Heparinized Gelatin-Based Hydrogels for Differentiation of Induced Pluripotent Stem Cells.

Chemically defined hydrogels are increasingly utilized to define the effects of extracellular matrix (ECM) components on cellular fate determination of human embryonic and induced pluripotent stem cell (hESC and hiPSCs). In particular, hydrogels cross-linked by orthogonal click chemistry, including thiol-norbornene photopolymerization and inverse electron demand Diels-Alder (iEDDA) reactions, are explored for 3D culture of hESC/hiPSCs owing to the specificity, efficiency, cytocompatibility, and modularity of the cross-linking reactions. In this work, we exploited the modularity of thiol-norbornene photopolymerization to create a biomimetic hydrogel platform for 3D culture and differentiation of hiPSCs. A cell-adhesive, protease-labile, and cross-linkable gelatin derivative, gelatin-norbornene (GelNB), was used as the backbone polymer for constructing hiPSC-laden biomimetic hydrogels. GelNB was further heparinized via the iEDDA click reaction using tetrazine-modified heparin (HepTz), creating GelNB-Hep. GelNB or GelNB-Hep was modularly cross-linked with either inert macromer poly(ethylene glycol)-tetra-thiol (PEG4SH) or another bioactive macromer-thiolated hyaluronic acid (THA). The formulations of these hydrogels were modularly tuned to afford biomimetic matrices with similar elastic moduli but varying bioactive components, enabling the understanding of each bioactive component on supporting hiPSC growth and ectodermal, mesodermal, and endodermal fate commitment under identical soluble differentiation cues.

Engineering Tools for Regulating Hypoxia in Tumour Models.

Major advances in the field of genomic technologies have led to an improvement in cancer diagnosis, classification and prognostication. However, many cancers remain incurable due to the development of drug resistance, minimal residual disease (MRD) and disease relapse, highlighting an incomplete understanding of the mechanisms underlying these processes. In recent years, the impact of non-genetic factors on neoplastic transformations has increasingly been acknowledged, and growing evidence suggests that low oxygen (O2 ) levels (ie hypoxia) in the tumour microenvironment play a critical role in the development and treatment of cancer. As a result, there is a growing need to develop research tools capable of reproducing physiologically relevant O2 conditions encountered by cancer cells in their natural environments in order to gain in-depth insight into tumour cell metabolism and function. In this review, the authors highlight the importance of hypoxia in the pathogenesis of malignant diseases and provide an overview of novel engineering tools that have the potential to further drive this evolving, yet technically challenging, field of cancer research.

Probing Osteocyte Functions in Gelatin Hydrogels with Tunable Viscoelasticity.

Bone is an attractive site for metastatic cancer cells and has been considered as "soil" for promoting tumor growth. However, accumulating evidence suggests that some bone cells (e.g., osteocytes) can actually suppress cancer cell migration and invasion via direct cell-cell contact and/or through cytokine secretion. Toward designing a biomimetic niche for supporting 3D osteocyte culture, we present here a gelatin-based hydrogel system with independently tunable matrix stiffness and viscoelasticity. In particular, we synthesized a bifunctional macromer, gelatin-norbornene-boronic acid (i.e., GelNB-BA), for covalent cross-linking with multifunctional thiol linkers [e.g., four-arm poly(ethylene glycol)-thiol or PEG4SH] to form thiol-NB hydrogels. The immobilized BA moieties in the hydrogel readily formed reversible boronate ester bonds with 1,3-diols on physically entrapped poly(vinyl alcohol) (PVA). Adjusting the compositions of GelNB-BA, PEG4SH, and PVA afforded hydrogels with independently tunable elasticity and viscoelasticity. With this new dynamic hydrogel platform, we investigated matrix mechanics-induced growth and cytokine secretion of encapsulated MLO-A5 pre-osteocytes. We discovered that more compliant or viscoelastic gels promoted A5 cell growth. On the other hand, cells encapsulated in stiffer gels secreted higher amounts of pro-inflammatory cytokines and chemokines. Finally, conditioned media (CM) collected from the encapsulated MLO-A5 cells (i.e., A5-CM) strongly inhibited breast cancer cell proliferation, invasion, and expression of tumor-activating genes. This new biomimetic hydrogel platform not only serves as a versatile matrix for investigating mechano-sensing in osteocytes but also provides a means to produce powerful anti-tumor CM.

