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Compositional changes in the chemical makeup of hydrogels offer a powerful strategy for fine tuning of mechanical properties, enabling specific targeting for different applications. The chemical versatility exhibited by the tunable system introduced here can be leveraged to address a broad range of characteristics across the field of tissue engineering-from blood vessels to cartilage, for example-which demands materials with very different mechanical profiles. Furthermore, we rely exclusively on dynamic, non-covalent cross-linking to provide opportunities for 3D printing and injectability. This work describes a highly tunable system based on hydrogen bonding and ionic interactions. Single network hydrogels were made by exploiting various acrylic monomers including N-acryloyl glycinamide (NAGA) and acrylic acid (AAc). Additionally, hybrid hydrogels were explored by combining these acrylic networks with an ionically cross-linked alginate network. By combining orthogonal cross-linking strategies and altering the ratio between different components in these hybrid gels, a broad range of mechanical properties is demonstrated. The characteristics were extensively investigated using tensile testing, compression testing, and rheological measurements. The final scaffolds were also shown to be non-cytotoxic in preliminary cell viability studies for human dermal fibroblasts.

Three-dimensional cell culture in engineered hydro gels is increasingly used in tissue engineering and regenerative medicine. The transfer of nutrients, gases, and waste materials through these hydrogels is of utmost importance for cell viability and response, yet the translation of diffusion coefficients into practical guidelines is not well established. Here, we combined mathematical modeling, fluorescent recovery after photobleaching, and hydrogel diffusion experiments on cell culture inserts to provide a multiscale practical approach for diffusion. We observed a dampening effect of the hydrogel that slowed the response to concentration changes and the creation of a diffusion gradient in the hydrogel by media refreshment. Our designed model combined with measurements provides a practical point of reference for diffusion coefficients in real-world culture conditions, enabling more informed choices on hydrogel culture conditions. This model can be improved in the future to simulate more complicated intrinsic hydrogel properties and study the effects of secondary interactions on the diffusion of analytes through the hydrogel.

Cartilage tissue presents low self-repair capability and lesions often undergo irreversible progression. Structures obtained by tissue engineering, such as those based in extrusion bioprinting of constructs loaded with stem cell spheroids may offer valuable alternatives for research and therapeutic purposes. Human mesenchymal stromal cell (hMSC) spheroids can be chondrogenically differentiated faster and more efficiently than single cells. This approach allows obtaining larger tissues in a rapid, controlled and reproducible way. However, it is challenging to control tissue architecture, construct stability, and cell viability during maturation. Herein, this work reports a reproducible bioprinting process followed by a successful post-bioprinting chondrogenic differentiation procedure using large quantities of hMSC spheroids encapsulated in a xanthan gum-alginate hydrogel. Multi-layered constructs are bioprinted, ionically crosslinked, and post chondrogenically differentiated for 28 days. The expression of glycosaminoglycan, collagen II and IV are observed. After 56 days in culture, the bioprinted constructs are still stable and show satisfactory cell metabolic activity with profuse extracellular matrix production. These results show a promising procedure to obtain 3D models for cartilage research and ultimately, an in vitro proof-of-concept of their potential use as stable chondral tissue implants.

Traditional synthetic covalent hydrogels lack the native tissue dynamics and hierarchical fibrous structure found in the extracellular matrix (ECM). These dynamics and fibrous nanostructures are imperative in obtaining the correct cell/material interactions. Consequently, the challenge to engineer functional dynamics in a fibrous hydrogel and recapitulate native ECM properties remains a bottle-neck to biomimetic hydrogel environments. Here, the molecular tuning of a supramolecular benzene-1,3,5-tricarboxamide (BTA) hydrogelator via simple modulation of hydrophobic substituents is reported. This tuning results in fibrous hydrogels with accessible viscoelasticity over 5 orders of magnitude, while maintaining a constant equilibrium storage modulus. BTA hydrogelators are created with systematic variations in the number of hydrophobic carbon atoms, and this is observed to control the viscoelasticity and stress-relaxation timescales in a logarithmic fashion. Some of these BTA hydrogels are shear-thinning, self-healing, extrudable, and injectable, and can be 3D printed into multiple layers. These hydrogels show high cell viability for chondrocytes and human mesenchymal stem cells, establishing their use in tissue engineering applications. This simple molecular tuning by changing hydrophobicity (with just a few carbon atoms) provides precise control over the viscoelasticity and 3D printability in fibrillar hydrogels and can be ported onto other 1D self-assembling structures. The molecular control and design of hydrogel network dynamics can push the field of supramolecular chemistry toward the design of new ECM-mimicking hydrogelators for numerous cell-culture and tissue-engineering applications and give access toward highly biomimetic bioinks for bioprinting.

