Hydrogel Stiffness Research Articles

DOP075 Tissue fibrosis modulates interleukin (IL)-13 signaling in intestinal epithelial cells

Abstract Background Inflammatory Bowel Diseases (IBDs) are characterized by chronic inflammation of the intestinal tissue and persistent disruption of the intestinal epithelial barrier. A common and dangerous complication of chronic inflammation during IBD is fibrosis, a mechanical stiffening of the intestinal tissue [1,2]. Previous work suggests that immune cell activity, for example Interleukin (IL)-13-mediated macrophage activation, can be mechanosensitive [3,4]. However, it is not currently well understood how fibrosis affects the inflammatory response in intestinal epithelial cells. Methods To address how tissue stiffness impacts intestinal inflammation, we studied the effects of inflammatory cytokine signaling in intestinal epithelial cells using a recently developed intestinal organoid monolayer model. We cultured intestinal organoid monolayers on soft (0.5kPa), intermediate (5kPa) or stiff (15kPa) hydrogels to mimic physiological or fibrotic conditions and studied the cellular response to IL-13, an inflammatory cytokine implicated in IBD and loss of barrier integrity. To investigate the impacts of substrate stiffness and IL-13 signaling on epithelial cell function, we combined high-resolution live microscopy, quantitative image analysis using AI-based models and biophysical measurements. Results We found that substrate mechanosensing and IL-13 signaling interact synergistically to modulate intestinal epithelial stem cell differentiation, leading to a dramatic increase in the frequency of secretory cells. Furthermore, organoids cultured on stiff substrates no longer respond to IL-13 treatment, suggesting that fibrotic conditions may promote an inflamed-like state even in the absence of inflammatory cytokines. To further investigate cross-talk between substrate mechanics and IL-13 signalling, we inhibited downstream IL-13 signalling and cell contractility. We found that both the stiffness- and IL-13-induced phenotypes could be rescued by either inhibiting myosin-2 activity or blocking downstream IL-13 signalling, further indicating a co-dependence and synergy of substrate stiffness and IL-13. Further experiments implicate the relocalization of myosin-2 to the cell cortex and increased nuclear accumulation of the mechanosensitive protein YAP in mediating this response. Experiments using mouse models of colitis further support a role for YAP in this process. Conclusion Taken together, this work reveals how tissue mechanics modulate the inflammatory response in the intestinal epithelium. Future translational work based on these findings will inspire new approaches to mitigate the effects of fibrosis during IBD.

Programming Green Light-Responsive Hydrogels: From Photo-Weakening to Photo-Unresponsiveness and Photo-Strengthening.

Light-responsive hydrogels are highly valued for their dynamic mechanical properties and biocompatibility. In this study, we present a hydrogel system that can either soften or strengthen on green light exposure, or remain unresponsive to light, depending on the addition of adenosyl cobalamin (AdoCbl) and Co2+. These protein-based hydrogels were formed using genetically encoded SpyTag-SpyCatcher chemistry and included green light-sensitive CarHc protein domains. As previously reported, these hydrogels formed in the dark with the addition of AdoCbl, due to the tetramerization of the CarHc domains. Upon green light exposure, the CarHc tetramers disassembled, leading to a rapid transition from gel to sol. Interestingly, we discovered that an excess of AdoCbl leads to photo-strengthening rather than photo-weakening. This occurred because light exposure induces interchain crosslinks between AdoCbl and poly-histidine tags (His6-tags) of the proteins. Furthermore, incorporating Co2+ ions enhanced hydrogel stiffness by coordinating to His6-tags. This not only suppressed photo-weakening but also promoted photo-strengthening behaviour. These findings highlight the role of His6-tags in photochemical crosslinking with excess AdoCbl and in coordination to Co2+ ions, providing a novel strategy for designing tuneable, light responsiveness in materials.

Unraveling the Atomistic Mechanism of Electrostatic Lateral Association of Peptide β-Sheet Structures and Its Role in Nanofiber Growth and Hydrogelation.

