In this study, atomic force microscopy-based dynamic oscillatory and force-relaxation indentation was employed to quantify the time-dependent nanomechanics of native (untreated) and proteoglycan ...(PG)-depleted cartilage disks, including indentation modulus Eind, force-relaxation time constant τ, magnitude of dynamic complex modulus |E∗|, phase angle δ between force and indentation depth, storage modulus E′, and loss modulus E″. At ∼2 nm dynamic deformation amplitude, |E∗| increased significantly with frequency from 0.22 ± 0.02 MPa (1 Hz) to 0.77 ± 0.10 MPa (316 Hz), accompanied by an increase in δ (energy dissipation). At this length scale, the energy dissipation mechanisms were deconvoluted: the dynamic frequency dependence was primarily governed by the fluid-flow-induced poroelasticity, whereas the long-time force relaxation reflected flow-independent viscoelasticity. After PG depletion, the change in the frequency response of |E∗| and δ was consistent with an increase in cartilage local hydraulic permeability. Although untreated disks showed only slight dynamic amplitude-dependent behavior, PG-depleted disks showed great amplitude-enhanced energy dissipation, possibly due to additional viscoelastic mechanisms. Hence, in addition to functioning as a primary determinant of cartilage compressive stiffness and hydraulic permeability, the presence of aggrecan minimized the amplitude dependence of |E∗| at nanometer-scale deformation.
Osteoarthritis (OA), the most common form of arthritis, is a multi-factorial disease that primarily affects cartilage as well as other joint tissues such as subchondral bone. The lack of effective ...drug delivery, due to the avascular nature of cartilage and the rapid clearance of intra-articularly delivered drugs via the synovium, remains a major challenge in the development of disease modifying drugs for OA. Cationic delivery carriers can significantly enhance the uptake, penetration and retention of drugs in cartilage by interacting with negatively charged matrix proteoglycans. In this study, we used ”supercharged” green fluorescent proteins (GFPs), engineered to have a wide range of net positive charge and surface charge distributions, to characterize the effects of carrier charge on transport into cartilage in isolation of other factors such as carrier size and shape. We quantified the uptake, extent of cartilage penetration and cellular uptake of the GFP variants into living human knee cartilage and bovine cartilage explants. Based on these results, we identified optimal net charges of GFP carriers for potential drug targets located within cartilage extracellular matrix as well as the resident live chondrocytes. These cationic GFPs did not have adverse effects on cartilage in terms of measured cell viability and metabolism, cartilage cell biosynthesis and matrix degradation at doses needed for drug delivery. In addition to quantifying the kinetics of GFP uptake, we developed a predictive mathematical model for transport of the GFP variants that exhibited the highest uptake and penetration into cartilage. This model was further used to predict the transport behavior of GFPs during scale-up to in vivo applications such as intra-articular injection into human knees. The insights gained from this study set the stage for development of cartilage-targeted delivery systems to prevent cartilage degeneration, improve tissue regeneration and reduce inflammation that may cause degradation of other joint tissues affected by OA.
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There are no drugs or treatment methods known to prevent the development of post-traumatic osteoarthritis (PTOA), a type of osteoarthritis (OA) that is triggered by traumatic joint ...injuries and accounts for ∼12% of the nearly 600 million OA cases worldwide. Lack of effective drug delivery techniques remains a major challenge in developing clinically effective treatments, but cationic delivery carriers can help overcome this challenge. Scaling up treatments that are effective in in vitro models to achieve success in preclinical in vivo models and clinical trials is also a challenging problem in the field. Here we use a cationic green fluorescent protein (GFP) as a carrier to deliver Insulin-Like Growth Factor 1 (IGF-1), a drug considered as a potential therapeutic for PTOA. GFP-IGF-1 conjugates were first synthesized as fusion proteins with different polypeptide linkers, and their transport properties were characterized in human cartilage explants. In vitro experimental data were used to develop a predictive mathematical transport model that was validated using an independent in vitro experimental data set. The model was used to predict the transport of these fusion proteins upon intra-articular injection into human knee joints. The predictions included results for the rate and extent of fusion protein penetration into cartilage, and the maximum levels of fusion proteins that would escape into systemic circulation through the joint capsule. Together, our transport measurements and model set the stage for translation of such explant culture studies to in vivo preclinical studies and potentially clinical application.
The lack of blood supply in cartilage and rapid clearance of drugs injected into human knees presents a major challenge in developing clinically effective treatments for osteoarthritis. Cationic delivery carriers can target negatively charged cartilage and help overcome this problem. Scaling up treatments that are effective in vitro to achieve success in vivo is also challenging. Here, we use a cationic green fluorescent protein (GFP) to deliver Insulin-Like Growth Factor-1 (IGF-1) into cartilage. Experiments measuring transport of GFP-IGF-1 fusion proteins in human cartilage explants were used to develop and validate a mathematical model to predict fusion protein transport upon injection into human knee joints. This work translates such explant culture studies to in vivo preclinical studies and potentially clinical application.
