Title Promoter Affiliations Abstract "EUROPEAN CALCIFIED TISSUE SOCIETY: Amgen bone biology research fellowship" "Bruno Lapauw" "Department of Internal Medicine and Pediatrics, Department of Internal medicine" "This research proposal aims at investigating the role of Wnt signaling molecules, RANKL and OPG in the regulation of bone metabolism and trabecular and cortical bone charachteristics during different stages of life in men, i.e. during puberty, at adult age and during senescence. In addition, possible determinants of the Wnt OPG/RANKL/RANK systerms will be explored, focusing on sex steroïd status." . "Jos Vander Sloten" "Biomechanics Section" "Recent developments in medical implant technology have seen a rise in devices designed for minimally invasive implantation in soft tissue. They allow to treat patients not considered for surgery, reduce the trauma caused by surgery, and thereby, reduce pain and improve the recovery time. For example, in the field of cardiology, transcatheter implants to treat aneurysms, vascular occlusion, and diseased heart valves are already available. However, the range of manipulations that can be performed during a minimally invasive surgery is limited. For example, in a transcatheter intervention, all instruments have to be flexible enough, and limited in diameter in order to pass through the vessels. This limits the ability to remove diseased tissue during the surgery, and to manipulate the tissue to fit closely around implant.  In a transcatheter aortic valve implantation the diseased valve is not removed. Instead, the implant is placed inside the native calcified valve. This can cause problems with the sealing around the new valve, resulting in a backflow of blood, alongside the implant, when the valve is closed. The limitations of the transcatheter approach increase the importance of device selection, the implant's size, and the implantation position. Computer aided surgical planning can provide additional information about the 3-dimensional geometry of the anatomy, and the interaction between the implant and the native tissue prior to the surgery. Therefore, the aim of this thesis is to develop computer aided surgical planning tools to improve device implantation in soft tissue.First, the aortic root of a population of transcatheter aortic valve implantation patients was characterised, based on the pre-interventional computed tomography images, to determine whether certain anatomical characteristics increased the risk of leakage after the implantation. A method was developed to calculate the calcification volume based on the images, and the geometry of the aortic root was characterised at two cross sections by multiple parameters such as diameter, area, etc. This investigation found that the patients suffering from moderate to severe aortic regurgitation post implantation, had a higher average calcification volume. However, there was a large overlap between the two patient groups. Whether an individual patient would develop severe aortic regurgitation could not be predicted based on the calcification volume.Next, a method was developed to include the 3-dimensional anatomical shape of the aortic root in the evaluation of the optimal implant size. A segmentation algorithm was developed to automatically extract the aortic root and the aortic valve leaflets from the pre-interventional images. Based on the models of the aortic root, a parametric description was created using the population average, and the principal modes of variation. In turn, the parametric description was used to develop and train a classification method that could automatically determine the optimal size of the implant. The result was an algorithm that assigned the same size implant as was implanted to 96% of the patients in the training set. When applied to the patients with moderate to severe aortic regurgitation, 54% of them were assigned a different size than was implanted. Further research is required to assess whether the different size implants would improve the outcome of those patients.In order to incorporate the interaction between the calcified native valve and the implant in the pre-interventional planning, a method for the patient specific simulation of the implantation was developed. The simulated stent was compared with post-intervention scans to validate the method. Based on the simulation result an algorithm was developed to estimate the post-interventional aortic regurgitation by detecting leakage paths between the modelled stent and the aortic root. The method was tested by simulating the implantation of 10 patients. The method could accurately predict the shape of the post-operative result, and the regurgitation estimation shows promising results that should be further validated on a larger patient set.Finally, the shape model of the aortic root and the simulation method were combined to develop a framework for virtual testing of the fit of a device in the patient population. This method could be used to optimise the design of the next generation of implants. Calcifications were assigned to instances of the shape model based on the pre-interventional scans of the patient population. These models were used to simulate the implantation. Currently, the results are less accurate than the patient specific simulations. Further research is needed to improve this method in order to realise its potential.In conclusion, this PhD thesis showed that computer aided surgical planning has the potential to improve the outcome for transcatheter aortic valve implantation patients by using the full potential of the 3-dimensional anatomical shape information, and by providing additional information about the interaction between the implant and the calcified aortic root. This will allow to make a more informed decision on the optimal size and position of the implant." "Mapping of skeletal progenitors in embryonic limb cartilage Their application in bone tissue engineering" "Frank Luyten" "BioMechanics (BMe), Skeletal Biology and Engineering Research Center" "Large bone defects can be caused by major trauma, infection, prosthetic revision, bone tumour resection or non-healing fractures and in clinical practice, their healing remains a therapeutic challenge. Current treatments such as iliac crest autografts or cadaver allografts require multiple and repetitive interventions and are associated with various risks resulting in