Kyle H. Vining

Abstract Periodontal disease is characterized by progressive degradation of the gingival extracellular matrix and loss of the physical confinement it imposes on resident stromal cells. In human periodontal tissue, ECM collagen integrity is inversely correlated with facultative nuclear histone acetylation in stromal cells. We hypothesized that matrix stiffness directly coordinates an epigenomic shift in stromal cells. We use a three-dimensional mechanically tunable hydrogel system to independently tune the storage moduli across the mechanical range of healthy and periodontitis-affected gingival tissue. Matrix stiffness drives a genome-wide response in donor-derived human gingival fibroblasts. Matrix-induced confinement leads to an isotropic nuclear geometry and a folded nuclear envelope architecture compared with more permissive, soft matrices. H3K27Ac is suppressed through a stiffness and actomyosin contractility-dependent mechanism. DNMT inhibition in stiff matrices restores the high-acetylation chromatin state with persistent nuclear envelope folding. At the genomic level, stiff matrix confinement drives global CpG methylation gain concentrated at pericentromeric satellite repeats and repeat-dense regions, while collagen synthesis gene promoters and CTCF binding sites are selectively hypomethylated. Non-canonical NF-κB inflammatory signaling is attenuated through promoter methylation of MAP3K14, and pharmacological NIK inhibition reduces TLR2-stimulated IL-6 secretion in soft-matrix fibroblasts to levels comparable to the stiff condition. These findings identify the gingival ECM as an active epigenomic regulator of stromal inflammatory competence and provide a mechanistic rationale for targeting matrix mechanics to restore stromal homeostasis in periodontitis. Graphical Abstract The mechanobiological state of human gingival fibroblasts differs between healthy, stiff extracellular matrices and degraded, soft matrices characteristic of periodontal disease. In a healthy environment, stiff matrices impose physical confinement that enforces an isotropic nuclear geometry, driving dense heterochromatin formation, high global CpG methylation, and reduced histone acetylation. Conversely, the loss of mechanical confinement in soft matrices enables cell spreading and an open euchromatin state, fundamentally rewiring the cellular epigenome to promote non-canonical NF-κB signaling and chronic inflammation.

Abstract The plasticity of dendritic cell (DC) functional state is a major hurdle in DC therapy, yet how DCs acquire distinct states independent of ontogeny remains poorly understood. Here, we demonstrate that changes in matrix stress relaxation mechanically educate DCs to adopt distinct, persistent functional states even after the removal of mechanical cues. Stem cell-derived DCs cultured in a fast-relaxing environment exhibited enhanced antigen presentation, faster migration, and higher expression of T cell-recruiting chemokines. Slow-relaxing DCs, biased towards pro-inflammatory cytokine secretion, were enriched for gene signatures associated with lipid accumulation and stress response. These mechanical responses were conserved across human and murine DCs. Using ovalbumin (OVA) as the model antigen, fast-relaxing DCs elicited a CD8+-biased response in vitro, with higher antigen-specific CD8+ T cell activation and proliferation. In vivo adoptive cell transfer of mechanically educated DCs demonstrated that the fast-relaxing matrix licensed DCs to induce a potent draining lymph node T cell response with more antigen-specific T cells and higher restimulation potential. We further showed that DCs sensed matrix stress relaxation through PI3K signaling and actin branching, mediated by the concerted signaling of IL-4 and GM-CSF. Together, these findings demonstrate the role of matrix stress relaxation on the functional state of DCs and suggest a novel approach to enhance ex vivo cellular engineering by targeting mechanical signaling. Graphical Abstract Stem cell-derived dendritic cells (DCs) generated ex vivo are engineered using biomaterial platform with tunable matrix stress relaxation. Mechanical education of DCs is licensed by cytokine signaling, actin branching, and PI3K signaling. Fast-relaxing DCs exhibit higher antigen presentation and faster migration, which enhances their capacity to prime and activate antigen-specific CD8+ T cells.

