Title Promoter Affiliations Abstract "Decellularized muscle as a scaffold for extraocular muscle tissue engineering" "Lieven Thorrez" "Development and Regeneration, Kulak Kortrijk Campus, Research Group Ophthalmology" "Injuries to skeletal muscle are dramatically impacting patient’s lives, limiting their daily activities. Depending on the defect, current treatment is either not possible or involves autologous tissue transfer, which leads to donor site morbidity and incomplete functional recovery. Tissue engineered muscle may provide an alternative for muscle repair and can also be used to study development, disease and drug effects in vitro. We propose to use decellularized skeletal muscle tissue as a scaffold for regeneration. In particular, we will focus on the extraocular muscles, due to their specific characteristics; these muscles are spared during ageing and development of Duchenne muscular dystrophy. The first goal will be to establish a novel decellularization protocol to efficiently decellularize thicker (>1 mm) muscle tissues. Second, extraocular muscle progenitor cells will be isolated. The obtained scaffold will be reseeded with extraocular myogenic cells by a novel seeding technique. The decellularized tissues as well as the recreated muscle tissues will be mechanically characterized. In this way, we will not only develop a transplantable construct, but also create an in vitro model for studying cell-matrix interactions." "Preclinical evaluation of decellularized abdominal muscles to repair complex abdominal wall defects" "Lieven Thorrez" "Development and Regeneration, Kulak Kortrijk Campus, Laboratory of Respiratory Diseases and Thoracic Surgery (BREATHE)" "Abdominal wall hernia repair is one of the most frequently performed surgical procedures. Unfortunately, primary repair using the inherently weakened native tissues or implantation of a synthetic mesh has a high risk of failure and often require reoperation on the long term. Other problems associated with synthetic meshes are pain and infection due to mesh degradation and impediment of autologous muscle repair. These problems could potentially be overcome by implanting biological scaffolds which support autologous repair mechanisms and are biodegradable. Such scaffolds may be provided through decellularization of abdominal muscle tissue harvested from donors. Decellularization is a process in which living cells and nuclear material are removed from tissues. Ideally, this occurs without affecting the structural integrity and desired composition of the extracellular matrix (ECM). Due to the high conservation of ECM components, the decellularized tissues may serve as an ideal support structure for migrating cells during regeneration. Furthermore, they are well suited to support the surrounding tissue especially if they have the same origin. In addition, these ECM scaffolds are largely free from immunogens and are believed to avoid immune rejection after transplantation. In a first work package, we aim to optimize a protocol for the decellularization of abdominal muscle tissue from animals. First, the focus will be on optimizing parameters such as composition of the decellularization agents, temperature of decellularization, time of decellularization, flow rate, rinsing buffer and duration. Next, it is important to accurately and quantitatively evaluate the efficiency of the decellularization process. Characterization of the decellularization will be based on several complementary methods: evaluation of removal of nuclear content, ECM characterization and mechanical characterization. The second work package aims to implant the obtained scaffolds in an autologous abdominal defect model. In pilot experiments we will assess mechanical integrity of the grafted tissue, morphological observations after abdominal wall defect repair (histological stains), immunohistological stains (neo-muscle fiber formation and angiogenesis) to evaluate the regenerative process and immunohistological stains (macrophages and lymphocytes) to evaluate the immunological response. Based on these short-term pilot experiments, the most optimal scaffold will be identified to use in one long-term experiment to evaluate herniation. Briefly, with this research we aim to optimize the decellularization protocol of abdominal muscle tissue and preclinically evaluate these decellularized scaffolds for abdominal defect repair." "Mechanical stimulation and incorporation of tissue-specific endothelial cells for tissue engineering of human skeletal muscle" "Lieven Thorrez" "Development and Regeneration, Kulak Kortrijk Campus, Urogenital, Abdominal and Plastic Surgery, Stem Cell and Developmental Biology" "In our lab, we create bio-artificial muscles (BAMs) made up of human aligned myofibers in an extracellular matrix starting from the adult stem cells present in skeletal muscle. Despite advances in the field, its applications are still hampered by the limited mechanical strength, size and stage of maturation of the BAMs. This justifies the need for mechanically stronger tissue-engineered muscle. The theme of this project is focused on advancing BAMs through hypertrophy. This is the process by which muscles grow and become stronger through exercise. We aim to mimick this exercise by applying mechanical stimulation to BAMs in culture. This is hypothesized to not only enhance fiber