Modular and Adaptable Tumor Niche Prepared from Visible Light Initiated Thiol-Norbornene Photopolymerization.

Photopolymerized biomimetic hydrogels with adaptable properties have been widely used for cell and tissue engineering applications. As a widely adopted gel cross-linking method, photopolymerization provides experimenters on-demand and spatial-temporal controls in gelation kinetics. Long wavelength ultraviolet (UV) light initiated photopolymerization is among the most popular methods in the fabrication of cell-laden hydrogels owing to its rapid and relatively mild gelation conditions. The use of UV light, however, still causes concerns regarding its potential negative impacts on cells. Alternatively, visible light based photopolymerization can be used to cross-link cell-laden hydrogels. The majority of visible light based gelation schemes involve photoinitiator, co-initiator, and comonomer. This multicomponent initiation system creates added challenges for optimizing hydrogel formulations. Here, we report a co-initiator/comonomer-free visible light initiated thiol-norbornene photopolymerization scheme to prepare modular biomimetic hydrogels suitable for in situ cell encapsulation. Eosin-Y was used as the sole initiator to initiate modular gelation between synthetic macromers (e.g., thiolated poly(vinyl alcohol) or poly(ethylene glycol)) and functionalized extracellular matrices (ECMs) including norbornene-functionalized gelatin (GelNB) or thiolated hyaluronic acid (THA). These components are modularly cross-linked to afford bioinert (i.e., purely synthetic), bioactive (i.e., using gelatin), and biomimetic (i.e., using gelatin and hyaluronic acid) hydrogels. The stiffness of the hydrogels can be easily tuned without affecting the contents of the bioactive components. Furthermore, the use of naturally derived biomacromolecules (e.g., gelatin and HA) renders these hydrogels susceptible to enzyme-mediated degradation. In addition to demonstrating efficient and tunable visible light mediated gelation, we also utilized this biomimetic modular gelation system to formulate artificial tumor niche and to study the effects of cell density and gel modulus on the formation of pancreatic ductal adenocarcinoma (PDAC) spheroids.

Photoclick Hydrogels Prepared from Functionalized Cyclodextrin and Poly(ethylene glycol) for Drug Delivery and in Situ Cell Encapsulation.

Polymers or hydrogels containing modified cyclodextrin (CD) are highly useful in drug delivery applications, as CD is a cytocompatible amphiphilic molecule that can complex with a variety of hydrophobic drugs. Here, we designed modular photoclick thiol-ene hydrogels from derivatives of ßCD and poly(ethylene glycol) (PEG), including ßCD-allylether (ßCD-AE), ßCD-thiol (ßCD-SH), PEG-thiol (PEGSH), and PEG-norbornene (PEGNB). Two types of CD-PEG hybrid hydrogels were prepared using radical-mediated thiol-ene photoclick reactions. Specifically, thiol-allylether hydrogels were formed by reacting multiarm PEGSH and ßCD-AE, and thiol-norbornene hydrogels were formed by cross-linking ßCD-SH and multiarm PEGNB. We characterized the properties of these two types of thiol-ene hydrogels, including gelation kinetics, gel fractions, hydrolytic stability, and cytocompatibility. Compared with thiol-allylether hydrogels, thiol-norbornene photoclick reaction formed hydrogels with faster gelation kinetics at equivalent macromer contents. Using curcumin, an anti-inflammatory and anticancer hydrophobic molecule, we demonstrated that CD-cross-linked PEG-based hydrogels, when compared with pure PEG-based hydrogels, afforded higher drug loading efficiency and prolonged delivery in vitro. Cytocompatibility of these CD-cross-linked hydrogels were evaluated by in situ encapsulation of radical sensitive pancreatic MIN6 ß-cells. All formulations and cross-linking conditions tested were cytocompatible for cell encapsulation. Furthermore, hydrogels cross-linked by ßCD-SH showed enhanced cell proliferation and insulin secretion as compared to gels cross-linked by either dithiothreitol (DTT) or ßCD-AE, suggesting the profound impact of both macromer compositions and gelation chemistry on cell fate in chemically cross-linked hydrogels.

Cell-cell communication mimicry with poly(ethylene glycol) hydrogels for enhancing beta-cell function.

