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The treatment of critical-sized bone defects poses significant challenges as these defects often require medical intervention. The gold standard treatment, autologous bone grafts, has drawbacks like donor site morbidity and limited tissue availability. In response, the field of bone tissue engineering (BTE) emerged, aiming to develop biological substitutes or scaffolds utilizing biomaterials, cells, and growth factors to regenerate bone. However, challenges such as insufficient vascularization still hinder clinical translation. Nonetheless, endochondral bone regeneration (EBR) is a BTE approach that holds promise. It involves mimicking a soft callus to serve as template for bone regeneration. This thesis focuses on advancing the EBR research of our group by developing a bioink containing chondrogenic micro pellets. A microwell system was selected to generate micro pellets, which were cultured across four mesenchymal stem cell donors. Seven different cell densities (1500, 3000, 6000, 12000, 24000, 36000, and 50,000 cells per micro pellet) and a control (250,000 cells per macro pellet) were analyzed for size and chondrogenic differentiation with a safranin-O, toluidine blue, and collagen II staining. In continuation, micro pellets were combined with Gelatin Methacryloyl (GelMA) to create a bioink, which was cast and volumetrically printed to demonstrate functionality by fabricating an incus bone shape. The microwell system of the Inge Zuhorn group was chosen for further optimization instead of microwell stamps. Micro pellets under 6000 cells displayed limited to no GAG and collagen II deposition. Above 6000 cells, micro pellets demonstrated unwanted aggregation but exhibited complete GAG and collagen II deposition. Due to these issues, a second bioink, composed of crushed macro pellets and GelMA, was formulated. The macro pellets were effectively crushed and integrated into bioink and used to volumetrically print an incus bone. In conclusion, a microwell system was se

Chondrogenic aggregates have emerged as promising building blocks for the development of implants intended to facilitate bone defect regeneration through the endochondral bone regeneration process. While most protocols rely on static culture systems to create these cell aggregates, scalability limitations have impeded their broader application. In this regard, dynamic culture systems offer an advantageous approach by enabling automated aggregate production, which not only ensures scalability and workload reduction but also aligns with good manufacturing practices (GMP) and facilitate clinical translation. In this study, we investigated the feasibility of collision-based self-assembly of human bone marrow-derived mesenchymal stromal cell (hBM-MSC) aggregates and their subsequent chondrogenic differentiation within the same spinner flask setup. Moreover, qualitative analyses of neural cell adhesion molecule (NCAM) protein expression were performed to study the effect of the hydrodynamic environment on cell aggregation in comparison to static culture. Given that stirring rate influences cell-cell collisions, in this study different velocities of the spinner flask were examined to determine the most favourable one for spontaneous hBM-MSC aggregate formation. The identification of appropriate stirring rate allowed dynamic spinner flask culture (1x10^6 cells/mL) of the spontaneously formed aggregates for up to 21 days using two different donors. Dynamically cultured aggregates showed significant variation in aggregates size and shape as opposed to static culture. Importantly, dynamic culture did not affect aggregate viability, although cell death was observed due to the large size of the aggregates. When assessing cell differentiation, staining of dynamically cultured aggregate sections for glycosaminoglycans (GAGs) and collagen II showed inter- and intra-aggregate heterogeneity in comparison to static culture. Moreover, NCAM protein expression was not modulated by the hy

The human body experiences a wide variety of stresses from loading produced by its own weight or external forces such as gravity. To prevent any damage to the bones, especially during movement, nature has designed special cushioning that is called hyaline cartilage. Hyaline is primarily found at the tips of long bones that make up our limbs, also referred to as articular cartilage. The major function of articular cartilage is to absorb shock and to lubricate the bones during movement. This specific function of hyaline is underlied by its composition. A major player in hyaline cartilage are large molecules that are called proteoglycans: ‘proteo’ meaning protein and ‘glyco’ meaning sugar. These molecules can attract and retain water inside the cartilage which gives it its shock-absorbing properties. In addition to the proteoglycans, there is a network of strands that can be aligned or randomly dispersed within the cartilage. This network provides structure and stiffness to the hyaline, and together with the proteoglycans determine cartilage functions. In this mini-review the major types of proteoglycans and their roles in hyaline were summarised. Moreover, hyaline proteoglycans were compared to proteoglycans in other types of cartilage such as mature or diseased cartilage.

Advanced in vitro human bone defect models can contribute to the evaluation of materials for in situ bone regeneration, addressing both translational and ethical concerns regarding animal models. In this study, we attempted to develop such a model to study material-driven regeneration, using a tissue engineering approach. By co-culturing human umbilical vein endothelial cells (HUVECs) with human bone marrow-derived mesenchymal stromal cells (hBMSCs) on silk fibroin scaffolds with in vitro critically sized defects, the growth of vascular-like networks and three-dimensional bone-like tissue was facilitated. After a model build-up phase of 28 days, materials were artificially implanted and HUVEC and hBMSC migration, cell-material interactions, and osteoinduction were evaluated 14 days after implantation. The materials physiologically relevant for bone regeneration included a platelet gel as blood clot mimic, cartilage spheres as soft callus mimics, and a fibrin gel as control. Although the in vitro model was limited in the evaluation of immune responses, hallmarks of physiological bone regeneration were observed in vitro. These included the endothelial cell chemotaxis induced by the blood clot mimic and the mineralization of the soft callus mimic. Therefore, the present in vitro model could contribute to an improved pre-clinical evaluation of biomaterials while reducing the need for animal experiments.