hypertophy but also blood vessel formation. In parallel, we will integrate elastomeric scaffolds in our model to serve as a temporary connective tissue scaffold for developing muscle. This is hypothesized to provide support to the developing muscle while autologous extracellular matrix develops." "Development of a 3D human skeletal muscle co-culture system to study atrophy and hypertrophy" "Lieven Thorrez" "Development and Regeneration, Kulak Kortrijk Campus" "Tissue engineering of 3D human skeletal muscle offers new perspectives for future regenerative therapies, basic science and drug testing. Currently, we are able to create aligned human muscle fibers in a 3−dimensional structure. In this proposal, we will further investigate the use of co-cultures of myogenic and other cell populations to create blood vessel structures in tissue-engineered human muscle. The influence of these additional cell populations on myofiber morphology and function will be studied over time and under influence of muscle stimulation. Furthermore, we will test this system as a model to study atrophic and hypertrophic responses." "Cultured stem cells for customized meat design" "Lieven Thorrez" "Development and Regeneration, Kulak Kortrijk Campus, Universiteit Gent" "Cellular agriculture is a promising avenue for sustainably contributing to the growing food demand. In this respect, cultured or in vitro meat receives increasing attention. Yet, there is currently no scientific evidence that animal myofibers equivalent to meat as a nutrient dense, highly structured food, can be made in vitro. This proposal will address several existing hurdles. A major challenge is to obtain a suitable cell type capable of both sufficient proliferation and robust differentiation. Muscle adult progenitor cells have a very good differentiation capacity, yet weak proliferation capacity, whereas mesenchymal and embryonic stem cells display the opposite behavior. First, we will compare and enhance the proliferation and differentiation capacity of these three cell types, resulting in an informed choice of cell type for myofiber generation. Secondly, we will design a ‘smart matrix’, including food-grade components that contribute to texture, flavor and a good nutritional profile. This involves a proper choice of materials which is mutually dependent on the technological approaches to create a 3D structure. Physicochemical stability of, and cellular adhesion and survival on these new matrices will be investigated. The best performing smart matrices will be tested on their potential to support muscle cell differentiation. These insights will be subsequently used to increase muscle fiber size and to study in vitro muscle development in a 3D architecture. In addition, strategies for stimulating the build-up of contractile proteins will be investigated. Finally, public acceptance, consumer attitudes, perceptions and intentions, as well as impact on sustainability will be assessed. This project will deliver a sound scientific basis and assessment of key technologies for advancing knowledge in this field and allowing informed decision making for downstream valorization strategies. " "Biofabrication of complex and vascularized tissues via hybrid bioprinting" "Heidi Declercq" "Development and Regeneration, Kulak Kortrijk Campus" "The biofabrication of 3D biomimetic tissue analogs, which accurately mimic the properties of native tissue, have an enormous potential in biomedical applications (drug discovery, cancer research, regenerative medicine,…). A basic prerequisite for the survival, maturation and function of 3D engineered tissues is the establishment of blood vessels. The most critical challenge in complex tissue engineering is the integration of a hierarchical vascular network. This project will integrate biomimetic approaches with bioprinting by combining knowledge of self-organizing vascularized spheroids with smart (cell instructive) biomaterials for generating vascularized tissues." "Targeting tendon-bone junction regeneration: development of a gradient scaffold." "Catharina De Schauwer" "Department of Organic and Macromolecular Chemistry, Department of Translational Physiology, Infectiology and Public Health" "The tendon-bone (T-B) junction is the transition zone from soft to hard tissue, prone to acute and overuse injuries in both human and equine athletes. Functional integration between tendon and bone remains a major challenge after injury, as the presence of inflammation affects the healing process, resulting in the formation of scar tissue. Tissue engineering strategies should be explored to support T-B regeneration. To this end, the characteristic gradient structure of the junction, i.e. the spatial distribution with a gradual decrease in collagen fiber organization while proteoglycan and mineral content is gradually increasing, should be mimicked. To develop such a functional scaffold, tailor-made photo-crosslinkable and biodegradable polymers (i.e. urea/urethane-based norbornene-endcapped precursors and thiolated decellularized extracellular matrix) will be synthesized and characterized. Then, the developed materials will be 3D printed into a continuous scaffold exhibiting a gradient in mechanical properties. Finally, by encapsulating undifferentiated mesenchymal stem cells (MSCs) during 3D-printing along with their differentiation