Injury-related cell death and proteoglycan loss in articular cartilage: Numerical model combining necrosis, reactive oxygen species, and inflammatory cytokines.

Osteoarthritis (OA) is a common musculoskeletal disease that leads to deterioration of articular cartilage, joint pain, and decreased quality of life. When OA develops after a joint injury, it is designated as post-traumatic OA (PTOA). The etiology of PTOA remains poorly understood, but it is known that proteoglycan (PG) loss, cell dysfunction, and cell death in cartilage are among the first signs of the disease. These processes, influenced by biomechanical and inflammatory stimuli, disturb the normal cell-regulated balance between tissue synthesis and degeneration. Previous computational mechanobiological models have not explicitly incorporated the cell-mediated degradation mechanisms triggered by an injury that eventually can lead to tissue-level compositional changes. Here, we developed a 2-D mechanobiological finite element model to predict necrosis, apoptosis following excessive production of reactive oxygen species (ROS), and inflammatory cytokine (interleukin-1)-driven apoptosis in cartilage explant. The resulting PG loss over 30 days was simulated. Biomechanically triggered PG degeneration, associated with cell necrosis, excessive ROS production, and cell apoptosis, was predicted to be localized near a lesion, while interleukin-1 diffusion-driven PG degeneration was manifested more globally. Interestingly, the model also showed proteolytic activity and PG biosynthesis closer to the levels of healthy tissue when pro-inflammatory cytokines were rapidly inhibited or cleared from the culture medium, leading to partial recovery of PG content. The numerical predictions of cell death and PG loss were supported by previous experimental findings. Furthermore, the simulated ROS and inflammation mechanisms had longer-lasting effects (over 3 days) on the PG content than localized necrosis. The mechanobiological model presented here may serve as a numerical tool for assessing early cartilage degeneration mechanisms and the efficacy of interventions to mitigate PTOA progression.

Charge shielding effects of PEG bound to NH2-terminated PAMAM dendrimers - an experimental approach.

Cationic poly(amido amine) (PAMAM) dendrimers exhibit great potential for use in drug delivery, but their high charge density leads to an inherent cytotoxicity. To increase biocompatibility, many studies have attached poly(ethylene glycol) (PEG) chains to the dendrimer surface. It is unclear how these tethered PEG chains influence the physicochemical properties of the dendrimer. Here, we develop a fluorescence-based assay utilizing anionic biological tissue to quantify the electrostatic binding affinity of a library of PEG-PAMAM conjugates with various PEG chain lengths and grafting densities. We find that covalently bound PEG chains reduce the electrostatic binding affinity more significantly than what can be achieved through covalent bonds only. Contrary to previous thought, this reduction is not explained by the steric hindrance effects of PEG chains, suggesting that other, non-covalent interactions between PEG and PAMAM are present. Using acetylated PAMAM conjugates, we convert electrostatic binding affinity to the number of charged amines accessible to the physiological environment. These data, coupled with 1H-NMR, allows us to study more closely the non-covalent interactions between PEG and PAMAM. We find that increasing PEG chain length increases the number of non-covalent interactions. Additionally, at low grafting densities, increasing the number of PEG chains on the PAMAM surface also increases the non-covalent interactions. At higher grafting densities, however, PEG chains sterically repel one another, forcing chains to elongate away from the surface and reducing the number of interactions between PAMAM and individual PEG chains. The data presented here provides a framework for a more precise mechanistic understanding of how the length and density of tethered PEG chains on PAMAM dendrimers influence drug delivery properties.

Spatial configuration of charge and hydrophobicity tune particle transport through mucus.