A biomimetic hydrogel platform was designed to signal encapsulated cells using immobilized cell-cell communication cues, with a focus on enhancing the survival and function of encapsulated pancreatic ß-cells to treat type 1 diabetes. When MIN6 cells, a pancreatic ß-cell line, were encapsulated in poly(ethylene glycol) (PEG) hydrogels, their survival and glucose responsiveness to insulin were highly dependent on the cell-packing density. A minimum packing density of 10(7) cells/mL was necessary to maintain the survival of encapsulated ß-cells without the addition of material functionalities (e.g., cell adhesion ligands). While single cell suspensions can improve diffusion-limited mass transfer, direct cell-cell interactions are limited. Thus, thiolated EphA5-Fc receptor and ephrinA5-Fc ligand were conjugated into PEG hydrogels via a thiol-acrylate photopolymerization to render an otherwise inert PEG hydrogel bioactive. The biomimetic hydrogels presented here can provide crucial cell-cell communication signals for dispersed ß-cells and improve their survival and proliferation. Together with the cell-adhesive peptide RGDS, the immobilized fusion proteins (EphA5-Fc and ephrinA5-Fc) synergistically increased the survival of both MIN6 ß-cells and dissociated islet cells, both at a very low cell-packing density (< 2 × 10(6) cells/mL). This unique gel platform demonstrates new strategies for tailoring biomimetic environments to enhance the encapsulation of cells that require cell-cell contact to survive and function.

Degradable and Multifunctional PEG-Based Hydrogels Formed by iEDDA Click Chemistry with Stable Click-Induced Supramolecular Interactions.

The inverse electron demand Diels-Alder (iEDDA) reactions are highly efficient click chemistry increasingly utilized in bioconjugation, live cell labeling, and the synthesis and modification of biomaterials. iEDDA click reactions have also been used to cross-link tetrazine (Tz) and norbornene (NB) modified macromers [e.g., multiarm poly(ethylene glycol) or PEG]. In these hydrogels, Tz-NB adducts exhibit stable supramolecular interactions with a high hydrolytic stability. Toward engineering a new class of PEG-based click hydrogels with highly adaptable properties, we previously reported a new group of NB-derivatized PEG macromers via reacting hydroxyl-terminated PEG with carbic anhydride (CA). In this work, we show that hydrogels cross-linked by PEGNBCA or its derivatives exhibited fast and tunable hydrolytic degradation. Here, we show that PEGNBCA (either mono- or octafunctional) and its dopamine or tyramine conjugated derivatives (i.e., PEGNB-D and PEGNB-T) readily cross-link with 4-arm PEG-Tz to form a novel class of multifunctional iEDDA click hydrogels. Through modularly adjusting the macromers with unstable and stable iEDDA click-induced supramolecular interactions (iEDDA-CSI), we achieved highly tunable degradation, with full degradation in less than 2 weeks to over two months. We also show that secondary enzymatic reactions could dynamically stiffen these hydrogels. These hydrogels could also be spatiotemporally photopatterned through visible light-initiated photochemistry. Finally, the iEDDA-CSI hydrogels post ester hydrolysis displayed shear-thinning and self-healing properties, enabling injectable delivery.

Viscoelastic stiffening of gelatin hydrogels for dynamic culture of pancreatic cancer spheroids.