The mechanical properties of the extracellular matrix (ECM) regulate tumor growth and invasion in the tumor microenvironment. Models of biopolymer networks have been used to investigate the impact of the elasticity and viscoelasticity of ECM on tumor behavior. Under tumor compression, these networks also show poroelastic behavior that is governed by the resistance to water flow through their pores. This work investigates the hypothesis that stress-dependent transport properties of biopolymer networks regulate tumor growth. Here, alginate hydrogels are used as a model ECM system with tunable ionic and hybrid ionic/covalent crosslinking. Hydrogel stiffness, viscoelasticity, and stress relaxation behavior were characterized using stepwise axial compression. Among these properties, we find that poroelastic fluid outflow dominates ECM stress relaxation, as the measured water flux was significantly affected under compression. Continuum mechanics-based modeling was developed to formulate and calculate the chemical potential gradients of water (solvent) in the hydrogels under compression. This framework was extended into an advection-diffusion framework to quantify growth factor (solute) distribution under varying strengths of stress and diffusion indexed by the relative strength of convective to diffusive transport, characterized by the Péclet number. An agent-based computational simulation showed that the Péclet numbers based on our experimental timescales strongly influenced tumor growth over longer, more physiologic timescales. Together, these results highlight the important role of water flux and transport in three-dimensional biopolymer networks.

Periodontal disease is characterized by inflamed gingival tissues and degradation of the gingival extracellular matrix (ECM), yet the role of mechanical cues remains poorly understood. Gingival ECM in periodontal disease showed reduced fibrillar collagen compared to healthy samples. We hypothesized that ECM softening in periodontal disease contributes to inflammation by dysregulating gingival fibroblasts (GFs). A mechanically tunable hydrogel model of the gingival ECM was developed to investigate the mechano-immune crosstalk. Stiff and soft collagen-alginate hydrogels matched the rheological properties of healthy and diseased gingival biopsies, respectively. Human donor GFs encapsulated in these stiff hydrogels showed significantly suppressed toll-like receptor-mediated inflammatory responses compared to those in soft hydrogels. The non-canonical NFκB pathway and epigenetic nuclear organization directed stiffness-dependent inflammatory responses of GFs. The direct impact of mechanical cues on immune responses was investigated ex vivo by co-culture of donor-derived human GFs with myeloid cells and in human gingival explants. Myeloid progenitors co-cultured with GFs in stiff hydrogels differentiated into immunomodulatory dendritic cells. Ex vivo crosslinking of human gingival tissue increased stiffness and reduced the production of inflammatory cytokines. Gingival mechano-immune regulation offers a novel approach to biomaterial-based treatments for periodontitis.

Natural biopolymer hydrogels often suffer from relatively low moduli and an inability to maintain structure and mechanics under cyclic loading, limiting their utility in dynamic mechanical environments. Here, a crosslinked collagen cryogel scaffold was fabricated by mechanical pre-compression to densify the network. Following lyophilization, the porous scaffolds maintained sustained >90% axial compressive strain with 200 cycles. Ogden hyperelastic modeling and second harmonic generation (SHG) imaging revealed that fiber alignment, densification, and strain-stiffening contributed to resilience under repetitive large-scale loading. After rehydration, crosslinked and densified hydrogels showed network stability and recoverability under cyclic loading, with a significantly reduced phase transition strain compared to non-crosslinked controls. The scaffolds supported cell encapsulation and maintained cell viability after 50 cycles of 90% compressive strain. Cyclic loading significantly densified the encapsulated cells in the loading direction, comparable to non-loaded controls. Overall, these results suggest that densified, shape memory collagen scaffolds provide a mechanically robust and biocompatible system for dynamic mechanical environments.

Collagen molecules self-assemble into supramolecular fibers within a molecularly crowded, polysaccharide-rich extracellular matrix (ECM). The ECM typically has fluid-like, viscoelastic properties that can be quantified rheologically. Here, we determine that the viscoelasticity of a polysaccharide alginate ECM regulates the assembly of type I collagen fibers. The viscoelasticity and shear moduli of the alginate network were tuned by the polymer weight percentage and degree of cooperative ionic and covalent norbornene-tetrazine crosslinking. Stepwise shear strain applied to covalently-crosslinked hydrogels generated higher stress than in ionic hydrogels. Hydrogels with reduced viscoelasticity also showed a reduction in water permeability. Second-harmonic generation confocal imaging revealed that decreasing viscoelasticity significantly suppressed collagen fiber self-assembly. Simulations demonstrated a mechanical coupling of the hydrogel network and the aggregate size of collagen molecules. Increased covalent crosslinking impaired the rate and magnitude of self-assembly in simulations and experimental results. These results suggest that ECM viscoelasticity plays a role in modulating the assembly and structural organization of collagen within the matrix. More broadly, they provide a framework for understanding how ECM mechanical properties can influence the assembly and organization of fibrillar macromolecules.

