Piperlongumine mediates amelioration of osteoarthritis via inhibition of chondrocyte senescence and inflammation in a goat ex vivo model.

In osteoarthritis (OA), chondrocytes manifest senescence, which results in a vicious signaling loop that aids the progression of the disease. More specifically, inflammation-associated senescence is one of the major regulators of the initiation and progression of OA. Therefore, we targeted senescence through inflammation with a pharmacological approach for OA amelioration. In this study, we first confirmed the suitability of the IL1ß-induced goat ex vivo OA model (emphasizing 3R's principle) for the screening of senotherapeutics, namely, ABT-263, ABT-737, and Piperlongumine (PL), wherein PL showed a positive outcome in the preliminary studies. Thereafter, we determined the cytocompatible concentrations of PL using live/dead staining. Further, treatment of ex vivo OA cartilage with PL exhibited a concentration-dependent increase in the retention of key cartilage matrix components. We then examined the effect of PL on chondrocyte senescence and observed a decreased expression of major senescence markers in the PL-treated groups. Interestingly, PL treatment reduced the expression of major downstream effectors of the chondrocyte senescence pathway in a concentration-dependent manner at both gene and protein levels. Moreover, IL1ß-induced elevated levels of oxidative stress and DNA damage in cartilage explants were rescued by all the tested concentrations of PL. In addition, PL also reduced the expression of major inflammatory markers of OA in the goat ex vivo OA model. Finally, we proposed a model for the mechanism of action of PL in the treatment of OA. Overall, PL showed a promising outcome as a senotherapeutic for the amelioration of OA in the goat ex vivo OA model.

Targeting multiple disease hallmarks using a synergistic disease-modifying drug combination ameliorates osteoarthritis via inhibition of senescence and inflammation.

AIMS: Osteoarthritis (OA), is a debilitating disease characterized by progressive cartilage degradation, synovial inflammation, and chondrocyte senescence. Various treatment agents independently targeting these hallmarks have been investigated. However, due to the complex multifaceted nature of OA, no disease-modifying osteoarthritis drugs are clinically available. In an attempt to overcome this, we developed a combinatorial approach and demonstrated the efficacy of TsC [Tissue inhibitor of metalloproteinase-3 (TIMP3) + sulfated carboxymethylcellulose (sCMC)] and piperlongumine (PL) combination for the amelioration of OA in a goat ex vivo OA model. MAIN METHODS: The efficacy of the drug combination was evaluated using the goat ex vivo OA explant model and results were validated in clinically relevant human OA cartilage explants. The chondroprotective effects were evaluated in terms of reduced inflammation and cartilage matrix loss, reduction in chondrosenescence, and reduced oxidative stress. KEY FINDINGS: A combination of TsC and PL (TsC-PL) significantly reduced inflammation, cartilage matrix loss, chondrosenescence, and oxidative stress in the goat ex vivo OA model and showed chondroprotective effects. Further, similar chondroprotective effects were observed in human OA cartilage. Additionally, the coefficient of drug interaction analysis indicated that the combination of TsC and PL had a synergistic effect in reducing matrix degrading proteases and inflammation (goat ex vivo OA model) and Reactive oxygen species (ROS) production (human OA cartilage). SIGNIFICANCE: Combinatorial treatment with TsC and PL demonstrated potential disease-modifying effects for the treatment of osteoarthritis via inhibition of inflammation and senescence and supports the usage of treatment strategies targeting multiple pathological factors of OA simultaneously.

Sulfated carboxymethylcellulose mediated enhancement of Timp3 efficacy synergistically attenuates osteoarthritis through inhibition of NFκB and JNK.