Guiding molecular assembly of peptides into rationally engineered nanostructures remains a major hurdle against the development of functional peptide-based nanomaterials. Various non-covalent interactions come into play to drive the formation and stabilization of these assemblies, of which electrostatic interactions are key. Here, the atomistic mechanisms by which electrostatic interactions contribute toward controlling self-assembly and lateral association of ultrashort β-sheet forming peptides are deciphered. Our results show that this is governed by charge distribution and ionic complementarity, both affecting the interaction patterns between charged residues: terminal, core, and/or terminal-to-core attraction/repulsion. Controlling electrostatic interactions enabled fine-tuning nanofiber morphology for the 16 examined peptides, resulting into versatile nanostructures ranging from extended thin fibrils and thick bundles to twisted helical "braids" and short pseudocrystalline nanosheets. This in turn affected the physical appearance and viscoelasticity of the formed materials, varying from turbid colloidal dispersions and viscous solutions to soft and stiff self-supportive hydrogels, as revealed from oscillatory rheology. Atomistic mechanisms of electrostatic interaction patterns were confirmed by molecular dynamic simulations, validating molecular and nanoscopic characterization of the developed materials. In essence, detailed mechanisms of electrostatic interactions emphasizing the impact of charge distribution and ionic complementarity on self-assembly, nanostructure formation, and hydrogelation are reported.

Improving the bioactivity and mechanical properties of poly(ethylene glycol)-based hydrogels through a supramolecular support network.

Most synthetic hydrogels are formed through radical polymerization to yield a homogenous covalent meshwork. In contrast, natural hydrogels form through mechanisms involving both covalent assembly and supramolecular interactions. In this communication, we expand the capabilities of covalent poly(ethylene glycol) (PEG) networks through co-assembly of supramolecular peptide nanofibers. Using a peptide hydrogelator derived from the tryptophan zipper (Trpzip) motif, we demonstrate how in situ formation of nanofiber networks can tune the stiffness of PEG-based hydrogels, while also imparting shear thinning, stress relaxation, and self-healing properties. The hybrid networks show enhanced toughness and durability under tension, providing scope for use in load bearing applications. A small quantity of Trpzip peptide renders the non-adhesive PEG network adhesive, supporting adipose derived stromal cell adhesion, elongation, and growth. The integration of supramolecular networks into covalent meshworks expands the versatility of these materials, opening up new avenues for applications in biotechnology and medicine.

Tumor-mimetic hydrogel stiffness regulates cancer stemness properties in H-Ras-transformed cancer model cells.

Cancer stem cells (CSCs) cause therapy-resistance and recurrence, therefore an establishment of therapeutic approaches targeting CSCs is essential for eradicating cancers; however, a lot of aspects of the mechanisms of CSCs generation remain unclear. We previously demonstrated that human glioblastoma cell lines cultured on double-network (DN) hydrogel were rapidly reprogrammed into CSCs. To elucidate molecular mechanisms underlying CSCs generation, we here focused on the elastic modulus of hydrogels mimicking the stiffness of tumor tissues. Mouse NIH3T3 fibroblasts transformed with representative oncogenes H-Ras and Src were employed as cancer model cells, and cultured on (2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel with different elastic modulus. The H-Ras-, but not Src-, transformed NIH3T3 cells induced stem cell properties on the PAMPS gel, particularly with elastic modulus of approximately 10kPa that mimics general tumor tissues. On the gels, expression levels of stemness markers such as Sox2, Nanog, and Oct4 were increased in the parental and H-Ras-transformed cells. Pull-down assay demonstrated that multiple proteins in H-Ras-transformed cells bound to the PAMPS gels with 9.3kPa stiffness, and desmoplakin was identified as one of the gel-bound proteins by LC-MS/MS analysis. Among Ca2+ channels functioning as mechanosensors, the expression levels of TRPC and TRPV families were efficiently increased on the gel with approximately 10kPa, as well as stemness markers. These data suggest that tumor tissues with the stiffness of 10kPa effectively trigger the signals for generating CSCs through alterations of membrane structures and Ca2+ influx, which will be beneficial for developing novel therapeutic applications targeting CSCs.

Survivin modulates stiffness-induced vascular smooth muscle cell motility.