Abstract Targeted control of gene expression in mineralized tissue would enable the use of nucleic acids to modulate the local microenvironment at diseased sites, ultimately promoting bone regeneration. Piperazine-linked bisphosphonate ionizable lipids provide a facile approach to targeting the transfection of mineralized tissue with lipid nanoparticles (LNPs). Here, we develop a Mineralized Tissue-Targeting Expression System (MiTEX) using bisphosphonate LNPs to locally target mineralized tissues by adsorption to mineral surfaces and bone graft materials. MiTEX demonstrated a significant increase in the adsorption of RNA onto hydroxyapatite substrates, which retained the ability to transfect bone mesenchymal cells via the adsorbed layer of mRNA LNPs. Bone graft scaffolds functionalized by adsorbed Cre mRNA-LNP were implanted to genetically label newly formed bone tissues in vivo. The surface affinity and adsorption of bisphosphonate lipids provided a local reservoir in mineralized tissues, sustaining the in vivo delivery of MiTEX. Furthermore, the targeted delivery of RNA therapeutics was demonstrated using STAT3 siRNA to modulate gene expression and proinflammatory cytokine release in ex vivo periodontal tissues. The design of this new RNA-functionalized delivery platform will promote the development of precision nucleic acid therapeutics for local anti-inflammatory treatments and bone regeneration at mineralized tissue interfaces.

Abstract The scarcity of primary conventional dendritic cells (cDCs) and the limited effectiveness of monocyte-derived dendritic cells (moDCs) have long hindered progress in human dendritic cell research and immunotherapy. We developed a feeder-free differentiation platform that generates CD1c + CD141 + hPSC-cDCs phenotypically aligned with CD141 + tissue-resident cDC2 subsets found in human tissues. We further optimized the differentiation process using a Design-of-Experiments framework to refine cytokine and serum conditions, enhancing differentiation efficiency while reducing cytokine demand. These hPSC-cDCs exhibit efficient antigen uptake, defined cytokine responses, and robust priming of antigen-specific CD8 + T cell proliferation and effector differentiation, outperforming moDCs in direct comparison. Together, this work establishes a robust, and generalizable platform for mechanistic studies and translational development of dendritic cell-based vaccines and standardized ex vivo T cell expansion.

Periodontal disease is associated with inflamed gingival tissues and degradation of the gingival extracellular matrix (ECM), yet the role of mechanical cues is poorly understood. Gingival ECM in periodontal disease showed a loss of fibrillar collagen compared to healthy samples. We hypothesized that ECM softening in periodontal disease contributes to inflammation due to dysregulated gingival fibroblasts (GFs). A mechanically tunable hydrogel model of the gingival ECM was developed to investigate the mechano-immune crosstalk. Stiff collagen-alginate hydrogels matched the rheological properties of gingival biopsies. Human donor GFs encapsulated in these stiff hydrogels showed significantly suppressed toll-like receptor inflammatory responses compared to soft. Stiffness-dependent inflammatory responses of GFs were directed by the non-canonical NFκB pathway and epigenetic nuclear organization. The direct impact of mechanical cues on immune responses was investigated with human donor cells ex vivo by co-culture of human GFs with myeloid cells and in human gingival explants. Myeloid progenitors co-cultured with GFs in stiff hydrogels differentiated into immunomodulatory dendritic cells. Ex vivo crosslinking of human gingival tissue increased stiffness and reduced inflammatory cytokines. Gingival mechano-immune regulation provides a new avenue for biomaterials-based treatments in periodontitis.

Biocompatible adhesive Resin A multi-functional polymeric resin system based on thiol-ene crosslinking provides a strong adhesive interface with dentin that composes the inside structures of teeth and is biocompatible with dental pulp cells residing in the dentinal tubules that extend from the dentin surface. More details can be found in the Research Article by Kyle H. Vining and co-workers (DOI: 10.1002/adhm.202501993).

The mechanical properties of the extracellular matrix (ECM) regulate tumor growth and invasion in the tumor microenvironment. Models of biopolymer networks have been used to investigate the impact of elasticity and viscoelasticity of ECM on tumor behavior. Under tumor compression, these networks also show poroelastic behavior that is governed by the resistance to water flow through their pores. This work investigates the hypothesis that poroelastic properties regulate tumor growth. Here, alginate hydrogels with tunable ionic and hybrid ionic/covalent crosslinking are used as a model biopolymer system. Hydrogel stiffness, viscoelasticity, and stress relaxation behavior were characterized using stepwise axial compression. Among these properties, we find poroelastic fluid outflow dominates ECM stress relaxation, as the measured water flux was significantly affected under compression. Continuum mechanics-based modeling was developed to formulate and calculate the chemical potential gradients of water (solvent) in the hydrogels under compression. This framework was extended into an advection-diffusion framework to quantify growth factor (solute) distribution under varying strengths of stress and diffusion indexed by the relative strength of convective to diffusive transport, characterized by the Péclet number. An agent-based computational simulation showed that tumor growth was affected by Péclet number. Together, these results highlight the role of the poroelastic properties of ECM on water flux and transport in the tumor microenvironment.

