The particular scientific focus of MatureTissue is to bundle existing technologies available at UASTW and TUW in the fields of bioreactors/biomaterials, microfluidics, 3D printing and microscaffolds, omics technologies and biomechanics towards establishing models of mature musculoskeletal tissues, specifically of tendon, muscle, cartilage and bone. The setup is truly collaborative and interdisciplinary, as each project will require technologies and know-how of several faculty members. As such, while each PhD student will have their own project, they will also need to work as a team with their peers, the faculty members and the groups of the faculty members to achieve the ultimate goals of MatureTissue.
PhD Topic #1: Micromechanical changes in tissue formed by mechanically manipulated tenocytes under inflammatory conditions
Hypothesis/Aim: In this topic tenocytes will be uniaxially strained using either a commercially available bioreactor system called Flexcell (Dunn labs) or a custom-built system from the University of Applied Sciences Technikum Wien (UASTW) . In previous studies, the synthesis of extracellular matrix (ECM) by tenocytes mainly consisting of collagen type I and III could already be induced. Our first aim is to determine parameters of the (micro-)mechanical stimulation to induce (1) alignment of cells and (2) synthesis of tendon-like collagenous tissue. The hypothesis is, that there is a sweet spot for the generation of tendon-like tissue matrix in terms of maximized mechanical properties regarding maximum force at failure as well as construct stiffness. To optimize parameter selection and obtain most matured tendon-like constructs an integrated approach including mechanical stimulation, gene expression, proteomics as well as micro- and nanobiomechanics will be chosen. After optimization and establishment of a protocol for the tendon-like tissue formation, the influence of inflammatory conditions by addition of inflammatory cytokines (TNFalpha, IL-1beta) will be investigated.
References:  Herchenhan et al. Dev Dyn. 2013 Jan;242(1):2-8.  Heher et al. Acta Biomater. 2015 Sep;24:251-65.
PhD Topic #2: Development of tailored silk-based hydrogels for creating a cartilage-on-a-chip model
Hypothesis/Aim: The aim of this project is to establish a living tissue analogue that resembles articular cartilage into a lab-on-a-chip system to analyze the interdependence of cellular reorganization and tissue function in the presence of controlled biomechanical forces. A cartilage-on-a-chip containing mechanical actuators will be adopted  und used to study (a) impact of silk-based hydrogels on tissue formation and (b) the combinatorial effect of shear and compression forces as well as matrix stiffness on tissue function, regeneration and repair following biochemical injury. Therefore different silk formulations or modifications will be tested in non-stimulated 3D chondrogenic cell cultures and the phenotype of entrapped chondrocytes will be evaluated (gene and protein expression, chondron formation etc.) . The microfluidic setup will generate a cartilage-on-a-chip system capable of emulating the complex biomechanical environment within an articular joint by incorporating dynamic hydraulic compression and shear forces designed to mimic daily movements. After successful generation of a physiologic cartilage-on-a-chip system the effects of biomechanical forces on cartilage regeneration and repair in the presence of inflammatory agents (e.g. Interleukin β, TNF-α) to induce osteoarthritic like conditions will be investigated .
References:  Sci Rep. 2020 Oct 1;10(1):16192.  Lab Chip. 2019 Jun 7;19(11):1916-1921.  Ziadlou et al. Mater. Sci. Eng. C 2020, recently accepted,  Vet J. 2016 Mar;209:40-9.
PhD Topic #3: Induction of endochondral ossification by acellular generated ROS using a hydrostatic pressure bioreactor systeme
Hypothesis/Aim: The aim of this study is to induce and enhance endochondral ossification (EO) of mesenchymal stem cells in pellet cultures or seeded on silk-based sponges via elevated levels of reactive oxygen species (ROS) generated via a custom-made hydrostatic pressure (HP) bioreactor system. Our hypothesis is that by defining acellular generated ROS levels we can induce/enhance hypertrophy in cell cultures by leading to mineralization and bone-like tissue formation. In this regard, ROS are mainly associated with cellular damage but their physiological role including chondrocyte hypertrophy in EC is well accepted [1-3]. In this topic a HP bioreactor system of the UASTW will be used that is capable of stimulating cell cultures with elevated ROS-levels . Dose-finding of ROS (timing and levels) will be crucial to identify optimal ROS levels that modulate the initiation of the hypertrophic changes. To follow cellular changes an integrated approach including gene expression, western blot, IF-stainings, histology as well as proteomics will be chosen. Of special interest will be the investigation of the tissue maturation process using molecular imaging (matrix-assisted laser desorption/ionisation mass spectrometric imaging (MALDI MSI)) in combination with elemental imaging (laser ablation inductively couple plasma mass spectrometry (LAICP MSI) and/or micro x-ray fluorescence (µXRF)).
