多巴胺輔助固定生物分子應用於抗污表面及骨植入物

Abstract

抗污表面 (anti-fouling surface) 在生醫器材中扮演重要的角色。在第一部分 實驗中,透過多巴胺 (dopamine) 聚合,我們發展出一種製作抗污表面,以及使 表面帶有生物素 (biotin) 用於生醫器材。聚乙烯亞胺接枝聚乙二醇( poly(ethylene - imine)-graft-poly(ethylene glycol), PEI-g-PEG ) 混合多巴胺鹼性溶液後,將欲處 理表面沉浸至該溶液兩小時,即製作出可以抵抗血清蛋白以及纖維母細胞 (fibroblast) 的吸附 (adsorption) 及貼附 (adhesion)。此外,透過多巴胺鹼性環境 聚合反應共沉積聚乙烯亞胺,接枝聚乙二醇和聚乙烯亞胺接枝生物素,可製作出 一以抗污表面做為背景,然而同時又可提供可反應之生物素分子。我們展示此表 面可透過卵白素-生物素 (avidin-biotin) 結合系統產生選擇性結合,之後再結合 精胺酸-甘胺酸-天冬胺酸-絲胺酸-生物素 (Arg-Gly- Asp-Ser-biotin, RGDS-biotin) 透過細胞-配合基 (cell-ligand) 交互作用而增加細胞貼附。 改植氫氧基磷灰石 (hydroxyapatite) 於骨科移植物上為一常見增加骨引導 (osteocondution) 之方法。在第二部分實驗中,透過多巴胺聚合反應我們發展出 一種簡易氫氧基磷灰石表面改質方法,以用於骨移植物上。氫氧基磷灰石奈米 粒子混入多巴胺鹼性溶液後,將移植物(鈦金屬)沉浸於該溶液中,即可於植入 物表面形成一層氫氧基磷灰石/多巴胺之膜。此氫氧基磷灰石/多巴胺之沉積膜可 增加骨母細胞 (osteoblast) 貼附、增生和骨礦化表現。此外,固定擁有精胺酸- 甘胺酸-天冬胺酸之胜肽序列於已將氫氧基磷灰石/多巴胺改植之表面,可促進 骨母細胞貼附及分化。 骨型態發生蛋白 (bone morphogenetic proteins, BMPs) 已被證實於骨科治療 下擁有骨誘導 (osteoinduction) 之功能。在第三部分實驗中,透過多巴胺聚合反 應我們發展出一種簡單將第二型骨型態發生蛋白和氫氧基磷灰石和精胺酸-甘胺 酸-天冬胺酸之胜肽序列同時改植於骨植物入表面之方法。將骨植入物(鈦金屬) 浸泡於此三生物分子之多巴胺鹼性溶液中,使此表面同時具有骨誘導、骨引導以 及增加細胞貼附之功能表面。此表面增加間葉幹細胞 (mesenchymal stem cell) 之 細胞貼附以及骨分化。此外,透過多巴胺沉積第二型骨型態發生蛋白 (BMP-2) 使間葉幹細胞分化成骨母細胞於無骨分化培養基下以及不使蛋白質變性。

Non-fouling coatings play a critical role in many biomedical applications. In the first part of this work, we have developed a facile one-step deposition method for preparation of non-fouling surfaces and biotinylated surfaces for biomedical applications. Poly(ethylene-imine)-graft-poly(ethylene glycol) copolymer (PEI-g-PEG) was mixed with an alkaline dopamine solution and then deposited onto substrates as a coating for 2h, which inhibited the adsorption of serum proteins and the attachment of fibroblasts. Furthermore, co-deposition of PEI-g-PEG and PEI-g-biotin in alkaline dopamine solutions provided a cell-resisting background surface while at the same time providing accessible biotin molecules. We demonstrated that this surface can be used for the selective binding of avidin, and the subsequent binding of RGDS-biotin enhanced cell attachment by specific cell-ligand interactions. Hydroxyapatite (HAp) coating on orthopaedic implants is a common strategy to increase osteointegration. In the second of this work, a facile deposition method based on dopamine polymerization was developed for preparation of HAp-coated titanium substrates for orthopaedic applications. Nano-structured HAp was mixed with an alkaline dopamine solution and then deposited onto titanium to form a dopamine/HAp ad-layer. The deposition of dopamine/HAp greatly enhanced the adhesion, proliferation and mineralization of osteoblasts. Furthermore, RGD-containing peptides were immobilized to dopamine/HAP coated titanium and further enhanced cell adhesion and osteogenic differentiation. Bone morphogenetic proteins (BMPs) have been proved to improve osteoinduction on orthopedic therapies. In the third part of this work, a facile deposition method based on dopamine polymerization was developed for preparation of RGD/HAp/BMP-2-coated substrates for orthopaedic applications. HAp, RGD and BMP-2 was mixed with an alkaline dopamine solution and then deposited onto titanium to form a dopamine/RGD/HAp/BMP-2 ad-layer. This layer improved mesenchymal stem cell (MSC) line adhesion and osteodifferentiation. Furthermore, the immobilization of BMP-2 via dopamine polymerization promote MSC differentiate into osteoblast without osteogenic supplement and occuration of protein denaturation.