Kindlin3 regulates biophysical properties and mechanics of membrane to cortex attachment.

Kindlin3 (K3), a FERM domain containing protein expressed in hematopoietic cells controls integrin activation and thus hemostatic and inflammatory responses. However, its role in the mechanics of plasma membrane remains unclear. Here, we show that genetic knockout of K3 in microglia and macrophages resulted in defective plasma membrane tension and membrane blebbing. Atomic force microscopy (AFM) of K3-deficient cells revealed a significant loss in membrane-to-cortex attachment (MCA), and consequently reduced membrane tension. This loss in MCA is amplified by the mislocalization of the cell cortex proteins-ezrin, radixin, and moesin (ERM)-to the plasma membrane of microglia and macrophages. Re-expression of K3 in K3-deficient macrophages rescued the defects and localization of ERMs implying a key role for K3 in MCA. Analysis of two K3 mutants, K3int affecting integrin binding and activation, and K3pxn/act disrupting binding to paxillin and actin but not integrin functions, demonstrated that the role of K3 in membrane mechanics is separate from integrin activation. The K3pxn/act mutant substantially diminished both membrane tension and Yes-associated protein (YAP) translocation to the nucleus, while preserving integrin activation, cell spreading, and migration. Together, our results show that K3 coordinates membrane mechanics, ERM protein recruitment to the membrane, and YAP translocation by linking integrin at the membrane to paxillin and actin of the cytoskeleton. This novel function of K3 is distinct from its role in integrin activation.

Alterations in phenotype and gene expression of adult human aneurysmal smooth muscle cells by exogenous nitric oxide.

Abdominal aortic aneurysms (AAA) are characterized by matrix remodeling, elastin degradation, absence of nitric oxide (NO) signaling, and inflammation, influencing smooth muscle cell (SMC) phenotype and gene expression. Little is known about the biomolecular release and intrinsic biomechanics of human AAA-SMCs. NO delivery could be an attractive therapeutic strategy to restore lost functionality of AAA-SMCs by inhibiting inflammation and cell stiffening. We aim to establish the differences in phenotype and gene expression of adult human AAA-SMCs from healthy SMCs. Based on our previous study which showed benefits of optimal NO dosage delivered via S-Nitrosoglutathione (GSNO) to healthy aortic SMCs, we tested whether such benefits would occur in AAA-SMCs. The mRNA expression of three genes involved in matrix degradation (ACE, ADAMTS5 and ADAMTS8) was significantly downregulated in AAA-SMCs. Total protein and glycosaminoglycans synthesis were higher in AAA-SMCs than healthy-SMCs (p < 0.05 for AAA-vs. healthy- SMC cultures) and was enhanced by GSNO and 3D cultures (p < 0.05 for 3D vs. 2D cultures; p < 0.05 for GSNO vs. non-GSNO cases). Elastin gene expression, synthesis and deposition, desmosine crosslinker levels, and lysyl oxidase (LOX) functional activity were lower, while cell proliferation, iNOS, LOX and fibrillin-1 gene expressions were higher in AAA-SMCs (p < 0.05 between respective cases), with differential benefits from GSNO exposure. GSNO and 3D cultures reduced MMPs -2, -9, and increased TIMP-1 release in AAA-SMC cultures (p < 0.05 for GSNO vs. non-GSNO cultures). AAA-SMCs were inherently stiffer and had smoother surface than healthy SMCs (p < 0.01 in both cases), but GSNO reduced stiffness (~25%; p < 0.01) and increased roughness (p < 0.05) of both cell types. In conclusion, exogenously-delivered NO offers an attractive strategy by providing therapeutic benefits to AAA-SMCs.

Sialylation status and mechanical properties of THP-1 macrophages upon LPS stimulation.

Cell surface receptors are the key contributors of macrophage function. Most macrophage cell surface receptors are glycoproteins with sialic acids at the terminal of their glycans. It is well recognized that lipopolysaccharide (LPS) induces cell surface sialylation changes that may in turn contribute to macrophage functions. In addition, cellular mechanics such as elasticity is also a major determinant of macrophage function, which in turn is modulated by LPS. In this report, we characterized the sialylation status of macrophages upon LPS stimulation and assessed the changes in its mechanical properties and function. Specifically, we confirmed that sialylation status is closely related to macrophage biomechanical characteristics (elastic modulus, tether force, tether radius, adhesion force, and membrane tension) and thus directly involved in macrophage function. Further, we modulated macrophage sialylation status by feeding the cell with exogenous free sialic acid (Neu5Ac, Neu5Gc) and sialidase inhibitors, and examined the resulting effects on cellular mechanics and function. A systematic recognition of sialylation status related to cellular mechanics of macrophages will contribute to defining their phenotypes and elucidate macrophage functional diversity.