Abstract Therapeutic T-cell engineering ex vivo from human hematopoietic stem cells (HSCs) focuses on recapitulating notch1-signaling and α4β1-integrin-mediated adhesion within the thymic niche with supportive stromal cell feeder-layers or surface-immobilized recombinant protein-based engineered thymic niches (ETNs). The relevant Notch1-DLL-4 and α4β1-integrin-VCAM-1 interactions are known to respond to mechanical forces that regulate their bond dissociation behaviors and downstream signal transduction, yet manipulating the mechanosensitive features of these key receptor-ligand interactions in thymopoiesis has been largely ignored in current ETN designs. Here, we demonstrate that human T-cell development from cord blood-derived CD34 + HSCs is regulated via molecular cooperativity in notch1 and integrin-mediated mechanotransduction. Mechanically confining interpenetrating network (IPN) hydrogel-based 3D cell culture comprised of collagen type I and alginate polysaccharides functionalized with DLL-4 and VCAM-1 is used as a model viscoelastic 3D ETN to manipulate human progenitor (pro)T-cell differentiation. This ETN enables orthogonal control of the mechanical and biomolecular features of the thymic niche, including thymopoietic ligand density, modulus, and viscoelastic properties (e.g., stress relaxation kinetics). We identify that soft, viscous matrices that enhance activation of the notch1-pathway, and subsequently notch1 intracellular domain (NICD) nuclear import sustain the T-cell development gene regulatory network during proT-cell differentiation. Conversely, stiff, elastic matrices inhibit HSC commitment to the T-lineage, and rather promotes Myeloid-cell differentiation. Our observations indicate mechanical reciprocity in signaling pathways indispensable to thymopoiesis, and highlights extracellular matrix mechanics as a variable in controlling hematopoietic stem cell fate decisions.

This study develops a biocompatible multifunctional thiol-ene resin system for adhesion to dentin mineralized tissue. Adhesive resins maintain the strength and longevity of dental composite restorations through chemophysical bonding to exposed dentin surfaces after cavity preparations. Monomers of conventional adhesive systems may result in inhomogeneous polymer networks and the release of residual monomers that cause cytotoxicity. In this study, a one-step multifunctional polymeric resin system by incorporating trimethylolpropane triacrylate (TMPTA) and bis[2-(methacryloyloxy)ethyl] phosphate (BMEP) is developed to enhance both mechanical properties and adhesion to dentin. Molecular dynamics simulations identify an optimal triacylate:trithiol ratio of 2.5:1, which is consistent with rheological and mechanical tests that yield a storage modulus of ≈30 MPa with or without BMEP. Shear bond tests demonstrate that the addition of BMEP significantly improves dentin adhesion, achieving a shear bond strength of 10.8 MPa, comparable to the commercial primer Clearfil SE Bond. Nanoindentation modulus mapping characterizes the hybrid layer and mechanical gradient of the adhesive resin system. Further, the triacrylate-BMEP resin shows biocompatibility with dental pulp cells and fibroblasts in vitro. These findings suggest that the triacrylate-trithiol crosslinking and chemophysical bonding of BMEP provide enhanced bond strength and biocompatibility for dental applications.