Mucus is a selectively permeable hydrogel that protects wet epithelia from pathogen invasion and poses a barrier to drug delivery. Determining the parameters of a particle that promote or prevent passage through mucus is critical, as it will enable predictions about the mucosal passage of pathogens and inform the design of therapeutics. The effect of particle net charge and size on mucosal transport has been characterized using simple model particles; however, predictions of mucosal passage remain challenging. Here, we utilize rationally designed peptides to examine the integrated contributions of charge, hydrophobicity, and spatial configuration on mucosal transport. We find that net charge does not entirely predict transport. Specifically, for cationic peptides, the inclusion of hydrophobic residues and the position of charged and hydrophobic residues within the peptide impact mucosal transport. We have developed a simple model of mucosal transport that predicts how previously unexplored amino acid sequences achieve slow versus fast passage through mucus. This model may be used as a basis to predict transport behavior of natural peptide-based particles, such as antimicrobial peptides or viruses, and assist in the engineering of synthetic sequences with desired transport properties.

Mechanical motion promotes expression of Prg4 in articular cartilage via multiple CREB-dependent, fluid flow shear stress-induced signaling pathways.

Lubricin is a secreted proteoglycan encoded by the Prg4 locus that is abundantly expressed by superficial zone articular chondrocytes and has been noted to both be sensitive to mechanical loading and protect against the development of osteoarthritis. In this study, we document that running induces maximal expression of Prg4 in the superficial zone of knee joint articular cartilage in a COX-2-dependent fashion, which correlates with augmented levels of phospho-S133 CREB and increased nuclear localization of CREB-regulated transcriptional coactivators (CRTCs) in this tissue. Furthermore, we found that fluid flow shear stress (FFSS) increases secretion of extracellular PGE2, PTHrP, and ATP (by epiphyseal chondrocytes), which together engage both PKA- and Ca(++)-regulated signaling pathways that work in combination to promote CREB-dependent induction of Prg4, specifically in superficial zone articular chondrocytes. Because running and FFSS both boost Prg4 expression in a COX-2-dependent fashion, our results suggest that mechanical motion may induce Prg4 expression in the superficial zone of articular cartilage by engaging the same signaling pathways activated in vitro by FFSS that promote CREB-dependent gene expression in this tissue.

Mechanobiological model for simulation of injured cartilage degradation via pro-inflammatory cytokines and mechanical stimulus.

Post-traumatic osteoarthritis (PTOA) is associated with cartilage degradation, ultimately leading to disability and decrease of quality of life. Two key mechanisms have been suggested to occur in PTOA: tissue inflammation and abnormal biomechanical loading. Both mechanisms have been suggested to result in loss of cartilage proteoglycans, the source of tissue fixed charge density (FCD). In order to predict the simultaneous effect of these degrading mechanisms on FCD content, a computational model has been developed. We simulated spatial and temporal changes of FCD content in injured cartilage using a novel finite element model that incorporates (1) diffusion of the pro-inflammatory cytokine interleukin-1 into tissue, and (2) the effect of excessive levels of shear strain near chondral defects during physiologically relevant loading. Cytokine-induced biochemical cartilage explant degradation occurs near the sides, top, and lesion, consistent with the literature. In turn, biomechanically-driven FCD loss is predicted near the lesion, in accordance with experimental findings: regions near lesions showed significantly more FCD depletion compared to regions away from lesions (p<0.01). Combined biochemical and biomechanical degradation is found near the free surfaces and especially near the lesion, and the corresponding bulk FCD loss agrees with experiments. We suggest that the presence of lesions plays a role in cytokine diffusion-driven degradation, and also predisposes cartilage for further biomechanical degradation. Models considering both these cartilage degradation pathways concomitantly are promising in silico tools for predicting disease progression, recognizing lesions at high risk, simulating treatments, and ultimately optimizing treatments to postpone the development of PTOA.

Regenerative Potential of Platelet Concentrate Lysate in Mechanically Injured Cartilage and Matrix-Associated Chondrocyte Implantation In Vitro.

