Photoclick Chemistry: A Bright Idea.

At its basic conceptualization, photoclick chemistry embodies a collection of click reactions that are performed via the application of light. The emergence of this concept has had diverse impact over a broad range of chemical and biological research due to the spatiotemporal control, high selectivity, and excellent product yields afforded by the combination of light and click chemistry. While the reactions designated as "photoclick" have many important features in common, each has its own particular combination of advantages and shortcomings. A more extensive realization of the potential of this chemistry requires a broader understanding of the physical and chemical characteristics of the specific reactions. This review discusses the features of the most frequently employed photoclick reactions reported in the literature: photomediated azide-alkyne cycloadditions, other 1,3-dipolarcycloadditions, Diels-Alder and inverse electron demand Diels-Alder additions, radical alternating addition chain transfer additions, and nucleophilic additions. Applications of these reactions in a variety of chemical syntheses, materials chemistry, and biological contexts are surveyed, with particular attention paid to the respective strengths and limitations of each reaction and how that reaction benefits from its combination with light. Finally, challenges to broader employment of these reactions are discussed, along with strategies and opportunities to mitigate such obstacles.

4D Printing of Extrudable and Degradable Poly(Ethylene Glycol) Microgel Scaffolds for Multidimensional Cell Culture.

Granular synthetic hydrogels are useful bioinks for their compatibility with a variety of chemistries, affording printable, stimuli-responsive scaffolds with programmable structure and function. Additive manufacturing of microscale hydrogels, or microgels, allows for the fabrication of large cellularized constructs with percolating interstitial space, providing a platform for tissue engineering at length scales that are inaccessible by bulk encapsulation where transport of media and other biological factors are limited by scaffold density. Herein, synthetic microgels with varying degrees of degradability are prepared with diameters on the order of hundreds of microns by submerged electrospray and UV photopolymerization. Porous microgel scaffolds are assembled by particle jamming and extrusion printing, and semi-orthogonal chemical cues are utilized to tune the void fraction in printed scaffolds in a logic-gated manner. Scaffolds with different void fractions are easily cellularized post printing and microgels can be directly annealed into cell-laden structures. Finally, high-throughput direct encapsulation of cells within printable microgels is demonstrated, enabling large-scale 3D culture in a macroporous biomaterial. This approach provides unprecedented spatiotemporal control over the properties of printed microporous annealed particle scaffolds for 2.5D and 3D tissue culture.

Engineering Native Biological Complexity from the Inside-out and Outside-in.

Focusing on engineering control over cell function and fate, this article examines the critical balance of 'outside-in' and 'inside-out signaling in tissue development and regeneration. It highlights emerging strategies to manipulate these interactions, including biomaterial design and synthetic biology to influence this delicate equilibrium and fine tune cellular responses.

2D co-culture model reveals a biophysical interplay between activated fibroblasts and cancer cells.

The tumor microenvironment (TME) comprises diverse cell types within an altered extracellular matrix (ECM) and plays a pivotal role in metastasis through intricate cell-cell and cell-ECM interactions. Fibroblasts, as key constituents of the TME, contribute significantly to cancer metastasis through their involvement in matrix deposition and remodeling mechanisms, modulated by their quiescent or activated states. Despite their recognized importance, the precise role of fibroblasts in cancer cell invasion remains incompletely understood. In this study, we investigated the impact of fibroblast activity on cancer cell progression using a 2D co-culture model. Michigan Cancer Foundation-7 (MCF7) breast cancer cells were co-cultured with normal human lung fibroblasts (NHLF), both with and without transforming growth factor ß (TGFß) treatment. Traction force microscopy (TFM) was employed to quantify traction and velocity forces associated with cellular migration. We observed that TGFß-activated fibroblasts form a distinctive ring around cancer cells in co-culture, with increased traction and tension at the cell island boundary. This force distribution is associated with the localization of force-related proteins at these boundary regions, including vinculin and E-cadherin. Metabolic profiling revealed a strong OXPHOS signal specific to the activated fibroblasts, in contrast to normal fibroblasts, which primarily display migratory behavior and a more heterogeneous pattern of forces and metabolic activity in co-culture. Our findings offer valuable insights into the mechanical forces and metabolic dynamics governing cellular migration in the tumor microenvironment, where our co-culture model could complement in vivo studies and enable researchers to explore specific microenvironmental cues for a deeper understanding of TME mechanisms. STATEMENT OF SIGNIFICANCE: Cancer models mimicking the dynamics of tumor microenvironment (TME) are an ideal tool to study cancer mechanisms and treatment. However, the full understanding of how cancer cells interact with their surroundings and other cells is still unknown. To tackle this, we developed a simple yet effective 2D co-culture model that allows us to control the arrangement of cell cultures precisely and use various imaging techniques to study interactions between cancer cells and fibroblasts. Here we could measure cell movements, force distribution, metabolic activity, and protein localization and interplay those factors in vitro. Our model helps us observe the underlying mechanisms between cancer cells and fibroblasts, contributing to our understanding of the dynamics in the TME.