This study developed a biocompatible multifunctional thiol-ene resin system for adhesion to dentin mineralized tissue. Adhesive resins maintain the strength and longevity of dental composite restorations through chemophysical bonding to exposed dentin surfaces after cavity preparations. Dental pulp cells are exposed to residual monomers transported through dentinal tubules. Monomers of conventional adhesive systems may result in inhomogeneous polymer networks and the release of residual monomers that cause cytotoxicity. In this study, we develop a one-step multi-functional polymeric resin system by incorporating trimethylolpropane triacrylate (TMPTA) and bis[2-(methacryloyloxy)ethyl] phosphate (BMEP) to enhance both mechanical properties and adhesion to dentin. Molecular dynamics simulations identified an optimal triacylate:trithiol ratio of 2.5:1, which was consistent with rheological and mechanical tests that yielded a storage modulus of ~30 MPa with or without BMEP. Shear bond tests demonstrated that the addition of BMEP significantly improved dentin adhesion, achieving a shear bond strength of 10.8 MPa, comparable to the commercial primer Clearfil SE Bond. Nanoindentation modulus mapping characterized the hybrid layer and mechanical gradient of the adhesive resin system. Further, the triacrylate-BMEP resin showed biocompatibility with dental pulp cells and fibroblasts in vitro. These findings suggest the triacrylate-trithiol crosslinking and chemophysical bonding of BMEP provide enhanced bond strength and biocompatibility for dental applications.

Natural biopolymer hydrogels often suffer from relatively low moduli and an inability to maintain structure and mechanics under cyclic loading, limiting their utility in dynamic mechanical environments. Here, a cross-linked collagen cryogel scaffold was fabricated by precompression to densify the network. Following lyophilization, the porous scaffolds sustained >90% axial compressive strain with 200 cycles. Ogden hyperelastic modeling and second harmonic generation (SHG) imaging revealed fiber alignment, densification, and strain-stiffening contributing to resilience under repetitive large-scale loading. After rehydration, cross-linked and densified hydrogels showed network stability and recoverability under cyclic loading, with significantly reduced phase transition strains compared to non-cross-linked controls. The scaffolds supported cell encapsulation and maintained cell viability after 50 cycles of 90% strain. Cyclic loading significantly densified the encapsulated cells in the loading direction, comparable to nonloaded controls. Overall, these results suggest that densified, shape memory collagen scaffolds provide a mechanically robust and biocompatible system for dynamic environments.

Immune cells experience a wide range of modes and magnitudes of mechanical forces as they infiltrate tissues and physically interact with other cells. Biophysical forces influence cell phenotypes through mechanosensing of the cytoskeleton, cell adhesion, catch and slip bonds, and mechanically gated ion channels. As a result, different mechanical environments impact the function and expression of immune cell receptors, which subsequently affects local and systemic immune responses. Mechanical coupling of immune cell receptors can be exploited in immuno-engineering applications such as adoptive cell transfer and artificial antigen-presenting cells through biomaterial systems with tunable mechanical properties that regulate receptor expression and cell activation. This review covers immune cell receptors in the adaptive and innate immune system that respond to mechanical forces and their potential to be applied for advancing current immunotherapies.

This study developed a biocompatible multifunctional thiol-ene resin system for adhesion to dentin mineralized tissue. Adhesive resins maintain the strength and longevity of dental composite restorations through chemophysical bonding to exposed dentin surfaces after cavity preparations. Dental pulp cells are exposed to residual monomers transported through dentinal tubules. Monomers of conventional adhesive systems may result in inhomogeneous polymer networks and the release of residual monomers that cause cytotoxicity. In this study, we develop a one-step multi-functional polymeric resin system by incorporating trimethylolpropane triacrylate (TMPTA) and bis[2-(methacryloyloxy)ethyl] phosphate (BMEP) to enhance both mechanical properties and adhesion to dentin. Molecular dynamics simulations identified an optimal triacylate:trithiol ratio of 2.5:1, which was consistent with rheological and mechanical tests that yielded a storage modulus of ~30 MPa with or without BMEP. Shear bond tests demonstrated that the addition of BMEP significantly improved dentin adhesion, achieving a shear bond strength of 10.8 MPa, comparable to the commercial primer Clearfil SE Bond. Nanoindentation modulus mapping characterized the hybrid layer and mechanical gradient of the adhesive resin system. Further, the triacrylate-BMEP resin showed biocompatibility with fibroblasts in vitro. These findings suggest the triacrylate-trithiol crosslinking and chemophysical bonding of BMEP provide enhanced bond strength and biocompatibility for dental applications.