Adjuvant therapy in autologous chondrocyte implantation (ACI) can control the post-traumatic environment and guide graft maturation to support cartilage repair. To investigate both aspects, we examined potential chondro-regenerative effects of lysed platelet concentrate (PC) and supplementary interleukin 10 (IL-10) on mechanically injured cartilage and on clinically used ACI scaffolds. ACI remnants and human cartilage explants, which were applied to an uniaxial unconfined compression as injury model, were treated with human IL-10 and/or PC from thrombocyte concentrates. We analyzed nuclear blebbing/TUNEL, sGAG content, immunohistochemistry, and the expression of COL1A1, COL2A1, COL10A1, SOX9, and ACAN. Post-injuriously, PC was associated with less cell death, increased COL2A1 expression, and decreased COL10A1 expression and, interestingly, the combination with Il-10 or Il-10 alone had no additional effects, except on COL10A1, which was most effectively decreased by the combination of PC and Il-10. The expression of COL2A1 or SOX9 was statistically not modulated by these substances. In contrast, in chondrocytes in ACI grafts the combination of PC and IL-10 had the most pronounced effects on all parameters except ACAN. Thus, using adjuvants such as PC and IL-10, preferably in combination, is a promising strategy for enhancing repair and graft maturation of autologous transplanted chondrocytes after cartilage injury.

Age-associated changes in the response of tendon explants to stress deprivation is sex-dependent.

Purpose of the Study: The incidence of tendon injuries increases dramatically with age, which presents a major clinical burden. While previous studies have sought to identify age-related changes in extracellular matrix structure and function, few have been able to explain fully why aged tissues are more prone to degeneration and injury. In addition, recent studies have also demonstrated that age-related processes in humans may be sex-dependent, which could be responsible for muddled conclusions in changes with age. In this study, we investigate short-term responses through an ex vivo explant culture model of stress deprivation that specifically questions how age and sex differentially affect the ability of tendons to respond to altered mechanical stimulus.Materials and Methods: We subjected murine flexor explants from young (4 months of age) and aged (22-24 months of age) male and female mice to stress-deprived culture conditions for up to 1 week and investigated changes in viability, cell metabolism and proliferation, matrix biosynthesis and composition, gene expression, and inflammatory responses throughout the culture period.Results and Conclusions: We found that aging did have a significant influence on the response to stress deprivation, demonstrating that aged explants have a less robust response overall with reduced metabolic activity, viability, proliferation, and biosynthesis. However, age-related changes appeared to be sex-dependent. Together, this work demonstrates that the aging process and the subsequent effect of age on the ability of tendons to respond to stress-deprivation are inherently different based on sex, where male explants favor increased activity, apoptosis, and matrix remodeling while female explants favor reduced activity and tissue preservation.

Size- and speed-dependent mechanical behavior in living mammalian cytoplasm.

Active transport in the cytoplasm plays critical roles in living cell physiology. However, the mechanical resistance that intracellular compartments experience, which is governed by the cytoplasmic material property, remains elusive, especially its dependence on size and speed. Here we use optical tweezers to drag a bead in the cytoplasm and directly probe the mechanical resistance with varying size a and speed V We introduce a method, combining the direct measurement and a simple scaling analysis, to reveal different origins of the size- and speed-dependent resistance in living mammalian cytoplasm. We show that the cytoplasm exhibits size-independent viscoelasticity as long as the effective strain rate V/a is maintained in a relatively low range (0.1 s-1 < V/a < 2 s-1) and exhibits size-dependent poroelasticity at a high effective strain rate regime (5 s-1 < V/a < 80 s-1). Moreover, the cytoplasmic modulus is found to be positively correlated with only V/a in the viscoelastic regime but also increases with the bead size at a constant V/a in the poroelastic regime. Based on our measurements, we obtain a full-scale state diagram of the living mammalian cytoplasm, which shows that the cytoplasm changes from a viscous fluid to an elastic solid, as well as from compressible material to incompressible material, with increases in the values of two dimensionless parameters, respectively. This state diagram is useful to understand the underlying mechanical nature of the cytoplasm in a variety of cellular processes over a broad range of speed and size scales.

Chemoproteomics of matrix metalloproteases in a model of cartilage degeneration suggests functional biomarkers associated with posttraumatic osteoarthritis.