Pediatric glioblastoma cells inhibit neurogenesis and promote astrogenesis, phenotypic transformation and migration of human neural progenitor cells within cocultures.

Neural progenitor cell (NPC) fate is influenced by a variety of biological cues elicited from the surrounding microenvironment and recent studies suggest their possible role in pediatric glioblastoma multiforme (GBM) development. Since a few GBM cells also display NPC characteristics, it is not clear whether NPCs transform to tumor cell phenotype leading to the onset of GBM formation, or NPCs migrate to developing tumor sites in response to paracrine signaling from GBM cells. Elucidating the paracrine interactions between GBM cells and NPCs in vivo is challenging due to the inherent complexity of the CNS. Here, we investigated the interactions between human NPCs (ReNcell) and human pediatric GBM-derived cells (SJ-GBM2) using a Transwell® coculture setup to assess the effects of GBM cells on ReNcells (cytokine and chemokine release, viability, phenotype, differentiation, migration). Standalone ReNcell or GBM cultures served as controls. Qualitative and quantitative results from ELISA®, Live/Dead® and BrdU assays, immunofluorescence labeling, western blot analysis, and scratch test suggests that although ReNcell viability remained unaffected in the presence of pediatric GBM cells, their morphology, phenotype, differentiation patterns, neurite outgrowth, migration patterns (average speed, distance, number of cells) and GSK-3ß expression were significantly influenced. The cumulative distance migrated by the cells in each condition was fit to Furth's formula, derived formally from Ornstein-Uhlenbeck process. ReNcell differentiation into neural lineage was compromised and astrogenesis promoted within cocultures. Such coculture platform could be extended to identify the specific molecules contributing to the observed phenomena, to investigate whether NPCs could be transplanted to replace lesions of excised tumor sites, and to elucidate the underlying molecular pathways involved in GBM-NPC interactions within the tumor microenvironment.

Biophysical and biomechanical properties of neural progenitor cells as indicators of developmental neurotoxicity.

Conventional in vitro toxicity studies have focused on identifying IC50 and the underlying mechanisms, but how toxicants influence biophysical and biomechanical changes in human cells, especially during developmental stages, remain understudied. Here, using an atomic force microscope, we characterized changes in biophysical (cell area, actin organization) and biomechanical (Young's modulus, force of adhesion, tether force, membrane tension, tether radius) aspects of human fetal brain-derived neural progenitor cells (NPCs) induced by four classes of widely used toxic compounds, including rotenone, digoxin, N-arachidonoylethanolamide (AEA), and chlorpyrifos, under exposure up to 36 h. The sub-cellular mechanisms (apoptosis, mitochondria membrane potential, DNA damage, glutathione levels) by which these toxicants induced biochemical changes in NPCs were assessed. Results suggest a significant compromise in cell viability with increasing toxicant concentration (p < 0.01), and biophysical and biomechanical characteristics with increasing exposure time (p < 0.01) as well as toxicant concentration (p < 0.01). Impairment of mitochondrial membrane potential appears to be the most sensitive mechanism of neurotoxicity for rotenone, AEA and chlorpyrifos exposure, but compromise in plasma membrane integrity for digoxin exposure. The surviving NPCs remarkably retained stemness (SOX2 expression) even at high toxicant concentrations. A negative linear correlation (R2 = 0.92) exists between the elastic modulus of surviving cells and the number of living cells in that environment. We propose that even subtle compromise in cell mechanics could serve as a crucial marker of developmental neurotoxicity (mechanotoxicology) and therefore should be included as part of toxicology assessment repertoire to characterize as well as predict developmental outcomes.

Three-dimensional collagenous niche and azacytidine selectively promote time-dependent cardiomyogenesis from human bone marrow-derived MSC spheroids.

