rhBMP-2 結合可斷鍵式氣相沉積鍍膜調控大鼠骨髓間質幹細胞之體外分化

Abstract

細胞生長因子在多種細胞的功能中扮演極重要的角色，例如細胞之間的訊息傳遞、增生、遷移以及分化等，但生長因子在生物體內降解速度極快，常在抵達目標物之前便已失去活性，因此導致結合生長因子修復組織的生醫材料相當缺乏效率且極其昂貴，需要大量且高頻度的施用才能達到預期的效果，然而此施用量往往伴隨著潛在的副作用。材料技術的進步，迄今已研發出能將生長因子固定在材料表面，以改善其在體內的穩定性與持續性，進而延長其對細胞和組織產生的刺激。除了能將生長因子固定在表面外，更進一步的研究希望能對改質後的表面進行動態地調控。本實驗設計了一種能夠固定生長因子，並帶有可斷鍵功能的鍍膜材料，再接上促進骨分化的誘導因子後在其上培養大鼠骨髓間質幹細胞並進行骨分化，並探討是否能夠成功斷鍵使生長因子被清除 ， 進而影響骨分化的程度。 骨形成蛋白 (BMPs) 在軟骨及硬骨的生成中具有極重要的地位，其中又以BMP-2 被證實具有極佳的骨誘導能力。本實驗利用自SD 大鼠骨髓中分離出來的間質幹細胞 (MSCs)，經CD11b 與 CD45 磁株抗體純化後，將其初代培養並繼代至第五代 (P5)，經流式細胞儀分析其表面抗體CD29、CD90、CD11b、CD45、CD34、CD31和CD86的表現，並測定其三向分化 (tri-lineage differentiation)以及聚落形成之能力以印證其具有間質幹細胞的特性。 試驗的第二部分對鍍膜的材料parylene A 進行測定，反射式吸收紅外光譜分析 (Infrared reflection-absorption spectroscopy，IRRAS) 以及石英晶體微天秤 (quartz crystal microbalancing，QCM) 的結果證實rhBMP-2 可以被成功地固定在parylene A 鍍膜的表面，並且由CCK-8 assay測定細胞生長所呈現的結果顯示parylene A 鍍膜對於MSCs 的生長情況並沒有顯著影響。更進一步的試驗將MSCs 分別培養於接有rhBMP-2 的培養皿與單只有parylene A 鍍膜的對照組進行骨分化能力比較，鹼性磷酸酶(alkaline phosphatase，ALP)的mRNA表現量分析以及Alizarin Red 染色分析，結果證實接上rhBMP-2 的表面確實能夠促進骨分化之程度。 第三部分的試驗則更進一步使用一種帶有可斷鍵機制的材料parylene S-S進行實驗。IRRAS 以及 QCM的分析結果同樣證實rhBMP-2 可以成功地固定在鍍膜表面，並且細胞生長測定一樣證明了對細胞生長並無顯著影響。經細胞存活率測定後所決定的斷鍵處理 : 10 mM GSH 一小時在試驗中亦已證實對於MSCs的分化及rhBMP-2 之活性皆無顯著影響，QCM 的實驗結果也印證此處理條件確實可藉由斷鍵的方式清除rhBMP-2，而非破壞其結構。最後在14天的分化過程中，我們分別於分化的第0天、第4天、第8天及第12天進行斷鍵處理並一同培養至第14天測定其分化程度，在ALP 的mRNA分析中可以顯現各組別的分化程度自第0天到對照組呈現逐漸上升的趨勢，證實細胞的分化程度確實可以受到此斷鍵機制的調控，但在統計分析上只有第0天與對照組之間具有顯著差異，與第4天、第8天和第12天三組之間則否。

Growth factors are important in regulating cellular functions, including cell signaling, proliferation, migration and differentiation. However, growth factors in body niche may be rapidly degraded or otherwise rendered inactive before reaching their target, and biomaterial systems designed to deliver growth factors for tissue repaired are therefore often ineffective and costly. Large amounts of growth factors are frequently needed to effect cellular outcomes, although such large quantities has the risk to damage cells and tissues. The method of immobilization of growth factors to biomaterial substrates has emerged as a way to improve the stability and persistence of growth factor delivered to cells or tissues, and even be more advance to enable the dynamic control chemical properties over material surface. In this study we designed the material surface with cleavable binding site to immobilized osteo-inductive growth factor, and cleave it to remove the growth factor. The outcome of osteogenesis related mRNA and phosphate calcium deposition will be done. BMPs play important roles in bone and cartilage regeneration. Among BMPs, BMP-2 has very strong osteo-induction activity. Previous studies have shown that BMP-2 can enhance the osteogenesis of mesenchymal stem cells (MSCs), and de novo orthotopic or ectopic bone formation. In this study we established the bone marrow derived MSCs from SD rats. MSCs were negatively selected by CD11b and CD45 antibody. The expression of CD29, CD90, CD11b, CD45, CD34, CD31 and CD86 on bone marrow derived MSCs was characterized using flow cytometry. Their ability to differentiate into adipocytes, chondrocytes, osteoblast, and colony formation were also proved. Additionally, we modified the surface of culture dishes with vapor-based synthesis parylene A. Then Immobilized with rhBMP-2. The results of Infrared reflection-absorption spectroscopy (IRRAS) and quartz crystal microbalancing (QCM) assay proved that rhBMP-2 is binding on the surface. Alizarin red staining and mRNA expression of alkaline phosphotase (ALP) show significantly higher (p<0.05) than control while MSCs were differentiated on it for 14 days. Furthermore, we modified the parylene A with disulfide bond (parylene S-S). Try to modulate the cells differentiation via redox thiol−disulfide interchange reaction with 10mM Glutathione (GSH) treatment. CCK-8-assay and alizarin red staining reveal that GSH treatment has no significant effect on cell viability and differentiated performance. In the experiment we separately treat GSH on day0, day4, day8, day12 in differentiation process to break the disulfide bond then replace differentiation medium to eliminate released rhBMP-2. The result of qPCR assay shows that the mRNA expression of ALP were getting higher from day0 to control, reveal that the differentiation level were affected by the treatment. This cleavable mechanism for attaching/detaching biomolecules has the potential to install growth factor on material surface and cleave it when deactivation is required.