Microinterfaces in biopolymer-based bicontinuous hydrogels guide rapid 3D cell migration.

Cell migration is critical for tissue development and regeneration but requires extracellular environments that are conducive to motion. Cells may actively generate migratory routes in vivo by degrading or remodeling their environments or instead utilize existing extracellular matrix microstructures or microtracks as innate pathways for migration. While hydrogels in general are valuable tools for probing the extracellular regulators of 3-dimensional migration, few recapitulate these natural migration paths. Here, we develop a biopolymer-based bicontinuous hydrogel system that comprises a covalent hydrogel of enzymatically crosslinked gelatin and a physical hydrogel of guest and host moieties bonded to hyaluronic acid. Bicontinuous hydrogels form through controlled solution immiscibility, and their continuous subdomains and high micro-interfacial surface area enable rapid 3D migration, particularly when compared to homogeneous hydrogels. Migratory behavior is mesenchymal in nature and regulated by biochemical and biophysical signals from the hydrogel, which is shown across various cell types and physiologically relevant contexts (e.g., cell spheroids, ex vivo tissues, in vivo tissues). Our findings introduce a design that leverages important local interfaces to guide rapid cell migration.

Hierarchical Design of Tissue-Mimetic Fibrillar Hydrogel Scaffolds.

Most tissues of the human body present hierarchical fibrillar extracellular matrices that have a strong influence over their physicochemical properties and biological behavior. Of great interest is the introduction of this fibrillar structure to hydrogels, particularly due to the water-rich composition, cytocompatibility and tunable properties of this class of biomaterials. Here, the main bottom-up fabrication strategies for the design and production of hierarchical biomimetic fibrillar hydrogels and their most representative applications in the fields of tissue engineering and regenerative medicine are reviewed. For example, the controlled assembly/arrangement of peptides, polymeric micelles, cellulose nanoparticles (NPs), and magnetically responsive nanostructures, among others, into fibrillar hydrogels is discussed, as well as their potential use as fibrillar-like hydrogels (e.g., those from cellulose NPs) with key biofunctionalities such as electrical conductivity or remote stimulation. Finally, major remaining barriers to the clinical translation of fibrillar hydrogels and potential future directions of research in this field are discussed. This article is protected by copyright. All rights reserved.

Multi-Material Volumetric Additive Manufacturing of Hydrogels using Gelatin as a Sacrificial Network and 3D Suspension Bath.

Volumetric additive manufacturing (VAM) is an emerging layerless method for the rapid processing of reactive resins into 3D structures, where printing is much faster (seconds) than other lithography and direct ink writing methods (minutes to hours). As a vial of resin rotates in the VAM process, patterned light exposure defines a 3D object and then resin that has not undergone gelation can be washed away. Despite the promise of VAM, there are challenges with the printing of soft hydrogel materials from non-viscous precursors, including multi-material constructs. To address this, sacrificial gelatin is used to modulate resin viscosity to support the cytocompatible VAM printing of macromers based on poly(ethylene glycol) (PEG), hyaluronic acid (HA), and polyacrylamide (PA). After printing, gelatin is removed by washing at an elevated temperature. To print multi-material constructs, the gelatin-containing resin is used as a shear-yielding suspension bath (including HA to further modulate bath properties) where ink can be extruded into the bath to define a multi-material resin that can then be processed with VAM into a defined object. Multi-material constructs of methacrylated HA (MeHA) and gelatin methacrylamide (GelMA) are printed (as proof-of-concept) with encapsulated mesenchymal stromal cells (MSCs), where the local hydrogel properties guide cell spreading behavior with culture.

Injectable MSC Spheroid and Microgel Granular Composites for Engineering Tissue.