Endogenous adult cardiac regenerative machinery is not capable of replacing the lost cells following myocardial infarction, often leading to permanent alterations in structure-function-mechanical properties. Regenerative therapies based on delivering autologous stem cells within an appropriate 3D milieu could meet such demand, by enabling homing and directed differentiation of the transplanted cells into lost specialized cell populations. Since type I collagen is the predominant cardiac tissue matrix protein, we here optimized the 3D niche which could promote time-dependent evolution of cardiomyogenesis from human bone marrow-derived mesenchymal stem cells (BM-MSC). 3D collagen gel physical and mechanical characteristics were assessed using SEM and AFM, respectively, while the standalone and combined effects of collagen concentration, culture duration, and 5-azacytidine (aza) dose on the phenotype and genotype of MSC spheroids were quantified using immunofluorescence labeling and RT-PCR analysis. Increasing collagen concentration led to a significant increase in Young's modulus (p < 0.01) but simultaneous decrease in the mean pore size, resulting in stiffer gels. Spheroid formation significantly modulated MSC differentiation and genotype, mostly due to better cell-cell interactions. Among the aza dosages tested, 10 µM appears to be optimal, while 3 mg/ml gels resulted in significantly lower cell viability compared to 1 or 2 mg/ml gels. Stiffer gels (2 and 3 mg/ml) and exposure to 10 µM aza upregulated early and late cardiac marker expressions in a time-dependent fashion. On the other hand, cell-cell signaling within the MSC spheroids seem to have a strong role in influencing mature cardiac markers expression, since neither aza nor gel stiffness seem to significantly improve their expression. Western blot analysis suggested that canonical Wnt/ß-catenin signaling pathway might be primarily mediating the observed benefits of aza on cardiac differentiation of MSC spheroids. In conclusion, 2 mg/ml collagen and 10 µM aza appears to offer optimal 3D microenvironment in terms of cell viability and time-dependent evolution of cardiomyogenesis from human BM-MSCs, with significant applications in cardiac tissue engineering and stem cell transplantation for regenerating lost cardiac tissue.

Common SYNE2 Genetic Variant Associated With Atrial Fibrillation Lowers Expression of Nesprin-2α1 With Downstream Effects on Nuclear and Electrophysiological Traits.

BACKGROUND: Atrial fibrillation GWAS (genome-wide association studies) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding Nesprin-2, which connects the nuclear membrane with the cytoskeleton. METHODS: Reporter gene vector transfection and CRISPR-Cas9 editing were used to identify the causal variant regulating the expression of SYNE2α1. After SYNE2 knockdown or SYNE2α1 overexpression in human stem cell-derived cardiomyocytes, nuclear phenotypes were assessed by imaging and atomic force microscopy. Gene expression was assessed by RNAseq and gene set enrichment analysis. Fura-2 AM staining assessed calcium transients. Optical mapping assessed action potential duration and conduction velocity. RESULTS: The risk allele of rs1152591 had lower promoter and enhancer activity and was significantly associated with lower expression of the short SYNE2α1 isoform in human stem cell-derived cardiomyocytes, without an effect on the expression of the full-length SYNE2 mRNA. SYNE2α1 overexpression had dominant negative effects on the nucleus with its overexpression or SYNE2 knockdown leading to increased nuclear area and decreased nuclear stiffness. Gene expression results from SYNE2α1 overexpression demonstrated both concordant and nonconcordant effects with SYNE2 knockdown. SYNE2α1 overexpression had a gain of function on electrophysiology, leading to significantly faster calcium reuptake and decreased assessed action potential duration, while SYNE2 knockdown showed both shortened assessed action potential duration and decreased conduction velocity. CONCLUSIONS: rs1152591 was identified as a causal atrial fibrillation variant, with the risk allele decreasing SYNE2α1 expression. Downstream effects of SYNE2α1 overexpression include changes in nuclear stiffness and electrophysiology, which may contribute to the mechanism for the risk allele's association with AF.

A prognostic molecular signature of hepatic steatosis is spatially heterogeneous and dynamic in human liver.