Active matrix metalloproteases (MMPs) play a significant role in the pathogenesis of many diseases including osteoarthritis (OA), which involves progressive proteolytic degradation of cartilage. Clinical success of OA interventions that target MMPs has been limited by a lack of information about the presence and activity of specific disease-related proteases. We therefore developed a chemoproteomics approach based on MS to characterize the release and activity of MMPs in an in vitro model of the early inflammatory phase of posttraumatic OA (PTOA). We designed and synthesized chemical activity-based probes (ABPs) to identify active MMPs in bovine cartilage explants cultured for 30 days with the proinflammatory cytokine, interleukin-1α. Using these probes in an activity-based protein profiling-multidimensional identification technology (ABPP-MudPIT) approach, we identified active MMP-1, -2, -3, -7, -9, -12, and -13 in the medium after 10 days of culture, the time at which irreversible proteolysis of the collagen network in the explant was detected using proteolytic activation of FRET-quenched MMP substrates. Total MMP levels were quantified by shotgun proteomics, which, taken with ABPP-MudPIT data, indicated the presence of predominantly inactive MMPs in the culture medium. The selectivity of the ABPP-MudPIT approach was further validated by detection of specific endogenous MMPs activated de novo with 4-aminophenylmurcuric acetate. The utility of the new ABPP-MudPIT approach for detecting molecular biomarkers of PTOA disease initiation and potential targets for therapeutics motivates possible application in other diseases involving MMP activity.

Nanoscale Poroelasticity of the Tectorial Membrane Determines Hair Bundle Deflections.

Stereociliary imprints in the tectorial membrane (TM) have been taken as evidence that outer hair cells are sensitive to shearing displacements of the TM, which plays a key role in shaping cochlear sensitivity and frequency selectivity via resonance and traveling wave mechanisms. However, the TM is highly hydrated (97% water by weight), suggesting that the TM may be flexible even at the level of single hair cells. Here we show that nanoscale oscillatory displacements of microscale spherical probes in contact with the TM are resisted by frequency-dependent forces that are in phase with TM displacement at low and high frequencies, but are in phase with TM velocity at transition frequencies. The phase lead can be as much as a quarter of a cycle, thereby contributing to frequency selectivity and stability of cochlear amplification.

Noncontact three-dimensional mapping of intracellular hydromechanical properties by Brillouin microscopy.

Current measurements of the biomechanical properties of cells require physical contact with cells or lack subcellular resolution. Here we developed a label-free microscopy technique based on Brillouin light scattering that is capable of measuring an intracellular longitudinal modulus with optical resolution. The 3D Brillouin maps we obtained of cells in 2D and 3D microenvironments revealed mechanical changes due to cytoskeletal modulation and cell-volume regulation.

Release of pro-inflammatory cytokines from muscle and bone causes tenocyte death in a novel rotator cuff in vitro explant culture model.

PURPOSE: Tendinopathy is a significant clinical problem thought to be associated with altered mechanical loading. Explant culture models allow researchers to alter mechanical loading in a controlled in vitro environment while maintaining tenocytes in their native matrix. However, current models do not accurately represent commonly injured tendons, ignoring contributions of associated musculature and bone, as well as regional collagen structure. This study details the characterization of amouse rotator cuff explant culture model, including bone, tendon, and muscle (BTM). MATERIALS AND METHODS: Following harvest, BTM explants were maintained in stress-deprived culture for one week and tendon was then assessed for changes in cell viability, metabolism, matrix structure and content. RESULTS: Matrix turnover occurred throughout culture as manifested in both gene expression and biosynthesis, but this did not translate to net changes in total collagen or sulfated glycosaminoglycan content. Furthermore, tendon structure was not significantly altered throughout culture. However, we found significant cell death in BTM tendons after 3 days in culture, which we hypothesize is cytokine-induced. Using a targeted multiplex assay, we found high levels of pro-inflammatory cytokines released to the culture medium from muscle and bone, levels that did cause cell deathin tendon-alone controls. CONCLUSIONS: Overall, this model presents an innovative approach to understandingrotator cuff injury and tenocyte mechanobiology in a clinically-relevant tendon structure. Our model can be a powerful tool to investigate how mechanical and biological stimuli can alter normal tendon health and lead to tendon degeneration, and may provide a testbed for therapeutics for tendon repair.

