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Decellularized extracellular matrix (dECM) hydrogels provide tissue-specific microenvironments which accommodate physiological cellular phenotypes in 3D in vitro cell cultures. However, their formation hinges on collagen fibrillogenesis, a complex process which limits regulation of physicochemical properties. Hence, achieving reproducible results with dECM hydrogels poses as a challenge. Here, we demonstrate that thiolation of solubilized liver dECM enables rapid formation of covalently crosslinked hydrogels via Michael-type addition, allowing for precise control over mechanical properties and superior organotypic biological activity. Investigation of various decellularization methodologies revealed that treatment of liver tissue with Triton X-100 and ammonium hydroxide resulted in near complete DNA removal with significant retention of the native liver proteome. Chemical functionalization of pepsin-solubilized liver dECMs via 1-ethyl-3(3-dimethylamino)propyl carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling of l-Cysteine created thiolated liver dECM (dECM-SH), which rapidly reacted with 4-arm polyethylene glycol (PEG)-maleimide to form optically clear hydrogels under controlled conditions. Importantly, Young's moduli could be precisely tuned between 1 - 7 kPa by varying polymer concentrations, enabling close replication of healthy and fibrotic liver conditions in in vitro cell cultures. Click dECM-SH hydrogels were cytocompatible, supported growth of HepG2 and HepaRG liver cells, and promoted liver-specific functional phenotypes as evidenced by increased metabolic activity, as well CYP1A2 and CYP3A4 activity and excretory function when compared to monolayer culture and collagen-based hydrogels. Our findings demonstrate that click-functionalized dECM hydrogels offer a highly controlled, reproducible alternative to conventional tissue-derived hydrogels for in vitro cell culture applications. STATEMENT OF SIGNIFICANCE: Traditional dECM hydrogels face challenges in reproducibility and mechanical property control due to variable crosslinking processes. We introduce a click hydrogel based on porcine liver decellularized extracellular matrix (dECM) that circumnavigates these challenges. After optimizing liver decellularization for ECM retention, we integrated thiol-functionalized liver dECM with polyethylene-glycol derivatives through Michael-type addition click chemistry, enabling rapid, room-temperature gelation. This offers enhanced control over the hydrogel's mechanical and biochemical properties. The resultant click dECM hydrogels mimic the liver's natural ECM and exhibit greater mechanical tunability and handling ease, facilitating their application in high-throughput and industrial settings. Moreover, these hydrogels significantly improve the function of HepaRG-derived hepatocytes in 3D culture, presenting an advancement for liver tissue cell culture models for drug testing applications., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Christoph Meinert reports a relationship with Gelomics Pty Ltd that includes: board membership, employment, and equity or stocks. Dietmar W. Hutmacher reports a relationship with Gelomics Pty Ltd that includes: equity or stocks. Jordan W. Davern reports a relationship with Gelomics Pty Ltd that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Extrusion-based bioprinting has gained widespread popularity in biofabrication due to its ability to assemble cells and biomaterials in precise patterns and form tissue-like constructs. To achieve this, bioinks must have rheological properties suitable for printing while maintaining cytocompatibility. However, many commonly used biomaterials do not meet the rheological requirements and therefore require modification for bioprinting applications. This study demonstrates the incorporation of Laponite-RD (LPN) into gelatin methacryloyl (GelMA) to produce highly customizable bioinks with desired rheological and mechanical properties for extrusion-based bioprinting. Bioink formulations were based on GelMA (5%-15% w/v) and LPN (0%-4% w/v), and a comprehensive rheological design was applied to evaluate key rheological properties necessary for extrusion-based bioprinting. The results showed that GelMA bioinks with LPN (1%-4% w/v) exhibited pronounced shear thinning and viscoelastic behavior, as well as improved thermal stability. Furthermore, a concentration window of 1%-2% (w/v) LPN to 5%-15% GelMA demonstrated enhanced rheological properties and printability required for extrusion-based bioprinting. Construct mechanical properties were highly tunable by varying polymer concentration and photocrosslinking parameters, with Young's moduli ranging from ∼0.2 to 75 kPa. Interestingly, at higher Laponite concentrations, GelMA cross-linking was inhibited, resulting in softer hydrogels. High viability of MCF-7 breast cancer cells was maintained in both free-swelling droplets and printed hydrogels, and metabolically active spheroids formed over 7 days of culture in all conditions. In summary, the addition of 1%-2% (w/v) LPN to gelatin-based bioinks significantly enhanced rheological properties and retained cell viability and proliferation, suggesting its suitability for extrusion-based bioprinting., Competing Interests: C.M. is a shareholder and the Chief Executive Officer of Gelomics Pty Ltd. T.J.K. is a shareholder of Gelomics Pty. Ltd and J.W.D. is an employee of Gelomics Pty Ltd., (© 2024 Author(s).)