References:  Morita et al. J. Exp. Med. 204, 1613–23 (2007).  Kronenberg, H. M. Nature 423, 332–6 (2003).  Kim et al. J. Biol. Chem. 285, 40294–302 (2010).  Rieder et al. Sci. Rep. 8, 17010 (2018).  J Intern Med. 2015 Jun;277(6):681-9.
PhD Topic #4: Co-Culture System of artificial muscle constructs with neuronal or endothelial cells
Hypothesis/Aim: Creating functional muscle-like tissue constructs with prevascular or neuronal networks could pose a breakthrough in skeletal muscle tissue engineering. The aim of this study will be to establish an effective co-culture model of human endothelial cells (EC) and/or neuronal cells (NC) with supporting cells (MSCs, fibroblasts) and C2C12 mouse myoblasts in a 3D fibrin matrix for the engineering of vascularized and/or innervated muscle-like tissue constructs under the application of mechanical strain. In this regard, co-cultures of EC with MSCs currently represent a promising approach in microvascular tissue engineering . Since vascular, neuronal and muscle tissue experience mechanical strain in vivo, we hypothesize that mechanical stimulation might improve development and differentiation of all three tissue types. In this topic a custom-made bioreactor will be used to create muscle-like constructs . Cells embedded into ring-shaped fibrin hydrogels, different ratios of myoblasts either with ECs/NCs or fibroblasts will be tested to identify most suitable set-ups. In a parallel approach the use of co-cultures of fibroblasts with the fibrin-based artificial muscle constructs will be investigated as fibroblasts might mechanically stabilize the fibrin-based constructs as they are responsibe for the production and maintainance of skeleteal muscle ECM. Beside analytical work-up using common molecular biology techniques such as PCR, western blot, IF-stainings, etc. additional analysis will include the testing of micromechanical properties of the matured muscle-like constructs. Moreover, the idea is to use mass spectrometry imaging to analyze spatially separted regions or possible generated tissue interfaces to verify cellular crosstalk via paracrine effects, etc.
References:  Vasc Biol. 2019 Apr 8;1(1):H17-H22.  Acta Biomater. 2015 Sep;24:251-65.
PhD Topic #5: Microscaffold-reinforced Stem Cell Based Spheroids as Building Blocks for Modular Tissue Engineering
Hypothesis/Aim: Due to high cell density and biomimetic conditions, spheroids are increasingly used as building blocks in tissue engineering. Nevertheless, merging multiple spheroids together results in final macrotissue assemblies, with poor control over their viability, their morphological shape and volume and their mechanical properties. Reinforcing them with biodegradable microscaffolds, produced by means of high-resolution 3D printing (i.e multi-photon polymerization [1,2]), provides unprecedented possibilities to precisely tune and dictate such properties. Multi-photon polymerization will be used to print microscaffolds of various designs in terms of diameter, porosity and shape. Stem cells suspension, such as human adipose derived stem cells, will be cultivated under low binding condition with and without microscaffolds until spheroids are formed. At the end of the differentiation period, cell viability (Live and Dead), ECM production (immunohistology, biochemical assays) and mechanical properties of the spheroids (AFM) will be tested. Then, the capacity of spheroids to self-assemble into larger macrotissues along with their stability and maturation over-time will be assessed. This self-assembly and fusion of single spheroids to larger tissue-like structures shall be enhanced by compression. Therefore the PhD candidate will use different hydrostatic pressure bioreactor systems available at UASTW (one published  and one unpublished custom-made system, and a commercial Flexcell® Compression system ) and investigate the impacts of HP on the functionality of those stem cell-based spheroids. We aim that with the addition of controlled mechanical stimulation via bioreactor systems we can achieve major progresses and stay pioneers in this “Third Tissue Strategy in Tissue Engineering”.
References:  Angew Chem Int Ed Engl. 2018  Adv Healthc Mater. 2020 Aug;9(15):e1900752.
 Biofabrication. 2020 Mar 31;12(2):025033  Sci Rep. 2018 Nov 19;8(1):17010.