Multiscale Poroviscoelastic Compressive Properties of Mouse Supraspinatus Tendons Are Altered in Young and Aged Mice.

Rotator cuff disorders are one of the most common causes of shoulder pain and disability in the aging population but, unfortunately, the etiology is still unknown. One factor thought to contribute to the progression of disease is the external compression of the rotator cuff tendons, which can be significantly increased by age-related changes such as muscle weakness and poor posture. The objective of this study was to investigate the baseline compressive response of tendon and determine how this response is altered during maturation and aging. We did this by characterizing the compressive mechanical, viscoelastic, and poroelastic properties of young, mature, and aged mouse supraspinatus tendons using macroscale indentation testing and nanoscale high-frequency AFM-based rheology testing. Using these multiscale techniques, we found that aged tendons were stiffer than their mature counterparts and that both young and aged tendons exhibited increased hydraulic permeability and energy dissipation. We hypothesize that regional and age-related variations in collagen morphology and organization are likely responsible for changes in the multiscale compressive response as these structural parameters may affect fluid flow. Importantly, these results suggest a role for age-related changes in the progression of tendon degeneration, and we hypothesize that decreased ability to resist compressive loading via fluid pressurization may result in damage to the extracellular matrix (ECM) and ultimately tendon degeneration. These studies provide insight into the regional multiscale compressive response of tendons and indicate that altered compressive properties in aging tendons may be a major contributor to overall tendon degeneration.

Near infrared spectroscopic assessment of developing engineered tissues: correlations with compositional and mechanical properties.

Articular cartilage degeneration causes pain and reduces the mobility of millions of people annually. Regeneration of cartilage is challenging, due in part to its avascular nature, and thus tissue engineering approaches for cartilage repair have been studied extensively. Current techniques to assess the composition and integrity of engineered tissues, including histology, biochemical evaluation, and mechanical testing, are destructive, which limits real-time monitoring of engineered cartilage tissue development in vitro and in vivo. Near infrared spectroscopy (NIRS) has been proposed as a non-destructive technique to characterize cartilage. In the current study, we describe a non-destructive NIRS approach for assessment of engineered cartilage during development, and demonstrate correlation of these data to gold standard mid infrared spectroscopic measurements, and to mechanical properties of constructs. Cartilage constructs were generated using bovine chondrocyte culture on polyglycolic acid (PGA) scaffolds for six weeks. BMP-4 growth factor and ultrasound mechanical stimulation were used to provide a greater dynamic range of tissue properties and outcome variables. NIR spectra were collected daily using an infrared fiber optic probe in diffuse reflectance mode. Constructs were harvested after three and six weeks of culture and evaluated by the correlative modalities of mid infrared (MIR) spectroscopy, histology, and mechanical testing (equilibrium and dynamic stiffness). We found that specific NIR spectral absorbances correlated with MIR measurements of chemical composition, including relative amount of PGA (R = 0.86, p = 0.02), collagen (R = 0.88, p = 0.03), and proteoglycan (R = 0.83, p = 0.01). In addition, NIR-derived water content correlated with MIR-derived proteoglycan content (R = 0.76, p = 0.04). Both equilibrium and dynamic mechanical properties generally improved with cartilage growth from three to six weeks. In addition, significant correlations between NIRS-derived parameters and mechanical properties were found for constructs that were not treated with ultrasound (PGA (R = 0.71, p = 0.01), water (R = 0.74, p = 0.02), collagen (R = 0.69, p = 0.04), and proteoglycan (R = 0.62, p = 0.05)). These results lay the groundwork for extension to arthroscopic engineered cartilage assessment in clinical studies.