Arterial stiffness is a key contributor to cardiovascular diseases, including atherosclerosis, restenosis, and coronary artery disease, it has been characterized to be associated with the aberrant migration of vascular smooth muscle cells (VSMCs). However, the underlying molecular mechanisms driving VSMC migration in stiff environments remain incompletely understood. We recently demonstrated that survivin, a member of the inhibitor of apoptosis protein family, is highly expressed in both mouse and human VSMCs cultured on stiff polyacrylamide hydrogels, where it modulates stiffness-mediated cell cycle progression and proliferation. However, its role in stiffness-dependent VSMC migration remains unknown. To assess its impact on migration, we performed time-lapse video microscopy on VSMCs seeded on fibronectin-coated soft and stiff polyacrylamide hydrogels, mimicking the physiological stiffness of normal and diseased arteries, with either survivin inhibition or overexpression. We observed that VSMC motility increased under stiff conditions, while pharmacologic or siRNA-mediated inhibition of survivin reduced stiffness-stimulated migration to rates similar to those observed under soft conditions. Further investigation revealed that cells on stiff hydrogels exhibited greater directional movement and robust lamellipodial protrusion compared to those on soft hydrogels. Interestingly, survivin-inhibited cells on stiff hydrogels showed reduced directional persistence and lamellipodial protrusion compared to control cells. We also examined whether survivin overexpression alone is sufficient to induce cell migration on soft hydrogels, and found that survivin overexpression modestly increased cell motility and partially rescued the lack of directional persistence compared to GFP-expressing control VSMCs on soft hydrogels. In conclusion, our findings demonstrate that survivin plays a key role in regulating stiffness-induced VSMC migration, suggesting that targeting survivin and its signaling pathways could offer therapeutic strategies for addressing arterial stiffness in cardiovascular diseases.

YAP and ECM Stiffness: Key Drivers of Adipocyte Differentiation and Lipid Accumulation.

ECM stiffness significantly influences the differentiation of adipose-derived stem cells (ADSCs), with YAP-a key transcription factor in the Hippo signaling pathway-playing a pivotal role. This study investigates the effects of ECM stiffness on ADSC differentiation and its relationship with YAP signaling. Various hydrogel concentrations were employed to simulate different levels of ECM stiffness, and their impact on ADSC differentiation was assessed through material properties, adipocyte-specific gene expression, lipid droplet staining, YAP localization, and protein levels. Our results demonstrated that increasing hydrogel stiffness enhanced adipocyte differentiation in a gradient manner. Notably, inhibiting YAP signaling further increased lipid droplet accumulation, suggesting that ECM stiffness influences adipogenesis by modulating YAP signaling and its cytoplasmic phosphorylation. This study elucidates the molecular mechanisms underlying ECM stiffness-dependent lipid deposition, highlighting YAP's regulatory role in adipogenesis. These findings provide valuable insights into the regulation of cell differentiation and have important implications for tissue engineering and obesity treatment strategies.

Cell response to extracellular matrix viscous energy dissipation outweighs high-rigidity sensing.

The mechanics of the extracellular matrix (ECM) determine cell activity and fate through mechanoresponsive proteins including Yes-associated protein 1 (YAP). Rigidity and viscous relaxation have emerged as the main mechanical properties of the ECM steering cell behavior. However, how cells integrate coexisting ECM rigidity and viscosity cues remains poorly understood, particularly in the high-stiffness regime. Here, we have exploited engineered stiff viscoelastic protein hydrogels to show that, contrary to current models of cell-ECM interaction, substrate viscous energy dissipation attenuates mechanosensing even when cells are exposed to higher effective rigidity. This unexpected behavior is however readily captured by a pull-and-hold model of molecular clutch-based cell mechanosensing, which also recapitulates opposite cellular response at low rigidities. Consistent with predictions of the pull-and-hold model, we find that myosin inhibition can boost mechanosensing on cells cultured on dissipative matrices. Together, our work provides general mechanistic understanding on how cells respond to the viscoelastic properties of the ECM.

Advanced Lignin‐Based Hydrogels with Superior Stiffness, Toughness, and Sensing Capabilities

AbstractHydrogels, known for their 3D polymer networks and high water content, are widely used in applications ranging from agriculture to tissue engineering and soft electronics. However, balancing toughness and stiffness in hydrogels remains a significant challenge due to the inverse relationship between these properties. In this study, a dual‐network hydrogel is developed composed of lignin/poly(N,N‐dimethylacrylamide) (PDMA) and sodium alginate/Ca2⁺ (SA/Ca2⁺) using a solvent exchange method. This hydrogel incorporates multi‐level energy dissipative structures, resulting in both high stiffness and toughness. Specifically, the DL/S0.1Ca0.5 hydrogel exhibited impressive mechanical performance, including a tensile stress of 3.7 MPa, a tensile strain of 1100%, and a tensile modulus of 8.7 MPa, along with remarkably high toughness of 97,000 J m−2 and work of extension of 25 MJ m−3. Additionally, it demonstrates exceptional rupture and collision resistance, outstanding conductivity of 19.7 S m−1, and high strain sensitivity with a gauge factor up to 7.78. These features highlight its potential for use in extreme sports protection and wearable sensors, representing a significant advancement in the development of multifunctional hydrogels.

