Enhancing volumetric muscle loss (VML) recovery in a rat model using super durable hydrogels derived from bacteria.

Bacteria can be programmed to deliver natural materials with defined biological and mechanical properties for controlling cell growth and differentiation. Here, we present an elastic, resilient and bioactive polysaccharide derived from the extracellular matrix of Pantoea sp. BCCS 001. Specifically, it was methacrylated to generate a new photo crosslinkable hydrogel that we coined Pantoan Methacrylate or put simply PAMA. We have used it for the first time as a tissue engineering hydrogel to treat VML injuries in rats. The crosslinked PAMA hydrogel was super elastic with a recovery nearing 100 %, while mimicking the mechanical stiffness of native muscle. After inclusion of thiolated gelatin via a Michaelis reaction with acrylate groups on PAMA we could also guide muscle progenitor cells into fused and aligned tubes - something reminiscent of mature muscle cells. These results were complemented by sarcomeric alpha-actinin immunostaining studies. Importantly, the implanted hydrogels exhibited almost 2-fold more muscle formation and 50 % less fibrous tissue formation compared to untreated rat groups. In vivo inflammation and toxicity assays likewise gave rise to positive results confirming the biocompatibility of this new biomaterial system. Overall, our results demonstrate that programmable polysaccharides derived from bacteria can be used to further advance the field of tissue engineering. In greater detail, they could in the foreseeable future be used in practical therapies against VML.

Hypothermic and cryogenic preservation of cardiac tissue-engineered constructs.

Cardiac tissue engineering (cTE) has already advanced towards the first clinical trials, investigating safety and feasibility of cTE construct transplantation in failing hearts. However, the lack of well-established preservation methods poses a hindrance to further scalability, commercialization, and transportation, thereby reducing their clinical implementation. In this study, hypothermic preservation (4 °C) and two methods for cryopreservation (i.e., a slow and fast cooling approach to -196 °C and -150 °C, respectively) were investigated as potential solutions to extend the cTE construct implantation window. The cTE model used consisted of human induced pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts embedded in a natural-derived hydrogel and supported by a polymeric melt electrowritten hexagonal scaffold. Constructs, composed of cardiomyocytes of different maturity, were preserved for three days, using several commercially available preservation protocols and solutions. Cardiomyocyte viability, function (beat rate and calcium handling), and metabolic activity were investigated after rewarming. Our observations show that cardiomyocytes' age did not influence post-rewarming viability, however, it influenced construct function. Hypothermic preservation with HypoThermosol® ensured cardiomyocyte viability and function. Furthermore, fast freezing outperformed slow freezing, but both viability and function were severely reduced after rewarming. In conclusion, whereas long-term preservation remains a challenge, hypothermic preservation with HypoThermosol® represents a promising solution for cTE construct short-term preservation and potential transportation, aiding in off-the-shelf availability, ultimately increasing their clinical applicability.

Experimentally-guided in silico design of engineered heart tissues to improve cardiac electrical function after myocardial infarction.

Engineered heart tissues (EHTs) built from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) showed promising results for cardiac function restoration following myocardial infarction. Nevertheless, human iPSC-CMs have longer action potential and lower cell-to-cell coupling than adult-like CMs. These immature electrophysiological properties favor arrhythmias due to the generation of electrophysiological gradients when hiPSC-CMs are injected in the cardiac tissue. Culturing hiPSC-CMs on three-dimensional (3D) scaffolds can promote their maturation and influence their alignment. However, it is still uncertain how on-scaffold culturing influences the overall electrophysiology of the in vitro and implanted EHTs, as it requires expensive and time consuming experimentation. Here, we computationally investigated the impact of the scaffold design on the EHT electrical depolarization and repolarization before and after engraftment on infarcted tissue. We first acquired and processed electrical recordings from in vitro EHTs, which we used to calibrate the modeling and simulation of in silico EHTs to replicate experimental outcomes. Next, we built in silico EHT models for a range of scaffold pore sizes, shapes (square, rectangular, auxetic, hexagonal) and thicknesses. In this setup, we found that scaffolds made of small (0.2 mm2), elongated (30° half-angle) hexagons led to faster EHT activation and better mimicked the cardiac anisotropy. The scaffold thickness had a marginal role on the not engrafted EHT electrophysiology. Moreover, EHT engraftment on infarcted tissue showed that the EHT conductivity should be at least 5% of that in healthy tissue for bidirectional EHT-myocardium electrical propagation. For conductivities above such threshold, the scaffold made of small elongated hexagons led to the lowest activation time (AT) in the coupled EHT-myocardium. If the EHT conductivity was further increased and the hiPSC-CMs were uniformly oriented parallel to the epicardial cells, the total AT and the repolarization time gradient decreased substantially, thus minimizing the likelihood for arrhythmias after EHT transplantation.