Metabolic dysfunction-associated steatotic liver disease (MASLD) prevalence is increasing in parallel with an obesity pandemic, calling for novel strategies for prevention and treatment. We defined a circulating proteome of human MASLD across ≈7000 proteins in ≈5000 individuals from diverse, at-risk populations across the metabolic health spectrum, demonstrating reproducible diagnostic performance and specifying both known and novel metabolic pathways relevant to MASLD (central carbon and amino acid metabolism, hepatocyte regeneration, inflammation, fibrosis, insulin sensitivity). A parsimonious proteomic signature of MASLD was associated with a protection from MASLD and its related multi-system metabolic consequences in >26000 free-living individuals, with an additive effect to polygenic risk. The MASLD proteome was encoded by genes that demonstrated transcriptional enrichment in liver, with spatial transcriptional activity in areas of steatosis in human liver biopsy and dynamicity for select targets in human liver across stages of steatosis. We replicated several top relations from proteomics and spatial tissue transcriptomics in a humanized "liver-on-a-chip" model of MASLD, highlighting the power of a full translational approach to discovery in MASLD. Collectively, these results underscore utility of blood-based proteomics as a dynamic "liquid biopsy" of human liver relevant to clinical biomarker and mechanistic applications.

Modeling mucus physiology and pathophysiology in human organs-on-chips.

The surfaces of human internal organs are lined by a mucus layer that ensures symbiotic relationships with commensal microbiome while protecting against potentially injurious environmental chemicals, toxins, and pathogens, and disruption of this layer can contribute to disease development. Studying mucus biology has been challenging due to the lack of physiologically relevant human in vitro models. Here we review recent progress that has been made in the development of human organ-on-a-chip microfluidic culture models that reconstitute epithelial tissue barriers and physiologically relevant mucus layers with a focus on lung, colon, small intestine, cervix and vagina. These organ-on-a-chip models that incorporate dynamic fluid flow, air-liquid interfaces, and physiologically relevant mechanical cues can be used to study mucus composition, mechanics, and structure, as well as investigate its contributions to human health and disease with a level of biomimicry not possible in the past.

Vaginal microbiome-host interactions modeled in a human vagina-on-a-chip.

BACKGROUND: A dominance of non-iners Lactobacillus species in the vaginal microbiome is optimal and strongly associated with gynecological and obstetric health, while the presence of diverse obligate or facultative anaerobic bacteria and a paucity in Lactobacillus species, similar to communities found in bacterial vaginosis (BV), is considered non-optimal and associated with adverse health outcomes. Various therapeutic strategies are being explored to modulate the composition of the vaginal microbiome; however, there is no human model that faithfully reproduces the vaginal epithelial microenvironment for preclinical validation of potential therapeutics or testing hypotheses about vaginal epithelium-microbiome interactions. RESULTS: Here, we describe an organ-on-a-chip (organ chip) microfluidic culture model of the human vaginal mucosa (vagina chip) that is lined by hormone-sensitive, primary vaginal epithelium interfaced with underlying stromal fibroblasts, which sustains a low physiological oxygen concentration in the epithelial lumen. We show that the Vagina Chip can be used to assess colonization by optimal L. crispatus consortia as well as non-optimal Gardnerella vaginalis-containing consortia, and to measure associated host innate immune responses. Co-culture and growth of the L. crispatus consortia on-chip was accompanied by maintenance of epithelial cell viability, accumulation of D- and L-lactic acid, maintenance of a physiologically relevant low pH, and down regulation of proinflammatory cytokines. In contrast, co-culture of G. vaginalis-containing consortia in the vagina chip resulted in epithelial cell injury, a rise in pH, and upregulation of proinflammatory cytokines. CONCLUSION: This study demonstrates the potential of applying human organ chip technology to create a preclinical model of the human vaginal mucosa that can be used to better understand interactions between the vaginal microbiome and host tissues, as well as to evaluate the safety and efficacy of live biotherapeutics products. Video Abstract.

Ectopic Lymphoid Follicle Formation and Human Seasonal Influenza Vaccination Responses Recapitulated in an Organ-on-a-Chip.

Lymphoid follicles (LFs) are responsible for generation of adaptive immune responses in secondary lymphoid organs and form ectopically during chronic inflammation. A human model of ectopic LF formation will provide a tool to understand LF development and an alternative to non-human primates for preclinical evaluation of vaccines. Here, it is shown that primary human blood B- and T-lymphocytes autonomously assemble into ectopic LFs when cultured in a 3D extracellular matrix gel within one channel of a two-channel organ-on-a-chip microfluidic device. Superfusion via a parallel channel separated by a microporous membrane is required for LF formation and prevents lymphocyte autoactivation. These germinal center-like LFs contain B cells expressing Activation-Induced Cytidine Deaminase and exhibit plasma cell differentiation upon activation. To explore their utility for seasonal vaccine testing, autologous monocyte-derived dendritic cells are integrated into LF Chips. The human LF chips demonstrate improved antibody responses to split virion influenza vaccination compared to 2D cultures, which are enhanced by a squalene-in-water emulsion adjuvant, and this is accompanied by increases in LF size and number. When inoculated with commercial influenza vaccine, plasma cell formation and production of anti-hemagglutinin IgG are observed, as well as secretion of cytokines similar to vaccinated humans over clinically relevant timescales.