The tumor microenvironment (TME) in pancreatic adenocarcinoma (PDAC) is a complex milieu of cellular and non-cellular components. Pancreatic cancer cells (PCC) and cancer-associated fibroblasts (CAF) are two major cell types in PDAC TME, whereas the non-cellular components are enriched with extracellular matrices (ECM) that contribute to high stiffness and fast stress-relaxation. Previous studies have suggested that higher matrix rigidity promoted aggressive phenotypes of tumors, including PDAC. However, the effects of dynamic viscoelastic matrix properties on cancer cell fate remain largely unexplored. The focus of this work was to understand the effects of such dynamic matrix properties on PDAC cell behaviors, particularly in the context of PCC/CAF co-culture. To this end, we engineered gelatin-norbornene (GelNB) based hydrogels with a built-in mechanism for simultaneously increasing matrix elastic modulus and viscoelasticity. Two GelNB-based macromers, namely GelNB-hydroxyphenylacetic acid (GelNB-HPA) and GelNB-boronic acid (GelNB-BA), were modularly mixed and crosslinked with 4-arm poly(ethylene glycol)-thiol (PEG4SH) to form elastic hydrogels. Treating the hybrid hydrogels with tyrosinase not only increased the elastic moduli of the gels (due to HPA dimerization) but also concurrently produced 1,2-diols that formed reversible boronic acid-diol bonding with the BA groups on GelNB-BA. We employed patient-derived CAF and a PCC cell line COLO-357 to demonstrate the effect of increasing matrix stiffness and viscoelasticity on CAF and PCC cell fate. Our results indicated that in the stiffened environment, PCC underwent epithelial-mesenchymal transition. In the co-culture PCC and CAF spheroid, CAF enhanced PCC spreading and stimulated collagen 1 production. Through mRNA-sequencing, we further showed that stiffened matrices, regardless of the degree of stress-relaxation, heightened the malignant phenotype of PDAC cells. STATEMENT OF SIGNIFICANCE: The pancreatic cancer microenvironment is a complex milieu composed of various cell types and extracellular matrices. It has been suggested that stiffer matrices could promote aggressive behavior in pancreatic cancer, but the effect of dynamic stiffening and matrix stress-relaxation on cancer cell fate remains largely undefined. This study aimed to explore the impact of dynamic changes in matrix viscoelasticity on pancreatic ductal adenocarcinoma (PDAC) cell behavior by developing a hydrogel system capable of simultaneously increasing stiffness and stress-relaxation on demand. This is achieved by crosslinking two gelatin-based macromers through orthogonal thiol-norbornene photochemistry and post-gelation stiffening with mushroom tyrosinase. The results revealed that higher matrix stiffness, regardless of the degree of stress relaxation, exacerbated the malignant characteristics of PDAC cells.

Orthogonally Crosslinked Gelatin-Norbornene Hydrogels for Biomedical Applications.

The thiol-norbornene photo-click reaction has exceptionally fast crosslinking efficiency compared with chain-growth polymerization at equivalent macromer contents. The orthogonal reactivity between norbornene and thiol/tetrazine permits crosslinking of synthetic and naturally derived macromolecules with modularity, including poly(ethylene glycol) (PEG)-norbornene (PEGNB), gelatin-norbornene (GelNB), among others. For example, collagen-derived gelatin contains both cell adhesive motifs (e.g., Arg-Gly-Asp or RGD) and protease-labile sequences, making it an ideal macromer for forming cell-laden hydrogels. First reported in 2014, GelNB is increasingly used in orthogonal crosslinking of biomimetic matrices in various applications. GelNB can be crosslinked into hydrogels using multi-functional thiol linkers (e.g., dithiothreitol (DTT) or PEG-tetra-thiol (PEG4SH) via visible light or longwave ultraviolet (UV) light step-growth thiol-norbornene reaction or through an enzyme-mediated crosslinking (i.e., horseradish peroxidase, HRP). GelNB-based hydrogels can also be modularly crosslinked with tetrazine-bearing macromers via inverse electron-demand Diels-Alder (iEDDA) click reaction. This review surveys the various methods for preparing GelNB macromers, the crosslinking mechanisms of GelNB-based hydrogels, and their applications in cell and tissue engineering, including crosslinking of dynamic matrices, disease modeling, and tissue regeneration, delivery of therapeutics, as well as bioprinting and biofabrication.

Fast-relaxing hydrogels with reversibly tunable mechanics for dynamic cancer cell culture.

The mechanics of the tumor microenvironment (TME) significantly impact disease progression and the efficacy of anti-cancer therapeutics. While it is recognized that advanced in vitro cancer models will benefit cancer research, none of the current engineered extracellular matrices (ECM) adequately recapitulate the highly dynamic TME. Through integrating reversible boronate-ester bonding and dithiolane ring-opening polymerization, we fabricated synthetic polymer hydrogels with tumor-mimetic fast relaxation and reversibly tunable elastic moduli. Importantly, the crosslinking and dynamic stiffening of matrix mechanics were achieved in the absence of a photoinitiator, often the source of cytotoxicity. Central to this strategy was Poly(PEGA-co-LAA-co-AAPBA) (PELA), a highly defined polymer synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. PELA contains dithiolane for initiator-free gel crosslinking, stiffening, and softening, as well as boronic acid for complexation with diol-containing polymers to give rise to tunable viscoelasticity. PELA hydrogels were highly cytocompatible for dynamic culture of patient-derived pancreatic cancer cells. It was found that the fast-relaxing matrix induced mesenchymal phenotype of cancer cells, and dynamic matrix stiffening restricted tumor spheroid growth. Moreover, this new dynamic viscoelastic hydrogel system permitted sequential stiffening and softening to mimic the physical changes of TME.

