Biophysics Group, Biomedical Research Institute, Hasselt University, Agoralaan Building C, 3590 Diepenbeek, Belgium.
Biomaterials & Tissue Engineering, UCL Eastman Dental Institute, University College London, 256 Gray's Inn Road, London WC1X 8LD, UK.
Acta Biomater. 2016 Jan;30:258-264. doi: 10.1016/j.actbio.2015.10.047. Epub 2015 Oct 29.
Hydrogels have emerged as promising biomaterials for regenerative medicine. Despite major advances, tissue engineers have faced challenges in studying the complex dynamics of cell-mediated hydrogel remodelling. Second harmonic generation (SHG) microscopy has been a pivotal tool for non-invasive visualization of collagen type I hydrogels. By taking into account the typical polarization SHG effect, we recently proposed an alternative image correlation spectroscopy (ICS) model to quantify characteristics of randomly oriented collagen fibrils. However, fibril alignment is an important feature in many tissues that needs to be monitored for effective assembly of anisotropic tissue constructs. Here we extended our previous approach to include the orientation distribution of fibrils in cellular hydrogels and show the power of this model in two biologically relevant applications. Using a collagen hydrogel contraction assay, we were able to capture cell-induced hydrogel modifications at the microscopic scale and link these to changes in overall gel dimensions over time. After 24h, the collagen density was about 3 times higher than the initial density, which was of the same order as the decrease in hydrogel area. We also showed that the orientation parameters recovered from our automated ICS model match values obtained from manual measurements. Furthermore, regions axial to cellular processes aligned at least 1.5 times faster compared with adjacent zones. Being able to capture minor temporal and spatial changes in hydrogel density and collagen fibril orientation, we demonstrated the sensitivity of this extended ICS model to deconstruct a complex environment and support its potential for tissue engineering research.
It is generally accepted that looking beyond bulk hydrogel composition is key in understanding the mechanisms that influence the mechanical and biological properties of artificial tissues. In this manuscript, we performed label-free non-invasive imaging and extended a robust automated analysis method to characterize the microstructural organisation of cellular hydrogel systems. We underpin the sensitivity of this technique by capturing minor changes in collagen density and fibril orientation in biologically relevant systems over time. Therefore, we believe that this method is applicable in fundamental cell-matrix research and has high-throughput potential in screening arrays of hydrogel scaffolds, making it an interesting tool for future tissue engineering research.
水凝胶已成为再生医学有前途的生物材料。尽管取得了重大进展，但组织工程师在研究细胞介导的水凝胶重塑的复杂动力学方面仍面临挑战。二次谐波产生（SHG）显微镜一直是用于非侵入性可视化 I 型胶原水凝胶的关键工具。考虑到典型的偏振 SHG 效应，我们最近提出了一种替代的图像相关光谱（ICS）模型来量化随机取向的胶原原纤维的特征。然而，纤维排列是许多组织中的一个重要特征，需要监测以有效地组装各向异性组织构建体。在这里，我们扩展了以前的方法，将其包括在细胞水凝胶中的纤维取向分布中，并展示了该模型在两个生物学相关应用中的强大功能。使用胶原水凝胶收缩测定法，我们能够在微观尺度上捕获细胞诱导的水凝胶变化，并将这些变化与随时间推移的整体凝胶尺寸变化联系起来。24 小时后，胶原密度比初始密度高约 3 倍，与水凝胶面积的减少量相同。我们还表明，从我们的自动 ICS 模型中恢复的取向参数与从手动测量中获得的值匹配。此外，与相邻区域相比，沿细胞突起的区域至少快 1.5 倍对齐。能够捕捉水凝胶密度和胶原原纤维取向的微小时间和空间变化，我们证明了这种扩展的 ICS 模型的敏感性，以解构复杂的环境，并支持其在组织工程研究中的潜力。
普遍认为，超越水凝胶组成的整体范围是理解影响人工组织的机械和生物学特性的机制的关键。在本文中，我们进行了无标记的非侵入性成像，并扩展了强大的自动分析方法来表征细胞水凝胶系统的微观结构组织。通过随时间在生物学相关系统中捕获胶原密度和纤维取向的微小变化，我们证明了该技术的敏感性。因此，我们相信该方法适用于基础细胞基质研究，并具有在水凝胶支架阵列中进行高通量筛选的潜力，使其成为未来组织工程研究的有趣工具。
