Jaw Bone Invasion of Oral Squamous Cell Carcinoma Is Associated with Osteoclast Count and Expression of Its Regulating Proteins in Patients and Organoids.

AIMS: Oral squamous cell carcinoma (OSCC) frequently invades the jaw. The exact mechanism of bone invasion remains unclear. This study investigates (premature) osteoclasts and the expression of its differentiation regulating proteins RANKL, OPG and RANK in patients with OSCC. METHODS: Resection specimens from OSCC patients were divided into NI group (No Invasion), E group (Erosion) or I group (bone Invasion). Tissue sections were stained with Cathepsin K (osteoclast-counting), RANKL, OPG and RANK. The staining intensity was scored on different regions of the tumor: front, center, back and normal mucosa. Immunohistochemistry and qPCR for RANKL/OPG/RANK were performed on five head and neck squamous cell carcinoma (HNSCC) organoids. RESULTS: The mean number of osteoclasts (I group) and premature osteoclasts (E group) was significantly higher compared to the NI group (p = 0.003, p = 0.036). RANKL expression was significantly higher in the tumor front and tumor center compared to normal mucosa (all groups). In the I group, RANKL and RANK expression was significantly higher in the tumor front compared to the tumor back and there was a trend of higher RANKL expression in the tumor front compared to the E group and NI group. qPCR showed a 20-43 times higher RANKL mRNA expression in three out of five tumor organoids compared to a normal squamous cell organoid line. There was no correlation between protein and mRNA expression in the HNSCC organoids. CONCLUSIONS: These findings suggest that OSCCs induce bone invasion by stimulating osteoclast activation by regulating the production of RANKL and RANK proteins.

Bone Regeneration in a Large Animal Model Featuring a Modular Off-the-Shelf Soft Callus Mimetic.

Implantation of engineered cartilage with soft callus features triggers remodeling to bone tissue via endochondral bone regeneration (EBR). Thus far, EBR has not progressed to the level of large animals on the axis of clinical translation. Herein, the feasibility of EBR is aimed for a critical-sized defect in a large animal model. Chondrogenesis is first induced in goat-derived multipotent mesenchymal stromal cells (MSCs) by fine-tuning the cellular differentiation process. Through a unique devitalization process, two off-the-shelf constructs aimed to recapitulate the different stages of the transient cartilaginous soft callus template in EBR are generated. To evaluate bone regeneration, the materials are implanted in an adapted bilateral iliac crest defect model in goats, featuring a novel titanium star-shaped spacer. After 3 months, the group at the more advanced differentiation stage shows remarkable regenerative capacity, with comparable amounts of bone regeneration as the autograft group. In contrast, while the biomaterial mimicking the earlier stages of chondrogenesis shows improved regeneration compared to the negative controls, this is subpar compared to the more advanced material. Concluding, EBR is attainable in large animals with a soft callus mimetic material that leads to fast conversion into centimeter-scale bone, which prospects successful implementation in the human clinics.

Osteoinductive calcium phosphate with submicron topography as bone graft substitute for maxillary sinus floor augmentation: A translational study.

OBJECTIVES: The aim of this study was the preclinical and clinical evaluation of osteoinductive calcium phosphate with submicron surface topography as a bone graft substitute for maxillary sinus floor augmentation (MSFA). MATERIAL AND METHODS: A preclinical sheep model of MSFA was used to compare a calcium phosphate with submicron needle-shaped topography (BCPN , MagnetOs Granules, Kuros Biosciences BV) to a calcium phosphate with submicron grain-shaped topography (BCPG ) and autologous bone graft (ABG) as controls. Secondly, a 10-patient, prospective, randomized, controlled trial was performed to compare BCPN to ABG in MSFA with two-stage implant placement. RESULTS: The pre-clinical study demonstrated that both BCPN and BCPG were highly biocompatible, supported bony ingrowth with direct bone apposition against the material, and exhibited bone formation as early as 3 weeks post-implantation. However, BCPN demonstrated significantly more bone formation than BCPG at the study endpoint of 12 weeks. Only BCPN reached an equivalent amount of bone formation in the available space and a greater proportion of calcified material (bone + graft material) in the maxillary sinus compared to the "gold standard" ABG after 12 weeks. These results were validated in a small prospective clinical study, in which BCPN was found comparable to ABG in implant stability, bone height, new bone formation in trephine core biopsies, and overall clinical outcome. CONCLUSION: This translational work demonstrates that osteoinductive calcium phosphates are promising bone graft substitutes for MSFA, whereas their bone-forming potential depends on the design of their surface features. Netherlands Trial Register, NL6436.