Many cell types require direct cell-cell interactions for differentiation and function; yet, this can be challenging to incorporate into 3-dimensional (3D) structures for the engineering of tissues. Here, a new approach is introduced that combines aggregates of cells (spheroids) with similarly-sized hydrogel particles (microgels) to form granular composites that are injectable, undergo interparticle crosslinking via light for initial stabilization, permit cell-cell contacts for cell signaling, and allow spheroid fusion and growth. One area where this is important is in cartilage tissue engineering, as cell-cell contacts are crucial to chondrogenesis and are missing in many tissue engineering approaches. To address this, granular composites are developed from adult porcine mesenchymal stromal cell (MSC) spheroids and hyaluronic acid microgels and simulations and experimental analyses are used to establish the importance of initial MSC spheroid to microgel volume ratios to balance mechanical support with tissue growth. Long-term chondrogenic cultures of granular composites produce engineered cartilage tissue with extensive matrix deposition and mechanical properties within the range of cartilage, as well as integration with native tissue. Altogether, a new strategy of injectable granular composites is developed that leverages the benefits of cell-cell interactions through spheroids with the mechanical stabilization afforded with engineered hydrogels.

Local and Sustained Baricitinib Delivery to the Skin through Injectable Hydrogels Containing Reversible Thioimidate Adducts.

Janus kinase (JAK) inhibitors are approved for many dermatologic disorders, but their use is limited by systemic toxicities including serious cardiovascular events and malignancy. To overcome these limitations, injectable hydrogels are engineered for the local and sustained delivery of baricitinib, a representative JAK inhibitor. Hydrogels are formed via disulfide crosslinking of thiolated hyaluronic acid macromers. Dynamic thioimidate bonds are introduced between the thiolated hyaluronic acid and nitrile-containing baricitinib for drug tethering, which is confirmed with 1 H and 13 C nuclear magnetic resonance (NMR). Release of baricitinib is tunable over six weeks in vitro and active in inhibiting JAK signaling in a cell line containing a luciferase reporter reflecting interferon signaling. For in vivo activity, baricitinib hydrogels or controls are injected intradermally into an imiquimod-induced mouse model of psoriasis. Imiquimod increases epidermal thickness in mice, which is unaffected when treated with baricitinib or hydrogel alone. Treatment with baricitinib hydrogels suppresses the increased epidermal thickness in mice treated with imiquimod, suggesting that the sustained and local release of baricitinib is important for a therapeutic outcome. This study is the first to utilize a thioimidate chemistry to deliver JAK inhibitors to the skin through injectable hydrogels, which has translational potential for treating inflammatory disorders.

Mechanical crosstalk between the intervertebral disc, facet joints, and vertebral endplate following acute disc injury in a rabbit model.

Background: Vertebral endplate sclerosis and facet osteoarthritis have been documented in animals and humans. However, it is unclear how these adjacent pathologies engage in crosstalk with the intervertebral disc. This study sought to elucidate this crosstalk by assessing each compartment individually in response to acute disc injury. Methods: Eleven New Zealand White rabbits underwent annular disc puncture using a 16G or 21G needle. At 4 and 10 weeks, individual compartments of the motion segment were analyzed. Discs underwent T 1 relaxation mapping with MRI contrast agent gadodiamide as well T 2 mapping. Both discs and facets underwent mechanical testing via vertebra-disc-vertebra tension-compression creep testing and indentation testing, respectively. Endplate bone density was quantified via µCT. Discs and facets were sectioned and stained for histology scoring. Results: Intervertebral discs became more degenerative with increasing needle diameter and time post-puncture. Bone density also increased in endplates adjacent to both 21G and 16G punctured discs leading to reduced gadodiamide transport at 10 weeks. The facet joints, however, did not follow this same trend. Facets adjacent to 16G punctured discs were less degenerative than facets adjacent to 21G punctured discs at 10 weeks. 16G facets were more degenerative at 4 weeks than at 10, suggesting the cartilage had recovered. The formation of severe disc osteophytes in 16G punctured discs between 4 and 10 weeks likely offloaded the facet cartilage, leading to the recovery observed. Conclusions: Overall, this study supports that degeneration spans the whole spinal motion segment following disc injury. Vertebral endplate thickening occurred in response to disc injury, which limited the diffusion of small molecules into the disc. This work also suggests that altered disc mechanics can induce facet degeneration, and that extreme bony remodeling adjacent to the disc may promote facet cartilage recovery through offloading of the articular cartilage.