Simulating drug concentrations in PDMS microfluidic organ chips.

Microfluidic organ-on-a-chip (Organ Chip) cell culture devices are often fabricated using polydimethylsiloxane (PDMS) because it is biocompatible, transparent, elastomeric, and oxygen permeable; however, hydrophobic small molecules can absorb to PDMS, which makes it challenging to predict drug responses. Here, we describe a combined simulation and experimental approach to predict the spatial and temporal concentration profile of a drug under continuous dosing in a PDMS Organ Chip containing two parallel channels separated by a porous membrane that is lined with cultured cells, without prior knowledge of its log P value. First, a three-dimensional finite element model of drug loss into the chip was developed that incorporates absorption, adsorption, convection, and diffusion, which simulates changes in drug levels over time and space as a function of potential PDMS diffusion coefficients and log P values. By then experimentally measuring the diffusivity of the compound in PDMS and determining its partition coefficient through mass spectrometric analysis of the drug concentration in the channel outflow, it is possible to estimate the effective log P range of the compound. The diffusion and partition coefficients were experimentally derived for the antimalarial drug and potential SARS-CoV-2 therapeutic, amodiaquine, and incorporated into the model to quantitatively estimate the drug-specific concentration profile over time measured in human lung airway chips lined with bronchial epithelium interfaced with pulmonary microvascular endothelium. The same strategy can be applied to any device geometry, surface treatment, or in vitro microfluidic model to simulate the spatial and temporal gradient of a drug in 3D without prior knowledge of the partition coefficient or the rate of diffusion in PDMS. Thus, this approach may expand the use of PDMS Organ Chip devices for various forms of drug testing.

Substrate stiffness induced mechanotransduction regulates temporal evolution of human fetal neural progenitor cell phenotype, differentiation, and biomechanics.

While the mechanotransduction-induced fate of adult neural stem/progenitor cells (NPCs) is relatively known, how substrate stiffness regulates the temporal evolution of the biomechanics and phenotype of developmentally relevant human fetal NPCs (hNPCs) and their mechanosensing pathways remain unknown. Here, we primed hNPCs on tissue-culture plastic (TCPS) for 3 days in non-differentiating medium before transferring to TCPS or Geltrex™ gels (<1 kPa) for 9-day cultures post-priming, and regularly assessed stemness, differentiation, and cell mechanics (Young's modulus, tether forces, apparent membrane tension, tether radius). hNPCs maintained stemness on TCPS while those on gels co-expressed stemness and neural/glial markers, 3-days post-priming. Biomechanical characteristics remained unchanged in cells on TCPS but were significantly altered in those on gels, 3-days post-priming. However, 9-days post-priming, hNPCs on gels differentiated, with significantly more neurons on softer gels and glia on stiffer gels, while those on TCPS maintained their native stemness. Withdrawal of bFGF and EGF in 9-day cultures induced hNPC differentiation and influenced cell mechanics. Cells on stiffer gels had higher biomechanical properties than those on softer gels throughout the culture period, with NPC-like > neural > glia subtypes. Higher stress fiber density in cells on stiffer gels explains their significantly different biomechanical properties on these gels. Blebbistatin treatment caused cell polarization, lowered elastic modulus, and enhanced tether forces, implicating the role of non-muscle myosin-II in hNPC mechanosensing, adaptability, and thereby mechanics. Such substrate-mediated temporal evolution of hNPCs guide design of smart scaffolds to investigate morphogenesis, disease modeling, stem cell biology, and biomaterials for tissue engineering.

Softening of the chronic hemi-section spinal cord injury scar parallels dysregulation of cellular and extracellular matrix content.

