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.

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.

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.

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.

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.

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.

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.

Osteosarcoma-enriched transcripts paradoxically generate osteosarcoma-suppressing extracellular proteins.

Osteosarcoma (OS) is the common primary bone cancer that affects mostly children and young adults. To augment the standard-of-care chemotherapy, we examined the possibility of protein-based therapy using mesenchymal stem cells (MSCs)-derived proteomes and OS-elevated proteins. While a conditioned medium (CM), collected from MSCs, did not present tumor-suppressing ability, the activation of PKA converted MSCs into induced tumor-suppressing cells (iTSCs). In a mouse model, the direct and hydrogel-assisted administration of CM inhibited tumor-induced bone destruction, and its effect was additive with cisplatin. CM was enriched with proteins such as calreticulin, which acted as an extracellular tumor suppressor by interacting with CD47. Notably, the level of CALR transcripts was elevated in OS tissues, together with other tumor-suppressing proteins, including histone H4, and PCOLCE. PCOLCE acted as an extracellular tumor-suppressing protein by interacting with amyloid precursor protein, a prognostic OS marker with poor survival. The results supported the possibility of employing a paradoxical strategy of utilizing OS transcriptomes for the treatment of OS.

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.

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.

Editorial on the Special Issue "Advances in Nanogels".

In recent decades, the rise of nanotechnology has led to the design of innovative nano-biomaterials which are used to improve pharmacological therapies and assist with disease diagnosis [...].

Erratum: Preventing tumor progression to the bone by induced tumor-suppressing MSCs: Erratum.

[This corrects the article DOI: 10.7150/thno.58779.].

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.

Hydrolytically Degradable PEG-Based Inverse Electron Demand Diels-Alder Click Hydrogels.

Hydrogels cross-linked by inverse electron demand Diels-Alder (iEDDA) click chemistry are increasingly used in biomedical applications. With a few exceptions in naturally derived and chemically modified macromers, iEDDA click hydrogels exhibit long-term hydrolytic stability, and no synthetic iEDDA click hydrogels can undergo accelerated and tunable hydrolytic degradation. We have previously reported a novel method for synthesizing norbornene (NB)-functionalized multiarm poly(ethylene glycol) (PEG), where carbic anhydride (CA) was used to replace 5-norbornene-2-carboxylic acid. The new PEGNBCA-based thiol-norbornene hydrogels exhibited unexpected fast yet highly tunable hydrolytic degradation. In this contribution, we leveraged the new PEGNBCA macromer for forming iEDDA click hydrogels with [methyl]tetrazine ([m]Tz)-modified macromers, leading to the first group of synthetic iEDDA click hydrogels with highly tunable hydrolytic degradation kinetics. We further exploited Tz and mTz dual conjugation to achieve tunable hydrolytic degradation with an in vitro degradation time ranging from 2 weeks to 3 months. Finally, we demonstrated the excellent in vitro cytocompatibility and in vivo biocompatibility of the new injectable PEGNBCA-based iEDDA click cross-linked hydrogels.

Correction: Sun et al. Generation of the Chondroprotective Proteomes by Activating PI3K and TNFα Signaling. Cancers 2022, 14, 3039.

In the original article [...].

Generation of the Chondroprotective Proteomes by Activating PI3K and TNFα Signaling.

PURPOSE: To develop a novel treatment option for Chondrosarcoma (CS) and inflammatory arthritis, we evaluated a counterintuitive approach of activating tumorigenic and inflammatory signaling for generating joint-protective proteomes. METHODS: We employed mesenchymal stem cells and chondrocytes to generate chondroprotective proteomes by activating PI3K signaling and the administration of TNFα. The efficacy of the proteomes was examined using human and mouse cell lines as well as a mouse model of CS. The regulatory mechanism was analyzed using mass spectrometry-based whole-genome proteomics. RESULTS: While tumor progression and inflammatory responses were promoted by activating PI3K signaling and the administration of TNFα to CS cells and chondrocytes, those cells paradoxically generated a chondroprotective conditioned medium (CM). The application of CM downregulated tumorigenic genes in CS cells and TNFα and MMP13 in chondrocytes. Mechanistically, Hsp90ab1 was enriched in the chondroprotective CM, and it immunoprecipitated GAPDH. Extracellular GAPDH interacted with L1CAM and inhibited tumorigenic behaviors, whereas intracellular GAPDH downregulated p38 and exerted anti-inflammatory effects. CONCLUSIONS: We demonstrated that the unconventional approach of activating oncogenic and inflammatory signaling can generate chondroprotective proteomes. The role of Hsp90ab1 and GAPDH differed in their locations and they acted as the uncommon protectors of the joint tissue from tumor and inflammatory responses.