Tissue-engineered constructs are currently limited by the lack of vascularization necessary for the survival and integration of implanted tissues. Hydrogen sulfide (H2S), an endogenous signaling gas (gasotransmitter), has been recently reported as a promising alternative to growth factors to mediate and promote angiogenesis in low concentrations. Yet, sustained delivery of H2S remains a challenge. Herein, we have developed angiogenic scaffolds by covalent attachment of an H2S donor to a polycaprolactone (PCL) electrospun scaffold. These scaffolds were engineered to include azide functional groups (on 1, 5, or 10% of the PCL end groups) and were modified using a straightforward click reaction with an alkyne-functionalized N-thiocarboxyanhydride (alkynyl-NTA). This created H2S-releasing scaffolds that rely on NTA ring-opening in water followed by conversion of released carbonyl sulfide into H2S. These functionalized scaffolds showed dose-dependent release of H2S based on the amount of NTA functionality within the scaffold. The NTA-functionalized fibrous scaffolds supported human umbilical vein endothelial cell (HUVEC) proliferation, formed more confluent endothelial monolayers, and facilitated the formation of tight cell-cell junctions to a greater extent than unfunctionalized scaffolds. Covalent conjugation of H2S donors to scaffolds not only promotes HUVEC proliferation in vitro, but also increases neovascularization in ovo, as observed in the chick chorioallantoic membrane assay. NTA-functionalized scaffolds provide localized control over vascularization through the sustained delivery of a powerful endogenous angiogenic agent, which should be further explored to promote angiogenesis in tissue engineering. Published version

Synthetically designed biomaterials strive to recapitulate and mimic the complex environment of natural systems. Using natural materials as a guide, the ability to create high-performance biomaterials that control cell fate, and support the next generation of cell- and tissue-based therapeutics, is starting to emerge. Supramolecular chemistry takes inspiration from the wealth of noncovalent interactions found in natural materials that are inherently complex, and using the skills of synthetic and polymer chemistry, recreates simple systems to imitate their features. Within the past decade, supramolecular biomaterials have shown utility in tissue engineering and the progress predicts a bright future. On this 30th anniversary of the Netherlands Biomaterials and Tissue Engineering society, we briefly recount the state of supramolecular biomaterials in the Dutch academic and industrial research and development context. This review provides the background, recent advances, industrial successes and challenges, as well as future directions of the field, as we see it. Throughout this work, we notice the intricate interplay between simplicity and complexity in creating more advanced solutions. We hope that the interplay and juxtaposition between these two forces can propel the field forward. Impact statement Supramolecular biomaterials based on noncovalent interactions hold the ability to rebuild some of the complexity of natural biomaterials in synthetic systems. While still in its infancy, the field is currently vigorously moving from fundamental experiments toward applications and products in the tissue engineering and regenerative medicine arena. Herein, we review the current state of the field in the Netherlands. While supramolecular biomaterials have incredible potential, systematic studies, balancing complexity and simplicity, efficient translation, and enhanced performance are all required for success of these strategies. As we move the field toward commercial solutions for clinical patients, we must also pay homage and remember the fundamental studies that allow these jumps in innovation.

