Crosslinking strategies govern the morphology and biological performance of decellularized extracellular matrix particle–tyramine-modified hyaluronic acid hydrogels
HRP/Eo crosslinking method is identified as a favorable strategy for engineering dECM particle–THA hydrogels for cartilage regeneration and improved mechanical performance and chondrocyte redifferentiation effect compared to SPS/Ru, attributed to the difference in hydrogel morphology.
Abstract
Decellularized extracellular matrix (dECM) particle-based hydrogels hold promise for cartilage repair. Due to its chondropermissive properties, tuneable viscoelastic profile, and the potential to form covalent bonds with ECM proteins, tyramine-modified hyaluronic acid (THA) is an ideal candidate as a binder for dECM particles. However, the incorporation of the dECM particles into a viscoelastic hydrogel may interfere with its crosslinking. The goal of this study was to compare two crosslinking methods: (1) horseradish peroxidase/Eosin (HRP/Eo) with H2O2 and (2) sodium persulfate/ruthenium (SPS/Ru) for fabricating THA hydrogels containing dECM particles, and to investigate their biological and biomechanical effect for cartilage tissue engineering. Incorporation of dECM markedly enhanced the mechanical properties of the composites in both crosslinking methods, increasing the storage modulus by 10-fold, while leaving the linear viscoelastic region unchanged. Chondrocyte-laden dECM hydrogels demonstrated sustained cytocompatibility over 14 days, evidenced by stable DNA content throughout the culture period. Compared with SPS/Ru, hydrogels showed more effective incorporation of dECM particles into the HRP/Eo-crosslinked network, as indicated by a lower I∼1630/I∼1030 ratio from FTIR analysis, clear morphological changes in confocal microscopy, higher glycosaminoglycan (GAG) and collagen retention, as well as slower GAG and collagen release. HRP/Eo-dECM hydrogels promoted stronger gene expression of type II collagen (COL2) and higher compressive modulus compared with SPS/Ru-dECM hydrogels. In summary, HRP/Eo crosslinked dECM hydrogels demonstrated improved mechanical performance and chondrocyte redifferentiation effect compared to SPS/Ru, attributed to the difference in hydrogel morphology. These findings identify the HRP/Eo crosslinking method as a favorable strategy for engineering dECM particle–THA hydrogels for cartilage regeneration.
Hydrogels combining the biochemical complexity of the native extracellular matrix (ECM) with the tunable properties of protein-based biomaterials are promising for neural tissue engineering. In this study, decellularized spinal cord meninges (dSCM) were combined with water-soluble hydrophilic silk fibroin (hSF) and enzymatically crosslinked using a horseradish peroxidase/H2O2 system to develop composite hydrogels. A detergent-free, sonication-assisted decellularization method effectively removed cellular components while preserving matrix integrity, reducing residual double-stranded DNA to below 50 ng mg-1 dry weight and retaining key ECM constituents, including collagen and glycosaminoglycans. Hydrogels prepared at different dSCM:hSF ratios showed composition-dependent structural and mechanical behavior, with the 1:0.5 and 1:1 formulations exhibiting the most favorable compressive stiffness and viscoelastic performance. Structural, thermal, and morphological analyses further indicated that hSF incorporation improved matrix stability and contributed to more controlled swelling and degradation behavior. Biological evaluation showed that the 1:0.5 formulation promoted neovascularization in the chorioallantoic membrane assay without evident adverse inflammatory response. In addition, SH-SY5Y cells maintained high viability and showed increased expression of the neuronal-associated markers β-III tubulin and MAP2 over time. Overall, these findings suggest that dSCM:hSF hydrogels provide a promising platform for neural tissue engineering.
Tugce Kurt, Burak Derkuş, Y. E. Arslan· ACS Biomaterials Science & E...· 0 citations
Results highlight the critical interplay between macromolecular composition, 3D microenvironment, and cell response, demonstrating that GelMA+TA systems represent a promising preliminary platform for the development of tunable bioactive scaffolds for wound-related applications.
Leonor Resina, M. M. Pérez‐Madrigal, Carlos Alemán· International Journal of Bio...· 0 citations
Three-dimensional (3D) cell culture using microcarriers is an effective strategy for scalable cell expansion; however, conventional enzymatic detachment can compromise cell viability, surface proteins, and native signalling. We report viscoelasticity-tuneable hyaluronic acid (HA)-gelatin microspheres as microcarriers, engineered with a thermoresponsive polymer coating to enhance cell attachment and enable gentle harvesting. Gelatin-only (GLA), gelatin-HA (G-HA), and gelatin-HA-L-lysine (G-HA-L) microspheres were fabricated. HA incorporation and lysine functionalization were used to tune microsphere mechanics and interfacial stability. Frequencysweep rheology revealed that HA-containing formulations exhibited higher elastic dominance (G' > G″) and a broader, more stable viscoelastic response than gelatin-only and a commercial gelatin microcarrier benchmark, with G-HA-L showing the most favorable balance of stiffness and damping (highest G'/G″ across the tested window). The microspheres were subsequently coated with poly (N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-AAc)), producing a temperature-responsive interface. Importantly, the thermoresponsive coating enhanced early cell attachment, particularly on G-HA-L (reaching ~70% within 4 h and approaching ~90% by 24 h), outperforming both coated G-HA and commercial microcarriers. For harvesting, lowtemperature conditioning markedly improved cell release and recovery compared to trypsinonly controls, consistent with temperature-triggered polymer swelling facilitating detachment. Collectively, these results demonstrate that coupling viscoelastic microcarrier design with thermoresponsive surface engineering provides a promising platform for efficient cell growth and gentle, process-friendly harvesting, with potential applications in tissue engineering and regenerative medicine.