Modeling IL-1 induced degradation of articular cartilage.

In this study, we develop a computational model to simulate the in vitro biochemical degradation of articular cartilage explants sourced from the femoropatellar grooves of bovine calves. Cartilage explants were incubated in culture medium with and without the inflammatory cytokine IL-1α. The spatio-temporal evolution of the cartilage explant's extracellular matrix components is modelled. Key variables in the model include chondrocytes, aggrecan, collagen, aggrecanase, collagenase and IL-1α. The model is first calibrated for aggrecan homeostasis of cartilage in vivo, then for data on (explant) controls, and finally for data on the IL-1α driven proteolysis of aggrecan and collagen over a 4-week period. The model was found to fit the experimental data best when: (i) chondrocytes continue to synthesize aggrecan during the cytokine challenge, (ii) a one to two day delay is introduced between the addition of IL-1α to the culture medium and subsequent aggrecanolysis, (iii) collagen degradation does not commence until the total concentration of aggrecan (i.e. both intact and degraded aggrecan) at any specific location within the explant becomes ≤ 1.5 mg/ml and (iv) degraded aggrecan formed due to the IL-1α induced proteolysis of intact aggrecan protects the collagen network while collagen degrades in a two-step process which, together, significantly modulate the collagen network degradation. Under simulated in vivo conditions, the model predicts increased aggrecan turnover rates in the presence of synovial IL-1α, consistent with experimental observations. Such models may help to infer the course of events in vivo following traumatic joint injury, and may also prove useful in quantitatively evaluating the efficiency of various therapeutic molecules that could be employed to avoid or modify the course of cartilage disease states.

Novel cartilage oligomeric matrix protein (COMP) neoepitopes identified in synovial fluids from patients with joint diseases using affinity chromatography and mass spectrometry.

To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDSPAGE followed by in-gel digestion and mass spectrometric identification and characterization.Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser77 were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided.

Articular cartilage of the knee 3 years after ACL reconstruction. A quantitative T2 relaxometry analysis of 10 knees.

BACKGROUND AND PURPOSE: T1ρ or T2 relaxation imaging has been increasingly used to evaluate the cartilage of the knee. We investigated the cartilage of ACL-reconstructed knees 3 years after surgery using T2 relaxation times. PATIENTS AND METHODS: 10 patients with a clinically successful unilateral ACL reconstruction were examined 3 years after surgery. Multiple-TE fast-spin echo sagittal images of both knees were acquired using a 3T MRI scanner for T2 mapping of the tibiofemoral cartilage. T2 values of the superficial and deep zones of the tibiofemoral cartilage were analyzed in sub-compartmental areas and compared between the ACL-reconstructed and uninjured contralateral knees. RESULTS: Higher T2 values were observed in 1 or more sub-compartmental areas of each ACL-reconstructed knee compared to the uninjured contralateral side. Most of the T2 increases were observed at the superficial zones of the cartilage, especially at the medial compartment. At the medial compartment of the ACL-reconstructed knee, the T2 values of the femoral and tibial cartilage were increased by 3-81% compared to the uninjured contralateral side, at the superficial zones of the weight-bearing areas. T2 values in the superficial zone of the central medial femoral condyle differed between the 2 groups (p = 0.002). INTERPRETATION: The articular cartilage of ACL-reconstructed knees, although clinically satisfactory, had higher T2 values in the superficial zone of the central medial femoral condyle than in the uninjured contralateral side 3 years after surgery. Further studies are warranted to determine whether these patients would undergo cartilage degeneration over time.

Impact of brain tissue filtering on neurostimulation fields: a modeling study.