Injectable Radiopaque Hyaluronic Acid Granular Hydrogels for Intervertebral Disc Repair.

Injectable hydrogels offer minimally-invasive treatment options for degenerative disc disease, a prevalent condition affecting millions annually. Many hydrogels explored for intervertebral disc (IVD) repair suffer from weak mechanical integrity, migration issues, and expulsion. To overcome these limitations, an injectable and radiopaque hyaluronic acid granular hydrogel is developed. The granular structure provides easy injectability and low extrusion forces, while the radiopacity enables direct visualization during injection into the disc and non-invasive monitoring after injection. The radiopaque granular hydrogel is injected into rabbit disc explants to investigate restoration of healthy disc mechanics following needle puncture injury ex vivo and then delivered in a minimally-invasive manner into the intradiscal space in a clinically-relevant in vivo large animal goat model of IVD degeneration initiated through degradation by chondroitinase. The radiopaque granular hydrogel successfully halted loss of disc height due to degeneration. Further, the hydrogel not only enhanced proteoglycan content and reduced collagen content in the nucleus pulposus (NP) region compared to degenerative discs, but also helped to maintain the structural integrity of the disc and promote healthy segregation of the NP and annulus fibrosus regions. Overall, this study demonstrates the great potential of an injectable radiopaque granular hydrogel for treatment of degenerative disc disease.

Facile Physicochemical Reprogramming of PEG-Dithiolane Microgels.

Granular biomaterials have found widespread applications in tissue engineering, in part because of their inherent porosity, tunable properties, injectability, and 3D printability. However, the assembly of granular hydrogels typically relies on spherical microparticles and more complex particle geometries have been limited in scope, often requiring templating of individual microgels by microfluidics or in-mold polymerization. Here, we use dithiolane-functionalized synthetic macromolecules to fabricate photopolymerized microgels via batch emulsion, and then harness the dynamic disulfide crosslinks to rearrange the network. Through unconfined compression between parallel plates in the presence of photoinitiated radicals, we transform the isotropic microgels are transformed into disks. Characterizing this process, we find that the areas of the microgel surface in contact with the compressive plates are flattened while the curvature of the uncompressed microgel boundaries increases. When cultured with C2C12 myoblasts, cells localize to regions of higher curvature on the disk-shaped microgel surfaces. This altered localization affects cell-driven construction of large supraparticle scaffold assemblies, with spherical particles assembling without specific junction structure while disk microgels assemble preferentially on their curved surfaces. These results represent a unique spatiotemporal process for rapid reprocessing of microgels into anisotropic shapes, providing new opportunities to study shape-driven mechanobiological cues during and after granular hydrogel assembly.

Engineered hydrogel reveals contribution of matrix mechanics to esophageal adenocarcinoma and identifies matrix-activated therapeutic targets.

Increased extracellular matrix (ECM) stiffness has been implicated in esophageal adenocarcinoma (EAC) progression, metastasis, and resistance to therapy. However, the underlying protumorigenic pathways are yet to be defined. Additional work is needed to develop physiologically relevant in vitro 3D culture models that better recapitulate the human tumor microenvironment and can be used to dissect the contributions of matrix stiffness to EAC pathogenesis. Here, we describe a modular, tumor ECM-mimetic hydrogel platform with tunable mechanical properties, defined presentation of cell-adhesive ligands, and protease-dependent degradation that supports robust in vitro growth and expansion of patient-derived EAC 3D organoids (EAC PDOs). Hydrogel mechanical properties control EAC PDO formation, growth, proliferation, and activation of tumor-associated pathways that elicit stem-like properties in the cancer cells, as highlighted through in vitro and in vivo environments. We also demonstrate that the engineered hydrogel serves as a platform for identifying potential therapeutic targets to disrupt the contribution of protumorigenic matrix mechanics in EAC. Together, these studies show that an engineered PDO culture platform can be used to elucidate underlying matrix-mediated mechanisms of EAC and inform the development of therapeutics that target ECM stiffness in EAC.