Evaluating material-driven regeneration in a tissue engineered human in vitro bone defect model.

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.

Biofabricating the vascular tree in engineered bone tissue.

The development of tissue engineering strategies for treatment of large bone defects has become increasingly relevant, given the growing demand for bone substitutes. Native bone is composed of a dense vascular network necessary for the regulation of bone development, regeneration and homeostasis. A major obstacle in fabricating living, clinically relevant-sized bone mimics (1-10 cm3) is the limited supply of nutrients, including oxygen to the core of the construct. Therefore, strategies to support vascularization are pivotal for the development of tissue engineered bone constructs. Creating a functional bone construct integrated with a vascular network, capable of delivering the necessary nutrients for optimal tissue development is imperative for translation into the clinics. The vascular system is composed of a complex network that runs throughout the body in a tree-like hierarchical branching fashion. A significant challenge for tissue engineering approaches lies in mimicking the intricate, multi-scale structures consisting of larger vessels (macro-vessels) which interconnect with multiple sprouting vessels (microvessels) in a closed network. The advent of biofabrication has enabled complex, out of plane channels to be generated and has laid the groundwork for the creation of multi-scale vasculature in recent years. This review highlights the key state-of-the-art achievements for the development of vascular networks of varying scales in the field of biofabrication with a particular focus for its application in developing a functional tissue engineered bone construct. STATEMENT OF SIGNIFICANCE: There is a growing need for bone substitutes to overcome the limited supply of patient-derived bone. Bone tissue engineering aims to overcome this by combining stem cells with scaffolds to restore missing bone. The current bottleneck in upscaling is the lack of an integrated vascular network, required for the delivery of nutrients to cells. 3D bioprinting techniques has enabled the creation of complex hollow structures of varying dimensions that resemble native blood vessels. The convergence of multiple materials, cell types and fabrication approaches, opens the possibility of developing clinically-relevant sized vascularized bone constructs. This review provides an up-to-date insight of the technologies currently available for the generation of complex vascular networks, with a focus on their application in bone tissue engineering.

An In Vitro Model to Test the Influence of Immune Cell Secretome on Mesenchymal Stromal Cell Osteogenic Differentiation.

Immune cells and their soluble factors have an important role in the bone healing process. Modulation of the immune response, therefore, offers a potential strategy to enhance bone formation. To investigate the influence of the immune system on osteogenesis, we developed and applied an in vitro model that incorporates both innate and adaptive immune cells. Human peripheral blood mononuclear cells (PBMCs) were isolated and cultured for 24 h and subsequently stimulated with immune-modulatory agents; C-class CpG oligodeoxynucleotide (CpG ODN C), polyinosinic acid-polycytidylic acid [Poly(I:C)], and lipopolysaccharide (LPS); all pathogen recognition receptor agonists, that target Toll-like receptors (TLRs) 9, 3, and 4, respectively. The conditioned medium (CM) obtained from PBMCs after 24 h was used to investigate its effects on the metabolic activity and osteogenic differentiation capacity of human bone marrow-derived mesenchymal stromal cells (MSCs). Conditioned media from unstimulated PBMCs did not affect the metabolic activity and osteogenic differentiation capacity of MSCs. The CM from CpG ODN C and LPS-stimulated PBMCs increased alkaline phosphatase activity (ALP) of MSCs by approximately threefold as compared with the unstimulated control, whereas Poly(I:C) CM did not enhance ALP activity of MSCs. Moreover, direct stimulation of MSCs with the immune-modulatory stimuli did not result in increased ALP. These results demonstrate that soluble factors present in CM from PBMCs stimulated with immune-modulatory factors enhance osteogenesis of MSCs. This in vitro model can serve as a tool in screening immune-modulatory stimulants from a broad variety of immune cells for (indirect) effects on osteogenesis and also to identify soluble factors from multiple immune cell types that may modulate bone healing.

Cardiovascular Tissue Engineering and Regeneration: A Plead for Further Knowledge Convergence.

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 and prevent the reinvention of technologies across the areas. Herein, we review the evolution of research and technologies in the individual focal areas and discuss how to enhance integration of-and collaboration between-these areas for improved scientific and clinical impact.

Acceleration of Bone Regeneration Induced by a Soft-Callus Mimetic Material.