Clinical implementation of endochondral bone regeneration (EBR) would benefit from the engineering of devitalized cartilaginous constructs of allogeneic origins. Nevertheless, development of effective devitalization strategies that preserves extracellular matrix (ECM) is still challenging. The aim of this study is to investigate EBR induced by devitalized, soft callus-mimetic spheroids. To challenge the translatability of this approach, the constructs are generated using an allogeneic cell source. Neo-bone formation is evaluated in an immunocompetent rat model, subcutaneously and in a critical size femur defect. Living spheroids are used as controls. Also, the effect of spheroid maturation towards hypertrophy is evaluated. The devitalization procedure successfully induces cell death without affecting ECM composition or bioactivity. In vivo, a larger amount of neo-bone formation is observed for the devitalized chondrogenic group both ectopically and orthotopically. In the femur defect, accelerated bone regeneration is observed in the devitalized chondrogenic group, where defect bridging is observed 4 weeks post-implantation. The authors' results show, for the first time, a dramatic increase in the rate of bone formation induced by devitalized soft callus-mimetics. These findings pave the way for the development of a new generation of allogeneic, "off-the-shelf" products for EBR, which are suitable for the treatment of every patient.

Endochondral bone regeneration, which consists of regenerating bone tissue based on a cartilage template, is a promising approach to treat bone defects. Cartilage constructs are engineered with mesenchymal stromal cells (MSCs) in vitro. To generate high quality cartilage tissue in vitro, the differentiation conditions for the MSCs are crucial. Among others, bone morphogenic protein 2 (BMP-2) is a common growth factor used to differentiate MSCs chondrogenically in vitro. However, it is not completely clear how BMP-2 affects chondrogenesis and extracellular matrix (ECM) production. Therefore, the effects of BMP-2 reported in literature so far on the chondrogenic differentiation of MSCs and therefore ECM composition are fundamental to be summarized to have a better understanding of the topic, in order to produce in vitro cartilage constructs which can effectively sustain bone tissue regeneration in vivo. This article aims to provide a broad overview on the effect of BMP-2 on MSCs chondrogenesis and on cartilage ECM composition. It also provides a collection of techniques helpful to detect BMP-2 remnants after cartilage ECM constructs engineering, since it could have a collateral impact on the therapeutical effects of the cartilage construct. It is reported that upon BMP-2 binding to its receptors, activation of different molecular pathways are ensued, where cell proliferation, cell survival and chondrogenesis are upregulated. Furthermore, the expression of chondrocyte-specific genes, such as collagen type II, aggrecan and glycosaminoglycans and hypertrophy-specific genes, such as collagen type X, alkaline phosphatase and matrix metalloproteinase are stimulated, influencing ECM composition. In detail, BMP-2 is a soluble extracellular protein that binds to BMP-2 receptor located on the surface of the target cell. Thereafter, upon the binding, two molecular pathways within the target cell are activated, namely the Smad and Non-Smad pathway. This results in a signalling ca

Cardiovascular tissue engineering and regeneration strive to provide long-term, effective solutions for a growing group of patients in need of myocardial repair, vascular (access) grafts, heart valves, and regeneration of organ microcirculation. In the past two decades, ongoing convergence of disciplines and multidisciplinary collaborations between cardiothoracic surgeons, cardiologists, bioengineers, material scientists, and cell biologists have resulted in better understanding of the problems at hand and novel regenerative approaches. As a side effect, however, the field has become strongly organized and differentiated around topical areas at risk of reinvention of technologies and repetition of approaches across the areas. A better integration of knowledge and technologies from the individual topical areas and regenerative approaches and technologies may pave the way toward faster and more effective treatments to cure the cardiovascular system. This review summarizes the evolution of research and regenerative approaches in the areas of myocardial regeneration, heart valve and vascular tissue engineering, and regeneration of microcirculations; and discusses previous and potential future integration of these individual areas and developed technologies for improved clinical impact. Finally, it provides a perspective on the further integration of research organization, knowledge implementation, and valorization as a contributor to advancing cardiovascular tissue engineering and regenerative medicine. Impact Statement Despite ongoing convergence of disciplines, research in the field of cardiovascular tissue engineering and regeneration is organized and differentiated around focal areas, including myocardial regeneration, heart valve tissue engineering, vascular tissue engineering, and engineering of microcirculations. Cross-area integration of knowledge, supported by a more holistic, overarching research approach, may lead to faster and more effective treatments an

