用於人體不易生長組織的生醫材料之研發

Development of innovative biomaterials for slow proliferating human tissues

汪郁信(Yu-Hsin Wang)

指導教授 ： 楊台鴻

國立臺灣大學/工學院/醫學工程學研究所/博士(2019年)

https://doi.org/10.6342/NTU201902344

摘要

人體組織損傷的修補，不可避免的需要靠細胞本身的複製，或是細胞外基質的分泌來達成。因為人體有些細胞in vivo複製緩慢，或是細胞外基質分泌不足，以致光靠自體修復無法完全復原，因此本研究想發展一些材料來改善這些組織的再生能力。
在本研究中，我們以神經細胞及角膜內皮細胞當成研究的重點，嘗試改善現有的生醫材料。在神經的方面，(N-(4-aminobutyl)-acrylamide) (P4Am)是一個正電高分子材料，和目前常用於神經培養的ploy-D-lysine(PDL)有很高的相似度。兩者主要差別是在胜肽鍵的結構，前者放在側鏈上，後者則是在主鏈上。我們假設神經細胞有足夠的敏感性可以分辨兩種材料的不同，所以我們用7天大的wistar大鼠小腦顆粒神經細胞，培養在將材料序列稀釋並且塗佈的培養基材上。在低濃度的塗佈條件下(< 0.16 µg/ml)，從細胞活性和型態分析可以發現神經細胞能夠區分兩種材料的結構差異。P4Am相比起PDL即使在低濃度下也能讓每個神經球結點保留平均超過3條神經束，維持神經網路的結構。這表示低濃度的P4Am胜肽鍵結構在和神經的交互作用有助於神經存活分化。
雖然成熟的神經細胞無法分裂複製，但新材料有助於神經存活和網路結構連結。對於嚴重的神經損傷，如何引導神經纖維的生長和維持結構是必要的。P4Am在神經組織工程上可當成新的生物基材。
在角膜內皮方面，我們目標放在分析chitosan/PCL混摻材料上的角膜內皮分化的機制和分析細胞外基質(ECM)成分，發現其用於組織工程的潛力。牛角膜細胞培養於此混摻材料上的貼附、複製、分化以及蛋白表現會和TCPS以及純chitosan作比較。此外我們還會特別分析幾種ECM的結果。從AFM發現PCL25比起純chitosan要粗糙，FTIR也證實了C=O官能基確實有進入膜材中。角膜內皮細胞在PCL25上面的型態和正常TCPS上都呈現一致的多角形以及相近的生長速度。此外在PCL25上的ECM分泌並沒有比TCPS要差，尤其collagen-IV表現更高，而TGFβ2表現則較低。
雖然神經系統和角膜內皮的損傷在人體很難完全復原，我們的研究找到具有發展潛力的材料，以及在組織工程應用上新的分析標的。

關鍵字

神經 ； 角膜內皮 ； 複製 ； 組織修補 ； 細胞外基質

並列摘要

Cell proliferation or extracellular matrix secretion is necessary for human tissues. Due to slow proliferating properties of cells in vivo, or insufficient extracellular secreting, some tissues cannot automatically repair after damage. Thus, we want to develop some biomaterials to improve the repairability of those tissues.
In this study, we focused on neurons and corneal endothelial cells and tried to improve existing biomaterials for tissue engineering. For neurons, (N-(4-aminobutyl)-acrylamide) (P4Am) is a positively charged material similar to well-known ploy-D-lysine(PDL), which is regularly used for neuron culture. The main difference is the peptide structure, which is in the backbone of PDL but locating at the side chain of P4Am. We assumed that neurons are sensitive enough to distinguish such structure difference, so these two cationic polymers were compared at serial coating concentrations for culturing cerebellar granule neurons from 7-day-old Wistar rats in this study. Cellular viability and morphology assay in the peptide structure between P4Am and PDL could be distinguished by neurons at low coating concentrations (< 0.16 µg/ml). P4Am at low coating concentration could keep aggregates with three or four thick processes to support more complete neural network with higher cellular viability than PDL. This suggests that the interaction between neurons and the specific peptide structure of P4Am at low coating concentration was able to improve survival and differentiation of cultured cerebellar granule neurons.
Although mature neurons cannot proliferate, new material increased cell viability and network connection. For severe neural damage, guiding neurite outgrowth and maintaining networks are essential. Therefore P4Am is a potential candidate for neural tissue engineering.
For cornea, we aimed to investigate the underlying mechanisms of the differentiation corneal endothelial cells (CECs) and to identify the compositions of extracellular matrix (ECM) using a chitosan/ polycaprolactone (PCL) blended membrane to explore the potential use of chitosan/PCL blends in tissue engineering of CECs. Bovine CECs were cultured on the blends and compared with TCPS and pure chitosan membrane. Cell behaviors in terms of cell attachment, proliferation, differentiation phenotype and expression of differentiation proteins were examined. Furthermore, the production of ECM proteins was also analyzed. Through experiments, we found that the topography (roughness) of PCL25 membrane examined by AFM was greater than that of pure chitosan membrane. FTIR results confirmed the C=O groups of PCL. The CECs displayed hexagonal morphology and a similar proliferation rate on both PCL25 membrane and TCPS. The production of ECM protein productions of CECs on PCL was not inferior to TCPS. Moreover, Western blot results verified the higher amount of collagen IV, and lower TGF-β2 expression on PCL25 membrane than that on TCPS substrate.
Although the nervous system and corneal endothelial cells hardly completely recover in the human body, our studies found not only a potential material but also new indicators to qualify our materials for tissue engineering.

並列關鍵字

neuron ； corneal endothelial cells ； proliferation ； tissue recovery ； extracellular matrix

參考文獻

[1] R.S. Cargill, 3rd, K.C. Dee, S. Malcolm, An assessment of the strength of NG108-15 cell adhesion to chemically modified surfaces, Biomaterials 20(23-24) (1999) 2417-25.

[2] S. Varon, The culture of chick embryo dorsal root ganglionic cells on polylysine-coated plastic, Neurochemical research 4(2) (1979) 155-73.

[3] J.M. Schakenraad, H.J. Busscher, C.R. Wildevuur, J. Arends, The influence of substratum surface free energy on growth and spreading of human fibroblasts in the presence and absence of serum proteins, Journal of biomedical materials research 20(6) (1986) 773-84.

[4] K. Smetana, Jr., J. Vacik, D. Souckova, Z. Krcova, J. Sulc, The influence of hydrogel functional groups on cell behavior, Journal of biomedical materials research 24(4) (1990) 463-70.

[5] A. Kishida, H. Iwata, Y. Tamada, Y. Ikada, Cell behaviour on polymer surfaces grafted with non-ionic and ionic monomers, Biomaterials 12(8) (1991) 786-92.