Rizka Musdalifah Amsar, Xiangqiang Lin, Jun-Sheng Wang et al.· Biofabrication· 0 citations
Semi-Interpenetrating Polymer Network (SIPN) hydrogels are promising soft materials for numerous applications, owing to their enhanced robustness and high swelling capacity. We designed biocompatible SIPNs by entangling linear hydroxyethyl cellulose (HEC) macromolecules within a covalently cross-linked poly(acrylic acid-co-2-hydroxyethyl methacrylate) matrix. We investigate the roles of different crosslinkers and the impact of accelerator amounts on the swelling behavior and mechanical performance. The hydrogels exhibit a strong pH-dependent response, with a maximum swelling capacity (%Qmax) reaching 2570% in neutral media, while showing significant deswelling at pH < 4. We find that maximum swelling decreases with increasing crosslinker concentration, which is paralleled by a significant increase in hydrogel stiffness. Interestingly, higher accelerator concentrations simultaneously increase both the swelling capacity and mechanical robustness under shear. The microstructure is investigated via histological techniques, and cell viability is tested using fibroblasts on optimal formulations. Significant cell viability, attachment, and migration were achieved by optimizing methacrylic acid (MAA) concentration to prevent medium acidification. Our results demonstrate the remarkable tunability of these SIPN hydrogels, a key advantage for applications ranging from soft robotics to tissue engineering. Furthermore, this work clearly illustrates the complex requirements for successful cell proliferation in hydrogels, emphasizing the need for not only biocompatible polymers, but also for optimal mechanical and precise microstructural properties. To evaluate biomedical utility, pH-triggered doxycycline release was characterized, achieving a 82% encapsulation efficiency. In gastrointestinal simulations, the SIPN protected the drug at gastric pH and triggered a fast, extensive release (89%) upon transition to intestinal conditions.
F. J. Vazquez-Perez, Alejandro Moltó-Ramírez, C. Cifuentes-Jiménez et al.· International Journal of Bio...· 0 citations
SUMMARY Hydrogels are promising for biomedical applications due to their tunable properties and extracellular matrix (ECM)-mimicking capabilities, but available chemistries limit customization potential. We present a modular bioconjugation system using polyphenol radical crosslinking, where gallic acid (GA)-modified glycosaminoglycans rapidly crosslink with diverse biomolecules under blue light and riboflavin. This method enables the creation of customizable, cell-compatible matrices. We validate conjugation chemistry using 1 H NMR, size-exclusion chromatography, and gel electrophoresis. As proof-of-concept, we have created injectable hydrogels from GA-modified hyaluronic acid (HA-GA) crosslinked with various proteins. Mechanical properties are tunable via riboflavin concentration and light exposure. HCT116 colon cancer cells form spheroids in HA-GA-BSA, while other protein-containing hydrogels support dispersed growth, indicating enhanced cell adhesion. HA-GA hydrogels upregulate stemness markers compared to 2D culture, while protein incorporation reduces this effect. Matrix-activated Wnt signaling confirms functional biomolecule integration using HA-GA-Wnt3A matrices. This strategy enables tailored ECM design for tissue engineering and 3D cell culture.
Austin D. Evans, R. P. Parvathaneni, Rolle Rahikainen et al.· Cell Reports Physical Scienc...· 0 citations
Existing Janus hydrogels for preventing postoperative tendon adhesion often neglect adequate mechanical protection and lubrication required for tendon regeneration. Inspired by the structural and functional synergy of berry exocarp with anti-adhesion and protection properties as well as mesocarp with adhesion, cushioning and antioxidant capacities, we fabricate a bilayer Janus hydrogel (PPDP) based on pectin and hyaluronic acid via a one-pot injectable in-situ sequential crosslinking strategy. The bottom layer is constructed with catechol-functionalized hyaluronic acid and adipic dihydrazide-modified pectin, which achieves stable tendon adhesion and favorable mechanical cushioning and over 90% reactive oxygen species scavenging capacity. The top layer, formed by photo-crosslinkable methacrylate-modified pectin and poly (ethylene glycol) diacrylate, robust hydrated anti-adhesive barrier with low friction coefficient of approximately 0.1 after more than 8000 friction cycles and its compressive strength is markedly enhanced in comparison with natural hydrogels up to 0.5 MPa. Remarkably, in the in vivo tendon injury model, PPDP integrates barrier protection, dynamic lubrication and anti-inflammation to achieve synergistic effects of structures and components. It accelerates tendon repair during the critical acute adhesion stage, outperforming the commercial Interceed™. This biomimetic design significantly inhibits tendon adhesion and facilitates functional recovery, providing a multifunctional integrated innovative strategy for clinical practice.
Tian Tian, Siyu Li, Li Wang et al.· Carbohydrate Polymers· 0 citations
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