Monocytes, key mediators of innate immunity, exhibit remarkable sensitivity to mechanical cues such as extracellular matrix (ECM) stiffness, substrate rigidity, shear stress, compression, and hydrostatic pressure, which shape their activation, differentiation, and functional polarization. Monocytes develop from the bone marrow and populate the vasculature throughout the body. During inflammation, they are recruited to injured or diseased tissues by chemokines and proinflammatory cytokines, modulating local immune responses during embryonic development and adulthood via mechanosensing and mechanotransduction pathways. This review synthesizes recent advances in monocyte mechanobiology. It highlights how the bone marrow ECM mechanics orchestrates myelopoiesis, the role of endothelium and hemodynamic forces in migration, and how tissue mechanics influences monocyte fate in chronic inflammation, fibrosis, and cancer. We discuss the mechanosensitive pathways that govern monocyte behavior in health and disease and therapeutic opportunities that emerge from targeting these mechanisms via biomaterial approaches. Additionally, future directions toward developing mechanotherapy for immune modulation are discussed. By bridging mechanobiology and immunology, this review underscores the potential of mechanical cues as therapeutic targets to reprogram monocyte behavior in disease.

The developing dentition comprises vital hard tissues of the craniofacial complex that undergo complex and distinct mineralization stages of development through changes in their physicochemical properties. This study investigates the mechanical and chemical properties of the developing enamel, dentin, and bone in mouse mandibles. We employ a multimodal, multiscale analysis of the developing postnatal incisor and first molar by integrating microcomputed tomography, nanoindentation, energy dispersive spectroscopy, and Raman spectroscopy. Our findings reveal patterns of mechanical, elemental, and chemical changes across the developing incisor in enamel and dentin. Magnesium, iron, and the carbon-to-phosphate ratio were significantly associated with enamel properties, while magnesium composition was associated with dentin. These results demonstrate that the mineral composition drives mechanical properties across these developing craniofacial hard tissues. The integrative multimodal approach provides a quantitative perspective on the early stages of enamel and dentin mineralization of the developing incisor.

Alginate biopolymers were modified with norbornene (Nb) and tetrazine (Tz) functional groups to generate hydrogel networks with tunable ionic and covalent cross-linking for modeling the strain-stiffening behavior of extracellular matrix. The mechanical properties of the hydrogels were investigated by oscillatory shear rheology, axial compression, and stress relaxation analysis. Introducing Nb-Tz irreversible covalent cross-links yielded dual-cross-linked hydrogels with stiffer and more elastic properties compared to purely ionically cross-linked alginate networks. The strain stiffening effect was observed under both shear amplitude sweeps and stepwise axial compression tests for the dual-cross-linked hydrogels. This study provides valuable insights into the structure-property relationship of dual-cross-linked biopolymer hydrogels for designing tunable extracellular matrix mimics of fibrotic tissues.

Soft biopolymer networks play pivotal roles in governing cellular mechanics, tissue structure, and physiological processes such as blood coagulation. Understanding their permeability and mechanical responses under compression is crucial for elucidating mass transport phenomena and their impact on extra- and intra-cellular behavior as well as processes affecting functionality of blood clots, cartilage and other fibrous tissues. The nonlinear responses of these networks to mechanical stresses prevent application of established linear poro-elasticity models. Despite extensive studies of fibrous network viscoelastic properties under shear deformations, their dynamic responses to compressive deformations remain poorly understood, particularly in physiological contexts of growth and collective migration of solid bodies. Conventional experimental techniques face challenges in accurately evaluating the permeability of these networks, hindering comprehensive understanding of their poromechanical behavior. In this study, we employ a novel poroelastic hybrid approach combining rheometer-based compression rheology with camera-facilitated sample shape detection to directly measure fluid flux and network permeability under controlled compressive strains. Accompanying experimental investigations, a continuum model implemented in finite elements, and an analytical model are developed to interpret the findings. The experimental data align well with the analytical model, revealing the emergence and disappearance of distinct densification regimes within the gel under mechanical stress. This study advances our understanding of the intricate interplay between mechanical forces, fluid flow, and structural properties in soft biopolymer networks, with a specific focus on fibrin- and collagen-based gels which represent the most abundant protein networks in the extracellular environment.

Craniofacial tissues undergo hard tissue development through mineralization and changes in physicochemical properties. This study investigates the mechanical and chemical properties of developing enamel, dentin, and bone in the mouse mandible. We employ a multi-modal, multi-scale analysis of the developing incisor and first molar at postnatal day 12 by integrating micro-computed tomography (microCT), nanoindentation (NI), energy dispersive spectroscopy (EDS), and Raman spectroscopy. Our findings demonstrate distinct patterns of mechanical, elemental, and chemical changes across mineralized tissues. These results suggest that mineral composition drives mechanical properties across different craniofacial hard tissues. Integrating multi-modal characterization of mineralized tissues opens new opportunities for investigating structure-function relationships in craniofacial biology and genetics.