Microinterfaces in bicontinuous hydrogels guide rapid 3D cell migration.

Cell migration is critical for tissue development and regeneration but requires extracellular environments that are conducive to motion. Cells may actively generate migratory routes in vivo by degrading or remodeling their environments or may instead utilize existing ECM microstructures or microtracks as innate pathways for migration. While hydrogels in general are valuable tools for probing the extracellular regulators of 3D migration, few have recapitulated these natural migration paths. Here, we developed a biopolymer-based (i.e., gelatin and hyaluronic acid) bicontinuous hydrogel system formed through controlled solution immiscibility whose continuous subdomains and high micro-interfacial surface area enabled rapid 3D migration, particularly when compared to homogeneous hydrogels. Migratory behavior was mesenchymal in nature and regulated by biochemical and biophysical signals from the hydrogel, which was shown across various cell types and physiologically relevant contexts (e.g., cell spheroids, ex vivo tissues, in vivo tissues). Our findings introduce a new design that leverages important local interfaces to guide rapid cell migration.

Facile Photopatterning of Perfusable Microchannels in Synthetic Hydrogels to Recreate Microphysiological Environments.

The fabrication of perfusable hydrogels is crucial for recreating in vitro microphysiological environments. Existing strategies to fabricate complex microchannels in hydrogels involve sophisticated equipment/techniques. A cost-effective, facile, versatile, and ultra-fast methodology is reported to fabricate perfusable microchannels of complex shapes in photopolymerizable hydrogels without the need of specialized equipment or sophisticated protocols. The methodology utilizes one-step ultraviolet (UV) light-triggered cross-linking and a photomask printed on inexpensive transparent films to photopattern PEG-norbornene hydrogels. Complex and intricate patterns with high resolution, including perfusable microchannels, can be fabricated in <1 s. The perfusable hydrogel is integrated into a custom-made microfluidic device that permits connection to external pump systems, allowing continuous fluid perfusion into the microchannels. Under dynamic culture, human endothelial cells form a functional and confluent endothelial monolayer that remains viable for at least 7 days and respond to inflammatory stimuli. Finally, approach to photopattern norbornene hyaluronic acid hydrogels is adapted, highlighting the versatility of the technique. This study presents an innovative strategy to simplify and reduce the cost of biofabrication techniques for developing functional in vitro models using perfusable three-dimensional (3D) hydrogels. The approach offers a novel solution to overcome the complexities associated with existing methods, allowing engineering advanced in vitro microphysiological environments.

Modulating design parameters to drive cell invasion into hydrogels for osteochondral tissue formation.

