Biomaterial composition and stiffness as decisive properties of 3D bioprinted constructs for type II collagen stimulation
Biomaterial composition and stiffness as decisive properties of 3D bioprinted constructs for type II collagen stimulation
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DOI：
10.1016/j.actbio.2022.08.058
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发表时间：
2022-10-12
影响因子：
9.7
通讯作者：
Kean, Thomas J.
中科院分区：
文献类型：
--
作者：
Martyniak, Kari;Lokshina, Alesia;Kean, Thomas J.
明胶甲基丙烯酸酯（GelMA）和透明质酸甲基丙烯酸酯（HAMA）是3D生物打印中常用的生物材料，具有各自成熟的材料特性。为了鉴定GelMA和HAMA用于软骨形成的理想组合，开发了一种新型的原代人软骨细胞COL 2A 1-Gaussia荧光素酶报告系统（HuCol 2gLuc）。通过这种非破坏性的高通量时间测定，Gaussia荧光素酶从细胞中分泌，并用作测量II型胶原蛋白产生的替代物。在进行3D生物打印之前，使用报告系统筛选GelMA：HAMA比率。该方法效率高，节省时间和材料，从而简化了生物材料的优化过程。筛选显示，与单独的GelMA（15%）相比，向GelMA中添加HAMA改善了软骨形成。使用相同GelMA：HAMA比率的动态力学分析测量储能模量，并建立类似于30 kPa的软骨形成的初始阈值。为了确定生物材料储能模量是否影响细胞移动性，将用绿色荧光蛋白（GFP）转导的人原代软骨细胞以1：1或2：1的比率进行3D生物打印，储能模量分别为32 kPa和57.9kPa。我们发现，在较硬的生物材料中，细胞活动性降低，与细胞活动性更高的较软材料相比，II型胶原蛋白表达更高。最后，在用HuCol 2gLuc细胞进行3D生物打印后，我们成功地确定了用于软骨形成的GelMA：HAMA的最佳组合（2：1）和光交联时间（38秒）。为了确定用于软骨形成的明胶甲基丙烯酸酯和透明质酸甲基丙烯酸酯的最佳组合，我们开发了原代人软骨细胞II型胶原高斯荧光素酶报告蛋白细胞（HuCol 2gLuc）。这种非破坏性的高通量测定使用分泌的Gaussia荧光素酶作为时间II型胶原蛋白产生的代表。该报告系统在3D生物打印之前简化了生物材料优化过程。我们还用它来确定软骨形成所需的硬度水平。这是我们第一次量化3D生物打印结构中的软骨细胞移动性。这些结果共同表明，具有较高储能模量和较低细胞迁移率的生物材料改善了软骨形成。(c)2022作者由爱思唯尔有限公司代表Acta Materialia Inc.出版。这是一个开放获取的文章，在CC BY-NC-ND许可证下（http://creativecommons.org/licenses/by-nc-nd/4.0/）
Gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) are frequently used biomaterials for 3D bioprinting, with individual well-established material characteristics. To identify an ideal combination of GelMA and HAMA for chondrogenesis, a novel, primary hu-man chondrocyte COL2A1-Gaussia luciferase reporter system (HuCol2gLuc) was developed. With this non-destructive, high-throughput temporal assay, Gaussia luciferase is secreted from the cells and used as a proxy for measuring type II collagen production. GelMA:HAMA ratios were screened using the reporter system before proceeding to 3D bioprinting. This method is efficient, saving on time and materials, re-sulting in a streamlined process of biomaterial optimization. The screen revealed that the addition of HAMA to GelMA improved chondrogenesis over GelMA (15%) alone. Storage moduli were measured us-ing dynamic mechanical analysis of the same GelMA:HAMA ratios and established an initial threshold for chondrogenesis of similar to 30kPa. To determine if biomaterial storage moduli impact cell mobility, human primary chondrocytes transduced with green fluorescent protein (GFP) were 3D bioprinted in either 1:1 or 2:1 ratios with storage moduli of 32kPa and 57.9kPa, respectively. We found that reduced cell mobil-ity, in the stiffer biomaterial, had higher type II collagen expression, than the softer material with more cell mobility. Finally, after 3D bioprinting with HuCol2gLuc cells we successfully identified an optimal combination (2:1) of GelMA:HAMA and photo-crosslinking time (38s) for chondrogenesis.Statement of significance One challenge of 3D bioprinting is identifying ideal biomaterials that stimulate articular cartilage devel-opment. To identify an optimal combination of gelatin methacrylate and hyaluronic acid methacrylate for chondrogenesis we developed a primary human chondrocyte type II collagen Gaussia luciferase re-porter cell (HuCol2gLuc). This non-destructive, high-throughput assay uses a secreted Gaussia luciferase as a proxy for temporal type II collagen production. This reporter system streamlines the biomaterial op-timization process before 3D bioprinting. We also used it to determine the level of stiffness required for chondrogenesis. And for the first time, we quantified chondrocyte mobility in a 3D bioprinted construct. Together these results indicate that a biomaterial with a higher storage modulus and less cell mobility, improves chondrogenesis.(c) 2022 The Authors. Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )