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標題: | 聚羥基烷酯膜材的表面性質對人類間葉幹細胞行為的影響 Effects of the surface characteristics of polyhydroxyalkanoate (PHA) films on the behaviors of human mesenchymal stem cells (hMSCs) |
作者: | Bo-Yi Yu 余柏毅 |
指導教授: | 楊台鴻 |
共同指導教授: | 孫一明 |
關鍵字: | 細胞與基材的相關性,細胞聚集,電紡絲膜材,幹細胞, cell-substrate interaction,cell aggregation,electrospun films,stem cells, |
出版年 : | 2008 |
學位: | 博士 |
摘要: | 聚羥基烷酯族(PHAs)是一系列新式的生醫材料,具有良好的潛能可被運用於組織工程的領域中,特別是第三代商業化產品PHBHHx,其機械性質與可加工性遠優於第一代的PHB以及第二代的PHBV。人類間葉幹細胞具有良好的再生與分化能力,而且沒有獲取上的道德問題,因此具有相當好的研究價值。此研究目的是瞭解人類間葉幹細胞與聚羥基烷酯膜材之間的關連性,換言之,就是研究並調控(modulate)人類間葉幹細胞在聚羥基烷酯膜材上的行為表現,以及評估此系列材料運用於再生醫學上的潛能。利用熱壓法、溶鑄法以及電紡絲法所製備的聚羥基烷酯族膜材,具有多樣化的表面性質,並可進一步塗佈(coat)透明質酸(hyaluronic acid)以及接枝(graft)聚丙稀酸(poly acrylic acid),以提升此材料對間葉幹細胞的生物相容性。
隨著HV比率的增加,PHBV膜材的結晶性與親水性會隨之下降,但間葉幹細胞在其膜材上的代謝表現則隨之上升。聚羥基烷酯族膜材塗佈上透明質酸後,可有效的降低間葉幹細胞的死亡率,並提升其代謝能力。間葉幹細胞在由溶鑄法所製備的膜材上(揮發溫度為18 ℃),不但會隨著時間而有貼覆、伸展(spread)以及遷移(migration)的表現,而且還有聚集成群(aggregation)的特殊行為。PHBHHx膜材的親水性以及表面粗糙度可以藉由調整丙稀酸的濃度以及紫外光的照射時間而控制。膜材親水性的增加有助於間葉幹細胞的伸展貼覆,而膜材表面裂痕的順向性也能有效地影響細胞內骨架(cytoskeleton)的排列。 利用不同製膜方式所製備的PHBHHx膜材,具有明顯不同的表面性質,而這些表現性質很明顯地可以影響人類間葉幹細胞的行為表現。利用溶鑄法來製備膜材,將揮發溫度由原本18 ℃提升至32 ℃時,膜材表面粗糙度不但有顯著的提升,同時表面還具有許多微小孔洞的結構。此時,人類間葉幹細胞在此膜材上,雖然仍會有貼覆、伸展與遷移的行為,但是需要更多的時間來完成細胞聚集(aggregation)。細胞在聚集的狀態時,除了仍保持與培養在TCPS上的細胞相同的表面標誌(surface markers),同時比處於伸展狀態(spread well)的細胞表現出更高的活性表現(viability),以及較低的分化能力(differentiation ability)。人類間葉幹細胞培養在電紡絲膜材上,不但細胞會有很好的貼覆表現,同時也會呈現相當一致的生長方向(orientation)。電紡絲膜材具有特殊孔洞性(不織布)結構有助於廢液、養分以及成長因子(growth factor)的擴散,雖然細胞因為尺寸的關係無法遷移進入膜材內部區域,但是細胞的行為仍會受到膜材內部孔洞結構的影響。人類間葉幹細胞在電紡絲膜材上會表現出最高的活性表現,但是不利於往細胞分化方向的進行,所以電紡絲膜材或許是一個很好的基材可以用來大規模的培養未分化的幹細胞。PHAs系列膜材不但對細胞有很好的生物相容性,是具有很好潛能的生醫材料,同時其多樣化表面性質可以很明顯地用來調控(modulate)人類間葉幹細胞的形態、成長與分化等行為表現。 Polyhydroxyalkanoates (PHAs) are a newer family of biomaterials for tissue engineering applications. Especially, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), the third generation commercializated product, attained lots of attention. Its mechanical and processible properties have been shown to better than that of PHB and PHBV. Human mesenchymal stem cells (hMSCs) have great proliferation and differentiation abilities without an ethic problem in obtaining. The aim of this study was to present a deeper picture of the relationship between hMSCs and the surface characteristics of PHAs films. In other words, it is to investigate the behaviors of human mesenchymal stem cells (hMSCs) grown on the surface of various PHA membranes and to evaluate if PHAs have potential application in regenerative medicine. The surface characteristics of PHA copolymer membranes were varied by the content of 3-hydroxyvalerate (HV) or 3-hydroxyhexanoate (HHx) and by the membrane preparation methods such as compression-molding, solvent-casting and electrospinning methods. Hyaluronic acid (HA) and poly acrylic acid (PAAc) were further applied to modify the surface properties of the PHA membranes. The acrylic acid molecules were grafted on PHBHHx membrane surface by UV irradiation. When the HV content increased, the crystallinity and the hydrophobicity of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) membranes reduced and the metabolic activity of hMSCs improved after being cultured for three days. Hyaluronic acid (HA) coating on PHA membranes could improve the metabolic activity and reduce the death rate of hMSCs. HMSCs could adhere to the surface, and then spread, migrated, aggregated and formed cellular clusters with time only on the solvent-cast PHBHHx films (the evaporation temperature was at 18 ℃). The hydrophilicity and surface roughness of various PHBHHx films were controlled by adjusting the acrylic acid concentration and the UV irradiation time. The hydrophilicity could effectively improve the spread of hMSCs, and the orientation of surface scars could guide the growth direction of cytoskeleton (actin) inside hMSCs. The behaviors of hMSCs were modulated by the surface characteristics of these films although the base material was all the same. HMSCs could also adhere to the surface, and then spread, migrate, aggregate and form cellular clusters with time on the solvent-cast PHBHHx films (the evaporation temperature was at 32 ℃). HMSCs needed more time to form cellular aggregation on the solvent-cast film with an arising degree of the surface roughness. HMSCs at the aggregative status on the solvent-cast film kept their original surface markers and presented obviously higher viability and lower differentiation ability than that at the spreading status on the compression-molded films or TCPS. HMSCs spread well on the surface and performed a regular orientation on the random electrospun fibrous films. Although hMSCs was not able to migrate into the interior of electrospun film, the interconnected porous structure of this film have an obvious influence on the behaviors of hMSCs. The chemical signals and mitogens produced by hMSCs itself or the growth factor in the medium could easily transport through the connected pores within the films. HMSCs on the electrospun films revealed the highest viability but the second lowest differentiation activity among that on all the tested films. The electrospun fibrous PHBHHx films are able to serve as suitable substrates for large quantity culturing of hMSCs when undifferentiated hMSCs are desired. The morphology, proliferation and differentiation of hMSCs were remarkably influenced by various surface characteristics of the PHA membranes. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42152 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學工程學研究所 |
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