Digital light processing (DLP) is an accurate and fast additive manufacturing technique to produce a variety of products, from patient-customized biomedical implants to consumer goods. However, DLP's use in tissue engineering has been hampered due to a lack of biodegradable resin development. Herein, a library of biodegradable poly(esters) capped with urethane acrylate (with variations in molecular weight) is investigated as the basis for DLP printable resins for tissue engineering. The synthesized oligomers show good printability and are capable of creating complex structures with mechanical moduli close to those of medium-soft tissues (1-3 MPa). While fabricated films from different molecular weight resins show few differences in surface topology, wettability, and protein adsorption, the adhesion and metabolic activity of NCTC clone 929 (L929) cells and human dermal fibroblasts (HDFs) are significantly different. Resins from higher molecular weight oligomers provide greater cell adhesion and metabolic activity. Furthermore, these materials show compatibility in a subcutaneous in vivo pig model. These customizable, biodegradable, and biocompatible resins show the importance of molecular tuning and open up new possibilities for the creation of biocompatible constructs for tissue engineering.

Melt extrusion-based additive manufacturing (AM) is often used to fabricate scaffolds for osteochondral (OC) regeneration. However, there are two shortcomings associated with this scaffold manufacturing technique for engineering of tissue interfaces: (a) most polymers used in the processing are bioinert, and (b) AM scaffolds often contain discrete (material) gradients accompanied with mechanically weak interfaces. The inability to mimic the gradual transition from cartilage to bone in OC tissue leads to poor scaffold performance and even failure. We hypothesized that introducing peptide gradients on the surface could gradually guide human mesenchymal stromal cell (hMSC) differentiation, from a chondrogenic towards on osteogenic phenotype. To work towards this goal, we initially manufactured poly(epsilon-caprolactone)-azide (PCLA) and PCL-maleimide (PCLM) scaffolds. The surface exposed click-type functional groups, with a surface concentration in the 10(2)pmol cm(-2) regime, were used to introduce bone morphogenic protein-2 or transforming growth factor-beta binding peptide sequences to drive hMSC differentiation towards osteogenic or chondrogenic phenotypes, respectively. After 3 weeks of culture in chondrogenic medium, we observed differentiation towards hypertrophic chondrogenic phenotypes with expression of characteristic markers such as collagen X. In osteogenic medium, we observed the upregulation of mineralization markers. In basic media, the chondro-peptide displayed a minor effect on chondrogenesis, whereas the osteo-peptide did not affect osteogenesis. In a subcutaneous rat model, we observed a minimal foreign body response to the constructs, indicating biocompatibility. As proof-of-concept, we finally used a novel AM technology to showcase its potential to create continuous polymer gradients (PCLA and PCLM) across scaffolds. These scaffolds did not display delamination and were mechanically stronger compared to discrete gradient scaffolds. Due to the versatility of the orthogonal chemistry applied, this approach provides a general strategy for the field; we could anchor other tissue specific cues on the clickable groups, making these gradient scaffolds interesting for multiple interfacial tissue applications. This work was supported by the European Research Council starting grant 'Cell Hybridge' for financial support under the Horizon2020 framework program (Grant #637308). The authors would like to acknowledge the Chemelot Innovation and Learning Labs (CHILL) located at Brightlands Chemelot Campus in Geleen (The Netherlands) for allowing us to use their GPC equipment.