In vitro bioprinted 3D model enhancing osteoblast-to-osteocyte differentiation

In vitrobone models are pivotal for understanding tissue behavior and cellular responses, particularly in unravelling certain pathologies' mechanisms and assessing the impact of new therapeutic interventions. A desirablein vitrobone model should incorporate primary human cells within a 3D environment that mimics the mechanical properties characteristics of osteoid and faithfully replicate all stages of osteogenic differentiation from osteoblasts to osteocytes. However, to date, no bio-printed model using primary osteoblasts has demonstrated the expression of osteocytic protein markers. This study aimed to develop bio-printedin vitromodel that accurately captures the differentiation process of human primary osteoblasts into osteocytes. Given the considerable impact of hydrogel stiffness and relaxation behavior on osteoblast activity, we employed three distinct cross-linking solutions to fabricate hydrogels. These hydrogels were designed to exhibit either similar elastic behavior with different elastic moduli, or similar elastic moduli with varying relaxation behavior. These hydrogels, composed of gelatin (5% w/v), alginate (1%w/v) and fibrinogen (2%w/v), were designed to be compatible with micro-extrusion bioprinting and proliferative. The modulation of their biomechanical properties, including stiffness and viscoelastic behavior, was achieved by applying various concentrations of cross-linkers targeting both gelatin covalent bonding (transglutaminase) and alginate chains' ionic cross-linking (calcium). Among the conditions tested, the hydrogel with a low elastic modulus of 8 kPa and a viscoelastic behavior over time exhibited promising outcomes regarding osteoblast-to-osteocyte differentiation. The cessation of cell proliferation coincided with a significant increase in alkaline phosphatase activity, the development of dendrites, and the expression of the osteocyte marker PHEX. Within this hydrogel, cells actively influenced their environment, as evidenced by hydrogel contraction and the secretion of collagen I. This bio-printed model, demonstrating primary human osteoblasts expressing an osteocyte-specific protein, marks a significant achievement. We envision its substantial utility in advancing research on bone pathologies, including osteoporosis and bone tumors.

DDDR-29. MATRIX STIFFNESS INDUCED PHENOTYPE IN BRAIN METASTATIC BREAST CANCER CELLS DETERMINES RESPONSE TO THERAPY

Abstract Breast cancer is the most prevalent form of cancer, representing 12.5% of all cancer cases diagnosed worldwide. Breast cancer cells can metastasize to distant organs (i.e., brain) and remain dormant for extended periods of time. This aspect makes it very difficult to treat these cells as they exhibit reduced growth as well as resistance to therapy. Despite advances in our understanding of breast cancer metastasis, the specific mechanisms that enable dormant cancer cells to resist therapeutics, particularly in brain metastatic breast cancer (BMBC), remain unclear. Herein, we employed brain tissue mimetic hyaluronic acid (HA) hydrogels of varying stiffness (i.e., ~0.4 kPa vs. ~4.5 kPa) to investigate the response to chemo or targeted therapy in dormant versus proliferative BMBC cells. It was revealed that BMBC cells grown on soft HA hydrogels (~0.4 kPa) displayed a non-proliferative (i.e., dormant) phenotype and exhibited resistance to Paclitaxel (PTX) or Lapatinib (LAP). However, BMBC cells grown on stiff HA hydrogels (~4.5 kPa) displayed a proliferative phenotype and sensitivity to PTX or LAP. Furthermore, we found that resistance to therapy was due to the enhanced expression of the serum/glucocorticoid regulated kinase 1 (SGK1) gene, mediated, in part, via the p38 mitogen-activated protein kinase (MAPK) pathway. Consequently, SGK1 inhibition using a SGK inhibitor in BMBC cells cultured on soft HA hydrogels resulted in a dormant-to-proliferative switch as well as response to PTX or LAP. In sum, our study demonstrated that matrix stiffness plays a major role in the dormancy-associated therapy response, mediated, in part, via the p38/SGK1 pathway.