Abstract Nucleic acid delivery with mRNA lipid nanoparticles are being developed for targeting a wide array of tissues and cell types. However, targeted delivery to the bone microenvironment remains a significant challenge in the field, due in part to low local blood flow and poor interactions between drug carriers and bone material. Here we report bone‐targeting ionizable lipids incorporating a piperazine backbone and bisphosphate moieties, which bind tightly with hydroxyapatite ([Ca 5 (PO 4 ) 3 OH]), a key component of mineralized tissues. These lipids demonstrate biocompatibility and low toxicity in both vitro and in vivo studies. LNP formulated with these lipids facilitated efficient cellular transfection and improved binding to hydroxyapatite in vitro, and targeted delivery to the bone microenvironment in vivo following systemic administration. Overall, our findings demonstrate the critical role of the piperazine backbone in a novel ionizable lipid, which incorporates a bisphosphonate group to enable efficient bone‐targeted delivery, highlighting the potential of rational design of ionizable lipids for next‐generation bone‐targeting delivery systems.

Abstract Nucleic acid delivery with mRNA lipid nanoparticles are being developed for targeting a wide array of tissues and cell types. However, targeted delivery to the bone microenvironment remains a significant challenge in the field, due in part to low local blood flow and poor interactions between drug carriers and bone material. Here we report bone‐targeting ionizable lipids incorporating a piperazine backbone and bisphosphate moieties, which bind tightly with hydroxyapatite ([Ca 5 (PO 4 ) 3 OH]), a key component of mineralized tissues. These lipids demonstrate biocompatibility and low toxicity in both vitro and in vivo studies. LNP formulated with these lipids facilitated efficient cellular transfection and improved binding to hydroxyapatite in vitro, and targeted delivery to the bone microenvironment in vivo following systemic administration. Overall, our findings demonstrate the critical role of the piperazine backbone in a novel ionizable lipid, which incorporates a bisphosphonate group to enable efficient bone‐targeted delivery, highlighting the potential of rational design of ionizable lipids for next‐generation bone‐targeting delivery systems.

Cell-matrix interactions in 3D environments significantly differ from those in 2D cultures. As such, mechanisms of mechanotransduction in 2D cultures are not necessarily applicable to cell-encapsulating hydrogels that resemble features of tissue architecture. Accordingly, the characterization of molecular pathways in 3D matrices is expected to uncover insights into how cells respond to their mechanical environment in physiological contexts, and potentially also inform hydrogel-based strategies in cell therapies. In this study, a bone marrow-mimetic hydrogel was employed to systematically investigate the stiffness-responsive transcriptome of mesenchymal stromal cells. High matrix rigidity impeded integrin-collagen adhesion, resulting in changes in cell morphology characterized by a contractile network of actin proximal to the cell membrane. This resulted in a suppression of extracellular matrix-regulatory genes involved in the remodeling of collagen fibrils, as well as the upregulation of secreted immunomodulatory factors. Moreover, an investigation of long noncoding RNAs revealed that the cytoskeleton regulator RNA (CYTOR) contributes to these 3D stiffness-driven changes in gene expression. Knockdown of CYTOR using antisense oligonucleotides enhanced the expression of numerous mechanoresponsive cytokines and chemokines to levels exceeding those achievable by modulating matrix stiffness alone. Taken together, our findings further our understanding of mechanisms of mechanotransduction that are distinct from canonical mechanotransductive pathways observed in 2D cultures.

Oral squamous cell carcinoma (OSCC) presents significant treatment challenges due to its poor survival and intense pain at the primary cancer site. Cancer pain is debilitating, contributes to diminished quality of life, and causes opioid tolerance. The stimulator of interferon genes (STING) agonism has been investigated as an anti-cancer strategy. We have developed STINGel, an extended-release formulation that prolongs the availability of STING agonists, which has demonstrated an enhanced anti-tumor effect in OSCC compared to STING agonist injection. This study investigates the impact of intra-tumoral STINGel on OSCC-induced pain using two separate OSCC models and nociceptive behavioral assays. Intra-tumoral STINGel significantly reduced mechanical allodynia in the orofacial cancer model and alleviated thermal and mechanical hyperalgesia in the hind paw model. To determine the cellular signaling cascade contributing to the antinociceptive effect, we performed an in-depth analysis of immune cell populations via single-cell RNA-seq. We demonstrated an increase in M1-like macrophages and N1-like neutrophils after STINGel treatment. The identified regulatory pathways controlled immune response activation, myeloid cell differentiation, and cytoplasmic translation. Functional pathway analysis demonstrated the suppression of translation at neuron synapses and the negative regulation of neuron projection development in M2-like macrophages after STINGel treatment. Importantly, STINGel treatment upregulated TGF-β pathway signaling between various cell populations and peripheral nervous system (PNS) macrophages and enhanced TGF-β signaling within the PNS itself. Overall, this study sheds light on the mechanisms underlying STINGel-mediated antinociception and anti-tumorigenic impact.