Background: The use of acellular hydrogels to repair osteochondral defects requires cells to first invade the biomaterial and then to deposit extracellular matrix for tissue regeneration. Due to the diverse physicochemical properties of engineered hydrogels, the specific properties that allow or even improve the behaviour of cells are not yet clear. The aim of this study was to investigate the influence of various physicochemical properties of hydrogels on cell migration and related tissue formation using in vitro, ex vivo and in vivo models. Methods: Three hydrogel platforms were used in the study: Gelatine methacryloyl (GelMA) (5% wt), norbornene hyaluronic acid (norHA) (2% wt) and tyramine functionalised hyaluronic acid (THA) (2.5% wt). GelMA was modified to vary the degree of functionalisation (DoF 50% and 80%), norHA was used with varied degradability via a matrix metalloproteinase (MMP) degradable crosslinker and THA was used with the addition of collagen fibrils. The migration of human mesenchymal stromal cells (hMSC) in hydrogels was studied in vitro using a 3D spheroid migration assay over 48h. In addition, chondrocyte migration within and around hydrogels was investigated in an ex vivo bovine cartilage ring model (three weeks). Finally, tissue repair within osteochondral defects was studied in a semi-orthotopic in vivo mouse model (six weeks). Results: A lower DoF of GelMA did not affect cell migration in vitro (p â€‹= â€‹0.390) and led to a higher migration score ex vivo (p â€‹< â€‹0.001). The introduction of a MMP degradable crosslinker in norHA hydrogels did not improve cell infiltration in vitro or in vivo. The addition of collagen to THA resulted in greater hMSC migration in vitro (p â€‹= â€‹0.031) and ex vivo (p â€‹< â€‹0.001). Hydrogels that exhibited more cell migration in vitro or ex vivo also showed more tissue formation in the osteochondral defects in vivo, except for the norHA group. Whereas norHA with a degradable crosslinker did not improve cell migration in vitro or ex vivo, it did significantly increase tissue formation in vivo compared to the non-degradable crosslinker (p â€‹< â€‹0.001). Conclusion: The modification of hydrogels by adapting DoF, use of a degradable crosslinker or including fibrillar collagen can control and improve cell migration and tissue formation for osteochondral defect repair. This study also emphasizes the importance of performing both in vitro and in vivo testing of biomaterials, as, depending on the material, the results might be affected by the model used.The translational potential of this article: This article highlights the potential of using acellular hydrogels to repair osteochondral defects, which are common injuries in orthopaedics. The study provides a deeper understanding of how to modify the properties of hydrogels to control cell migration and tissue formation for osteochondral defect repair. The results of this article also highlight that the choice of the used laboratory model can affect the outcome. Testing hydrogels in different models is thus advised for successful translation of laboratory results to the clinical application.

A fetal wound healing program after intrauterine bile duct injury may contribute to biliary atresia.

BACKGROUND & AIMS: Biliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause. Fetal tissues have increased levels of hyaluronic acid (HA), which plays an integral role in fetal wound healing. The objective of this study was to determine whether a program of fetal wound healing is part of the response to fetal EHBD injury. METHODS: Mouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes. RESULTS: A wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device. CONCLUSION: The fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with prolonged high levels of HA typical of fetal wound healing. The expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD. IMPACT AND IMPLICATIONS: Biliary atresia is a pediatric cholangiopathy associated with high morbidity and mortality rates; although multiple etiologies have been proposed, the fetal response to bile duct damage is largely unknown. This study explores the fetal pathogenesis after extrahepatic bile duct damage, thereby opening a completely new avenue to study therapeutic targets in the context of biliary atresia.

Evaluation of surgical fixation methods for the implantation of melt electrowriting-reinforced hyaluronic acid hydrogel composites in porcine cartilage defects.

The surgical repair of articular cartilage remains an ongoing challenge in orthopedics. Tissue engineering is a promising approach to treat cartilage defects; however, scaffolds must (i) possess the requisite material properties to support neocartilage formation, (ii) exhibit sufficient mechanical integrity for handling during implantation, and (iii) be reliably fixed within cartilage defects during surgery. In this study, we demonstrate the reinforcement of soft norbornene-modified hyaluronic acid (NorHA) hydrogels via the melt electrowriting (MEW) of polycaprolactone to fabricate composite scaffolds that support encapsulated porcine mesenchymal stromal cell (pMSC, three donors) chondrogenesis and cartilage formation and exhibit mechanical properties suitable for handling during implantation. Thereafter, acellular MEW-NorHA composites or MEW-NorHA composites with encapsulated pMSCs and precultured for 28 days were implanted in full-thickness cartilage defects in porcine knees using either bioresorbable pins or fibrin glue to assess surgical fixation methods. Fixation of composites with either biodegradable pins or fibrin glue ensured implant retention in most cases (80%); however, defects treated with pinned composites exhibited more subchondral bone remodeling and inferior cartilage repair, as evidenced by micro-computed tomography (micro-CT) and safranin O/fast green staining, respectively, when compared to defects treated with glued composites. Interestingly, no differences in repair tissue were observed between acellular and cellularized implants. Additional work is required to assess the full potential of these scaffolds for cartilage repair. However, these results suggest that future approaches for cartilage repair with MEW-reinforced hydrogels should be carefully evaluated with regard to their fixation approach for construct retention and surrounding cartilage tissue damage.

