Multiphoton Imaging of Maturation in Tissue Engineering

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 (alpha-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. Impact statement Fluorescence imaging is a cornerstone for monitoring tissue formation and remodeling processes during tissue maturation. Conventional tissue sectioning, staining, and imaging is considered the gold standard, but it has several drawbacks that preclude its use for longitudinal tissue maturation monitoring. Multiphoton microscopy offers opportunities for non-invasive tissue imaging in 3D. This non-invasive nature ultimately would allow imaging without interfering with the conditioning process. As a first step in this process, this study explains where multiphoton microscopy can outperform conventional tissue sectioning methods in non-invasive biomedical imaging of tissue engineered samples and thus highlights the opportunities in tissue engineering.