Supramolecular materials based on the self-assembly of benzene-1,3,5-tricarboxamide (BTA) offer an approach to mimic fibrous self-assembled proteins found in numerous natural systems. Yet, synthetic methods to rapidly build complexity, scalability, and multifunctionality into BTA-based materials are needed. The diversity of BTA structures is often hampered by the limited flexibility of existing desymmetrization routes and the purification of multifunctional BTAs. To alleviate this bottleneck, we have developed a desymmetrization method based on activated ester coupling of a symmetric synthon. We created a small library of activated ester synthons and found that a pentafluorophenol benzene triester (BTE) enabled effective desymmetrization and creation of multifunctional BTAs in good yield with high reaction fidelity. This new methodology enabled the rapid synthesis of a small library of BTA monomers with hydrophobic and/or orthogonal reactive handles and could be extended to create polymeric BTA hydrogelators. These BTA hydrogelators self-assembled in water to create fiber and fibrous sheet-like structures as observed by cryo-TEM, and the identity of the BTA conjugated can tune the mechanical properties of the hydrogel. These hydrogelators display high cytocompatibility for chondrocytes, indicating potential for the use of these systems in 3D cell culture and tissue engineering applications. This newly developed synthetic strategy facilitates the simple and rapid creation of chemically diverse BTA supramolecular polymers, and the newly developed and scalable hydrogels can unlock exploration of BTA based materials in a wider variety of tissue engineering applications.

There is increasing evidence that cells cultured in three-dimensional (3D) settings have superior performance compared to their traditional counterparts in monolayers. This has been attributed to cell-cell and cell-extracellular matrix interactions that more closely resemble the in vivo tissue architecture. The rapid adoption of 3D cell culture systems as experimental tools for diverse applications has not always been matched by an improved understanding of cell behavior in different 3D environments. Here, we studied human mesenchymal stem/stromal cells (hMSCs) as scaffold-free self-assembled aggregates of low and high cell number and compared them to cell-laden alginate hydrogels with and without arginine-glycine-aspartic acid peptides. We observed a significant decrease in the size of cell-only aggregates over 14 days in culture compared to the cells encapsulated in alginate hydrogels. Alginate hydrogels had persistently more living cells for a longer period (14 days) in culture as measured by total DNA content. Proliferation studies revealed that a weeklong culture of hMSCs in 3D culture, whether as aggregates or cell-laden alginate hydrogels, reduced their proliferation over time. Cell cycle analysis found no significant differences between days 1 and 7 for the different culture systems. The findings of this study improve our understanding of how aggregate cultures differ with or without a hydrogel carrier, and whether aggregation itself is important when it comes to the 3D culture of hMSCs.

Shape morphing materials, especially those fabricated by 4D printing, are gaining much attention due to their versatility of actuation and capability of being programmed in advance. These materials become particularly interesting for biomedical applications where implant materials could be remotely actuated, exerting a force on the surrounding tissues and cells. However, applications in this field have been restricted due to the biocompatibility of the materials and the character of the required stimuli, generally not compatible with physiological environments. Magnetic nanoparticles (MNPs) represent a great opportunity to this end; however, the actuation results in a uniform movement toward the magnet that requires anchoring of the object. Here, for the first time, the application of anisotropic Fe3O4 MNPs is described, and synthesized by a novel and easy route, that can be aligned on pre-defined patterns within objects printed by digital light processing, resulting in materials that can be actuated remotely (4D printing). These nanoparticles (178 nm x 55 nm), show good biocompatibility when directly seeded on top of human mesenchymal stem cells, despite being uptaken. Most importantly, the alignment of the MNPs can tune the movement of fabricated nanocomposite materials, resulting in complex movements of attraction or repulsion depending on the direction of the applied magnetic field. T.K., and S.C.-E. contributed equally to this work. This work was supported financially by the Province of Limburg and the Brightlands Materials Center. The authors were also grateful to the research program Innovation Fund Chemistry, which was partly financed by the Netherlands Organization for Scientific Research (NWO) under TA grant agreement 731.016.202 ("DynAM"). The authors acknowledge the Texas A&M Health Science Center College of Medicine Institute for Regenerative Medicine at Scott & White who isolated and provided the cells through a grant from NCRR of the NIH (Grant #P40RR017447).

