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 (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.

Two-Photon Endoscopy: State of the Art and Perspectives.

In recent years, the demand for non-destructive deep-tissue imaging modalities has led to interest in multiphoton endoscopy. In contrast to bench top systems, multiphoton endoscopy enables subcellular resolution imaging in areas not reachable before. Several groups have recently presented their development towards the goal of producing user friendly plug and play system, which could be used in biological research and, potentially, clinical applications. We first present the technological challenges, prerequisites, and solutions in two-photon endoscopic systems. Secondly, we focus on the applications already found in literature. These applications mostly serve as a quality check of the built system, but do not answer a specific biomedical research question. Therefore, in the last part, we will describe our vision on the enormous potential applicability of adult two-photon endoscopic systems in biological and clinical research. We will thus bring forward the concept that two-photon endoscopy is a sine qua non in bringing this technique to the forefront in clinical applications.

Towards technically controlled bioreactor maturation of tissue-engineered heart valves.

Bioreactors are important tools for the pre-conditioning of tissue-engineered heart valves. The current state of the art mostly provides for timed, physical and biochemical stimulation in the bioreactor systems according to standard protocols (SOP). However, this does not meet to the individual biological variability of living tissue-engineered constructs. To achieve this, it is necessary to implement (i) sensory systems that detect the actual status of the implant and (ii) controllable bioreactor systems that allow patient-individualized pre-conditioning. During the maturation process, a pulsatile transvalvular flow of culture medium is generated within the bioreactor. For the improvement of this conditioning procedure, the relationship between the mechanical and biochemical stimuli and the corresponding tissue response has to be analyzed by performing reproducible and comparable experiments. In this work, a technological framework for maturation experiments of tissue-engineered heart valves in a pulsating bioreactor is introduced. The aim is the development of a bioreactor system that allows for continuous control and documentation of the conditioning process to increase reproducibility and comparability of experiments. This includes hardware components, a communication structure and software including online user communication and supervision. Preliminary experiments were performed with a tissue-engineered heart valve to evaluate the function of the new system. The results of the experiment proof the adequacy of the setup. Consequently, the concept is an important step for further research towards controlled maturation of tissue-engineered heart valves. The integration of molecular and histological sensor systems will be the next important step towards a fully automated, self-controlled preconditioning system.