Enhancing anti-tumor potential: low-intensity vibration suppresses osteosarcoma progression and augments MSCs' tumor-suppressive abilities.

Rationale: Osteosarcoma (OS), a common malignant bone tumor, calls for the investigation of novel treatment strategies. Low-intensity vibration (LIV) presents itself as a promising option, given its potential to enhance bone health and decrease cancer susceptibility. This research delves into the effects of LIV on OS cells and mesenchymal stem cells (MSCs), with a primary focus on generating induced tumor-suppressing cells (iTSCs) and tumor-suppressive conditioned medium (CM). Methods: To ascertain the influence of vibration frequency, we employed numerical simulations and conducted experiments to determine the most effective LIV conditions. Subsequently, we generated iTSCs and CM through LIV exposure and assessed the impact of CM on OS cells. We also explored the underlying mechanisms of the tumor-suppressive effects of LIV-treated MSC CM, with a specific focus on vinculin (VCL). We employed cytokine array, RNA sequencing, and Western blot techniques to investigate alterations in cytokine profiles, transcriptomes, and tumor suppressor proteins. Results: Numerical simulations validated LIV frequencies within the 10-100 Hz range. LIV induced notable morphological changes in OS cells and MSCs, confirming its dual role in inhibiting OS cell progression and promoting MSC conversion into iTSCs. Upregulated VCL expression enhanced MSC responsiveness to LIV, significantly bolstering CM's efficacy. Notably, we identified tumor suppressor proteins in LIV-treated CM, including procollagen C endopeptidase enhancer (PCOLCE), histone H4 (H4), peptidylprolyl isomerase B (PPIB), and aldolase A (ALDOA). Consistently, cytokine levels decreased significantly in LIV-treated mouse femurs, and oncogenic transcript levels were downregulated in LIV-treated OS cells. Moreover, our study demonstrated that combining LIV-treated MSC CM with chemotherapy drugs yielded additive anti-tumor effects. Conclusions: LIV effectively impeded the progression of OS cells and facilitated the transformation of MSCs into iTSCs. Notably, iTSC-derived CM demonstrated robust anti-tumor properties and the augmentation of MSC responsiveness to LIV via VCL. Furthermore, the enrichment of tumor suppressor proteins within LIV-treated MSC CM and the reduction of cytokines within LIV-treated isolated bone underscore the pivotal tumor-suppressive role of LIV within the bone tumor microenvironment.

Effect of 3D matrix compositions on the efficacy of EGFR inhibition in pancreatic ductal adenocarcinoma cells.

Therapeutics to inhibit signaling of epidermal growth factor receptor (EGFR) has been suggested as a potential treatment for pancreatic cancers, and two-dimensional (2D) cell culture techniques are commonly used to identify and/or verify the therapeutic efficacy of EGFR inhibitors. However, drug targets identified from conventional cell culture techniques may not exhibit desired functions when these drugs are tested in animal studies, in large part due to the complicated tumor microenvironments. Hence, it is crucial to develop a biomimetic cell culture system capable of recapitulating aspects of tumor niches for studying cancer cell fate processes under the influence of various environmental stimuli. In this study, we utilized a versatile PEG-peptide hydrogel system to demonstrate the influence of matrix properties and EGFR inhibition on the growth of a pancreatic ductal adenocarcinoma cell line (PANC-1). PANC-1 cells were encapsulated in 8-arm PEG-norbornene (PEG8NB) hydrogels cross-linked by matrix metalloproteinase (MMP) sensitive peptide (MMP(Linker)) using thiol-ene photoclick chemistry. In soft hydrogels (G' ~ 2 kPa), cells retained high initial viability and formed clusters after prolonged culture, whereas cells encapsulated in stiff hydrogels (G' ~ 12 kPa) exhibited lower initial viability and reduced proliferation. While the immobilization of an EGFR peptide inhibitor, Asn-Tyr-Gln-Gln-Asn or NYQQN, in soft hydrogels did not cause cell death, this peptide induced significant cell apoptosis when immobilized in stiff hydrogels. Western blotting results showed that cell death was due to reduced expression of EGFR and Akt in stiff hydrogels under the influence of immobilized NYQQN peptide. These results shed light on the importance and non-negligible role of matrix properties on the efficacy of antitumor drugs.