Modeling development using hydrogels.

The development of multicellular complex organisms relies on coordinated signaling from the microenvironment, including both biochemical and mechanical interactions. To better understand developmental biology, increasingly sophisticated in vitro systems are needed to mimic these complex extracellular features. In this Primer, we explore how engineered hydrogels can serve as in vitro culture platforms to present such signals in a controlled manner and include examples of how they have been used to advance our understanding of developmental biology.

Improvement in cardiac function and regional LV strain following intramyocardial injection of a theranostic hydrogel early postmyocardial infarction in a porcine model.

Myocardial infarction (MI) is often complicated by left ventricular (LV) remodeling and heart failure. We evaluated the feasibility of a multimodality imaging approach to guide delivery of an imageable hydrogel and assessed LV functional changes with therapy. Yorkshire pigs underwent surgical occlusions of branches of the left anterior descending and/or circumflex artery to create an anterolateral MI. We evaluated the hemodynamic and mechanical effects of intramyocardial delivery of an imageable hydrogel in the central infarct area (Hydrogel group, n = 8) and a Control group (n = 5) early post-MI. LV and aortic pressure and ECG were measured and contrast cineCT angiography was performed at baseline, 60 min post-MI, and 90 min post-hydrogel delivery. LV hemodynamic indices, pressure-volume measures, and normalized regional and global strains were measured and compared. Both Control and Hydrogel groups demonstrated a decline in heart rate, LV pressure, stroke volume, ejection fraction, and pressure-volume loop area, and an increase in myocardial performance (Tei) index and supply/demand (S/D) ratio. After hydrogel delivery, Tei index and S/D ratio were reduced to baseline levels, diastolic and systolic functional indices either stabilized or improved, and radial strain and circumferential strain increased significantly in the MI regions (ENrr: +52.7%, ENcc: +44.1%). However, the Control group demonstrated a progressive decline in all functional indices to levels significantly below those of Hydrogel group. Thus, acute intramyocardial delivery of a novel imageable hydrogel to MI region resulted in rapid stabilization or improvement in LV hemodynamics and function.NEW & NOTEWORTHY Our study demonstrates that contrast cineCT imaging can be used to evaluate the acute effects of intramyocardial delivery of a therapeutic hydrogel to the central MI region early post MI, which resulted in a rapid stabilization of LV hemodynamics and improvement in regional and global LV function.

Tumor-restrictive type III collagen in the breast cancer microenvironment: prognostic and therapeutic implications.

Collagen plays a critical role in regulating breast cancer progression and therapeutic resistance. An improved understanding of both the features and drivers of tumor-permissive and -restrictive collagen matrices are critical to improve prognostication and develop more effective therapeutic strategies. In this study, using a combination of in vitro, in vivo and in silico experiments, we show that type III collagen (Col3) plays a tumor-restrictive role in human breast cancer. We demonstrate that Col3-deficient, human fibroblasts produce tumor-permissive collagen matrices that drive cell proliferation and suppress apoptosis in noninvasive and invasive breast cancer cell lines. In human TNBC biopsy samples, we demonstrate elevated deposition of Col3 relative to type I collagen (Col1) in noninvasive compared to invasive regions. Similarly, in silico analyses of over 1000 breast cancer patient biopsies from The Cancer Genome Atlas BRCA cohort revealed that patients with higher Col3:Col1 bulk tumor expression had improved overall, disease-free and progression-free survival relative to those with higher Col1:Col3 expression. Using an established 3D culture model, we show that Col3 increases spheroid formation and induces formation of lumen-like structures that resemble non-neoplastic mammary acini. Finally, our in vivo study shows co-injection of murine breast cancer cells (4T1) with rhCol3-supplemented hydrogels limits tumor growth and decreases pulmonary metastatic burden compared to controls. Taken together, these data collectively support a tumor-suppressive role for Col3 in human breast cancer and suggest that strategies that increase Col3 may provide a safe and effective modality to limit recurrence in breast cancer patients.