factors, the MSCs will gradually differentiate towards the desired phenotypes. Undifferentiated MSC will secrete anti-inflammatory factors, which is beneficial to counter the inflammation observed immediately after injury. Once the MSC are differentiating, trophic factors will be produced, which is beneficial to support T-B regeneration." "Research at the interface between human genetics and reproduction." "Karen Sermon" "Biology of the Testis, Department of Embryology and Genetics" "Today, numerous mitochondrial and nuclear pathogenic gene defects causing mitochondrial disease have already been identified. More recently, our attention has turned towards protein synthesis in mitochondria. Malformations of cortical development (MCD) represent a major cause of developmental disabilities and severe epilepsy. Polymicrogyria (PMG) is a heterogeneous condition, with respect to both phenotype and genotype. In the first part of the project the genetic basis of PMG is addressed. The second part of the study aims at improving the phenotypic characterization of MCD in order to facilitate further genetic research. The research carried out under Inge Liebaers has traditionally had a strong focus on all aspects of reproductive genetics.. Within reproductive genetics, the genetic causes of male infertility is a first topic of interest. To elucidate genetic causes of male infertility, genes with a testis-specific expression pattern will be examined and the whole genome will be investigated for the presence of deletions/duplications. Treatment of male infertility is a logical complement of the former topic. A specific subpopulation of male infertile patients are survivors of childhood cancers who underwent aggressive treatment including chemo- and radiotherapy and bone marrow transplantation. Studies to investigate the genetic and epigenetic aspects of spermatogonial stem cell (SSC) transplantation as well as cryopreservation and tissue expansion are mandatory before a clinical application becomes feasible. Another route to mature sperm would be by differentiation of human embryonic stem cells (hESC) into spermatozoa. As a way to elucidating the pathways controlling differentiation into SSC, dedifferentiation of SSC into pluripotent cells (PC) will be attempted. Safety of assisted reproductive technology (ART), and more specifically at the level of epigenetics and genomic imprinting, is another firm link between reproduction and genetics. We have developed in-house methods for the analysis of DNA methylation on small samples, typically one embryo. Although the majority of children born after assisted reproductive technology (ART) are healthy, recent years have seen the publication of several papers that report on the association of ART and imprinting disorders. Data obtained will subsequently be linked in an anonymous way to the neonatal data obtained from the follow-up study of IVF/ICSI children. Understanding totipotency at the cellular and molecular level will have a great impact on the knowledge of preimplantation development and may have important implications for the current practice in the IVF lab. Although PGD has been part of everyday clinical routine at UZ Brussel since the mid-nineties, we still aim to improve the technology. The combination of several emerging technologies will be evaluated and clinically implemented. Concurrently, data mining of the past 15 years of clinical PGD, will allow for retrospective analysis of PGD data. Clinical research on children born after different ART techniques was started since the introduction of IVF at the UZ Brussel (1983). In the future, studies on (inherited) male infertility in boys born after ICSI will be performed once they reach 18 years in the next few years and the safety of many new techniques (PGD, cryopreservation and vitrification) will be evaluated through the health of the offspring. Other possible risks of ART techniques for the offspring will be examined on the older children of the cohort. Human embryonic stem cell research (HESC) has been established in our group in 2002 and has already led to interesting results and publications. We have currently derived 26 different HESC lines, of which 16 carry a SGD. The great value of these cells for research into fundamental embryology and the molecular basis of the SGD they carry is beyond doubt. We wish to further expand our possibilities for the procurement of pluripotent cells, such as induced pluripotent cells, from different sources. Preliminary results have shown that our hESCs accumulate different types of chromosomal abnormalities, ranging from the amplification of known ""stemness genes"", ie genes involved in pluripotency, to large deletions and duplications. We will explore the causes of these abnormalities by the study of the gene expression. Differentiation into endodermal and mesodermal derivatives will for a large part be carried out in collaboration with other groups. In our own lab, we will attempt to differentiate hESC into mature lung cells, using a liquid-air interface culture system that we have previously developed. Differentiation in normal hESC will be compared with differentiation in hESC carrying a SGD affecting primarily muscle (DM1, FSHD, DMD) or lung (CF). The CRM vitrification project will encompass three fields of application: embryos, oocytes and in extension, pluripotent cells."