摘要:關節軟骨因其自我修補能力以及再生能力的不足,在受傷後自我復元能力很差。目前臨床上針對軟骨組織的損傷, 是採用軟骨自體移植(autologous chondrocytetransplantation, ACT)或異體的方式治療,利用平時較少受力點的正常軟骨或是捐贈之軟骨移植到受損的軟骨部分;但是,此方法會因為軟骨細胞取得不易而受限。骨髓間葉幹細胞 (Mesenchymal stem cells, MSCs) 是一群具分化生長能力的細胞,具分化成軟骨細胞之潛力,因此可以作為軟骨修復之細胞來源。如何將骨髓間葉幹細胞成功的誘導成軟骨細胞並具有其軟骨細胞特性為當前最主要之問題。一般皆認為軟骨分化過程中需要細胞生長因子TGF-β1、BMPs 及細胞周遭的環境,例如:細胞胞外基質 (extracellularmatrix ,ECM)。玻尿酸 (Hyaluronic acid)是關節軟骨主要成分之一,主要包圍在軟骨細胞外,是由葡萄醣醛酸(glucuronic acid)及N-乙醯葡萄糖胺(N-acetylglucosamine)聚合成的葡萄醣胺聚醣(glycosaminoglycan)。在體內其分子量約在5,000 - 20,000,000Da 之間。在關節軟骨內會與aggrecan 結合形成一帶負電基團,它能幫助軟骨細胞在關節處抵抗運動時產生的壓擠力。有研究指出玻尿酸可當作罹癌標記,臨床上經常用來治療退化性關節炎。玻尿酸可與軟骨細胞表面受體CD44 、RHAMM (hyaluronan-mediatedmotility receptor) 和 ICAM-1 (inter-celllular adhesion molecule-1)作用可影響細胞的移動及黏附性;經由與CD44 表面受體與免疫細胞作用可降低發炎反應。軟骨素(Chondroitin sulfate) 為一長鏈無分支的硫酸鹽葡萄糖醣胺 (sulfatedglycosaminoglycan )會與蛋白質結合成蛋白多醣 (proteoglycan),與玻尿酸共同存在於關節軟骨中, 扮演幫助軟骨維持韌性之角色。聚乳酸– 甘醇酸( poly( lactic-co-glycolic acid, PLGA)為一良好的生物材料,具有生物相容性及生物可分解性,而且是唯一被美國食物藥物管理局(FDA)所核准使用的醫材。我們在動物實驗上成功地將自體軟骨種植於PLGA 支架上用以修復關節軟骨損傷,並建立出標準手術流程。但是尚有些問題懸而未決,例如:1. 如何提高細胞在 PLGA 支架上的種植率2. PLGA 的疏水性降低了細胞對於PLGA 支架吸附力3. PLGA 降解後會引發細胞免疫接下來我們將著手改良PLGA 支架以期能運用於臨床上之軟骨修復。第一年, 我們將改良3D-PLGA 支架,添加玻尿酸及軟骨素期望能增進細胞之貼附力及促進細胞生長。第二年, 我們將把骨髓間葉幹細胞種植於改良後的PLGA 支架中,探討玻尿酸及軟骨素對於骨髓間葉幹細胞分化成軟骨之影響。這結果可以幫助我們了解胞外基質對於骨髓間葉幹細胞分化之影響。第三年, 我們將利用動物模式來探討改良後的PLGA 支架對於體內軟骨修復之影響。這些結果不僅可幫助我們了解胞外基質對於細胞命運決定之影響而且可以幫助我們建立更好的手術治療流程。
Abstract: Articular cartilage defect is an unsolved clinical problem due to its poor repair capacity.At present, the defect is mainly repaired by autograft or allograft transplantation usingengineered cartilage fragments or chondrocytes. The source of chondrocytes is limited.Mesenchymal stem cells (MSCs) have multipotential for differentiation and could serve as agood cell source. Induction of MSCs differentiation into chondrocytes and secretion ofextracellular matrix are important issues for cartilage repair. It needs the cellular growthfactors (TGF-or BMPs) to stimulate the MSCs differentiate into chondrogenic cells. Inaddition, the environmental factors including extracellular matrix (ECM) could serve as animportant stimulator for maintaining the properties of cells or inducing MSC differentiation.Hyaluronic acid (HA) is one of the major components of articular cartilage, where it is presentas a coat around each chondrocyte. It is a polymer of repeated N-acetylglucosamine andD-glucuronic acid disaccharide and the size of the polymer ranged from 5,000 to 20,000,000Da in vivo. Binding of HA with aggrecan monomer in the presence of link protein results inlarge highly negatively-charged aggregates form. These aggregates adsorb water that rendersthe cartilage resistance to compression. HA is a potential tumor marker and also used to treatosteoarthritis of the knee in clinic. The effects of HA are mediated through cell surfacereceptors, such as CD44, RHAMM (hyaluronan-mediated motility receptor) and ICAM-1(inter-celllular adhesion molecule-1). These molecules are associated with cell motility andadhesion. In addition, HA could reduce inflammatory by binding receptor CD44. Chondroitinsulfate is a long, unbranched chain of sulfated glycosaminoglycan. It is usually found attachedto proteins as part of a proteoglycan. It is an important structural component of cartilage andtogether with HA provides the resilience of cartilage. PLGA is a good biomaterial for itsbiodegradability and biocompatibility and is approved by Food and Drug Administration(FDA) in therapeutic devices. We have developed a suitable PLGA scaffold and establishedthe standard surgical operation protocols in the animal model to treat cartilage defects withautologous chondrocyte implantation (ACI). However, challenges remain with this approach,such as:1. The difficulty in uniformly seeding cells in the scaffold2. The adhesion of cells on PLGA scaffold is poor because of the hydrophobic propertyof PLGA3. The degradation of PLGA could induce inflammationHere we propose to improve our scaffold for cartilage repair. In the first year, we willmodify the 3D-PLGA scaffolds and coat them with various sizes of hyaluronic acid andchondroitin sulfate and monitor the cell adhesion and growth. In the second year, we will seedMSCs in these scaffolds and investigate the effects of HA and chondroitin on chondrogenesis.The results could let us further understand the relationship between extracellular matrix andthe cell fate of MSCs. In the third year, we will apply the improved scaffold in animal modelto explore the cartilage repair in vivo. These results will not only let us further understand therole of extracellular matrix in cell fate determination, but also lead us to develop bettertherapeutic protocol for treatment.