Interpenetrating Polymer Network Hydrogels with Tunable Viscoelasticity and Proteolytic Cleavability to Direct Stem Cells In Vitro.

The dynamic nature of cellular microenvironments, regulated by the viscoelasticity and enzymatic cleavage of the extracellular matrix, remains challenging to emulate in engineered synthetic biomaterials. To address this, a novel platform of cell-instructive hydrogels is introduced, composed of two concurrently forming interpenetrating polymer networks (IPNs). These IPNs consist of the same basic building blocks - four-armed poly(ethylene glycol) and the sulfated glycosaminoglycan (sGAG) heparin - are cross-linked through either chemical or physical interactions, allowing for precise and selective tuning of the hydrogel's stiffness, viscoelasticity, and proteolytic cleavability. The studies of the individual and combined effects of these parameters on stem cell behavior revealed that human mesenchymal stem cells exhibited increased spreading and Yes-associated protein transcriptional activity in more viscoelastic and cleavable sGAG-IPN hydrogels. Furthermore, human induced pluripotent stem cell (iPSC) cysts displayed enhanced lumen formation, growth, and pluripotency maintenance when cultured in sGAG-IPN hydrogels with higher viscoelasticity. Inhibition studies emphasized the pivotal roles of actin dynamics and matrix metalloproteinase activity in iPSC cyst morphology, which varied with the viscoelastic properties of the hydrogels. Thus, the introduced sGAG-IPN hydrogel platform offers a powerful methodology for exogenous stem cell fate control.

Frontal polymerization of acrylamide/GelMA/gelatin hydrogels with controlled mechanical properties and inherent self-recovery

Low mechanical resistance represents one of the significant problems of hydrogels, limiting their applicability in many fields. One approach to overcome this issue is synthesizing interpenetrating polymeric networks. In this work, the frontal polymerization technique was used to synthesize two series of novel hydrogels: (i) poly(acrylamide) (PAAm)-based hydrogels copolymerized/crosslinked with methacrylate gelatin (GelMA) (AAm-GelMA copolymer networks), and (ii) semi-IPN made of AAm-GelMA copolymer networks and a physically crosslinked gelatin network. With the final objective of improving the rheological, mechanical, morphological, thermal, and swelling properties of PAAm hydrogels, GelMA with two different degrees of methacrylation (30 and 75 mol%) was used. Interactions between GelMA chains, which give rise to physical network formation (i.e., GelMA-GelMA interactions), resulted in very efficient crosslinking for PAAm-based hydrogels, requiring a significantly lower methacrylic group concentration (0.04 mol%) for hydrogel formation compared to N,N′-methylene-bis-acrylamide (1 mol%), which is the agent typically used as a crosslinker for PAAm. Furthermore, the degree of GelMA methacrylation markedly affected the properties of the hydrogels. For example, regarding the swelling degree, hydrogels containing 22 wt% of GELMA30 had an SR% of 2870, while those containing the same amount of GELMA75 swelled much less (870 %). The introduction of gelatin as a secondary network in semi-IPNs influenced the rheological and mechanical properties, resulting in increased hydrogel modulus and stiffness attributed to enhanced physical interactions within the network. Finally, dynamic rheological shear strain and cyclic loading compression tests demonstrated exceptional recovery capabilities in all hydrogel formulations: samples subjected to alternating low (0.1 %) and high (300 % or 10 %) shear strain demonstrated a complete and prompt recovery of G′ and G″ values.

Single-layer graphene oxide nanosheets induce proliferation and Osteogenesis of single-cell hBMSCs encapsulated in Alginate Microgels