Dissolvable microgel-templated macroporous hydrogels for controlled cell assembly.

Mesenchymal stem cells (MSCs)-based therapies have been widely used to promote tissue regeneration and to modulate immune/inflammatory response. The therapeutic potential of MSCs can be further improved by forming multi-cellular spheroids. Meanwhile, hydrogels with macroporous structures are advantageous for improving mass transport properties for the cell-laden matrices. Herein, we report the fabrication of MSC-laden macroporous hydrogel scaffolds through incorporating rapidly dissolvable spherical cell-laden microgels. Dissolvable microgels were fabricated by tandem droplet-microfluidics and thiol-norbornene photopolymerization using a novel fast-degrading macromer poly(ethylene glycol)-norbornene-dopamine (PEGNB-Dopa). The cell-laden PEGNB-Dopa microgels were subsequently encapsulated within another bulk hydrogel matrix, whose porous structure was generated efficiently by the rapid degradation of the PEGNB-Dopa microgels. The cytocompatibility of this in situ pore-forming approach was demonstrated with multiple cell types. Furthermore, adjusting the stiffness and cell adhesiveness of the bulk hydrogels afforded the formation of solid cell spheroids or hollow spheres. The assembly of solid or hollow MSC spheroids led to differential activation of AKT pathway. Finally, MSCs solid spheroids formed in situ within the macroporous hydrogels exhibited robust secretion of HGF, VEGF-A, IL-6, IL-8, and TIMP-2. In summary, this platform provides an innovative method for forming cell-laden macroporous hydrogels for a variety of future biomedical applications.

Injectable Acylhydrazone-Linked RAFT Polymer Hydrogels for Sustained Protein Release and Cell Encapsulation.

A new class of temperature responsive polymer, termed PADO, is synthesized by reversible addition-fragmentation chain-transfer polymerization. Synthesized from copolymerization of diacetone acrylamide (DAAM), di(ethylene glycol) ethyl ether acrylate, and oligo(ethylene glycol) methyl ether acrylate, PADO polymer phase separates at temperature above its lower critical solution temperature (36-42 °C) due to enhanced hydrophobic interactions between the short ethylene glycol side chains. Solution of PADO polymers exhibit injectable shear-thinning properties and reach sol-gel transition rapidly (<5 min) at 37 °C. When the ketone moieties on DAAM are linked by adipic acid dihydrazdie, PADO polymers form crosslinked and injectable acylhydrazone hydrogels, which are hydrolytically degradable at a mild acidic environment owing to the pH sensitive acylhydrazone bonds. The pH-responsive degradation kinetics can be controlled by tuning polymer contents and ketone/hydrazide ratio. Importantly, the injectable PADO hydrogels are highly cytocompatible and can be easily formulated for pH-responsive sustained protein delivery.

Norbornene-functionalized methylcellulose as a thermo- and photo-responsive bioink.

Three-dimensional (3D) bioprinting has emerged as an important tool to fabricate scaffolds with complex structures for tissue engineering and regenerative medicine applications. For extrusion-based 3D bioprinting, the success of printing complex structures relies largely on the properties of bioink. Methylcellulose (MC) has been exploited as a potential bioink for 3D bioprinting due to its temperature-dependent rheological properties. However, MC is highly soluble and has low structural stability at room temperature, making it suboptimal for 3D bioprinting applications. In this study, we report a one-step synthesis protocol for modifying MC with norbornene (MCNB), which serves as a new bioink for 3D bioprinting. MCNB preserves the temperature-dependent reversible sol-gel transition and readily reacts with thiol-bearing linkers through light-mediated step-growth thiol-norbornene photopolymerization. Furthermore, we rendered the otherwise inert MC network bioactive through facile conjugation of integrin-binding ligands (e.g. CRGDS) or via incorporating cell-adhesive and protease-sensitive gelatin-based macromer (e.g. GelNB). The adaptability of the new MCNB-based bioink offers an attractive option for diverse 3D bioprinting applications.