Injurious overloading and inflammation perturbate homeostasis of articular cartilage, leading to abnormal tissue-level loading during post-traumatic osteoarthritis. Our objective was to gain time- ...and cartilage depth-dependent insights into the early-stage disease progression with an in vitro model incorporating for the first time the coaction of (1) mechanical injury, (2) pro-inflammatory interleukin-1 challenge, and (3) cyclic loading mimicking walking and considered beneficial for cartilage health. Cartilage plugs (n = 406) were harvested from the patellofemoral grooves of young calves (N = 6) and subjected to injurious compression (50% strain, rate 100%/s; INJ), interleukin-1α-challenge (1 ng/ml; IL), and cyclic loading (intermittent 1 h loading periods, 15% strain, 1 Hz; CL). Plugs were assigned to six groups (control, INJ, IL, INJ-IL, IL-CL, INJ-IL-CL). Bulk and localized glycosaminoglycan (GAG) content (DMMB assay, digital densitometry), aggrecan biosynthesis (35S-sulfate incorporation), and chondrocyte viability (fluorescence microscopy) were assessed on days 3–12. The INJ, IL, and INJ-IL groups exhibited rapid early (days 2–4) GAG loss in contrast to CL groups. On day 3, deep cartilage of INJ-IL-CL group had higher GAG content than INJ group (p < 0.05). On day 12, INJ-IL-CL group showed more accumulated GAG loss (normalized with control) than INJ-IL group (average fold changes 1.97 95% CI: 1.23–2.70; 1.66 1.42–1.89; p = 0.007). Aggrecan biosynthesis increased in CL groups on day 12 compared to day 0. Despite promoting aggrecan biosynthesis, this cyclic loading protocol seems to be beneficial early-on to deep cartilage, but later becoming incapable of restricting further degradation triggered by marked but non-destructive injury and cytokine transport.
The goal of this study was to examine the effects of mechanical compression on chondrocyte biosynthesis of extracellular matrix (ECM) components during culture in a new alginate disk culture system. ...Specifically, we have examined chondrocyte biosynthesis rates, and the structure of aggrecan core protein species present in the cell-associated matrix (CM), in the further removed matrix (FRM) and in the surrounding culture medium. In this alginate disk culture system, chondrocytes can be subjected to mechanical deformations similar to those experienced in vivo. Our results show that over an 8-week culture period, chondrocytes synthesize a functional ECM and can respond to mechanical forces similarly to chondrocytes maintained in native cartilage. In the alginate disk system, static compression was shown to decrease and dynamic compression to increase synthesis of aggrecan of bovine chondrocytes. Western blot analysis of the core proteins of aggrecan molecules identified a number of different species that were present in different relative amounts in the CM, FRM, and medium. Over 21 days of culture, the predominant form of aggrecan found in the ECM was a full-length link-stabilized species. In addition, our data show that the application of 40 h of static compression caused an increase in the proportion of newly synthesized aggrecan molecules released into the medium. However, this was not accompanied by a significant change in the size and composition of aggrecan and aggrecan fragments in the different compartments, suggesting that mechanical compression did not alter the catabolic pathways. Together, these data show that chondrocyte function is maintained in an alginate disk culture system and that this culture system is a useful model to examine chondrocyte ECM assembly and some aspects of catabolism normally found in vivo.
Little is known regarding the impact of socioeconomic factors on the use of evidence-based therapies and outcomes in patients with heart failure with reduced ejection fraction across Asia.
We ...investigated the association of both patient-level (household income, education levels) and country-level (regional income level by World Bank classification, income disparity by Gini index) socioeconomic indicators on use of guideline-directed therapy and clinical outcomes (composite of 1-year mortality or HF hospitalization, quality of life) in the prospective multinational ASIAN-HF study (Asian Sudden Cardiac Death in Heart Failure).
Among 4540 patients (mean age: 60±13 years, 23% women) with heart failure with reduced ejection fraction, 39% lived in low-income regions; 34% in regions with high-income disparity (Gini ≥42.8%); 64.4% had low monthly household income (<US$1000); and 29.5% had no/only primary education. The largest disparity in treatment across regional income levels pertained to β-blocker and device therapies, with patients from low-income regions being less likely to receive these treatments compared with those from high-income regions and even greater disparity among patients with lower education status and lower household income within each regional income strata. Higher country- and patient-level socioeconomic indicators related to higher quality of life scores and lower risk of the primary composite outcome. Notably, we found a significant interaction between regional income level and both household income and education status (
<0.001 for both), where the association of low household income and low education status with poor outcomes was more pronounced in high-income compared with lower income regions.
These findings highlight the importance of socioeconomic determinants among patients with heart failure in Asia and suggest that attention should be paid to address disparities in access to care among the poor and less educated, including those from wealthy regions. Registration: URL: https://clinicaltrials.gov; Unique Identifier: NCT01633398.
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