Hydrogel Stiffness Research Articles

Bioactive hydrogels based on lysine dendrigrafts as crosslinkers: tailoring elastic properties to influence hMSC osteogenic differentiation.

Dendrigrafts are multivalent macromolecules with less ordered topology and higher branching than dendrimers. Exhibiting a high density of terminal amines, poly-L-lysine dendrigrafts of the fifth generation (DGL G5) allow hydrogel formation with tailorable crosslinking density and surface modification. This work presents DGL G5 as multifunctional crosslinkers in biomimetic PEG hydrogels to favour the osteogenic differentiation of human mesenchymal stem cells (hMSCs). DGL G5 reaction with dicarboxylic-acid PEG chains yielded amide networks of variable stiffness, measured at the macro and surface nanoscale. Oscillatory rheometry and compression afforded consistent values of Young's modulus, increasing from 8 to more than 30 kPa and correlating with DGL G5 concentration. At the surface level, AFM measurements showed the same tendency but higher E values, from approximately 15 to more than 100 kPa, respectively. To promote cell adhesion and differentiation, the hydrogels were functionalised with a GRGDSPC peptide and a biomimetic of the bone morphogenetic protein 2 (BMP-2), ensuring the same grafting concentrations (between 2.15 ± 0.54 and 2.28 ± 0.23 pmols mm-2) but different hydrogel stiffness. 6 h after seeding on functionalised hydrogels in serum-less media, hMSC showed nascent adhesions on the stiffer gels and greater spreading than on glass controls with serum. After two weeks in osteogenic media, hMSC seeded on the stiffer gels showed greater spreading, more polygonal morphologies and increased levels of osteopontin, an osteoblast marker, compared to controls, which peaked on 22 kPa-gels. Together, these results demonstrate that DGL G5-PEG hydrogel bioactivity can influence the adhesion, spreading and early commitment of hMSCs.

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.

Hydrogel Films with Impact Resistance by Sacrificial Micelle-Assisted-Alignment.

Various strategies are developed to engineer aligned hierarchical architectures in polymer hydrogels for enhanced mechanical performance. However, chain alignment remains impeded by the presence of hydrogen bonds between adjacent chains. Herein, a facile sacrificial micelle-assisted-alignment strategy is proposed, leading to well-aligned, strong and tough pure chitosan hydrogels. The sacrificial sodium dodecyl sulfate micelles electrostatically interact with the protonated chitosan chains, enabling chain sliding and alignment under uniaxial forces. Subsequently, sacrificial micelles can be easily removed via NaOH treatment, causing the reforming of H-bond in the chain networks. The strength of the pure chitosan hydrogels increases 140-fold, reaching 58.9±3.4MPa; the modulus increases 595-fold, reaching 226.4±42.8MPa. After drying-rehydration, the strength and modulus further rise to 70.3±2.4 and 403.5±76.3MPa, marking a significant advancement in high-strength pure chitosan hydrogel films. Furthermore, the designed multiscale architectures involving enhanced crystallinity, well-aligned fibers, strong interfaces, robust multilayer Bouligand assembly contribute to the exact replica of lobster underbelly with impact resistance up to 6.8±1.0kJ m-1. This work presents a promising strategy for strong, tough, stiff and impact-resistant polymer hydrogels via well-aligned hierarchical design.

Geometric constraint of mechanosensing by modification of hydrogel thickness prevents stiffness-induced differentiation in bone marrow stromal cells.

Extracellular matrix (ECM) stiffness is fundamental in cell division, movement and differentiation. The stiffness that cells sense is determined not only by the elastic modulus of the ECM material but also by ECM geometry and cell density. We hypothesized that these factors would influence cell traction-induced matrix deformations and cellular differentiation in bone marrow stromal cells (BMSCs). To achieve this, we cultivated BMSCs on polyacrylamide hydrogels that varied in elastic modulus and geometry and measured cell spreading, cell-imparted matrix deformations and differentiation. At low cell density BMSCs spread to a greater extent on stiff compared with soft hydrogels, or on thin compared with thick hydrogels. Cell-imparted matrix deformations were greater on soft compared with stiff hydrogels or thick compared with thin hydrogels. There were no significant differences in osteogenic differentiation relative to hydrogel elastic modulus and thickness. However, increased cell density and/or prolonged culture significantly reduced matrix deformations on soft hydrogels to levels similar to those on stiff substrates. This suggests that at high cell densities cell traction-induced matrix displacements are reduced by both neighbouring cells and the constraint imposed by an underlying stiff support. This may explain observations of the lack of difference in osteogenic differentiation as a function of stiffness.

Matrix Stiffness of GelMA Hydrogels Regulates Lymphatic Endothelial Cells toward Enhanced Lymphangiogenesis.