Synthetic biodegradable materials are commonly used to create constructs for medical devices and tissue engineered constructs. However, many of the homopolymers used in FDA approved devices such as poly(ε-caprolactone) (PCL), poly(lactic acid), or poly(carbonates) lack biogically relevant functional groups to steer biological responses in a controlled fashion. Commonly, an interconversion of the end groups is required to insert addressable moieties for the attachment of biologically active groups. In this study, the activation of the hydroxyl groups of a low molecular weight PCL-diol to the corresponding p-toluene sulfonate ester using p-toluenesulfonyl chloride was performed in both dichloromethane (DCM) and dimethylformamide (DMF). To our initial surprise, we only yielded the chlorinated product in DMF, while in DCM the tosylate ester was obtained. In a small series of reactions, we studied the solvent dependent switchability between tosylation and chlorination on PCL. We concluded that in polar aprotic solvents (DMF and dimethylsulfoxide), we rapidly and efficiently converted the hydroxyl into the chloride group, whereas in inert solvents (DCM and chloroform) we yielded the tosylated product. The data suggested that solvation effects of the polar aprotic solvents led to a Sn2 reaction of the tosyl group by the chloride. Furthermore, we utilized a polyethylene glycol (PEG) polymer to show translatability of the chlorination reaction to other (biomedical) polymers. This work highlights a new reaction pathway during the tosylation of a polymer end group, and presents a new useful strategy to insert clickable groups on synthetic polymers that are only soluble in polar aprotic solvents. We thank the CORE IMS lab, Division of Imaging Mass Spectrometry at The Maastricht Multimodal Molecular Imaging Institute (M4i), for the ToF-SIMS experiments. We are also grateful to the European Research Council starting grant “Cell Hybridge” for financial support under the Horizon2020 framework program (Grant #637308).

Additive manufacturing and injection are essential tools in the rapidly developing field of personalized medicine and are particularly promising for applications in regenerative medicine. One of the biggest challenges in this vibrant research domain remains the processing of complex formulations with robust mechanical properties. Mimicking the native extracellular matrix associated with many tissues requires materials that have high degrees of functionality for performing the complex array of functions within the cellular environment. Furthermore, native tissues often possess outstanding mechanical properties, particularly in connective tissues. These exceptional mechanics are a challenge to emulate in their own right, especially considering the accompanying demands for additional functionality. Double-network hydrogels have emerged as strong candidates for tissue engineering because of the impressive mechanics and versatility in terms of chemical makeup. Combining advances in processing (i.e., additive manufacturing and injection) with dual-network hydrogel formulations has led to an impressive collection of results, making great strides toward systems capable of addressing the demanding environment surrounding tissues while being amenable to personalized fabrication techniques. This review provides a brief summary of the most contemporary trends collected from the literature describing dual-network hydrogels being demonstrated in additive manufacturing and injectable applications.

Few synthetic hydrogels can mimic both the viscoelasticity and supramolecular fibrous structure found in the naturally occurring extracellular matrix (ECM). Furthermore, the ability to control the viscoelasticity of fibrous supramolecular hydrogel networks to influence cell culture remains a challenge. Here, we show that modular mixing of supramolecular architectures with slow and fast exchange dynamics can provide a suitable environment for multiple cell types and influence cellular aggregation. We employed modular mixing of two synthetic benzene-1,3,5-tricarboxamide (BTA) architectures: a small molecule water-soluble BTA with slow exchange dynamics and a telechelic polymeric BTA-PEG-BTA with fast exchange dynamics. Copolymerisation of these two supramolecular architectures was observed, and all tested formulations formed stable hydrogels in water and cell culture media. We found that rational tuning of mechanical and viscoelastic properties is possible by mixing BTA with BTA-PEG-BTA. These hydrogels showed high viability for both chondrocyte (ATDC5) and human dermal fibroblast (HDF) encapsulation (>80%) and supported neuronal outgrowth (PC12 and dorsal root ganglion, DRG). Furthermore, ATDC5s and human mesenchymal stem cells (hMSCs) were able to form spheroids within these viscoelastic hydrogels, with control over cell aggregation modulated by the dynamic properties of the material. Overall, this study shows that modular mixing of supramolecular architectures enables tunable fibrous hydrogels, creating a biomimetic environment for cell encapsulation. These materials are suitable for the formation and culture of spheroids in 3D, critical for upscaling tissue engineering approaches towards cell densities relevant for physiological tissues.