Regeneration following spinal cord injury (SCI) is challenging in part due to the modified tissue composition and organization of the resulting glial and fibrotic scar regions. Inhibitory cell types and biochemical cues present in the scar have received attention as therapeutic targets to promote regeneration. However, altered Young's modulus of the scar as a readout for potential impeding factors for regeneration are not as well-defined, especially in vivo. Although the decreased Young's modulus of surrounding tissue at acute stages post-injury is known, the causation and outcomes at chronic time points remain largely understudied and controversial, which motivates this work. This study assessed the glial and fibrotic scar tissue's Young's modulus and composition (scar morphometry, cell identity, extracellular matrix (ECM) makeup) that contribute to the tissue's stiffness. The spatial Young's modulus of a chronic (~18-wks, post-injury) hemi-section, including the glial and fibrotic regions, were significantly less than naïve tissue (~200 Pa; p < 0.0001). The chronic scar contained cystic cavities dispersed in areas of dense nuclei packing. Abundant CNS cell types such as astrocytes, oligodendrocytes, and neurons were dysregulated in the scar, while epithelial markers such as vimentin were upregulated. The key ECM components in the CNS, namely sulfated proteoglycans (sPGs), were significantly downregulated following injury with concomitant upregulation of unsulfated glycosaminoglycans (GAGs) and hyaluronic acid (HA), likely altering the foundational ECM network that contributes to tissue stiffness. Our results reveal the Young's modulus of the chronic SCI scar as well as quantification of contributing elastic components that can provide a foundation for future study into their role in tissue repair and regeneration.

Microglia control vascular architecture via a TGFß1 dependent paracrine mechanism linked to tissue mechanics.

Tissue microarchitecture and mechanics are important in development and pathologies of the Central Nervous System (CNS); however, their coordinating mechanisms are unclear. Here, we report that during colonization of the retina, microglia contacts the deep layer of high stiffness, which coincides with microglial bipolarization, reduction in TGFß1 signaling and termination of vascular growth. Likewise, stiff substrates induce microglial bipolarization and diminish TGFß1 expression in hydrogels. Both microglial bipolarization in vivo and the responses to stiff substrates in vitro require intracellular adaptor Kindlin3 but not microglial integrins. Lack of Kindlin3 causes high microglial contractility, dysregulation of ERK signaling, excessive TGFß1 expression and abnormally-patterned vasculature with severe malformations in the area of photoreceptors. Both excessive TGFß1 signaling and vascular defects caused by Kindlin3-deficient microglia are rescued by either microglial depletion or microglial knockout of TGFß1 in vivo. This mechanism underlies an interplay between microglia, vascular patterning and tissue mechanics within the CNS.

Characterizing viscoelasticity of unhydrolyzed chicken sternal cartilage extract suspensions: Towards development of injectable therapeutics formulations.

Exogenous delivery of cartilage extract is being explored as a promising candidate for knee arthritis treatment as it biomimics native cartilage tissue characteristics. In this study, we report on the rheological characterization of aqueous suspensions constituted from a powdered form of unhydrolyzed chicken sternum extract. The effect of particle size (as-received vs. milled), suspension fluid (water vs. PBS), and temperature (37°C vs. 4°C), on the viscoelastic properties of the sternum extract based particulate suspensions were evaluated. Results showed that these suspensions exhibit shear-thinning characteristics as shear rate (γ̇) increases, while viscosity (η), storage (G'), and loss (G″) moduli of the suspensions increased with increasing particulate loading (ϕ: 2.5-10wt%). Reducing the as-received particle size by milling decreased G', G, and η of the suspensions and increased the influence of ϕ on these properties, possibly due to improved particle packing. Replacing water with PBS had no significant effect on the rheological properties, but temperature reduction from 37°C to 4°C increased G', G", and η of the suspensions and lowered the impact of powder loading on viscoelastic properties. The suspension's time-dependent response was typical of viscoelastic materials, characterized by an asymptotical approach to a final stress (stress relaxation) or strain (creep). Results were fit to a power-law model for creep, a general relaxation model for exponential decay in stress, Carreau-Yasuda models for flow curves, and a two-parameter Liu model to identify the maximum powder loading (ϕm). Among the various forces involved in particle-particle interactions within these suspensions, electrostatic forces appeared to dominate the most. Such characterization of the viscoelastic nature of these suspensions would help in formulating stable injectable cartilage extract based therapeutics for in vivo applications.