Visible-light-mediated thiol-ene hydrogelation using eosin-Y as the only photoinitiator.

The utility of visible-light-mediated polymerization in tissue engineering has been limited due to the necessary use of potentially cytotoxic coinitiator and comonomer. Here, we report a visible-light-mediated thiol-ene hydrogelation scheme using eosin-Y as the only photoinitiator. Under visible light exposure, rapid and highly tunable step-growth gelation is achieved using PEG-norbornene and a model cross-linker dithiothreitol. In addition to investigating the gelation kinetics and properties of thiol-ene hydrogels formed by this new gelation scheme, we also report high cytocompatibility of these hydrogels using human mesenchymal stem cells (hMSCs) and pancreatic MIN6 ß-cells.

Facile preparation of photodegradable hydrogels by photopolymerization.

Photodegradable hydrogels have emerged as a powerful material platform for studying and directing cell behaviors, as well as for delivering drugs. The premise of this technique is to use a cytocompatible light source to cleave linkers within a hydrogel, thus causing reduction of matrix stiffness or liberation of matrix-tethered biomolecules in a spatial-temporally controlled manner. The most commonly used photodegradable units are molecules containing nitrobenzyl moieties that absorb light in the ultraviolet (UV) to lower visible wavelengths (~280 to 450 nm). Because photodegradable linkers and hydrogels reported in the literature thus far are all sensitive to UV light, highly efficient UV-mediated photopolymerizations are less likely to be used as the method to prepare these hydrogels. As a result, currently available photodegradable hydrogels are formed by redox-mediated radical polymerizations, emulsion polymerizations, Michael-type addition reactions, or orthogonal click chemistries. Here, we report the first photodegradable poly(ethylene glycol)-based hydrogel system prepared by step-growth photopolymerization. The model photolabile peptide cross-linkers, synthesized by conventional solid phase peptide synthesis, contained terminal cysteines for step-growth thiol-ene photo-click reactions and a UV-sensitive 2-nitrophenylalanine residue in the peptide backbone for photo-cleavage. Photolysis of this peptide was achieved through adjusting UV light exposure time and intensity. Photopolymerization of photodegradable hydrogels containing photolabile peptide cross-linkers was made possible via a highly efficient visible light-mediated thiol-ene photo-click reaction using a non-cleavage type photoinitiator eosin-Y. Rapid gelation was confirmed by in situ photo-rheometry. Flood UV irradiation at controlled wavelength and intensity was used to demonstrate the photodegradability of these photopolymerized hydrogels.

Knee loading reduces MMP13 activity in the mouse cartilage.

BACKGROUND: Moderate loads with knee loading enhance bone formation, but its effects on the maintenance of the knee are not well understood. In this study, we examined the effects of knee loading on the activity of matrix metalloproteinase13 (MMP13) and evaluated the role of p38 MAPK and Rac1 GTPase in the regulation of MMP13. METHODS: Knee loading (0.5-3 N for 5 min) was applied to the right knee of surgically-induced osteoarthritis (OA) mice as well as normal (non-OA) mice, and MMP13 activity in the femoral cartilage was examined. The sham-loaded knee was used as a non-loading control. We also employed primary non-OA and OA human chondrocytes as well as C28/I2 chondrocyte cells, and examined MMP13 activity and molecular signaling in response to shear at 2-20 dyn/cm². RESULTS: Daily knee loading at 1 N for 2 weeks suppressed cartilage destruction in the knee of OA mice. Induction of OA elevated MMP13 activity and knee loading at 1 N suppressed this elevation. MMP13 activity was also increased in primary OA chondrocytes, and this increase was attenuated by applying shear at 10 dyn/cm². Load-driven reduction in MMP13 was associated with a decrease in the phosphorylation level of p38 MAPK (p-p38) and NFκB (p-NFκB). Molecular imaging using a fluorescence resonance energy transfer (FRET) technique showed that Rac1 activity was reduced by shear at 10 dyn/cm² and elevated by it at 20 dyn/cm². Silencing Rac1 GTPase significantly reduced MMP13 expression and p-p38 but not p-NFκB. Transfection of a constitutively active Rac1 GTPase mutant increased MMP13 activity, while a dominant negative mutant decreased it. CONCLUSIONS: Knee loading reduces MMP13 activity at least in part through Rac1-mediated p38 MAPK signaling. This study suggests the possibility of knee loading as a therapy not only for strengthening bone but also preventing tissue degradation of the femoral cartilage.