Osteoarthritis (OA) is a prevalent degenerative joint condition with no effective disease modifying treatments. In this study, we aimed to address multiple OA hallmarks using a combination of pro-chondrogenic sulfated carboxymethylcellulose (sCMC) and anti-catabolic tissue inhibitor of metalloproteases 3 (Timp3) in relevant disease systems. Firstly, we chemically sulfated carboxymethylcellulose to impart a negative charge and improve the stability of cationic Timp3. The modified sCMC exhibited a molecular weight of 10 kDa and a degree of sulfation of ∼10 %. We further demonstrated that sulfation of CMC confers pro-chondrogenic characteristics. Subsequently, we demonstrated that the combination of sCMC and Timp3 effectively reduced key OA hallmarks, such as matrix degradation, inflammation, and protease expression, in a goat ex vivo OA model compared to individual treatments. We further demonstrated that the anti-OA effect of sCMC and Timp3 is mediated through the suppression of NFκB and JNK activation. To validate the clinical potential and mechanism of action, we conducted experiments on human OA explants. The combination treatment synergistically reduced the expression of MMP13 and NFκB in human OA explants. Overall, sCMC-mediated enhancement of Timp3 efficacy synergistically reduced OA-like traits and demonstrates the potential for OA amelioration.

Sulfated carboxymethylcellulose-based scaffold mediated delivery of Timp3 alleviates osteoarthritis.

Osteoarthritis (OA) is a debilitating progressive joint disease with high incidence and socioeconomic burden. However, no disease-modifying treatment is currently available for OA. Here, we report a sulfated carboxymethylcellulose-based scaffold mediated delivery of tissue inhibitor of metalloprotease 3 (Timp3) as a disease-modifying therapeutic strategy for OA. First, we chemically modified carboxymethylcellulose (CMC) to sulfated carboxymethylcellulose (sCMC) to impart native-like electrostatic interaction-based binding of cationic proteins. We then fabricated cartilage ECM mimicking sCMC-gelatin scaffolds which showed preferential binding and sustained delivery of Timp3. This scaffold-mediated delivery of Timp3 demonstrated a reduction in matrix degradation, protease expression and inflammatory markers in the goat ex vivo OA model leading to enhanced retention of cartilage ECM markers when compared to OA control. Further, similar results were obtained when sCMC-gelatin scaffolds were evaluated using human OA samples, supporting its clinical potential. Overall, the Timp3 loaded sCMC-gelatin scaffold shows potential as a treatment approach for OA.

A human osteoarthritis mimicking goat cartilage explant-based disease model for drug screening.

Although osteoarthritis (OA) is the most prevalent human joint disease with a large socioeconomic burden, it remains a neglected disease with no clinically approved disease modifying therapies. One of the key reasons for this is that the available disease models poorly recapitulate human OA-like traits, possibly because of the challenge of mimicking the disease in an ECM-rich cartilage tissue. In this study, we report the establishment and validation of a clinically relevant ex vivo OA model using IL1ß-treated goat articular cartilage explants. Treatment with IL1ß induced OA-like traits in goat cartilage explants and caused a shift in cartilage homeostasis towards enhanced catabolism, resulting in higher matrix degradation, overexpression of degradative and inflammatory mediators, and chondrocyte hypertrophy. We then validated the developed disease model for drug response using the drugs celecoxib, BMP7, and rapamycin, all of which demonstrated concentration-dependent disease amelioration in the model. Finally, we evaluated the translational relevance of the developed ex vivo OA model by comparing it with late-stage OA patient samples and observed a striking resemblance in terms of matrix degradation, expression of degradative enzymes, chondrocyte hypertrophy, and inflammation. Overall, the goat ex vivo OA model elicited a biological response to cytokine treatment that mirrors human OA-like traits and may reduce discordance between preclinical and clinical studies in OA drug development.

Pore Alignment in Gelatin Scaffolds Enhances Chondrogenic Differentiation of Infrapatellar Fat Pad Derived Mesenchymal Stromal Cells.

One of the major strategies in tissue engineering is the biomimetic scaffold-based approach that aims at providing a near-native-like environment for cells to facilitate the regeneration of damaged/lost tissue. The extracellular matrix in native articular cartilage contains aligned collagen fibrils in the superficial (parallel to the articular surface) and deep zones (perpendicular to articular surface) of the tissue. Therefore, we hypothesized that scaffolds with aligned pore architecture may offer aligned collagen deposition upon cell seeding, and as a result, may enable enhanced chondrogenesis. We tested this hypothesis by comparing gelatin scaffolds with random and aligned pore architecture for their ability to differentiate infrapatellar fat pad derived mesenchymal stromal cells (IFP-MSCs) toward the chondrogenic lineage. The fabricated scaffolds with random and aligned pore architecture were comparable in terms of pore size, degree of cross-linking, equilibrium swelling ratio, and in vitro degradation behavior. However, scaffolds with aligned pore architecture demonstrated higher compressive modulus along with cellular infiltration and alignment in comparison to the scaffolds with random pore architecture. An in vitro chondrogenesis study of IFP-MSCs seeded in the developed scaffold systems revealed that scaffolds with aligned pore architecture supported better chondrogenesis in terms of sGAG and total collagen (histology and biochemical) and cartilage specific matrix deposition (immunofluorescence). Further, scaffolds with aligned pore architecture also supported oriented deposition of cell secreted collagen. Taken together, these results suggest that scaffolds with aligned pore architecture enhance in vitro chondrogenic differentiation of IFP-MSCs as compared to scaffolds with random pore architecture and hence could be a potential design criterion in the development of scaffolds for cartilage regeneration.

