製備富含高濃度血小板血漿之多孔組織工程支架及其在幹細胞培養與分化之應用

Parylene-Based Porous Scaffold with Functionalized Encapsulation of PRP for Stem Cells Culture and Differentiation

曾鈞昱(Jun-Yu Zeng)

指導教授 ： 陳賢燁

國立臺灣大學/工學院/化學工程學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202000934

摘要

富含血小板的血漿（Platelet-rich plasma, PRP）由於有高濃度的血小板，富含自然生長因子，具有刺激有絲分裂、血管生成、趨化性的功能，因此可以幫助軟組織癒合、血管增生、傷口癒合和骨骼再生。在這項研究中，利用先前報導的基於使用聚對二甲苯（parylene）的化學氣相沉積（CVD）的多孔材料的新穎製造方法來構建封裝PRP的支架。攜帶自體生長因子的富含高濃度血小板血漿被應用於所引入的昇華/沉積過程中做為水相，從而形成了包裹PRP的三維聚對二甲苯多孔支架。氣相通過基於質譜的實時氣體分析儀來表徵，暗示所引入的昇華/沉積製程在進行。SEM顯微照片顯示的多孔結構，EDS表徵的元素分佈以及IRRAS表徵的官能基支持了封裝PRP的聚對二甲苯多孔支架的成功構建。根據裝載的hASC(人類脂肪幹細胞) 第7天的ALP活動（指示成骨分化的開始）和第14天鈣沉積物的形成（通過定量評估新分化的成骨細胞形成的基於鈣的礦化），評估其骨分化的表現。這些結果表明封裝在聚對二甲苯基支架中的PRP成分可以顯著增強成骨誘導培養基中培養的被裝載hASC的骨分化。基於在第14天的脂質油滴形成來評估被裝載hASC脂肪分化的表現，顯示無論在脂肪形成誘導培養基或生長培養基中培養，PRP成分均可以有效地增強被裝載hASC的脂肪分化。PRP成分在引導人類脂肪幹細胞（hASCs）的成骨和成脂分化途徑中的有益作用表明了該製備方法的可行性，並且聚對二甲苯多孔支架可能可以作為與PRP結合使用的合適基質並允許細胞形態發生。該複合材料和引入的製造方法具有眾多本質的有魅力的特性，展現出了未來應用和發展的無限潛力。

關鍵字

多孔支架 ； 富血小板血漿 ； 聚對二甲苯 ； 化學氣相沉積 ； 人類脂肪幹細胞

並列摘要

Platelet-rich plasma (PRP), with high concentration of platelet, is rich in natural growth factors, possessing the function of stimulating mitogenesis, angiogenesis, chemotaxis, and thus can help soft tissue healing, vascular proliferation, wound healing, and bone regeneration. In this study, a novel fabrication method for porous material based on chemical vapor deposition (CVD) of poly-para-xylylene (parylene) previously reported was utilized to construct PRP-encapsulating scaffold. Platelet-rich plasma (PRP), which carried autologous growth factors, was applied in the introduced sublimation/deposition process as water phase, resulting in 3D parylene porous scaffold encapsulating PRP. The vapor phase was characterized by a mass spectrometry-based, real-time gas analyzer, implying the proceeding of the introduced sublimation/deposition fabrication process. The successful construction of parylene porous scaffold encapsulating PRP was supported by the porous structures demonstrated by SEM micrographs, the element distributions characterized by EDS, and the functional groups characterized by IRRAS. The osteogenesis activities of loaded hASCs (adipose-derived stem cells) were examined based on their ALP expression, which indicates the initiation of osteogenic differentiation, at day 7 and on calcium deposit formation for quantitative evaluation of calcium-based mineralization by freshly differentiated osteoblasts at day 14. Those results exhibited that the PRP ingredient encapsulated into parylene scaffolds could significantly enhance the osteogenesis of loaded hASCs cultured in osteogenesis induction medium. The adipogenesis activities of loaded hASCs were examined based on lipid droplets formation at day 14, showing that the PRP ingredient could effectively enhance the adipogenesis of loaded hASCs whether cultured in adipogenesis induction medium or growth medium. The beneficial performance of PRP ingredient on guiding hASCs differentiation pathways of osteogenesis and adipogenesis suggested the feasibility of the fabrication method and that the parylene porous scaffold may function as a suitable matrix used in combination with PRP allowing cell morphogenesis. The numerous intrinsic appealing properties of the composite material and introduced fabrication method exhibit unlimited potential for future applications and developments.

並列關鍵字

porous scaffold ； Platelet-rich plasma ； poly p-xylylene ； parylene ； chemical vapor deposition ； human adipose-derived stem cells

參考文獻

1. Alves, R. and R. Grimalt, A Review of Platelet-Rich Plasma: History, Biology, Mechanism of Action, and Classification. Skin Appendage Disord, 2018. 4(1): p. 18-24.

2. Foster, T.E., et al., Platelet-rich plasma: from basic science to clinical applications. Am J Sports Med, 2009. 37(11): p. 2259-72.

3. Yamada, Y., et al., Autogenous injectable bone for regeneration with mesenchymal stem cells and platelet-rich plasma: tissue-engineered bone regeneration. Tissue engineering, 2004. 10(5-6): p. 955-964.

4. Andia, I. and M. Abate, Platelet-rich plasma: underlying biology and clinical correlates. Regenerative medicine, 2013. 8(5): p. 645-658.

5. Kutlu, B., et al., Platelet-rich plasma-loaded chitosan scaffolds: preparation and growth factor release kinetics. J Biomed Mater Res B Appl Biomater, 2013. 101(1): p. 28-35.