Digital Light Processing 3D Bioprinting of Gelatin-Norbornene Hydrogel for Enhanced Vascularization.

Digital light processing (DLP) bioprinting can be used to fabricate volumetric scaffolds with intricate internal structures, such as perfusable vascular channels. The successful implementation of DLP bioprinting in tissue fabrication requires using suitable photo-reactive bioinks. Norbornene-based bioinks have emerged as an attractive alternative to (meth)acrylated macromers in 3D bioprinting owing to their mild and rapid reaction kinetics, high cytocompatibility for in situ cell encapsulation, and adaptability for post-printing modification or conjugation of bioactive motifs. In this contribution, the development of gelatin-norbornene (GelNB) is reported as a photo-cross-linkable bioink for DLP 3D bioprinting. Low concentrations of GelNB (2-5 wt.%) and poly(ethylene glycol)-tetra-thiol (PEG4SH) are DLP-printed with a wide range of stiffness (G' ≈120 to 4000 Pa) and with perfusable channels. DLP-printed GelNB hydrogels are highly cytocompatible, as demonstrated by the high viability of the encapsulated human umbilical vein endothelial cells (HUVECs). The encapsulated HUVECs formed an interconnected microvascular network with lumen structures. Notably, the GelNB bioink permitted both in situ tethering and secondary conjugation of QK peptide, a vascular endothelial growth factor (VEGF)-mimetic peptide. Incorporation of QK peptide significantly improved endothelialization and vasculogenesis of the DLP-printed GelNB hydrogels, reinforcing the applicability of this bioink system in diverse biofabrication applications.

Poly(ethylene glycol)-Norbornene as a Photoclick Bioink for Digital Light Processing 3D Bioprinting.

Digital light processing (DLP) bioprinting is an emerging technology for three-dimensional bioprinting (3DBP) owing to its high printing fidelity, fast fabrication speed, and higher printing resolution. Low-viscosity bioinks such as poly(ethylene glycol) diacrylate (PEGDA) are commonly used for DLP-based bioprinting. However, the cross-linking of PEGDA proceeds via chain-growth photopolymerization that displays significant heterogeneity in cross-linking density. In contrast, step-growth thiol-norbornene photopolymerization is not oxygen inhibited and produces hydrogels with an ideal network structure. The high cytocompatibility and rapid gelation of thiol-norbornene photopolymerization have lent itself to the cross-linking of cell-laden hydrogels but have not been extensively used for DLP bioprinting. In this study, we explored eight-arm PEG-norbornene (PEG8NB) as a bioink/resin for visible light-initiated DLP-based 3DBP. The PEG8NB-based DLP resin showed high printing fidelity and cytocompatibility even without the use of any bioactive motifs and high initial stiffness. In addition, we demonstrated the versatility of the PEGNB resin by printing solid structures as cell culture devices, hollow channels for endothelialization, and microwells for generating cell spheroids. This work not only expands the selection of bioinks for DLP-based 3DBP but also provides a platform for dynamic modification of the bioprinted constructs.

Photo-click hydrogels for 3D in situ differentiation of pancreatic progenitors from induced pluripotent stem cells.

BACKGROUND: Induced pluripotent stem cells (iPSC) can be differentiated to cells in all three germ layers, as well as cells in the extraembryonic tissues. Efforts in iPSC differentiation into pancreatic progenitors in vitro have largely been focused on optimizing soluble growth cues in conventional two-dimensional (2D) culture, whereas the impact of three-dimensional (3D) matrix properties on the morphogenesis of iPSC remains elusive. METHODS: In this work, we employ gelatin-based thiol-norbornene photo-click hydrogels for in situ 3D differentiation of human iPSCs into pancreatic progenitors (PP). Molecular analysis and single-cell RNA-sequencing were utilized to elucidate on the distinct identities of subpopulations within the 2D and 3D differentiated cells. RESULTS: We found that, while established soluble cues led to predominately PP cells in 2D culture, differentiation of iPSCs using the same soluble factors led to prominent branching morphogenesis, ductal network formation, and generation of diverse endoderm populations. Through single-cell RNA-sequencing, we found that 3D differentiation resulted in enrichments of pan-endodermal cells and ductal cells. We further noted the emergence of a group of extraembryonic cells in 3D, which was absent in 2D differentiation. The unexpected emergence of extraembryonic cells in 3D was found to be associated with enrichment of Wnt and BMP signaling pathways, which may have contributed to the emergence of diverse cell populations. The expressions of PP signature genes PDX1 and NKX6.1 were restored through inhibition of Wnt signaling at the beginning of the posterior foregut stage. CONCLUSIONS: To our knowledge, this work established the first 3D hydrogel system for in situ differentiation of human iPSCs into PPs.