Photothermal Actuation of Thick 3D-Printed Liquid Crystalline Elastomer Nanocomposites.

Liquid crystalline elastomers (LCEs) are stimuli-responsive materials that transduce an input energy into a mechanical response. LCE composites prepared with photothermal agents, such as nanoinclusions, are a means to realize wireless, remote, and local control of deformation with light. Amongst photothermal agents, gold nanorods (AuNRs) are highly efficient converters when the irradiation wavelength matches the longitudinal surface plasmon resonance (LSPR) of the AuNRs. However, AuNR aggregation broadens the LSPR which also reduces photothermal efficiency. Here, the surface chemistry of AuNRs is engineered via a well-controlled two-step ligand exchange with a monofunctional poly(ethylene glycol) (PEG) thiol that greatly improves the dispersion of AuNRs in LCEs. Accordingly, LCE-AuNR nanocomposites with very low PEG-AuNR content (0.01 wt%) prepared by 3D printing are shown to be highly efficient photothermal actuators with rapid response (>60% strain s-1) upon irradiation with near-infrared (NIR; 808 nm) light. Because of the excellent dispersion of PEG-AuNR within the LCE, unabsorbed NIR light transmits through the nanocomposites and can actuate a series of samples. Further, the dispersion also allows for the optical deformation of millimeter-thick 3D printed structures without sacrificing actuation speed. The realization of well-dispersed nanoinclusions to maximize the stimulus-response of LCEs can benefit functional implementation in soft robotics or medical devices.

PTEN Regulates Myofibroblast Activation in Valvular Interstitials Cells based on Subcellular Localization.

Aortic valve stenosis (AVS) is characterized by altered mechanics of the valve leaflets, which disrupts blood flow through the aorta and can cause left ventricle hypotrophy. These changes in the valve tissue result in activation of resident valvular interstitial cells (VICs) into myofibroblasts, which have increased levels of αSMA in their stress fibers. The persistence of VIC myofibroblast activation is a hallmark of AVS. In recent years, the tumor suppressor gene phosphatase and tensin homolog (PTEN) has emerged as an important player in the regulation of fibrosis in various tissues (e.g., lung, skin), which motivated us to investigate PTEN as a potential protective factor against matrix-induced myofibroblast activation in VICs. In aortic valve samples from humans, we found high levels of PTEN in healthy tissue and low levels of PTEN in diseased tissue. Then, using pharmacological inducers to treat VIC cultures, we observed PTEN overexpression prevented stiffness-induced myofibroblast activation, whereas genetic and pharmacological inhibition of PTEN further activated myofibroblasts. We also observed increased nuclear PTEN localization in VICs cultured on stiff matrices, and nuclear PTEN also correlated with smaller nuclei, altered expression of histones and a quiescent fibroblast phenotype. Together, these results suggest that PTEN not only suppresses VIC activation, but functions to promote quiescence, and could serve as a potential pharmacological target for the treatment of AVS.

Radical-Mediated Degradation of Thiol-Maleimide Hydrogels.

Michael addition between thiol- and maleimide-functionalized molecules is a long-standing approach used for bioconjugation, hydrogel crosslinking, and the functionalization of other advanced materials. While the simplicity of this chemistry enables facile synthesis of hydrogels, network degradation is also desirable in many instances. Here, the susceptibility of thiol-maleimide bonds to radical-mediated degradation is reported. Irreversible degradation in crosslinked materials is demonstrated using photoinitiated and chemically initiated radicals in hydrogels and linear polymers. The extent of degradation is shown to be dependent on initiator concentration. Using a model linear polymer system, the radical-mediated mechanism of degradation is elucidated, in which the thiosuccinimide crosslink is converted to a succinimide and a new thioether formed with an initiator fragment. Using laser stereolithography, high-fidelity spatiotemporal control over degradation in crosslinked gels is demonstrated. Ultimately, this work establishes a platform for controllable, radical-mediated degradation in thiol-maleimide hydrogels, further expanding their versatility as functional materials.

