the rheological test on different concentrations of injectable ADM, the influence of blending α-hydroxy acids and additives with injectable ADM was examined. Overall, when 2 % glycolic acid blending with the injectable ADM showed the longest retardation time (τ), indicating that the rigidity of this blend is in a higher extent to demonstrate the best persistence among all. This is because the higher the τ value, the more “solid-like” the material will be, and vice versa. With respect to serving as soft tissue replacement material, the extent of rigidity is one of the critical factors to consider. On the other hand, temperature also affects the rheological characteristics of injectable ADM.
The biocompatibility of injectable ADM was confirmed with the evaluation of in vitro cytotoxicity test using fibroblast (3T3) as a qualitative indicator based on the
morphological examination of cell damage and growth rate when in direct contact with the materials. No significant morphologic changes were observed for these cells in contact with ADM for all the studied time periods. It concludes that ADM is highly compatible to be used to prepare materials for soft tissue augmentation
