Abstract：
Donor cell-specific tissue-engineered (TE) implants are a promising therapy for personalized treatment of cardiovascular diseases, but current development protocols lack a stable longitudinal assessment of tissue development at subcellular resolution. As a first step toward such an assessment approach, in this study we establish a generalized labeling and imaging protocol to obtain quantified maturation parameters of TE constructs in three dimensions (3D) without the need of histological slicing, thus leaving the tissue intact. Focusing on intracellular matrix (ICM) and extracellular matrix (ECM) networks, multiphoton laser scanning microscopy (MPLSM) was used to investigate TE patches of different conditioning durations of up to 21 days. We show here that with a straightforward labeling procedure of whole-mount samples (so without slicing into thin histological sections), followed by an easy-to-use multiphoton imaging process, we obtained high-quality images of the tissue in 3D at various time points during development. The stacks of images could then be further analyzed to visualize and quantify the volume of cell coverage as well as the volume fraction and network of structural proteins. We showed that collagen and alpha-smooth muscle actin (α-SMA) volume fractions increased as normalized to full tissue volume and proportional to the cell count, with a converging trend to the final density of (4.0% ± 0.6%) and (7.6% ± 0.7%), respectively. The image analysis of ICM and ECM revealed a developing and widely branched interconnected matrix. We are currently working on the second step, that is, to integrate MPLSM endoscopy into a dynamic bioreactor system to monitor the maturation of intact TE constructs over time, thus without the need to take them out.
摘要：
供体细胞特异性组织工程（TE）植入物是心血管疾病个性化治疗的一种有前途的疗法，但是目前的开发方案缺乏对亚细胞分辨率下的组织发育的稳定纵向评估。作为这种评估方法的第一步，在这项研究中，我们建立了一个通用的标记和成像方案，以获得三维（3D）中TE构建体的定量成熟参数，而不需要组织学切片，从而使组织完好无损。专注于细胞内基质（ICM）和细胞外基质（ECM）网络，多光子激光扫描显微镜（MPLSM）用于研究不同持续时间长达21天的TE斑块。我们在这里表明，通过对整个安装样本进行简单的标记程序（因此无需切成薄的组织学切片），其次是易于使用的多光子成像过程，我们在发育过程中的各个时间点获得了高质量的3D组织图像.然后可以进一步分析图像的堆叠以可视化和量化细胞覆盖的体积以及结构蛋白的体积分数和网络。我们表明，胶原蛋白和α-平滑肌肌动蛋白（α-SMA）体积分数随着归一化到整个组织体积而增加，并且与细胞计数成正比。最终密度为(4.0±0.6)%和(7.6±0.7)%，分别。ICM和ECM的图像分析揭示了一个发展的和广泛分支的互连基质。我们目前正在进行第二步工作，即，将MPLSM内窥镜集成到动态生物反应器系统中，以监测完整TE构建体随时间的成熟，这样就不需要把它们拿出来了。