Modulus-dependent effects on neurogenic, myogenic, and chondrogenic differentiation of human mesenchymal stem cells in three-dimensional hydrogel cultures.

Human mesenchymal stromal cells (hMSCs) are of significant interest as a renewable source of therapeutically useful cells. In tissue engineering, hMSCs are implanted within a scaffold to provide enhanced capacity for tissue repair. The present study evaluates how mechanical properties of that scaffold can alter the phenotype and genotype of the cells, with the aim of augmenting hMSC differentiation along the myogenic, neurogenic or chondrogenic linages. The hMSCs were grown three-dimensionally (3D) in a hydrogel comprised of poly(ethylene glycol) (PEG)-conjugated to fibrinogen. The hydrogel's shear storage modulus (G'), which was controlled by increasing the amount of PEG-diacrylate cross-linker in the matrix, was varied in the range of 100-2000 Pascal (Pa). The differentiation into each lineage was initiated by a defined culture medium, and the hMSCs grown in the different modulus hydrogels were characterized using gene and protein expression. Materials having lower storage moduli (G' = 100 Pa) exhibited more hMSCs differentiating to neurogenic lineages. Myogenesis was favored in materials having intermediate modulus values (G' = 500 Pa), whereas chondrogenesis was favored in materials with a higher modulus (G' = 1000 Pa). Enhancing the differentiation pathway of hMSCs in 3D hydrogel scaffolds using simple modifications to mechanical properties represents an important achievement toward the effective application of these cells in tissue engineering.

Suspension bath bioprinting and maturation of anisotropic meniscal constructs.

Due to limited intrinsic healing capacity of the meniscus, meniscal injuries pose a significant clinical challenge. The most common method for treatment of damaged meniscal tissues, meniscectomy, leads to improper loading within the knee joint, which can increase the risk of osteoarthritis. Thus, there is a clinical need for the development of constructs for meniscal repair that better replicate meniscal tissue organization to improve load distributions and function over time. Advanced three-dimensional bioprinting technologies such as suspension bath bioprinting provide some key advantages, such as the ability to support the fabrication of complex structures using non-viscous bioinks. In this work, the suspension bath printing process is utilized to print anisotropic constructs with a unique bioink that contains embedded hydrogel fibers that align via shear stresses during printing. Constructs with and without fibers are printed and then cultured for up to 56 din vitroin a custom clamping system. Printed constructs with fibers demonstrate increased cell and collagen alignment, as well as enhanced tensile moduli when compared to constructs printed without fibers. This work advances the use of biofabrication to develop anisotropic constructs that can be utilized for the repair of meniscal tissue.

Biophysical forces mediated by respiration maintain lung alveolar epithelial cell fate.

Lungs undergo mechanical strain during breathing, but how these biophysical forces affect cell fate and tissue homeostasis are unclear. We show that biophysical forces through normal respiratory motion actively maintain alveolar type 1 (AT1) cell identity and restrict these cells from reprogramming into AT2 cells in the adult lung. AT1 cell fate is maintained at homeostasis by Cdc42- and Ptk2-mediated actin remodeling and cytoskeletal strain, and inactivation of these pathways causes a rapid reprogramming into the AT2 cell fate. This plasticity induces chromatin reorganization and changes in nuclear lamina-chromatin interactions, which can discriminate AT1 and AT2 cell identity. Unloading the biophysical forces of breathing movements leads to AT1-AT2 cell reprogramming, revealing that normal respiration is essential to maintain alveolar epithelial cell fate. These data demonstrate the integral function of mechanotransduction in maintaining lung cell fate and identifies the AT1 cell as an important mechanosensor in the alveolar niche.