Electrical neurostimulation techniques, such as deep brain stimulation (DBS) and transcranial magnetic stimulation (TMS), are increasingly used in the neurosciences, e.g., for studying brain function, and for neurotherapeutics, e.g., for treating depression, epilepsy, and Parkinson's disease. The characterization of electrical properties of brain tissue has guided our fundamental understanding and application of these methods, from electrophysiologic theory to clinical dosing-metrics. Nonetheless, prior computational models have primarily relied on ex-vivo impedance measurements. We recorded the in-vivo impedances of brain tissues during neurosurgical procedures and used these results to construct MRI guided computational models of TMS and DBS neurostimulatory fields and conductance-based models of neurons exposed to stimulation. We demonstrated that tissues carry neurostimulation currents through frequency dependent resistive and capacitive properties not typically accounted for by past neurostimulation modeling work. We show that these fundamental brain tissue properties can have significant effects on the neurostimulatory-fields (capacitive and resistive current composition and spatial/temporal dynamics) and neural responses (stimulation threshold, ionic currents, and membrane dynamics). These findings highlight the importance of tissue impedance properties on neurostimulation and impact our understanding of the biological mechanisms and technological potential of neurostimulatory methods.

Prediction of cartilage compressive modulus using multiexponential analysis of T(2) relaxation data and support vector regression.

Evaluation of mechanical characteristics of cartilage by magnetic resonance imaging would provide a noninvasive measure of tissue quality both for tissue engineering and when monitoring clinical response to therapeutic interventions for cartilage degradation. We use results from multiexponential transverse relaxation analysis to predict equilibrium and dynamic stiffness of control and degraded bovine nasal cartilage, a biochemical model for articular cartilage. Sulfated glycosaminoglycan concentration/wet weight (ww) and equilibrium and dynamic stiffness decreased with degradation from 103.6 ± 37.0 µg/mg ww, 1.71 ± 1.10 MPa and 15.3 ± 6.7 MPa in controls to 8.25 ± 2.4 µg/mg ww, 0.015 ± 0.006 MPa and 0.89 ± 0.25MPa, respectively, in severely degraded explants. Magnetic resonance measurements were performed on cartilage explants at 4 °C in a 9.4 T wide-bore NMR spectrometer using a Carr-Purcell-Meiboom-Gill sequence. Multiexponential T2 analysis revealed four water compartments with T2 values of approximately 0.14, 3, 40 and 150 ms, with corresponding weight fractions of approximately 3, 2, 4 and 91%. Correlations between weight fractions and stiffness based on conventional univariate and multiple linear regressions exhibited a maximum r(2) of 0.65, while those based on support vector regression (SVR) had a maximum r(2) value of 0.90. These results indicate that (i) compartment weight fractions derived from multiexponential analysis reflect cartilage stiffness and (ii) SVR-based multivariate regression exhibits greatly improved accuracy in predicting mechanical properties as compared with conventional regression.

Molecular adhesion between cartilage extracellular matrix macromolecules.

In this study, we investigated the molecular adhesion between the major constituents of cartilage extracellular matrix, namely, the highly negatively charged proteoglycan aggrecan and the type II/IX/XI fibrillar collagen network, in simulated physiological conditions. Colloidal force spectroscopy was applied to measure the maximum adhesion force and total adhesion energy between aggrecan end-attached spherical tips (end radius R ≈ 2.5 µm) and trypsin-treated cartilage disks with undamaged collagen networks. Studies were carried out in various aqueous solutions to reveal the physical factors that govern aggrecan-collagen adhesion. Increasing both ionic strength and [Ca(2+)] significantly increased adhesion, highlighting the importance of electrostatic repulsion and Ca(2+)-mediated ion bridging effects. In addition, we probed how partial enzymatic degradation of the collagen network, which simulates osteoarthritic conditions, affects the aggrecan-collagen interactions. Interestingly, we found a significant increase in aggrecan-collagen adhesion even when there were no detectable changes at the macro- or microscales. It is hypothesized that the aggrecan-collagen adhesion, together with aggrecan-aggrecan self-adhesion, works synergistically to determine the local molecular deformability and energy dissipation of the cartilage matrix, in turn, affecting its macroscopic tissue properties.