a high socio-economic burden. Several tissue engineering strategies have been developed to overcome these challenges and one of them is based on bone developmental engineering. This approach involves the in vitro manufacturing of a living cartilage tissue construct that upon implantation forms bone in vivo by mimicking the process of endochondral ossification as it takes place during embryonic development.Briefly, during that process, Prrx1 expressing limb mesenchymal cells condense and differentiate into Sox9+ chondrocytes. These chondrocytes proliferate, organize in columns and enter hypertrophy under the control of an Ihh/PTHrP loop. After cell maturation into Runx2+ hypertrophic chondrocytes, a shift in matrix synthesis occurs from collagen type II to type X. This matrix calcifies and is replaced by bone by invading osteoblasts and transdifferentiating non-apoptotic hypertrophic chondrocytes, both characterized by Osterix expression and secretion of osteoid matrix.The cell sources to engineer cartilage intermediates for in vivo bone healing can be diverse with the periosteum currently considered an excellent cell source. Lineage tracing experiments in mice have shown that during bone repair, osteoblasts and osteoclasts originate from the bone marrow, endosteum and periosteum, but that callus chondrocytes are primarily derived from the periosteum. More recently, it has been shown that human periosteal cells can be primed in vitro, by using conditioned medium and cell aggregation, to a cartilaginous intermediate tissue able to develop in vivo into bone ectopically and facilitate healing in an orthotopic long bone defect. However, these cells still generated excessive fibrous tissue. Enrichment for osteochondrogenic precursors is expected to result in an enhanced bone forming potential and improved purity of cell-based treatments.Several studies in mice focused on the identification and contribution of skeletal stem cells and osteochondroprogenitors in bone development, homeostasis and fracture healing. These cells were found either in the zone underneath the growth plate, blood vessel niches or the periosteum. Several molecular markers have been associated with these cells such as Nestin, Gremlin1, Leptin Receptor, Gli1 and Periostin. In another study, “rainbow” adult mice displayed a high frequency of clonal regions in the growth plate, characterized by Alpha V Integrin (CD51) expression but negative for CD45 and TER119. This population was subsequently divided into eight subpopulations based on differential expression of CD105, CD90.2, CD200 and 6C3 cell surface markers. By combining this strategy with in vivo and in vitro approaches, they mapped bone, cartilage and stromal development from a postnatal mouse skeletal stem cell to its downstream progenitors in a hierarchical program similar to hematopoiesis.In this thesis we hypothesized that enrichment for the skeletal progenitors leads to enhanced bone formation, and the possibility to reduce the cell dosage without losing potency of the cell-based construct. To test this hypothesis, we optimized the prospective isolation of stem and progenitor cell populations from the mouse embryonic hind limb cartilage 14.5 dpc.We showed that primary mouse embryonic cartilage cells continued their developmental program and formed a bone organoid in an in vivo ectopic bone forming assay when encapsulated in collagen I hydrogel. Tracking experiments with eGFP+ embryonic cartilage cells revealed the contribution of donor cells to the osseous tissue. To purify the progenitors from the limb cartilage, we designed a polychromatic flow cytometry protocol, based on the previously described markers indicative of skeletal stem cells markers. We purified from the embryonic cartilage cells two cell populations, namely the mouse skeletal stem cell and a pre-progenitor, a direct descendent of the skeletal stem cell. Both populations were able to form bone in the collagen I hydrogel, and quantification of bone volume indicated more tissue formed in comparison to a pool of embryonic cartilage cells, even at lower cell density. In addition, the observed bone tissue was predominantly formed by endochondral ossification, as after one-week hypertrophic cartilage was observed. We noticed however that the potency of the progenitors was affected by the hydrogel encapsulating the cells, since a significant reduction in bone formation was observed when the cells were encapsulated in alginate hydrogel.Next, we aimed to investigate the possibility to expand the primary embryonic cartilage cells, and simultaneously tried to enrich for progenitors during this expansion phase. For this, we culture expanded the embryonic cartilage cells in the presence of FGF2, a standard ligand used in stem cells expansion protocols. Gene expression analysis revealed no dedifferentiation of FGF2 expanded cells in comparison to cells expanded in foetal bovine serum containing growth medium as determined by the expression of ACTA2. Flow cytometry analysis of the CD marker set revealed an enrichment for stem cells and progenitors after two passages. However, when the stem cells and progenitors from expanded cells were implanted in collagen I, a major loss in in vivo bone formation was observed. Retrieved explants were 50% smaller, bone tissue volume was reduced, and the explants contained fibrotic tissue.In this thesis we showed that purification of progenitors has a beneficial effect on bone formation upon in vivo implantation, and that it enabled reduction of cell dosage without negatively affecting the in vivo bone forming potency. However, prospective validation of CD markers identifying skeletal progenitors is required and may depend on the cell source. Our findings indicate that the relation between in vitro cell identity and in vivo tissue formation can be altered by in vitro manipulations." "iPSC Technology and Bone Regeneration" "Frank Luyten" "Skeletal Biology and Engineering Research Center" "Bone is a calcified tissue with multiple functions. It allows movement by providing attachment points for skeletal muscle, supports haematopoiesis, stores minerals and protects soft tissues and organs. Additionally, bone has the unique capacity to regenerate upon damage without scar formation or loss of function.Bone fracture repair recapitulates embryonic endochondral bone formation. This process is characterized by the formation of a cartilaginous template (soft callus) that precedes bone formation. This template is considered to be one of the most important tissue intermediates during endochondral ossification as it provides a structural framework for osteogenesis and it allows recruitment of osteogenic cells and blood vessels.Despite this regenerative capacity, bone (fracture) healing is often impaired due to local and congenital factors. 