Photo-click hydrogels for 3D in situ differentiation of pancreatic progenitors from induced pluripotent stem cells.

Background Induced pluripotent stem cells (iPSC) can be differentiated to cells in all three germ layers, as well as cells in the extraembryonic tissues. Efforts in iPSC differentiation into pancreatic progenitors in vitro have largely been focused on optimizing soluble growth cues in conventional two-dimensional (2D) culture, whereas the impact of three-dimensional (3D) matrix properties on the morphogenesis of iPSC remains elusive. Methods In this work, we employ gelatin-based thiol-norbornene photo-click hydrogels for in situ 3D differentiation of human iPSCs into pancreatic progenitors (PP). Molecular analysis and single cell RNA-sequencing were utilized to elucidate on the distinct identities of subpopulations within the 2D and 3D differentiated cells. Results We found that, while established soluble cues led to predominately PP cells in 2D culture, differentiation of iPSCs using the same soluble factors led to prominent branching morphogenesis, ductal network formation, and generation of diverse endoderm populations. Through single-cell RNA-sequencing, we found that 3D differentiation resulted in enrichments of pan-endodermal cells and ductal cells. We further noted the emergence of a group of extraembryonic cells in 3D, which was absent in 2D differentiation. The unexpected emergence of extraembryonic cells in 3D was found to be associated with enrichment of Wnt and BMP signaling pathways, which may have contributed to the emergence of diverse cell populations. The expressions of PP signature genes PDX1 and NKX6.1 were restored through inhibition of Wnt signaling at the beginning of the posterior foregut stage. Conclusions To our knowledge, this work established the first 3D hydrogel system for in situ differentiation of human iPSCs into PPs. Ongoing work focuses on enhancing pancreatic differentiation efficiency through modulating physicochemical properties of the iPSC-laden matrices.

Viscoelastic hydrogels for interrogating pancreatic cancer-stromal cell interactions.

The tumor microenvironment (TME) is known to direct cancer cell growth, migration, invasion into the matrix and distant tissues, and to confer drug resistance in cancer cells. While multiple aspects of TME have been studied using in vitro, ex vivo, and in vivo tumor models and engineering tools, the influence of matrix viscoelasticity on pancreatic cancer cells and its associated TME remained largely unexplored. In this contribution, we synthesized a new biomimetic hydrogel with tunable matrix stiffness and stress-relaxation for evaluating the effect of matrix viscoelasticity on pancreatic cancer cell (PCC) behaviors in vitro. Using three simple monomers and Reverse-Addition Fragmentation Chain-Transfer (RAFT) polymerization, we synthesized a new class of phenylboronic acid containing polymers (e.g., poly (OEGA-s-HEAA-s-APBA) or PEHA). Norbornene group was conjugated to HEAA on PEHA via carbic anhydride, affording a new NB and BA dually modified polymer - PEHNBA amenable for orthogonal thiol-norbornene photopolymerization and boronate ester diol complexation. The former provided tunable matrix elasticity, while the latter gave rise to matrix stress-relaxation (or viscoelasticity). The new PEHNBA polymers were shown to be highly cytocompatible for in situ encapsulation of PCCs and cancer-associated fibroblasts (CAFs). Furthermore, we demonstrated that hydrogels with high stress-relaxation promoted spreading of CAFs, which in turns promoted PCC proliferation and spreading in the viscoelastic matrix. Compared with elastic matrix, viscoelastic gels upregulated the secretion of soluble proteins known to promote epithelial-mesenchymal transition (EMT). This study demonstrated the crucial influence of matrix viscoelasticity on pancreatic cancer cell fate and provided an engineered viscoelastic matrix for future studies and applications related to TME.