Pluripotent stem cell-derived kidney organoids offer a promising solution to renal failure, yet current organoid protocols often lead to off-target cells and phenotypic alterations, preventing maturity. Here, various dynamic hydrogel architectures are created, conferring a controlled and biomimetic environment for organoid encapsulation. How hydrogel stiffness and stress relaxation affect renal phenotype and undesired fibrotic markers are investigated. The authors observe that stiff hydrogel encapsulation leads to an absence of certain renal cell types and signs of an epithelial-mesenchymal transition (EMT), whereas encapsulation in soft, stress-relaxing hydrogels leads to all major renal segments, fewer fibrosis or EMT associated proteins, apical proximal tubule polarization, and primary cilia formation, representing a significant improvement over current approaches to culture kidney organoids. The findings show that engineering hydrogel mechanics and dynamics have a decided benefit for organoid culture. These structure-property-function relationships can enable the rational design of materials, bringing us closer to functional engraftments and disease-modeling applications.

Biomaterial matrices must permit tissue growth and maturation for the success of tissue regeneration strategies. Naturally, this accommodation is achieved via the dynamic remodeling of a cell's extracellular matrix (ECM). Synthetically, hydrolytic or enzymatic degradation are often engineered into materials for this purpose. More recently, supramolecular interactions have been used to provide a biomimetic and tunable mechanism to facilitate tissue formation via their dynamic and reversible non-covalent interactions. By engineering the mechanical and bioactive properties of a material, supramolecular chemists are able to design permissivity into the construct and facilitate tissue integration in-vivo . Furthermore, via the reversibility of non-covalent interactions, injectability and responsiveness can be designed for enhanced delivery and spatio-temporal control. In this review, we delineate the basic considerations needed when designing permissive supramolecular hydrogels for tissue engineering with an eye toward tissue growth and integration. We highlight three archetypal hydrogel systems that have shown well-documented tissue integration in vivo, and provide avenues to assess tissue in-growth. Careful design and assessment of the biomedical potential of a supramolecular hydrogels can inspire the creation of robust and dynamic implants for new tissue engineering applications. Statement of significance Tissue engineering and regeneration require careful design of materials to coax the tissue formation process. The materials must provide the cells with a proper environment, but also get out of the way to allow new tissue to form. Normally, biomaterial hydrogels are designed to chemically degrade to permit tissue formation; however, dynamic and reversible supramolecular interactions can be used to facilitate tissue formation and remodelability. Herein we review the common design parameters for supramolecular hydrogels and highlight applications where they have shown the ability to facilitate tissue formation via their dynamic interactions. The review also briefly provides insight into the functionality and control enabled by supramolecular design and the methods one can use to start to assess cell migration and tissue integration. (c) 2021 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

Time-to-solution for structure-based screening of massive chemical databases for COVID-19 drug discovery has been decreased by an order of magnitude, and a virtual laboratory has been deployed at scale on up to 27,612 GPUs on the Summit supercomputer, allowing an average molecular docking of 19,028 compounds per second. Over one billion compounds were docked to two SARS-CoV-2 protein structures with full optimization of ligand position and 20 poses per docking, each in under 24 hours. GPU acceleration and high-throughput optimizations of the docking program produced 350× mean speedup over the CPU version (50× speedup per node). GPU acceleration of both feature calculation for machine-learning based scoring and distributed database queries reduced processing of the 2.4 TB output by orders of magnitude. The resulting 50× speedup for the full pipeline reduces an initial 43 day runtime to 21 hours per protein for providing high-scoring compounds to experimental collaborators for validation assays.