Nonlinear Elastic Bottlebrush Polymer Hydrogels Modulate Actomyosin Mediated Protrusion Formation in Mesenchymal Stromal Cells.

The nonlinear elasticity of many tissue-specific extracellular matrices is difficult to recapitulate without the use of fibrous architectures, which couple strain-stiffening with stress relaxation. Herein, bottlebrush polymers are synthesized and crosslinked to form poly(ethylene glycol)-based hydrogels and used to study how strain-stiffening behavior affects human mesenchymal stromal cells (hMSCs). By tailoring the bottlebrush polymer length, the critical stress associated with the onset of network stiffening is systematically varied, and a unique protrusion-rich hMSC morphology emerges only at critical stresses within a biologically accessible stress regime. Local cell-matrix interactions are quantified using 3D traction force microscopy and small molecule inhibitors are used to identify cellular machinery that plays a critical role in hMSC mechanosensing of the engineered, strain-stiffening microenvironment. Collectively, this study demonstrates how covalently crosslinked bottlebrush polymer hydrogels can recapitulate strain-stiffening biomechanical cues at biologically relevant stresses and be used to probe how nonlinear elastic matrix properties regulate cellular processes.

Fully synthetic hydrogels promote robust crypt formation in intestinal organoids.

Initial landmark studies in the design of synthetic hydrogels for intestinal organoid culture identified precise matrix requirements for differentiation, namely decompression of matrix-imposed forces and supplementation of laminin. But beyond stating the necessity of laminin, organoid-laminin interactions have gone largely unstudied, as this ubiquitous requirement of exogenous laminin hinders investigation. In this work, we exploit a fast stress relaxing, boronate ester based synthetic hydrogel for the culture of intestinal organoids, and fortuitously discover that unlike all other synthetic hydrogels to date, laminin does not need to be supplemented for crypt formation. This highly defined material provides a unique opportunity to investigate laminin-organoid interactions and how it influences crypt evolution and organoid function. Via fluorescent labeling of non-canonical amino acids, we further show that adaptable boronate ester bonds increase deposition of nascent proteins, including laminin. Collectively, these results advance the understanding of how mechanical and matricellular signaling influence intestinal organoid development.

Reversible Intracellular Gelation of MCF10A Cells Enables Programmable Control Over 3D Spheroid Growth.

In nature, some organisms survive extreme environments by inducing a biostatic state wherein cellular contents are effectively vitrified. Recently, a synthetic biostatic state in mammalian cells is achieved via intracellular network formation using bio-orthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) reactions between functionalized poly(ethylene glycol) (PEG) macromers. In this work, the effects of intracellular network formation on a 3D epithelial MCF10A spheroid model are explored. Macromer-transfected cells are encapsulated in Matrigel, and spheroid area is reduced by ≈50% compared to controls. The intracellular hydrogel network increases the quiescent cell population, as indicated by increased p21 expression. Additionally, bioenergetics (ATP/ADP ratio) and functional metabolic rates are reduced. To enable reversibility of the biostasis effect, a photosensitive nitrobenzyl-containing macromer is incorporated into the PEG network, allowing for light-induced degradation. Following light exposure, cell state, and proliferation return to control levels, while SPAAC-treated spheroids without light exposure (i.e., containing intact intracellular networks) remain smaller and less proliferative through this same period. These results demonstrate that photodegradable intracellular hydrogels can induce a reversible slow-growing state in 3D spheroid culture.

Stiffness anisotropy coordinates supracellular contractility driving long-range myotube-ECM alignment.

The ability of cells to organize into tissues with proper structure and function requires the effective coordination of proliferation, migration, polarization, and differentiation across length scales. Skeletal muscle is innately anisotropic; however, few biomaterials can emulate mechanical anisotropy to determine its influence on tissue patterning without introducing confounding topography. Here, we demonstrate that substrate stiffness anisotropy coordinates contractility-driven collective cellular dynamics resulting in C2C12 myotube alignment over millimeter-scale distances. When cultured on mechanically anisotropic liquid crystalline polymer networks (LCNs) lacking topography, C2C12 myoblasts collectively polarize in the stiffest direction. Cellular coordination is amplified through reciprocal cell-ECM dynamics that emerge during fusion, driving global myotube-ECM ordering. Conversely, myotube alignment was restricted to small local domains with no directional preference on mechanically isotropic LCNs of the same chemical formulation. These findings provide valuable insights for designing biomaterials that mimic anisotropic microenvironments and underscore the importance of stiffness anisotropy in orchestrating tissue morphogenesis.