Sulfated polysaccharide mediated TGF-ß1 presentation in pre-formed injectable scaffolds for cartilage tissue engineering.

In this work, a plant-derived polysaccharide carboxymethylcellulose (CMC) was chemically modified to incorporate sulfate groups to facilitate binding of cationic growth factors. The sulfated CMC (heparin mimic) was then used with CMC (glycosaminoglycan mimic) and gelatin (collagen mimic) to fabricate injectable pre-formed, macroporous scaffolds for cartilage tissue engineering. These scaffolds demonstrated high resilience and shape memory, thereby making them injectable through a 14G needle for up to 4-6 aspiration and injection cycles. Further, the scaffolds could sequester cationic proteins and growth factors (TGF-ß1) through affinity-based interactions. When seeded with infrapatellar fat pad derived MSCs, the scaffolds demonstrated enhanced chondrogenesis after 28 days of in vitro culture when compared to controls. Taken together; these results demonstrate a polysaccharide-based minimally-invasive and translatable pre-formed injectable scaffold-based cell and growth factor delivery system for cartilage regeneration.

Alcohols as molecular probes in ionic liquids: evidence for nanostructuration.

A comprehensive study of the solution and solvation of linear alcohols (propan-1-ol, butan-1-ol and pentan-1-ol) in ionic liquids (ILs) is presented. The effect of the alkyl chain size of both alcohols and ILs (1-alkyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, [CnC1im][NTf2], ionic liquid series) on the thermodynamic properties of solution and solvation was used to obtain insight into the interactions between alcohols and ILs. Alcohols were used as molecular probes to ascertain whether their solvation in ILs would reflect IL nanostructuration. A trend shift was found in the values of enthalpy of solution and solvation for the [CnC1im][NTf2] series at a critical alkyl size (CAS) of C6. Further, the effect of the hydrogen bond basicity of the anion in the solvation of alcohols was explored based on the comparative study of the solvation of propan-1-ol in two different IL series, 1-alkyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [CnC1im][NTf2] and hexafluorophosphate [CnC1im][PF6]. The results obtained provide experimental support for the strength of hydrogen bonds between the alcohols and the NTf2 and PF6 anions, providing insights into the IL intermolecular interactions, namely by indicating the ability of the alcohols to discriminate the IL anion hydrogen bond basicity.

Fabrication and characterization of Pluronic modified poly(hydroxybutyrate) fibers for potential wound dressing applications.

Electrospun poly(hydroxybutyrate) (PHB) fiber meshes have shown some success in wound dressing applications, however, their use is limited by their high hydrophobicity and brittle nature. In this study we investigated the effect of hydrophilization of electrospun PHB fibers by blending with Pluronic F-108 (PF) for use as a wound dressing material. Blending of PHB with different concentrations of PF (0.5%PF-PHB and 1.0% PF-PHB) before electrospinning led to a significant increase in the water wettability and swelling properties of fibers as compared to pristine PHB fibers. Further, it was observed that though the tensile moduli of PF blended PHB fibers were relatively lower as compared to PHB fibers, they show higher resistance to failure measured in terms of strain to failure and energy to failure. Moreover, PF blending significantly improved the in vitro blood clotting rate on PHB fibers when compared to control PHB fibers. Furthermore, the fabricated fiber systems were found to be cytocompatible and supported adhesion of fibroblasts in vitro. Finally, it was demonstrated that the PF blended fiber systems were suitable for the encapsulation of an antibiotic (doxycycline) to render them with antibacterial properties. Taken together, this study demonstrates that PF blending can be used to significantly improve properties of PHB fibers for wound dressing applications.

