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Biomomentum多軸機(jī)械測(cè)試儀Mach-1應(yīng)用：立體光刻3D生物打印方法制作人角膜基質(zhì)等效物。

生物醫(yī)學(xué)工程學(xué)年鑒，生物材料-工程細(xì)胞行為，已出版：2020年6月5日3D生物打印技術(shù)是用于角膜基質(zhì)組織再生的一種有前途的方法。在這項(xiàng)研究中，與角膜基質(zhì)細(xì)胞混合的甲基丙烯酸明膠（GelMA）被用作生物墨水。基于可見光的立體光刻（SLA）3D生物打印方法用于打印人類角膜基質(zhì)的解剖學(xué)相似的圓頂形結(jié)構(gòu)。測(cè)試了兩種不同濃度的GelMA大分子單體（7.5和12.5％）的角膜基質(zhì)生物印記。由于高聚物濃度高，12.5％的GelMA比7.5％的GelMA堅(jiān)硬，這使其更易于處理。就生物印刷支架的水含量和透光率而言，我們觀察到具有12.5％GelMA濃度的支架更接近于天然角膜基質(zhì)組織。回到頂端|提供反饋信息的人隨后，細(xì)胞增殖，研究了在生物打印支架中封裝的人角膜基質(zhì)細(xì)胞的基因和蛋白質(zhì)表達(dá)。在第1天和第7天，觀察到在12.5％GelMA支架中的細(xì)胞相容性分別為81.86和156.11％，顯著高于7.5％GelMA支架中的細(xì)胞相容性。7天后在12.5％的GelMA樣品中觀察到角膜基質(zhì)細(xì)胞伸長(zhǎng)，表明細(xì)胞附著，生長(zhǎng)和在支架內(nèi)的整合。與在塑料組織培養(yǎng)板上培養(yǎng)的細(xì)胞相比，在12.5％GelMA支架中培養(yǎng)的細(xì)胞的I型膠原蛋白，盧米肯和硫酸角質(zhì)素的基因表達(dá)隨時(shí)間增加。28天后，還使用免疫組織化學(xué)法觀察了I型膠原蛋白和lumican的表達(dá)。使用垂直軸的微機(jī)械測(cè)試儀（測(cè)定交聯(lián)時(shí)間MACH-c，Biomomentum，加拿大）。

Abstract

3D bioprinting technology is a promising approach for corneal stromal tissue regeneration. In this study, gelatin methacrylate (GelMA) mixed with corneal stromal cells was used as a bioink. The visible light-based stereolithography (SLA) 3D bioprinting method was utilized to print the anatomically similar dome-shaped structure of the human corneal stroma. Two different concentrations of GelMA macromer (7.5 and 12.5%) were tested for corneal stroma bioprinting. Due to high macromer concentrations, 12.5% GelMA was stiffer than 7.5% GelMA, which made it easier to handle. In terms of water content and optical transmittance of the bioprinted scaffolds, we observed that scaffold with 12.5% GelMA concentration was closer to the native corneal stroma tissue. Subsequently, cell proliferation, gene and protein expression of human corneal stromal cells encapsulated in the bioprinted scaffolds were investigated. Cytocompatibility in 12.5% GelMA scaffolds was observed to be 81.86 and 156.11% at day 1 and 7, respectively, which were significantly higher than those in 7.5% GelMA scaffolds. Elongated corneal stromal cells were observed in 12.5% GelMA samples after 7 days, indicating the cell attachment, growth, and integration within the scaffold. The gene expression of collagen type I, lumican and keratan sulfate increased over time for the cells cultured in 12.5% GelMA scaffolds as compared to those cultured on the plastic tissue culture plate. The expression of collagen type I and lumican were also visualized using immunohistochemistry after 28 days. These findings imply that the SLA 3D bioprinting method with GelMA hydrogel bioinks is a promising approach for corneal stroma tissue biofabrication.

 

The crosslinking time was determined using a vertical axis micromechanical tester (Mach-1 v500c, Biomomentum Inc., Montreal, QC, Canada) by recording in situ gelation kinetics.