Photochemical Control of Network Topology in PEG Hydrogels.

Hydrogels are often synthesized through photoinitiated step-, chain-, and mixed-mode polymerizations, generating diverse network topologies and resultant material properties that depend on the underlying network connectivity. While many photocrosslinking reactions are available, few afford controllable connectivity of the hydrogel network. Herein, a versatile photochemical strategy is introduced for tuning the structure of poly(ethylene glycol) (PEG) hydrogels using macromolecular monomers functionalized with maleimide and styrene moieties. Hydrogels are prepared along a gradient of topologies by varying the ratio of step-growth (maleimide dimerization) to chain-growth (maleimide-styrene alternating copolymerization) network-forming reactions. The initial PEG content and final network physical properties (e.g., modulus, swelling, diffusivity) are tailored in an independent manner, highlighting configurable gel mechanics and reactivity. These photochemical reactions allow high-fidelity photopatterning and 3D printing and are compatible with 2D and 3D cell culture. Ultimately, this photopolymer chemistry allows facile control over network connectivity to achieve adjustable material properties for broad applications.

In situ modulation of intestinal organoid epithelial curvature through photoinduced viscoelasticity directs crypt morphogenesis.

Spatiotemporally coordinated transformations in epithelial curvature are necessary to generate crypt-villus structures during intestinal development. However, the temporal regulation of mechanotransduction pathways that drive crypt morphogenesis remains understudied. Intestinal organoids have proven useful to study crypt morphogenesis in vitro, yet the reliance on static culture scaffolds limits the ability to assess the temporal effects of changing curvature. Here, a photoinduced hydrogel cross-link exchange reaction is used to spatiotemporally alter epithelial curvature and study how dynamic changes in curvature influence mechanotransduction pathways to instruct crypt morphogenesis. Photopatterned curvature increased membrane tension and depolarization, which was required for subsequent nuclear localization of yes-associated protein 1 (YAP) observed 24 hours following curvature change. Curvature-directed crypt morphogenesis only occurred following a delay in the induction of differentiation that coincided with the delay in spatially restricted YAP localization, indicating that dynamic changes in curvature initiate epithelial curvature-dependent mechanotransduction pathways that temporally regulate crypt morphogenesis.

Osteopontin activity modulates sex-specific calcification in engineered valve tissue mimics.

Patients with aortic valve stenosis (AVS) have sexually dimorphic phenotypes in their valve tissue, where male valvular tissue adopts a calcified phenotype and female tissue becomes more fibrotic. The molecular mechanisms that regulate sex-specific calcification in valvular tissue remain poorly understood. Here, we explored the role of osteopontin (OPN), a pro-fibrotic but anti-calcific bone sialoprotein, in regulating the calcification of female aortic valve tissue. Recognizing that OPN mediates calcification processes, we hypothesized that aortic valvular interstitial cells (VICs) in female tissue have reduced expression of osteogenic markers in the presence of elevated OPN relative to male VICs. Human female valve leaflets displayed reduced and smaller microcalcifications, but increased OPN expression relative to male leaflets. To understand how OPN expression contributes to observed sex dimorphisms in valve tissue, we employed enzymatically degradable hydrogels as a 3D cell culture platform to recapitulate male or female VIC interactions with the extracellular matrix. Using this system, we recapitulated sex differences observed in human tissue, specifically demonstrating that female VICs exposed to calcifying medium have smaller mineral deposits within the hydrogel relative to male VICs. We identified a change in OPN dynamics in female VICs in the presence of calcification stimuli, where OPN deposition localized from the extracellular matrix to perinuclear regions. Additionally, exogenously delivered endothelin-1 to encapsulated VICs increased OPN gene expression in male cells, which resulted in reduced calcification. Collectively, our results suggest that increased OPN in female valve tissue may play a sex-specific role in mitigating mineralization during AVS progression.

Photoinduced Dithiolane Crosslinking for Multiresponsive Dynamic Hydrogels.