The urgent search for drugs to combat SARS-CoV-2 has included the use of supercomputers. The use of general-purpose graphical processing units (GPUs), massive parallelism, and new software for high-performance computing (HPC) has allowed researchers to search the vast chemical space of potential drugs faster than ever before. We developed a new drug discovery pipeline using the Summit supercomputer at Oak Ridge National Laboratory to help pioneer this effort, with new platforms that incorporate GPU-accelerated simulation and allow for the virtual screening of billions of potential drug compounds in days compared to weeks or months for their ability to inhibit SARS-COV-2 proteins. This effort will accelerate the process of developing drugs to combat the current COVID-19 pandemic and other diseases.

Rational design of hydrogels that balance processability and extracellular matrix (ECM) biomimicry remains a challenge for tissue engineering and biofabrication. Hydrogels suitable for biofabrication techniques, yet tuneable to match the mechanical (static and dynamic) properties of native tissues remain elusive. Dynamic covalent hydrogels possessing shear-thinning/self-healing (processability) and time-dependent cross-links (mechanical properties) provide a potential solution, yet can be difficult to rationally control. Here, the straightforward modular mixing of dynamic cross-links with different timescales (hydrazone and oxime) is explored using rheology, self-healing tests, extrusion printing, and culture of primary human dermal fibroblasts. Maintaining a constant polymer content and cross-linker concentration, the stiffness and stress relaxation can be tuned across two orders of magnitude. All formulations demonstrate a similar flow profile after network rupture, allowing the separation of initial mechanical properties from flow behavior during printing. Furthermore, the self-healing nature of hydrogels with high hydrazone content enables recyclability of printed structures. Last, a distinct threshold for cell spreading and morphology is observed within this hydrogel series, even in multi-material constructs. Simple cross-linker mixing enables fine control and is of general interest for bioink development, targeting viscoelastic properties of specific cellular niches, and as an accessible and flexible platform for designing dynamic networks.

Bioprinting techniques have been flourishing in the field of biofabrication with pronounced and exponential developments in the past years. Novel biomaterial inks used for the formation of bioinks have been developed, allowing the manufacturing of in vitro models and implants tested preclinically with a certain degree of success. Furthermore, incredible advances in cell biology, namely, in pluripotent stem cells, have also contributed to the latest milestones where more relevant tissues or organ-like constructs with a certain degree of functionality can already be obtained. These incredible strides have been possible with a multitude of multidisciplinary teams around the world, working to make bioprinted tissues and organs more relevant and functional. Yet, there is still a long way to go until these biofabricated constructs will be able to reach the clinics. In this review, we summarize the main bioprinting activities linking them to tissue and organ development and physiology. Most bioprinting approaches focus on mimicking fully matured tissues. Future bioprinting strategies might pursue earlier developmental stages of tissues and organs. The continuous convergence of the experts in the fields of material sciences, cell biology, engineering, and many other disciplines will gradually allow us to overcome the barriers identified on the demanding path toward manufacturing and adoption of tissue and organ replacements.

Supramolecular and dynamic biomaterials hold promise to recapitulate the time-dependent properties and stimuli-responsiveness of the native extracellular matrix (ECM). Host-guest chemistry is one of the most widely studied supramolecular bonds, yet the binding characteristics of host-guest complexes (beta-CD/adamantane) in relevant biomaterials have mostly focused on singular host-guest interactions or nondiscrete multivalent pendent polymers. The stepwise synergistic effect of multivalent host-guest interactions for the formation of dynamic biomaterials remains relatively unreported. In this work, we study how a series of multivalent adamantane (guest) cross-linkers affect the overall binding affinity and ability to form supramolecular networks with alginate-CD (Alg-CD). These binding constants of the multivalent cross-linkers were determined via NMR titration and showed increases in binding constants occurring with multivalent constructs. The higher multivalent cross-linkers enabled hydrogel formation; furthermore, an increase in binding and gelation was observed with the inclusion of a phenyl spacer to the cross-linker. A preliminary screen shows that only cross-linking Alg-CD with an 8-arm-multivalent guest results in robust gel formation. These cytocompatible hydrogels highlight the importance of multivalent design for dynamically cross-linked hydrogels. These materials hold promise for development toward cell- and small molecule-delivery platforms and allow discrete and fine-tuning of network properties.

