Multiphoton Imaging of Maturation in Tissue Engineering

Tissue Engineering Part C Methods, vol. 30, pp. 38–48

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.

Authors 7

1. Maximilian P. Werner corresponding Aachen Department of Biohybrid and Medical Textiles (BioTex) Institute of Applied Medical Engineering

   Maastricht University · RWTH Aachen University

   Affiliation as printed

   Aachen-Maastricht-Institute for Biobased Materials (AMIBM), Maastricht University, Geleen, The Netherlands

   Department of Biohybrid & Medical Textiles (BioTex), Institute of Applied Medical Engineering (AME), Helmholtz Institute, RWTH Aachen University, Aachen, Germany

2. Vytautas Kučikas Aachen Institute of Molecular Cardiovascular Research

   RWTH Aachen University

   Affiliation as printed

   Institute of Molecular Cardiovascular Research (IMCAR), RWTH Aachen University, Aachen, Germany

3. Kirsten Voß Aachen Institute of Automatic Control (IRT)

   RWTH Aachen University

   Affiliation as printed

   Institute of Automatic Control (IRT), RWTH Aachen University, Aachen, Germany

4. Dirk Abel Aachen Institute of Automatic Control (IRT)

   RWTH Aachen University

   Affiliation as printed

   Institute of Automatic Control (IRT), RWTH Aachen University, Aachen, Germany

5. Stefan Jockenhoevel Aachen Department of Biohybrid and Medical Textiles (BioTex) Institute of Applied Medical Engineering

   Maastricht University · RWTH Aachen University

   Affiliation as printed

   Aachen-Maastricht-Institute for Biobased Materials (AMIBM), Maastricht University, Geleen, The Netherlands

   Department of Biohybrid & Medical Textiles (BioTex), Institute of Applied Medical Engineering (AME), Helmholtz Institute, RWTH Aachen University, Aachen, Germany

6. Marc A. M. J. van Zandvoort Aachen Institute of Molecular Cardiovascular Research

   Maastricht University · RWTH Aachen University

   Affiliation as printed

   Department of Genetics and Cell Biology, Cardiovascular Research Institute Maastricht (CARIM), School for Oncology and Developmental Biology (GROW), Maastricht, The Netherlands

   Institute of Molecular Cardiovascular Research (IMCAR), RWTH Aachen University, Aachen, Germany

7. Thomas Schmitz‐Rode Aachen Department of Biohybrid and Medical Textiles (BioTex) Institute of Applied Medical Engineering

   RWTH Aachen University

   Affiliation as printed

   Department of Biohybrid & Medical Textiles (BioTex), Institute of Applied Medical Engineering (AME), Helmholtz Institute, RWTH Aachen University, Aachen, Germany