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.

Development and Characterization of Gelatin-Norbornene Bioink to Understand the Interplay between Physical Architecture and Micro-Capillary Formation in Biofabricated Vascularized Constructs.

The principle challenge for engineering viable, cell-laden hydrogel constructs of clinically-relevant size, is rapid vascularization, in order to moderate the finite capacity of passive nutrient diffusion. A multiscale vascular approach, with large open channels and bulk microcapillaries may be an admissible approach to accelerate this process, promoting overall pre-vascularization for long-term viability of constructs. However, the limited availability of bioinks that possess suitable characteristics that support both fabrication of complex architectures and formation of microcapillaries, remains a barrier to advancement in this space. In this study, gelatin-norbornene (Gel-NOR) is investigated as a vascular bioink with tailorable physico-mechanical properties, which promoted the self-assembly of human stromal and endothelial cells into microcapillaries, as well as being compatible with extrusion and lithography-based biofabrication modalities. Gel-NOR constructs containing self-assembled microcapillaries are successfully biofabricated with varying physical architecture (fiber diameter, spacing, and orientation). Both channel sizes and cell types affect the overall structural changes of the printed constructs, where cross-signaling between both human stromal and endothelial cells may be responsible for the reduction in open channel lumen observed over time. Overall, this work highlights an exciting three-way interplay between bioink formulation, construct design, and cell-mediated response that can be exploited towards engineering vascular tissues.

High-resolution lithographic biofabrication of hydrogels with complex microchannels from low-temperature-soluble gelatin bioresins.

Biofabrication via light-based 3D printing offers superior resolution and ability to generate free-form architectures, compared to conventional extrusion technologies. While extensive efforts in the design of new hydrogel bioinks lead to major advances in extrusion methods, the accessibility of lithographic bioprinting is still hampered by a limited choice of cell-friendly resins. Herein, we report the development of a novel set of photoresponsive bioresins derived from ichthyic-origin gelatin, designed to print high-resolution hydrogel constructs with embedded convoluted networks of vessel-mimetic channels. Unlike mammalian gelatins, these materials display thermal stability as pre-hydrogel solutions at room temperature, ideal for bioprinting on any easily-accessible lithographic printer. Norbornene- and methacryloyl-modification of the gelatin backbone, combined with a ruthenium-based visible light photoinitiator and new coccine as a cytocompatible photoabsorber, allowed to print structures resolving single-pixel features (∼50 â€‹µm) with high shape fidelity, even when using low stiffness gels, ideal for cell encapsulation (1-2 â€‹kPa). Moreover, aqueous two-phase emulsion bioresins allowed to modulate the permeability of the printed hydrogel bulk. Bioprinted mesenchymal stromal cells displayed high functionality over a month of culture, and underwent multi-lineage differentiation while colonizing the bioresin bulk with tissue-specific neo-deposited extracellular matrix. Importantly, printed hydrogels embedding complex channels with perfusable lumen (diameter <200 â€‹µm) were obtained, replicating anatomical 3D networks with out-of-plane branches (i.e. brain vessels) that cannot otherwise be reproduced by extrusion bioprinting. This versatile bioresin platform opens new avenues for the widespread adoption of lithographic biofabrication, and for bioprinting complex channel-laden constructs with envisioned applications in regenerative medicine and hydrogel-based organ-on-a-chip devices.

Impact of Endotoxins in Gelatine Hydrogels on Chondrogenic Differentiation and Inflammatory Cytokine Secretion In Vitro.

Gelatine methacryloyl (GelMA) hydrogels are widely used in studies aimed at cartilage regeneration. However, the endotoxin content of commercially available GelMAs and gelatines used in these studies is often overlooked, even though endotoxins may influence several cellular functions. Moreover, regulations for clinical use of biomaterials dictate a stringent endotoxin limit. We determined the endotoxin level of five different GelMAs and evaluated the effect on the chondrogenic differentiation of equine mesenchymal stromal cells (MSCs). Cartilage-like matrix production was evaluated by biochemical assays and immunohistochemistry. Furthermore, equine peripheral blood mononuclear cells (PBMCs) were cultured on the hydrogels for 24 h, followed by the assessment of tumour necrosis factor (TNF)-α and C-C motif chemokine ligand (CCL)2 as inflammatory markers. The GelMAs were found to have widely varying endotoxin content (two with >1000 EU/mL and three with <10 EU/mL), however, this was not a critical factor determining in vitro cartilage-like matrix production of embedded MSCs. PBMCs did produce significantly higher TNF-α and CCL2 in response to the GelMA with the highest endotoxin level compared to the other GelMAs. Although limited effects on chondrogenic differentiation were found in this study, caution with the use of commercial hydrogels is warranted in the translation from in vitro to in vivo studies because of regulatory constraints and potential inflammatory effects of the content of these hydrogels.