While many hydrogels are elastic networks crosslinked by covalent bonds, viscoelastic hydrogels with adaptable crosslinks are increasingly being developed to better recapitulate time and position-dependent processes found in many tissues. In this work, 1,2-dithiolanes are presented as dynamic covalent photocrosslinkers of hydrogels, resulting in disulfide bonds throughout the hydrogel that respond to multiple stimuli. Using lipoic acid as a model dithiolane, disulfide crosslinks are formed under physiological conditions, enabling cell encapsulation via an initiator-free light-induced dithiolane ring-opening photopolymerization. The resulting hydrogels allow for multiple photoinduced dynamic responses including stress relaxation, stiffening, softening, and network functionalization using a single chemistry, which can be supplemented by permanent reaction with alkenes to further control network properties and connectivity using irreversible thioether crosslinks. Moreover, complementary photochemical approaches are used to achieve rapid and complete sample degradation via radical scission and post-gelation network stiffening when irradiated in the presence of reactive gel precursor. The results herein demonstrate the versatility of this material chemistry to study and direct 2D and 3D cell-material interactions. This work highlights dithiolane-based hydrogel photocrosslinking as a robust method for generating adaptable hydrogels with a range of biologically relevant mechanical and chemical properties that are varied on demand.

Stiffness anisotropy coordinates supracellular contractility driving long-range myotube-ECM alignment.

In skeletal muscle tissue, injury-related changes in stiffness activate muscle stem cells through mechanosensitive signaling pathways. Functional muscle tissue regeneration also requires the effective coordination of myoblast proliferation, migration, polarization, differentiation, and fusion across multiple length scales. Here, we demonstrate that substrate stiffness anisotropy coordinates contractility-driven collective cellular dynamics resulting in C2C12 myotube alignment over millimeter-scale distances. When cultured on mechanically anisotropic liquid crystalline polymer networks (LCNs) lacking topographic features that could confer contact guidance, C2C12 myoblasts collectively polarize in the stiffest direction of the substrate. Cellular coordination is amplified through reciprocal cell-ECM dynamics that emerge during fusion, driving global myotube-ECM ordering. Conversely, myotube alignment was restricted to small local domains with no directional preference on mechanically isotropic LCNs of same chemical formulation. These findings reveal a role for stiffness anisotropy in coordinating emergent collective cellular dynamics, with implications for understanding skeletal muscle tissue development and regeneration.

Facile Physicochemical Reprogramming of PEG-Dithiolane Microgels.

Granular biomaterials have found widespread applications in tissue engineering, in part because of their inherent porosity, tunable properties, injectability, and 3D printability. However, the assembly of granular hydrogels typically relies on spherical microparticles and more complex particle geometries have been limited in scope, often requiring templating of individual microgels by microfluidics or in-mold polymerization. Here, we use dithiolane-functionalized synthetic macromolecules to fabricate photopolymerized microgels via batch emulsion, and then harness the dynamic disulfide crosslinks to rearrange the network. Through unconfined compression between parallel plates in the presence of photoinitiated radicals, we transform the isotropic microgels are transformed into disks. Characterizing this process, we find that the areas of the microgel surface in contact with the compressive plates are flattened while the curvature of the uncompressed microgel boundaries increases. When cultured with C2C12 myoblasts, cells localize to regions of higher curvature on the disk-shaped microgel surfaces. This altered localization affects cell-driven construction of large supraparticle scaffold assemblies, with spherical particles assembling without specific junction structure while disk microgels assemble preferentially on their curved surfaces. These results represent a unique spatiotemporal process for rapid reprocessing of microgels into anisotropic shapes, providing new opportunities to study shape-driven mechanobiological cues during and after granular hydrogel assembly.

Surface-Enforced Alignment of Reprogrammable Liquid Crystalline Elastomers.

Liquid crystalline elastomers (LCEs) are stimuli-responsive materials capable of undergoing large deformations. The thermomechanical response of LCEs is attributable to the coupling of polymer network properties and disruption of order between liquid crystalline mesogens. Complex deformations have been realized in LCEs by either programming the nematic director via surface-enforced alignment or localized mechanical deformation in materials incorporating dynamic covalent chemistries. Here, the preparation of LCEs via thiol-Michael addition reaction is reported that are amenable to surface-enforced alignment. Afforded by the thiol-Michael addition reaction, dynamic covalent bonds are uniquely incorporated in chemistries subject to surface-enforce alignment. Accordingly, LCEs prepared with complex director profiles are able to be programmed and reprogrammed by (re)activating the dynamic covalent chemistry to realize distinctive shape transformations.

