Temporal and spatial presentations of biological cues are critical for tissue engineering. There is a great need in improving the incorporation of bioagent(s) (specifically growth factor(s) [GF(s)]) onto three-dimensional scaffolds. In this study, we developed a process to combine additive manufacturing (AM) technology with acoustic droplet ejection (ADE) technology to control GF distribution. More specifically, we implemented ADE to control the distribution of recombinant human bone morphogenetic protein-2 (rhBMP-2) onto polycaprolactone (PCL)-based tissue engineering constructs (TECs). Three substrates were used in this study: (1) succinimide-terminated PCL (PCL-N-hydroxysuccinimide [NHS]) as model material, (2) alkali-treated PCL (PCL-NaOH) as first control material, and (3) fibrin-coated PCL (PCL-Fibrin) as second control material. It was shown that our process enables a pattern of BMP-2 spots of ∼250 μm in diameter with ∼700 μm center-to-center spacing. An initial concentration of BMP-2 higher than 300 μg/L was required to retain a detectable amount of GF on the substrate after a wash with phosphate-buffered solution. However, to obtain detectable osteogenic differentiation of C2C12 cells, the initial concentration of BMP-2 higher than 750 μg/L was needed. The cells on PCL-NHS samples showed spatial alkaline phosphatase staining correlating with local patterns of BMP-2, although the intensity was lower than the controls (PCL-NaOH and PCL-Fibrin). Our results have demonstrated that the developed AM-ADE process holds great promise in creating TECs with highly controlled GF patterning.

中文翻译：

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三维组织工程生长因子的声学模式。


生物线索的时间和空间呈现对于组织工程至关重要。非常需要改进生物剂（特别是生长因子[GF(s)]）在三维支架上的掺入。在这项研究中，我们开发了一种将增材制造 (AM) 技术与声学液滴喷射 (ADE) 技术相结合来控制 GF 分布的工艺。更具体地说，我们实施了 ADE 来控制重组人骨形态发生蛋白 2 (rhBMP-2) 在基于聚己内酯 (PCL) 的组织工程构建体 (TEC) 上的分布。本研究使用了三种底物：(1) 琥珀酰亚胺封端的 PCL（PCL-N-羟基琥珀酰亚胺 [NHS]）作为模型材料，(2) 碱处理的 PCL (PCL-NaOH) 作为第一对照材料，以及 (3)纤维蛋白涂层 PCL（PCL-Fibrin）作为第二对照材料。结果表明，我们的工艺能够形成直径约 250 μm、中心间距约 700 μm 的 BMP-2 斑点图案。在用磷酸盐缓冲溶液洗涤后，需要高于 300 μg/L 的 BMP-2 初始浓度才能在基质上保留可检测量的 GF。然而，为了获得可检测的C2C12细胞成骨分化，BMP-2的初始浓度需要高于750 μg/L。 PCL-NHS 样品上的细胞显示出与 BMP-2 局部模式相关的空间碱性磷酸酶染色，尽管强度低于对照（PCL-NaOH 和 PCL-Fibrin）。我们的结果表明，开发的 AM-ADE 工艺在创建具有高度控制的 GF 图案的 TEC 方面具有巨大的前景。