Lymphatic vessel regeneration is crucial for various tissue engineering strategies, particularly in resolving inflammation and restoring tissue homeostasis. In our study, we focused on investigating how hydrogel matrix stiffness influences lymphatic endothelial cells (LECs) in promoting lymphatic vessel regeneration. Gelatin methacrylate (GelMA) was chosen as our biomaterial due to its versatility in tissue engineering and biofabrication. We fabricated GelMA hydrogels at concentrations of 5, 7.5, and 15% (w/v) with corresponding Young's modulus values of 1.55 kPa (soft matrix), 12.02 kPa (medium matrix), and 48.50 kPa (stiff matrix). Among these, the 7.5% GelMA hydrogel exhibited optimal stiffness for promoting lymphangiogenesis. LECs seeded either on the hydrogel surface or within spontaneously formed a more stable lymphatic capillary network compared with other GelMA formulations. Furthermore, we investigated the enhancement of lymphangiogenesis by incorporating VEGF-C into the GelMA hydrogel, leveraging the synergistic effects of mechanical and chemical cues. Our results underscored the critical role of FAK-phosphorylation in this process; treatment with an FAK-specific inhibitor prevented the formation of tube-like structures by LECs and attenuated the expression of lymphatic markers. Overall, our findings highlight how the mechanical and chemical cues provided by GelMA hydrogels can effectively regulate LEC behavior toward enhanced lymphangiogenesis via the integrin/FAK mechanotransduction pathway. This study proposes a promising strategy for developing hydrogel-based scaffolds or bioinks tailored to promote lymphatic vessel regeneration in therapeutic applications.

Controlled release of microorganisms from engineered living materials.

Probiotics offer therapeutic benefits and have the potential to treat complex diseases, but their persistence at the target site is often required for effective treatment. Although probiotic persistence can be achieved by repeated delivery, no biomaterial that releases metabolically active probiotics in a sustained manner has been developed yet. This work demonstrates a generic mechanism where stiff probiotics encapsulated within relatively less stiff hydrogels proliferate and induce hydrogel fracture. This allows a zero-order release of probiotics which can be easily controlled by adjusting the properties of the encapsulating matrices. This generic mechanism is applicable for a wide range of probiotics with different synthetic matrices and has the potential to be used in the treatment of a broad range of diseases.

Abstract A054: Collagen fiber architecture modulates PDAC cell and organoid phenotype

Abstract Introduction Collagen plays a significant role in the development of pancreatic ductal adenocarcinoma (PDAC). Disease progression is mediated by a dense collagenous stroma, which acts as a barrier to drug delivery and immune cell access. Current in vitro models do not well represent the collagenous stroma seen in PDAC. The aim of this study is to develop a fibrillar collagen-based hydrogel system which mimics the tumour microenvironment seen in PDAC and assess cell-mediated collagen remodeling. Methods Metastatic SUIT2-007 pancreatic cancer cells, non-metastatic BxPC3 pancreatic cancer cells, and human pancreatic stellate cells (hPSCs) were encapsulated in bovine type I collagen gels of various concentrations. KPC mouse derived tumour organoids were also encapsulated in bovine type I collagen gels of 2 mg/ml and 6 mg/ml concentrations, basement membrane extract (BME), and a combination of BME and collagen gel. Cell viability and proliferation was assessed using Live/Dead imaging and CellTitre-Glo 3D respectively. Cell mediated collagen remodeling was assessed via monitoring of gel contraction on the mm scale, and confocal reflectance microscopy on the cellular scale. Collagen contraction around cells was assessed by measuring the pixel intensity of collagen fibrils within the vicinity of the cell. Cell phenotype was assessed via immunofluorescent staining and qPCR. Results Collagen concentration alters the hydrogel structure and stiffness, with 3 mg/ml collagen resulting in straighter, shorter and thicker fibrils. Metastatic SUIT-007 cells contracted collagen gels more rapidly than non-metastatic BxPC3 cells, with both achieving similar contraction by day 7. SUIT2-007 cells remodel and align soft 1 mg/ml gels while 3 mg/ml gels proved to be sufficiently stiff to resist remodeling. Cellular collagen association was increased in more elongated SUIT-007 cells than in BxPC3 cells. Organoid proliferation varies with hydrogel collagen concentration. Conclusion Metastatic SUIT2-007 cells were able to remodel collagen more rapidly and to a greater extent than non-metastatic BxPC3 cells. Collagen supports an invasive organoid phenotype. Collagen gels combined with confocal reflectance microscopy provide a platform for the quantitative assessment of cell-matrix interactions in PDAC. Acknowledgements This project was funded by a Medical Traineeship from UCD School of Medicine, and summer student scholarships from Irish Cancer Society and Breakthrough Cancer Research. We acknowledge the Conway Imaging Core Facility for assistance with microscopy. Citation Format: Ciara L Doyle, Shanu Xavier, Jarren Y.S Lu, Leah Fallon, Anwesha Sarkar, Jan J Baguyo, Maela Gars, Stephen D Thorpe. Collagen fiber architecture modulates PDAC cell and organoid phenotype [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A054.