Microfluidics cell encapsulation offers a way to mimic a 3D microenvironment that supports cell growth and proliferation, while also protecting cells from environmental stress. This technique has found extensive applications in tissue engineering and cell therapies. Several studies have demonstrated the advantages of graphene oxide (GO) as an osteogenic inducer; however, the significance of GO on stem cell fate in the single-cell state is still unclear. Here, a microfluidics-based approach is developed for continuous encapsulation of mesenchymal stem cells (MSCs) at the single-cell level using alginate microgels. So, single-layer graphene oxide (slGO) nanosheet is used to be encapsulated inside the alginate droplets. The results of AFM and SEM show that slGO can increase the roughness and reduce the stiffness of alginate hydrogels. The Young’s modulus of the alginate and alginate-slGO was obtained as 1414 kPa and 985.9 kPa, respectively. Live/dead assay and fluorescence microscopy images illustrate that slGO can maintain the viability and proliferation of microencapsulated hBMSCs. The obtained results show that slGO increases the mineralization of the encapsulated hBMSCs, so that microgels containing hBMSCs gradually became opaque during 21 days of culture. RT-qPCR results indicate that the expression of OCN, Runx2, and ALP in the alginate-slGO microgels is significantly higher than in the alginate microgels. The expression of OCN and Runx2 in the alginate-slGO microgels is 4.27 and 5.87-fold higher than in the alginate microgels, respectively. It can be concluded that low doses of slGO nanosheets have the potential to be utilized in the development of tissue engineering and bone regeneration. This finding offers a new method for creating injectable tissue transplants that are minimally invasive.

Controlled stiffness and diffusivity of poly(ethylene glycol) hydrogel formed with cellulose-nanofiber framework

Hydrogels composed of polymer networks are widely used in industry and scientific research for high water retention and unique mechanical properties. Nevertheless, in ordinary hydrogel formation, the trade-off relationship between stiffness and mesh size remains a crucial consideration for practical applications. This study describes a facile approach to controlling hydrogel stiffness and mesh size by hybridizing poly (ethylene glycol) diacrylate (PEGDA) and methacrylated TEMPO-oxidized cellulose nanofibers (T-CNFMA). After disintegrating T-CNF by ultrasonication, T-CNFMA was synthesized resulting in a degree of substitution of 2.04 mmol/g. Incorporation of T-CNFMA in the PEGDA network allowed for independent control of hydrogel stiffness and mesh size by reinforcing the whole hydrogel network as a framework. Consequently, the swelling ratio and shear modulus could be manipulated by controlling the PEGDA/T-CNFMA ratio. Structural analyses revealed that an increase in the T-CNFMA content in the presence of a low amount of PEGDA resulted in a large mesh size with a constant stiffness. The diffusivity test was also consistent with the properties of the hydrogels. This result indicates that the incorporation of T-CNF in hydrogel network is useful to control the physical property of the hydrogel, especially for varying mesh size, regardless of stiffness alteration.

Tunable Blended Collagen I/II and Collagen I/III Hydrogels as Tissue Mimics.

Collagen (Col) is commonly used as a natural biomaterial for biomedical applications. Although Col I is the most prevalent col type employed, many collagen types work together in vivo to confer function and biological activity. Thus, blending collagen types can better recapitulate many native environments. This work investigates how hydrogel properties can be tuned through blending collagen types (col I/II and col I/III) and by varying polymerization temperatures. Col I/II results in poorly developed fibril networks, which softened the gels, especially at lower polymerization temperatures. Conversely, col I/III hydrogels exhibit well-connected fibril networks with localized areas of fine fibrils and result in stiffer hydrogels. A decreased molecular mass recovery rate is observed in blended hydrogels. The altered fibril morphologies, mechanical properties, and biological signals of the blended gels can be leveraged to alter cell responses and can be used as models for different tissue types (e.g., healthy vs fibrotic tissue). Furthermore, the biomimetic hydrogel properties are a tool that can be used to modulate the transport of drugs, nutrients, and wastes in tissue engineering applications.

Direct M2 macrophage co-culture overrides viscoelastic hydrogel mechanics to promote fibroblast activation.

Fibroblast activation drives fibrotic diseases such as pulmonary fibrosis. However, the complex interplay of how tissue mechanics and macrophage signals combine to influence fibroblast activation is not well understood. Here, we use hyaluronic acid hydrogels as a tunable cell culture system to mimic lung tissue stiffness and viscoelasticity. We applied this platform to investigate the influence of macrophage signaling on fibroblast activation. Fibroblasts cultured on stiff (50 kPa) hydrogels mimicking fibrotic tissue exhibit increased activation as measured by spreading as well as type I collagen and cadherin-11 expression compared to fibroblasts cultured on soft (1 kPa) viscoelastic hydrogels mimicking normal tissue. These trends were unchanged in fibroblasts cultured with macrophage-conditioned media. However, fibroblasts directly co-cultured with M2 macrophages show increased activation, even on soft viscoelastic hydrogels that normally suppress activation. Inhibition of interleukin 6 (IL6) signaling does not change activation in fibroblast-only cultures but ameliorates the pro-fibrotic effects of M2 macrophage co-culture. These results underscore the ability of direct M2 macrophage co-culture to override hydrogel viscoelasticity to promote fibroblast activation in an IL6-dependent manner. This work also highlights the utility of using hydrogels to deconstruct complex tissue microenvironments to better understand the interplay between microenvironmental mechanical and cellular cues.