Thermophysical properties of two ammonium-based protic ionic liquids.

Experimental data for density, viscosity, refractive index and surface tension are reported, for the first time, in the temperature range between 288.15 K and 353.15 K and at atmospheric pressure for two protic ionic liquids, namely 2-(dimethylamino)-N,N-dimethylethan-1-ammonium acetate, [N11{2(N11)}H][CH3CO2], and N-ethyl-N,N-dimethylammonium phenylacetate, [N112H][C7H7CO2]. The effect of the anion aromaticity and the cation's aliphatic tails on the studied properties is discussed. From the measured properties temperature dependency the derived properties, such as the isobaric thermal expansion coefficient, the surface entropy and enthalpy, and the critical temperature, were estimated.

Thermophysical properties of phosphonium-based ionic liquids.

Experimental data for density, viscosity, refractive index and surface tension of four phosphonium-based ionic liquids were measured in the temperature range between (288.15 and 353.15) K and at atmospheric pressure. The ionic liquids considered include tri(isobutyl) methylphosphonium tosylate, [P i(444)1][Tos], tri(butyl)methylphosphonium methylsulfate, [P4441][CH3SO4], tri(butyl)ethylphosphonium diethylphosphate, [P4442][(C2H5O)2PO2], and tetraoctylphosphonium bromide, [P8888][Br]. Additionally, derivative properties, such as the isobaric thermal expansion coefficient, the surface thermodynamic properties and the critical temperatures for the investigated ionic liquids were also estimated and are presented and discussed. Group contribution methods were evaluated and fitted to the density, viscosity and refractive index experimental data.

Thermophysical properties of sulfonium- and ammonium-based ionic liquids.

Experimental data for the density, viscosity, refractive index and surface tension of four sulfonium- and ammonium-based Ionic Liquids (ILs) with the common bis(trifluoromethylsulfonyl)imide anion were measured in the temperature range between 288.15 and 353.15 K and at atmospheric pressure. The ILs considered include butyltrimethylammonium bis(trifluoromethylsulfonyl)imide, [N4111][NTf2], tributylmethylammonium bis(trifluoromethylsulfonyl)imide, [N4441][NTf2], diethylmethylsulfonium bis(trifluoromethylsulfonyl)imide, [S221][NTf2], and triethylsulfonium bis(trifluoromethylsulfonyl)imide, [S222][NTf2]. Based on the gathered results and on data taken from literature, the impact of the cation isomerism and of the size of the aliphatic tails, as well as the effect resulting from the substitution of a nitrogen by a sulfur atom as the cation central atom, on the thermophysical properties of sulfonium- and ammonium-based ILs is here discussed. Remarkably, more symmetric cations present a lower viscosity for the same, and sometimes even for higher, alkyl chain lengths at the cation. Additional derivative properties, such as the isobaric thermal expansion coefficient, the surface thermodynamic properties and the critical temperature for the investigated ILs were also estimated and are presented and discussed.

Ion association and solvation behavior of tetraalkylammonium iodides in binary mixtures of dichloromethane + N,N-dimethylformamide probed by a conductometric study.

Precise measurements on electrical conductances of tetraalkylammonium iodides, R(4)NI (R = n-butyl to n-heptyl), in different mass% (0-100) of dichloromethane (DCM) + N,N-dimethylformamide (DMF) at 298.15 K have been performed. Limiting molar conductances (Λ(0)), association constants (K(A)) and co-sphere diameter (R) for ion-pair formation in the mixed solvent mixtures have been evaluated using the Fuoss conductance-concentration equation. However, the deviation of the conductometric curves (Λ versus √c) from linearity for the electrolytes in 100 mass% of DCM indicated triple ion formation, and therefore corresponding conductance data have been analyzed by the Fuoss-Kraus theory of triple ions. Limiting ionic molar conductances λ have been calculated by the reference electrolyte method along with a numerical evaluation of ion pair and triple ion formation constants (K(P)≈K(A) and K(T)). The results have been interpreted in terms of ion-solvent interactions and structural changes in the mixed solvents.