Covalent Grafting of Functionalized MEW Fibers to Silk Fibroin Hydrogels to Obtain Reinforced Tissue Engineered Constructs.

Hydrogels are ideal materials to encapsulate cells, making them suitable for applications in tissue engineering and regenerative medicine. However, they generally do not possess adequate mechanical strength to functionally replace human tissues, and therefore they often need to be combined with reinforcing structures. While the interaction at the interface between the hydrogel and reinforcing structure is imperative for mechanical function and subsequent biological performance, this interaction is often overlooked. Melt electrowriting enables the production of reinforcing microscale fibers that can be effectively integrated with hydrogels. Yet, studies on the interaction between these micrometer scale fibers and hydrogels are limited. Here, we explored the influence of covalent interfacial interactions between reinforcing structures and silk fibroin methacryloyl hydrogels (silkMA) on the mechanical properties of the construct and cartilage-specific matrix production in vitro. For this, melt electrowritten fibers of a thermoplastic polymer blend (poly(hydroxymethylglycolide-co-ε-caprolactone):poly(ε-caprolactone) (pHMGCL:PCL)) were compared to those of the respective methacrylated polymer blend pMHMGCL:PCL as reinforcing structures. Photopolymerization of the methacrylate groups, present in both silkMA and pMHMGCL, was used to generate hybrid materials. Covalent bonding between the pMHMGCL:PCL blend and silkMA hydrogels resulted in an elastic response to the application of torque. In addition, an improved resistance was observed to compression (∼3-fold) and traction (∼40-55%) by the scaffolds with covalent links at the interface compared to those without these interactions. Biologically, both types of scaffolds (pHMGCL:PCL and pMHMGCL:PCL) showed similar levels of viability and metabolic activity, also compared to frequently used PCL. Moreover, articular cartilage progenitor cells embedded within the reinforced silkMA hydrogel were able to form a cartilage-like matrix after 28 days of in vitro culture. This study shows that hybrid cartilage constructs can be engineered with tunable mechanical properties by grafting silkMA hydrogels covalently to pMHMGCL:PCL blend microfibers at the interface.

Orthotopic equine study confirms the pivotal importance of structural reinforcement over the pre-culture of cartilage implants.

In articular cartilage (AC), the collagen arcades provide the tissue with its extraordinary mechanical properties. As these structures cannot be restored once damaged, functional restoration of AC defects remains a major challenge. We report that the use of a converged bioprinted, osteochondral implant, based on a gelatin methacryloyl cartilage phase, reinforced with precisely patterned melt electrowritten polycaprolactone micrometer-scale fibers in a zonal fashion, inspired by native collagen architecture, can provide long-term mechanically stable neo-tissue in an orthotopic large animal model. The design of this novel implant was achieved via state-of-the-art converging of extrusion-based ceramic printing, melt electrowriting, and extrusion-based bioprinting. Interestingly, the cell-free implants, used as a control in this study, showed abundant cell ingrowth and similar favorable results as the cell-containing implants. Our findings underscore the hypothesis that mechanical stability is more determining for the successful survival of the implant than the presence of cells and pre-cultured extracellular matrix. This observation is of great translational importance and highlights the aptness of advanced 3D (bio)fabrication technologies for functional tissue restoration in the harsh articular joint mechanical environment.

Proinflammatory phenotype of iPS cell-derived JAK2 V617F megakaryocytes induces fibrosis in 3D in vitro bone marrow niche.

The myeloproliferative disease polycythemia vera (PV) driven by the JAK2 V617F mutation can transform into myelofibrosis (post-PV-MF). It remains an open question how JAK2 V617F in hematopoietic stem cells induces MF. Megakaryocytes are major players in murine PV models but are difficult to study in the human setting. We generated induced pluripotent stem cells (iPSCs) from JAK2 V617F PV patients and differentiated them into megakaryocytes. In differentiation assays, JAK2 V617F iPSCs recapitulated the pathognomonic skewed megakaryocytic and erythroid differentiation. JAK2 V617F iPSCs had a TPO-independent and increased propensity to differentiate into megakaryocytes. RNA sequencing of JAK2 V617F iPSC-derived megakaryocytes reflected a proinflammatory, profibrotic phenotype and decreased ribosome biogenesis. In three-dimensional (3D) coculture, JAK2 V617F megakaryocytes induced a profibrotic phenotype through direct cell contact, which was reversed by the JAK2 inhibitor ruxolitinib. The 3D coculture system opens the perspective for further disease modeling and drug discovery.