10% of all fractures are healing poorly or result in a non-union. Bone tissue engineering aims to provide a solution by combining stem cell biology with engineering science. Typically, skeletal stem cells are combined with growth factors and seeded on scaffolds to induce bone formation. However, current therapeutic strategies are being hampered by the lack of robust and reproducible cell populations, which display favourable differentiation and proliferative properties. Fortunately, cellular reprogramming has provided an alternative and powerful strategy for the derivation of robust cell populations for medicinal research and clinical therapies.Cellular reprogramming is a strategy in which cells can be converted to alternative cell types through the forced expression of transcription factors that control cell fate. Activation of these factors can lead to the reintroduction of embryonic pluripotency in adult terminally differentiated cell types thus resulting in the derivation of induced pluripotent stem cells. These stem cells can then be further specialized into skeletal cells for bone regeneration (indirect target cell derivation). However, instead of reacquiring pluripotency, cellular reprogramming strategies can be adapted whereby the cell of choice can be directly obtained through the forced expression of cell type specific transcription factors (direct cell reprogramming). Both strategies have been explored within this PhD project and different skeletal cell types have been derived for bone augmentation.Murine skin fibroblast cells were directly reprogrammed to cartilage cells (chondrocytes) by using Sox9, KLF4 and cMYC. Two chondrogenic cells lines were derived through either constitutive or doxycycline inducible expression of these transcription factors. Although both cell lines displayed in vitro chondrogenic differentiation capacities, cells reprogrammed with inducible transcription factors were able to undergo hypertrophic maturation and could induce bone formation.To translate these findings towards human cells, (induced) pluripotent stem cells were differentiated towards cartilaginous aggregates. Subsequently upon hypertrophic stimulation, soft callus-like tissue could be obtained. When implanted, these tissue intermediates allowed progressive healing of critical size long bone defects. These results support the concept of developmental tissue engineering strategies for bone regeneration.In the last part of this PhD, it has been demonstrated that embryonic limb progenitors could be in vitro expanded. When implanted, these cells spontaneouslygave rise to endochondral ossification. To achieve potential clinical translation, culture and differentiation conditions have been established which allowed us to derive these progenitors from induced pluripotent stem cells. These experiments further highlight the promise of cellular reprogramming for the creation of skeletal progenitor cells for novel bone tissue engineering strategies.Collectively, the findings presented within this dissertation are not only contributing to the in depth knowledge of skeletal tissue regeneration and skeletal stem cell biology, but also demonstrate the potential of cellular reprogramming for the derivation of novel (stem) cell populations capable of triggering bone repair. " "Investigating the role of autophagy in arterial calcification and arterial stiffness." "Pieter-Jan Guns" "Physiopharmacology (PHYSPHAR)" "Cardiovascular calcification significantly contributes to cardiovascular disease, which is the leading cause of mortality and a major cause of morbidity in Europe. Cardiovascular calcification occurs in both rare monogenic (e.g. pseudoxanthoma elasticum) and in common acquired diseases (e.g. atherosclerosis and chronic kidney disease). Importantly, cardiovascular calcification is an active but incompletely understood process regulated by a variety of (epi)genetic and environmental factors, acting both systemically and locally. Despite its major clinical impact, no specific therapeutic strategies targeting cardiovascular calcification are applied in current clinical practice. In 2019, the Physiopharmacology and Pathophysiology research groups of the University of Antwerp were both part of the ""eRaDiCal"" consortium (H2020-MSCA-ITN call) aimed to investigate risk factors and underlying mechanisms across rare and common ectopic calcification disorders for the development of adequate diagnostic, preventive and therapeutic solutions to target cardiovascular calcification. eRaDiCal received an excellent reviewer score (94.8%, reserve list), but was not funded. As part of its strategic research plan, the University of Antwerp provides funding for excellent H2020 proposals to facilitate and encourage a successful resubmission. The teams of Physiopharmacology and Pathophysiology will use the budget to substantiate the evidence base on the role of autophagy in arterial calcification and stiffness. The intention is to recruit a (part-time) postdoc and/or PhD student to obtain preliminary data during the next year." "Study on the effect of phytate (inositol hexakisphosphate) on the development of vascular calcification in rats with chronic renal failure" "Ellen Neven" Pathophysiology "Vascular calcification is the major risk factor for the high cardiovascular mortality in patients with end-stage renal disease. Current therapies, mainly focusing on disturbances in mineral metabolism such as hyperphosphatemia and secondary hyperparathyroidism, reduce the progression of vascular calcification, but these agents can not completely block the calcification process and suffer from a poor compliance. A new therapeutic approach consists of direct interference with the formation of the calcified lesions. Therefore, the current project aims to investigate the effect of phytic acid, which prevents the formation and growth of calcium-phosphate microcrystals in soft tissue, on the development of vascular calcification in rats with chronic renal failure."