Endochondral Bone Regeneration by Non-autologous Mesenchymal Stem Cells.

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 alternative to autologous transplantation.

Gel Casting as an Approach for Tissue Engineering of Multilayered Tubular Structures.

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. Impact Statement 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 multichambered polydimethylsiloxane mold was casted with cell-loaded, fiber-reinforced hydrogels, with the fiber mesh as guidance, into a tubular construct. A graft produced by this technique can improve reconstructive surgery by providing subepithelial vascularization and thereby can reduce graft failure.

Bio-ink development for three-dimensional bioprinting of hetero-cellular cartilage constructs.

Bioprinting is a promising tool to fabricate organized cartilage. This study aimed to investigate the printability of gelatin-methacryloyl/gellan gum (gelMA/gellan) hydrogels with and without methacrylated hyaluronic acid (HAMA), and to explore (zone-specific) chondrogenesis of chondrocytes, articular cartilage progenitor cells (ACPCs), and multipotent mesenchymal stromal cells (MSCs) embedded in these bio-inks.The incorporating of HAMA in gelMA/gellan bio-ink increased filament stability, as measured using a filament collapse assay, but did not influence (zone-specific) chondrogenesis of any of the cell types. Highest chondrogenic potential was observed for MSCs, followed by ACPCs, which displayed relatively high proteoglycan IV mRNA levels. Therefore, two-zone constructs were printed with gelMA/gellan/HAMA containing ACPCs in the superficial region and MSCs in the middle/deep region. Chondrogenic differentiation was confirmed, however, printing influence cellular differentiation.ACPC- and MSC-laden gelMA/gellan/HAMA hydrogels are of interest for the fabrication of cartilage constructs. Nevertheless, this study underscores the need for careful evaluation of the effects of printing on cellular differentiation.

Contrast enhanced computed tomography for real-time quantification of glycosaminoglycans in cartilage tissue engineered constructs.

Tissue engineering and regenerative medicine are two therapeutic strategies to treat, and to potentially cure, diseases affecting cartilaginous tissues, such as osteoarthritis and cartilage defects. Insights into the processes occurring during regeneration are essential to steer and inform development of the envisaged regenerative strategy, however tools are needed for longitudinal and quantitative monitoring of cartilage matrix components. In this study, we introduce a contrast-enhanced computed tomography (CECT)-based method using a cationic iodinated contrast agent (CA4+) for longitudinal quantification of glycosaminoglycans (GAG) in cartilage-engineered constructs. CA4+ concentration and scanning protocols were first optimized to ensure no cytotoxicity and a facile procedure with minimal radiation dose. Chondrocyte and mesenchymal stem cell pellets, containing different GAG content were generated and exposed to CA4+. The CA4+ content in the pellets, as determined by micro computed tomography, was plotted against GAG content, as measured by 1,9-dimethylmethylene blue analysis, and showed a high linear correlation. The established equation was used for longitudinal measurements of GAG content over 28 days of pellet culture. Importantly, this method did not adversely affect cell viability or chondrogenesis. Additionally, the CA4+ distribution accurately matched safranin-O staining on histological sections. Hence, we show proof-of-concept for the application of CECT, utilizing a positively charged contrast agent, for longitudinal and quantitative imaging of GAG distribution in cartilage tissue-engineered constructs. STATEMENT OF SIGNIFICANCE: Tissue engineering and regenerative medicine are promising therapeutic strategies for different joint pathologies such as cartilage defects or osteoarthritis. Currently, in vitro assessment on the quality and composition of the engineered cartilage mainly relies on destructive methods. Therefore, there is a need for the development of techniques that allow for longitudinal and quantitative imaging and monitoring of cartilage-engineered constructs. This work harnesses the electrostatic interactions between the negatively-charged glycosaminoglycans (GAGs) and a positively-charged contrast agent for longitudinal and non-destructive quantification of GAGs, providing valuable insight on GAG development and distribution in cartilage engineered constructs. Such technique can advance the development of regenerative strategies, not only by allowing continuous monitoring but also by serving as a pre-implantation screening tool.