Mimicking endochondral bone formation is a promising strategy for bone regeneration. To become a successful therapy, the cell source is a crucial translational aspect. Typically, autologous cells are used. The use of non-autologous mesenchymal stromal cells (MSCs) represents an interesting alternative. Nevertheless, non-autologous, differentiated MSCs may trigger an undesired immune response, hampering bone regeneration. The aim of this study was to unravel the influence of the immune response on endochondral bone regeneration, when using xenogeneic (human) or allogeneic (Dark Agouti) MSCs. To this end, chondrogenically differentiated MSCs embedded in a collagen carrier were implanted in critical size femoral defects of immunocompetent Brown Norway rats. Control groups were included with syngeneic/autologous (Brown Norway) MSCs or a cell-free carrier. The amount of neo-bone formation was proportional to the degree of host-donor relatedness, as no full bridging of the defect was observed in the xenogeneic group whereas 2/8 and 7/7 bridges occurred in the allogeneic and the syngeneic group, respectively. One week post-implantation, the xenogeneic grafts were invaded by pro-inflammatory macrophages, T lymphocytes, which persisted after 12 weeks, and anti-human antibodies were developed. The immune response toward the allogeneic graft was comparable to the one evoked by the syngeneic implants, aside from an increased production of alloantibodies, which might be responsible for the more heterogeneous bone formation. Our results demonstrate for the first time the feasibility of using non-autologous MSC-derived chondrocytes to elicit endochondral bone regeneration in vivo. Nevertheless, the pronounced immune response and the limited bone formation observed in the xenogeneic group undermine the clinical relevance of this group. On the contrary, although further research on how to achieve robust bone formation with allogeneic cells is needed, they may represent an alterna

Several urological structures, such as the male urethra, have a tubular organization consisting of different layers. However, in severe urethral disease, urologists are limited to replacing solely the epithelial layer. In case of severe hypospadias and urethral stricture disease, the underlying supporting structure (the corpus spongiosum) is either absent or fibrotic, causing suboptimal vascularization and therefore increasing the risk of graft failure. Recapitulating the multilayered architecture of the urethra, including supporting structure with tissue engineering, might minimize urethral graft failure. However, current tissue engineering applications for complex multilayered tubular constructs are limited. We describe a gel casting method to tissue engineer multilayered tubular constructs based on fiber-reinforced cell-laden hydrogels. For this, a multichambered polydimethylsiloxane mold was casted with fiber-reinforced hydrogels containing smooth muscle cells (SMCs) and a coculture of endothelial cells and pericytes. The cell-loaded hydrogels were rolled, with the fiber mesh as guidance, into a tubular construct. In the lumen, urothelial cells were seeded and survived for 2 weeks. In the tubular construct, the cells showed good viability and functionality: endothelial cells formed capillary-like structures supported by pericytes and SMCs expressed elastin. With a graft produced by this technique, supported with subepithelial vascularization, urethral reconstructive surgery can be improved. This approach toward tissue engineering of multilayered tubular structures can also be applied to other multilayered tubular structures found in the human body. Recapitulating the multilayered architecture of tubular structures found in the human body might minimize graft failure. Current tissue engineering applications for complex multilayered tubular constructs are limited. Here we describe a gel casting approach based on fiber-reinforced cell-laden hydrogels. A multicha

Bioengineered grafts have the potential to overcome the limitations of autologous and non-resorbable synthetic vessels as vascular substitutes. However, one of the challenges in creating these living grafts is to induce and maintain multiple cell phenotypes with a biomimetic organization. Our biomimetic grafts with heterotypic design hold promises for functional neovessel regeneration by guiding the layered cellular and tissue organization into a native-like structure. In this study, a perfusable two-compartment bioreactor chamber was designed for the further maturation of these vascular grafts, with a compartmentalized exposure of the graft's luminal and outer layer to cell-specific media. We used the system for a co-culture of endothelial colony forming cells and multipotent mesenchymal stromal cells (MSCs) in the vascular grafts, produced by combining electrospinning and melt electrowriting. It was demonstrated that the targeted cell phenotypes (i.e., endothelial cells (ECs) and vascular smooth muscle cells (vSMCs), respectively) could be induced and maintained during flow perfusion. The confluent luminal layer of ECs showed flow responsiveness, as indicated by the upregulation of COX-2, KLF2, and eNOS, as well as through stress fiber remodeling and cell elongation. In the outer layer, the circumferentially oriented, multi-layered structure of MSCs could be successfully differentiated into vSM-like cells using TGFβ, as indicated by the upregulation of αSMA, calponin, collagen IV, and (tropo)elastin, without affecting the endothelial monolayer. The cellular layers inhibited diffusion between the outer and the inner medium reservoirs. This implies tightly sealed cellular layers in the constructs, resulting in truly separated bioreactor compartments, ensuring the exposure of the inner endothelium and the outer smooth muscle-like layer to cell-specific media. In conclusion, using this system, we successfully induced layer-specific cell differentiation with a nativ