Cell-based regenerative constructs provide hope for the restoration of tissue function in compromised biological conditions such as complex bone defects. A strategy mimicking the cascade of events of postnatal fracture healing suggests an implant design where progenitor cells provide the driving force for the construct's tissue forming capacity, while framing biomaterials provide cells with 3D cues to direct cellular processes. Large bone defects mainly heal through the formation of an intermediate endochondral fracture callus. The authors aimed to develop an in vitro engineered fracture callus manufactured by bioprinting to provide a spatially organized tissue construct based on: i) in vitro 3D primed human periosteum derived cells and ii) biocompatible thiol-ene alginate hydrogels, mimicking the cells and extracellular matrix present in the different zones of the callus. Cell viability and maintained osteochondrogenic differentiation upon bioprinting is confirmed in vitro. In vivo assessment displays that the developed biomaterials provided essential 3D cues that further guided the cells in their tissue forming process in the absence of additional stimulatory molecules. The reported findings confirm the appeal of a biomimetic approach to steer tissue development of in vitro engineered constructs and illustrate the suitability of bioprinting methodologies for the fabrication of living regenerative implants.

Digital light processing (DLP) is a versatile additive manufacturing technique able to quickly create complex 3D constructs. DLP has been used successfully for many years in dental applications and rapid prototyping and has gained recent attention in the field of tissue regeneration. Especially the advances in fabricating soft tissue constructs have favored the development of biocompatible and bioresorbable resins. However, to be printable, a resin needs a viscosity of < 10 Pa.s. Thus at ambient temperatures, these resins often require the use of added solvents or reactive diluents (RD) to lower the formulation viscosity to enable printing. Alternatively, the temperature of the resins can also be increased to lower their viscosity. However, most commercial DLP printers come without a thermally controlled bath, and even lack the possibility of adding this functionality without purchasing a new machine. In this work, we report the development of a transparent indium-tin-oxide (ITO) based heating stage that is low-cost and can be easily implemented by other research groups with an existing DLP printer. We then demonstrate that this heating solution ensures a constant, controllable temperature for each layer that is photocured. Furthermore, we showed the effectiveness of this approach by fabricating complex 3D structures using different biocompatible poly(ester) and poly(ester-co-carbonate) urethane acrylate based resins that are unprintable at ambient temperature. The heating stage reported herein represents a small, but impactful improvement that can be applied to any commercial DLP printer, expanding the scope and fidelity of printable resins.

Differentiated kidney organoids from induced pluripotent stem cells hold promise as a treatment for patients with kidney diseases. Before these organoids can be translated to the clinic, shortcomings regarding their cellular, extracellular compositions and developmental plateau needs to be overcome. We performed a proteomic analysis on kidney organoids cultured for a prolonged culture time and we found a specific change in the extracellular matrix composition with increased expression of types 1a1, 2 and 6a1 collagen. Such an excessive accumulation of specific collagen types is a hallmark of renal fibrosis that causes a life-threatening pathological condition by compromising key functions of the human kidney. Here we hypothesized the need for a three-dimensional environment to grow the kidney organoids, which could better mimic the in vivo surroundings of the developing kidney than standard culture on a transwell filter. Encapsulating organoids for four days in a soft, thiol-ene cross-linked alginate hydrogel resulted in decreased type 1a1 collagen expression. Furthermore, the encapsulation did not result in any changes of organoid structural morphology. Using a biomaterial to modulate collagen expression allows for a prolonged kidney organoid culture in vitro and a reduction of abnormal type 1a1 collagen expression bringing kidney organoids closer to clinical application.HIGHLIGHTSProlonging kidney organoid culture results in a developmental plateau instead of improved in vitro maturation.Proteomic analyses point to an increased expression of specific collagen subtypes associated with renal fibrosis.Encapsulating kidney organoids using a soft thiol-ene cross-linked alginate hydrogel reduces collagen type 1a1 and αSMA deposition.