Layer-specific cell differentiation in bi-layered vascular grafts under flow perfusion.

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 TGFß, 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 native-like cell organization. This co-culture system enables the creation of biomimetic neovessels, and as such can be exploited to investigate and improve bioengineered vascular grafts.

A Versatile Biosynthetic Hydrogel Platform for Engineering of Tissue Analogues.

For creating functional tissue analogues in tissue engineering, stem cells require very specific 3D microenvironments to thrive and mature. Demanding (stem) cell types that are used nowadays can find such an environment in a heterogeneous protein mixture with the trade name Matrigel. Several variations of synthetic hydrogel platforms composed of poly(ethylene glycol) (PEG), which are spiked with peptides, have been recently developed and shown equivalence to Matrigel for stem cell differentiation. Here a clinically relevant hydrogel platform, based on PEG and gelatin, which even outperforms Matrigel when targeting 3D prevascularized bone and liver organoid tissue engineering models is presented. The hybrid hydrogel with natural and synthetic components stimulates efficient cell differentiation, superior to Matrigel models. Furthermore, the strength of this hydrogel lies in the option to covalently incorporate unmodified proteins. These results demonstrate how a hybrid hydrogel platform with intermediate biological complexity, when compared to existing biological materials and synthetic PEG-peptide approaches, can efficiently support tissue development from human primary cells.

Visible Light Cross-Linking of Gelatin Hydrogels Offers an Enhanced Cell Microenvironment with Improved Light Penetration Depth.

In this study, the cyto-compatibility and cellular functionality of cell-laden gelatin-methacryloyl (Gel-MA) hydrogels fabricated using a set of photo-initiators which absorb in 400-450 nm of the visible light range are investigated. Gel-MA hydrogels cross-linked using ruthenium (Ru) and sodium persulfate (SPS), are characterized to have comparable physico-mechanical properties as Gel-MA gels photo-polymerized using more conventionally adopted photo-initiators, such as 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one (Irgacure 2959) and lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP). It is demonstrated that the Ru/SPS system has a less adverse effect on the viability and metabolic activity of human articular chondrocytes encapsulated in Gel-MA hydrogels for up to 35 days. Furthermore, cell-laden constructs cross-linked using the Ru/SPS system have significantly higher glycosaminoglycan content and re-differentiation capacity as compared to cells encapsulated using I2959 and LAP. Moreover, the Ru/SPS system offers significantly greater light penetration depth as compared to the I2959 system, allowing thick (10 mm) Gel-MA hydrogels to be fabricated with homogenous cross-linking density throughout the construct. These results demonstrate the considerable advantages of the Ru/SPS system over traditional UV polymerizing systems in terms of clinical relevance and practicability for applications such as cell encapsulation, biofabrication, and in situ cross-linking of injectable hydrogels.

Effect of donor variation on osteogenesis and vasculogenesis in hydrogel cocultures.

To introduce a functional vascular network into tissue-engineered bone equivalents, human endothelial colony forming cells (ECFCs) and multipotent mesenchymal stromal cells (MSCs) can be cocultured. Here, we studied the impact of donor variation of human bone marrow-derived MSCs and cord blood-derived ECFCs on vasculogenesis and osteogenesis using a 3D in vitro coculture model. Further, to make the step towards cocultures consisting of cells derived from a single donor, we tested how induced pluripotent stem cell (iPSC)-derived human endothelial cells (iECs) performed in coculture models. Cocultures with varying combinations of human donors of MSCs, ECFCs, or iECs were prepared in Matrigel. The constructs were cultured in an osteogenic differentiation medium. Following a 10-day culture period, the length of the prevascular structures and osteogenic differentiation were evaluated for up to 21 days of culture. The particular combination of MSC and ECFC donors influenced the vasculogenic properties significantly and induced variation in osteogenic potential. In addition, the use of iECs in the cocultures resulted in prevascular structure formation in osteogenically differentiated constructs. Together, these results showed that close attention to the source of primary cells, such as ECFCs and MSCs, is critical to address variability in vasculogenic and osteogenic potential. The 3D coculture model appeared to successfully generate prevascularized constructs and were sufficient in exceeding the ~200 µm diffusion limit. In addition, iPSC-derived cell lineages may decrease variability by providing a larger and potentially more uniform source of cells for future preclinical and clinical applications.