Hydrogels provide a promising platform for cartilage repair and regeneration. Although hydrogels have shown some efficacy, they still have shortcomings including poor mechanical properties and suboptimal integration with surrounding cartilage. Herein, hydrogels that are injectable, cytocompatible, mechanically robust, and highly adhesive to cartilage are developed. This approach uses GelMA-glycol chitosan (GelMA-GC) that is crosslinkable with visible light and photoinitiators (lithium acylphosphinate and tris (2,2'-bipyridyl) dichlororuthenium (II) hexahydrate ([RuII(bpy) 3 ] 2+ and sodium persulfate (Ru/SPS)). Ru/SPS-cross-linked hydrogels have higher compressive and tensile modulus, and most prominently higher adhesive strength with cartilage, which also depends on inclusion of GC. Tensile and push-out tests of the Ru/SPS-cross-linked GelMA-GC hydrogels demonstrate adhesive strength of ≈100 and 46 kPa, respectively. Hydrogel precursor solutions behave in a Newtonian manner and are injectable. After injection in focal bovine cartilage defects and in situ cross-linking, this hydrogel system remains intact and integrated with cartilage following joint manipulation ex vivo. Cells remain viable (>85%) in the hydrogel system and further show tissue regeneration potential after three weeks of in vitro culture. These preliminary results provide further motivation for future research on bioadhesive hydrogels for cartilage repair and regeneration., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Untreated osteochondral defects are a leading cause of osteoarthritis, a condition that places a heavy burden on both patients and orthopedic surgeons. Although tissue engineering has shown promise for creating mechanically similar cartilage-like constructs, their integration with cartilage remains elusive. Therefore, a formulation of biodegradable, biocompatible biomaterial with sufficient mechanical and adhesive properties for cartilage repair is required. To accomplish this, we prepared biocompatible, photo-curable, mechanically robust, and highly adhesive GelMA-glycol chitosan (GelMA-GC) hydrogels. GelMA-GC hydrogels had a modulus of 283 kPa and provided a biocompatible environment (>70% viability of embedded chondrocytes) in long-term culture within a bovine cartilage ring. The adhesive strength of bovine chondrocyte-laden GelMA-GC hydrogel to bovine cartilage increased from 38 to 52 kPa over four weeks of culture. Moreover, intermittent uniaxial mechanical stimulation enhanced the adhesive strength to ∼60 kPa, indicating that the cartilage-hydrogel integration could remain secure and functional under dynamic loading conditions. Furthermore, gene expression data and immunofluorescence staining revealed the capacity of chondrocytes in GelMA-GC hydrogel to synthesize chondrogenic markers (COL2A1 and ACAN), suggesting the potential for tissue regeneration. The promising in vitro results of this work motivate further exploration of the potential of photo-curable GelMA-GC bioadhesive hydrogels for cartilage repair and regeneration., Competing Interests: T. J. Klein and C. Meinert are co-founders and shareholders of Gelomics Pty Ltd. C. Meinert is also the Chief Executive Officer of Gelomics Pty Ltd. A. Weekes and J. W. Davern are employees of Gelomics Pty Ltd., (© 2023 Author(s).)

Basement membrane extracts (BME) derived from Engelbreth-Holm-Swarm (EHS) mouse sarcomas such as Matrigel ® remain the gold standard extracellular matrix (ECM) for three-dimensional (3D) cell culture in cancer research. Yet, BMEs suffer from substantial batch-to-batch variation, ill-defined composition, and lack the ability for physichochemical manipulation. Here, we developed a novel 3D cell culture system based on thiolated gelatin (Gel-SH), an inexpensive and highly controlled raw material capable of forming hydrogels with a high level of biophysical control and cell-instructive bioactivity. We demonstrate the successful thiolation of gelatin raw materials to enable rapid covalent crosslinking upon mixing with a synthetic poly(ethylene glycol) (PEG)-based crosslinker. The mechanical properties of the resulting gelatin-based hydrogels were readily tuned by varying precursor material concentrations, with Young's moduli ranging from ~2.5 to 5.8 kPa. All hydrogels of varying stiffnesses supported the viability and proliferation of MDA-MB-231 and MCF-7 breast cancer cell lines for 14 and 21 days of cell culture, respectively. Additionally, the gelatin-based hydrogels supported the growth, viability, and osteogenic differentiation of patient-derived preosteoblasts over 28 days of culture. Collectively, our data demonstrate that gelatin-based biomaterials provide an inexpensive and tunable 3D cell culture platform that may overcome the limitations of traditional BMEs.