4D Materials with Photoadaptable Properties Instruct and Enhance Intestinal Organoid Development.

Intestinal organoids are self-organized tissue constructs, grown in vitro, that resemble the structure and function of the intestine and are often considered promising as a prospective platform for drug testing and disease modeling. Organoid development in vitro is typically instructed by exogenous cues delivered from the media, but cellular responses also depend on properties of the surrounding microenvironmental niche, such as mechanical stiffness and extracellular matrix (ECM) ligands. In recent years, synthetic hydrogel platforms have been engineered to resemble the in vivo niche, with the goal of generating physiologically relevant environments that can promote mature and reproducible organoid development. However, a few of these approaches consider the importance of intestinal organoid morphology or how morphology changes during development, as cues that may dictate organoid functionality. For example, intestinal organoids grown in vitro often lack the physical boundary conditions found in vivo that are responsible for shaping a collection of cells into developmentally relevant morphologies, resulting in organoids that often differ in structure and cellular organization from the parent organ. This disconnect relates, in part, to a lack of appropriate adaptable and programmable materials for cell culture, especially those that enable control over colony growth and differentiation in space and time (i.e., 4D materials). We posit that the future of organoid culture platforms may benefit from advances in photoadaptable chemistries and integration into biomaterials scaffolds, thereby allowing greater user-directed control over both the macro- and microscale material properties. In this way, synthetic materials can begin to better replicate changes in the ECM during development or regeneration in vivo. Recapitulation of cellular and tissue morphological changes, along with an appreciation for the appropriate developmental time scales, should help instruct the next generation of organoid models to facilitate predictable outcomes.

Granular PEG hydrogels mediate osteoporotic MSC clustering via N-cadherin influencing the pro-resorptive bias of their secretory profile.

Postmenopausal osteoporosis results from a pro-resorptive bone environment, which decreases bone mineral density causing increased fracture risk. Bone marrow derived mesenchymal stem/stromal cells (MSCs) secrete factors involved in bone homeostasis, but osteoporosis mediated changes to their secretions remain understudied. Herein, we examined the secretome of MSCs isolated from ovariectomized rats (OVX rMSCs), a model of post-menopausal osteoporosis, as a function of cell-cell interactions. Specifically, we controlled clustering of OVX and SHAM rMSCs by assembling them in granular hydrogels synthesized from poly(ethylene glycol) microgels with average diameters of ∼10, 100, and 200 µm. We directed both the sizes of rMSC clusters (single cells to ∼30 cells/cluster) and the percentages of cells within clusters (∼20-90%) by controlling the scaffold pore dimensions. Large clusters of OVX rMSCs had a pro-resorptive secretory profile, with increased concentrations of Activin A, CXCL1, CX3CL1, MCP-1, TIMP-1, and TNF-ɑ, compared to SHAM rMSCs. As this pro-resorptive bias was only observed in large cell clusters, we characterized the expression of several cadherins, mediators of cell-cell contacts. N-cadherin expression was elevated (∼4-fold) in OVX relative to SHAM rMSCs, in both cell clusters and single cells. Finally, TIMP-1 and MCP-1 secretion was only decreased in large cell clusters of OVX rMSCs when N-cadherin interactions were blocked, highlighting the dependence of OVX rMSC secretion of pro-resorptive cytokines on N-cadherin mediated cell-cell contacts. Further elucidation of the N-cadherin mediated osteoporotic MSC secretome may have implications for developing therapies for postmenopausal osteoporosis. STATEMENT OF SIGNIFICANCE: Postmenopausal osteoporosis is a prevalent bone disorder that affects tens of millions of women worldwide. This disease is characterized by severe bone loss resulting from a pro-resorptive bone marrow environment, where the rates of bone resorption outpace the rates of bone deposition. The paracrine factors secreted by bone marrow MSCs can influence cell types responsible for bone homeostasis, but the osteoporosis-mediated changes to MSC secretory properties remains understudied. In this study, we used PEG-based porous granular scaffolds to study the influence of cell clustering on the secretory properties of osteoporotic MSCs. We observed increased secretion of several pro-resorptive factors by osteoporotic MSCs in large clusters. Further, we explored the dependence of this altered secretion profile on N-cadherin mediated cell-cell contacts.