Cross-Linker Architectures Impact Viscoelasticity in Dynamic Covalent Hydrogels.

Dynamic covalent cross-linked (DCC) hydrogels represent a significant advance in biomaterials for regenerative medicine and mechanobiology, offering viscoelasticity, and self-healing properties that more closely mimic in vivo tissue mechanics than traditional, predominantly elastic, covalent hydrogels. However, the effects of varying cross-linker architecture on DCC hydrogel viscoelasticity have not been thoroughly investigated. This study introduces hydrazone-based alginate hydrogels to explore how cross-linker architectures impact stiffness and viscoelasticity. In hydrogels with side-chain cross-linker (SCX), higher cross-linker concentrations enhance stiffness and decelerate stress relaxation, while an off-stoichiometric hydrazine-to-aldehyde ratio reduces stiffness and shortens relaxation time. In hydrogels with telechelic cross-linking, maximal stiffness and relaxation time occurs at intermediate cross-linker mixing ratio for both linear cross-linker (LX) and star cross-linker (SX), with higher cross-linker valency further enhancing these properties. Further, the ranges of stiffness and viscoelasticity accessible with the different cross-linker architectures are found to be distinct, with SCX hydrogels leading to slower stress relaxation relative to the other architectures, and SX hydrogels providing increased stiffness and slower stress relaxation versus LX hydrogels. This research underscores the pivotal role of cross-linker architecture in defining hydrogel stiffness and viscoelasticity, providing insights for designing DCC hydrogels with tailored mechanical properties for specific biomedical applications.

Formulation-Property Effects in Novel Injectable and Resilient Natural Polymer-Based Hydrogels for Soft Tissue Regeneration

The development of injectable hydrogels for soft tissue regeneration has gained significant attention due to their minimally invasive application and ability to conform precisely to the shape of irregular tissue cavities. This study presents a novel injectable porous scaffold based on natural polymers that undergoes in situ crosslinking, forming a highly resilient hydrogel with tailorable mechanical and physical properties to meet the specific demands of soft tissue repair. By adjusting the formulation, we achieved a range of stiffness values that closely mimic the mechanical characteristics of native tissues while maintaining very high resilience (>90%). The effects of gelatin, alginate, and crosslinker concentrations, as well as porosity, on the hydrogel’s properties were elucidated. The main results indicated a compression modulus range of 2.7–89 kPa, which fits all soft tissues, and gelation times ranging from 5 to 30 s, which enable the scaffold to be successfully used in various operations. An increase in gelatin and crosslinker concentrations results in a higher modulus and lower gelation time, i.e., a stiffer hydrogel that is created in a shorter time. In vitro cell viability tests on human fibroblasts were performed and indicated high biocompatibility. Our findings demonstrate that these injectable hydrogel scaffolds offer a promising solution for enhancing soft tissue repair and regeneration, providing a customizable and resilient framework that is expected to support tissue integration and healing with minimal surgical intervention.

Designing highly tunable anion responsive Cardin-motif peptide based self-assembled nanostructures for accessing diverse cellular response

Several anions present in the extracellular matrix (ECM) not only have significant physiological functions in ECM but also play an important role in regulating peptide-based self-assembly. Herein, we have employed a non-conventional approach to overcome the limitations of the positively charged Cardin-motif peptide that failed to self-assemble at physiological pH. We used a simple and elegant strategy by employing different anions such as HPO42-, Cl- and I- to mask the overall surface charge of peptide. Interestingly, these anions were utilized to modulate the nanostructure formation and mechanical stiffness of peptide hydrogels owing to their differential interactions with water molecules according to the Hofmeister series. Interestingly, these anions induced hydrogels showed diverse cellular responses on two different cell lines, fibroblast and neuronal, indicating diverse application potential of the new scaffold. Thus, this study emphasizes the importance of anions to regulate the self-assembly of Cardin-motif peptide and this approach can be utilized in developing the ideal biomimetic model of ECM for futuristic applications.