High Resolution Image Download MS PowerPoint Slide Metal surgical pins and screws are employed in millions of orthopedic surgical procedures every year worldwide, but their usability is limited in the case of complex, comminuted fractures or in surgeries on smaller bones. Therefore, replacing such implants with a bone adhesive material has long been considered an attractive option. However, synthesizing a biocompatible bone adhesive with a high bond strength that is simple to apply presents many challenges. To rapidly identify candidate polymers for a biocompatible bone adhesive, we employed a high-throughput screening strategy to assess human mesenchymal stromal cell (hMSC) adhesion toward a library of polymers synthesized via thiol–ene click chemistry. We chose thiol–ene click chemistry because multifunctional monomers can be rapidly cured via ultraviolet (UV) light while minimizing residual monomer, and it provides a scalable manufacturing process for candidate polymers identified from a high-throughput screen. This screening methodology identified a copolymer (1-S2-FT01) composed of the monomers 1,3,5-triallyl-1,3,5-triazine-2,4,6(1 H,3 H,5 H )-trione (TATATO) and pentaerythritol tetrakis (3-mercaptopropionate) (PETMP), which supported highest hMSC adhesion across a library of 90 polymers. The identified copolymer (1-S2-FT01) exhibited favorable compressive and tensile properties compared to existing commercial bone adhesives and adhered to bone with adhesion strengths similar to commercially available bone glues such as Histoacryl. Furthermore, this cytocompatible polymer supported osteogenic differentiation of hMSCs and could adhere 3D porous polymer scaffolds to the bone tissue, making this polymer an ideal candidate as an alternative bone adhesive with broad utility in orthopedic surgery.

Functionally distinct T-cell populations can be generated from T cells that received the same stimulation by altering the viscoelasticity of their surrounding extracellular matrix. The efficacy of adoptive T-cell therapies largely depends on the generation of T-cell populations that provide rapid effector function and long-term protective immunity. Yet it is becoming clearer that the phenotypes and functions of T cells are inherently linked to their localization in tissues. Here we show that functionally distinct T-cell populations can be generated from T cells that received the same stimulation by altering the viscoelasticity of their surrounding extracellular matrix (ECM). By using a model ECM based on a norbornene-modified collagen type I whose viscoelasticity can be adjusted independently from its bulk stiffness by varying the degree of covalent crosslinking via a bioorthogonal click reaction with tetrazine moieties, we show that ECM viscoelasticity regulates T-cell phenotype and function via the activator-protein-1 signalling pathway, a critical regulator of T-cell activation and fate. Our observations are consistent with the tissue-dependent gene-expression profiles of T cells isolated from mechanically distinct tissues from patients with cancer or fibrosis, and suggest that matrix viscoelasticity could be leveraged when generating T-cell products for therapeutic applications.

Oral lichen planus (OLP) and recurrent aphthous stomatitis (RAS) are common chronic inflammatory conditions, manifesting as painful oral lesions that negatively affect patients’ quality of life. Current treatment approaches are mainly palliative and often ineffective due to inadequate contact time of the therapeutic agent with the lesions. Here, we developed the Dental Tough Adhesive (DenTAl), a bioinspired adhesive patch with robust mechanical properties, capable of strong adhesion against diverse wet and dynamically moving intraoral tissues, and extended drug delivery of clobetasol-17-propionate, a first-line drug for treating OLP and RAS. DenTAl was found to have superior physical and adhesive properties compared to existing oral technologies, with ~2 to 100× adhesion to porcine keratinized gingiva and ~3 to 15× stretchability. Clobetasol-17-propionate incorporated into the DenTAl was released in a tunable sustained manner for at least 3 wk and demonstrated immunomodulatory capabilities in vitro, evidenced by reductions in several cytokines, including TNF-α, IL-6, IL-10, MCP-5, MIP-2, and TIMP-1. Our findings suggest that DenTAl may be a promising device for intraoral delivery of small-molecule drugs applicable to the management of painful oral lesions associated with chronic inflammatory conditions.