Loss Of Glycosaminoglycans Research Articles

Kinetics and Retention of Polystyrenesulfonate for Proteoglycan Replacement in Cartilage.

Tissue hydration provides articular cartilage with dynamic viscoelastic properties. Early stage osteoarthritis (OA) is marked by loss of proteoglycans and glycosaminoglycans (GAG), lowering fixed charge density, and impairing tissue osmotic function. The most common GAG replacement, chondroitin sulfate (CS), has failed to show effectiveness. Here, we investigated a synthetic polyelectrolyte, poly(styrenesulfonate) (PSS), both as a model compound to investigate polyelectrolyte transport in cartilage, and as a potential candidate to restore bulk fixed charge density in cartilage with GAG loss. Through bovine explants and histology, we determined zonal-based effective diffusion coefficients for three different molecular weights of PSS. Compared to CS, PSS was retained longer in GAG-depleted cartilage in static and compression-based desorption experiments. We explained enhanced solute performance of PSS by its more compact morphology and higher charge density by small-angle X-ray scattering. This study may improve design of GAG mimetic molecules for repairing osmotic function in OA cartilage.

Increased femoral cartilage thickness in acne patients using isotretinoin: could it be a sign of early osteoarthritis?

Vitamin A derivatives have inhibitory effects on cartilage tissue, such as decreasing chondrocyte proliferation and collagen synthesis, and increasing the loss of glycosaminoglycans and proteoglycans. Therefore, isotretinoin (a vitamin A derivative) may play a role in the pathogenesis of cartilage-related diseases like osteoarthritis by affecting the balance of cartilage tissue. The aim of this study was to evaluate the distal femoral cartilage thickness in acne patients under the systemic isotretinoin therapy and to determine whether it constitutes a risk factor for the development of osteoarthritis. The study included 52 patients (42 female, 10 male, mean age 23.31 ± 3.89 years) who were prescribed systemic isotretinoin for acne and completed at least 3 months of treatment, along with 45 healthy controls ((35 female, 10 male, mean age 23.85 ± 4.77 years). Bilateral distal femoral cartilage thickness was measured by ultrasonography before isotretinoin treatment and after the completion of the third month of treatment. After treatment, a statistically significant increase was found in the thickness of the right medial, right lateral, left medial, left lateral, and left intercondylar cartilage (p = 0.014, 0.012, 0.019, 0.027, 0.002, respectively). There was also an increase in the right intercondylar cartilage thickness, but this was not statistically significant (p = 0.1). Systemic isotretinoin seems to make cartilage thicker. The increase in femoral cartilage thickness observed after short-term isotretinoin treatment might be an indicator of very early-stage osteoarthritis. Extended follow-up studies with larger participant pools are necessary to substantiate this result.

Single Injection AAV2-FGF18 Gene Therapy Reduces Cartilage Loss and Subchondral Bone Damage in a Mechanically Induced Model of Osteoarthritis.

Osteoarthritis (OA) is a highly debilitating, degenerative pathology of cartilaginous joints affecting over 500 million people worldwide. The global economic burden of OA is estimated at $260-519 billion and growing, driven by aging global population and increasing rates of obesity. To date, only the multi-injection chondroanabolic treatment regimen of Fibroblast Growth Factor 18 (FGF18) has demonstrated clinically meaningful disease-modifying efficacy in placebo-controlled human trials. Our work focuses on the development of a novel single injection disease-modifying gene therapy, based on FGF18's chondroanabolic activity. OA was induced in Sprague-Dawley rats using destabilization of the medial meniscus (DMM) (3 weeks), followed by intra-articular treatment with 3 dose levels of AAV2-FGF18, rh- FGF18 protein, and PBS. Durability, redosability, and biodistribution were measured by quantifying nLuc reporter bioluminescence. Transcriptomic analysis was performed by RNA-seq on cultured human chondrocytes and rat knee joints. Morphological analysis was performed on knee joints stained with Safranin O/Fast Green and anti-PRG antibody. Dose-dependent reductions in cartilage defect size were observed in the AAV2-FGF18- treated joints relative to the vehicle control. Total defect width was reduced by up to 76% and cartilage thickness in the thinnest zone was increased by up to 106%. Morphologically, the vehicle- treated joints exhibited pronounced degeneration, ranging from severe cartilage erosion and bone void formation, to subchondral bone remodeling and near-complete subchondral bone collapse. In contrast, AAV2-FGF18-treated joints appeared more anatomically normal, with only regional glycosaminoglycan loss and marginal cartilage erosion. While effective at reducing cartilage lesions, treatment with rhFGF18 injections resulted in significant joint swelling (19% increase in diameter), as well as a decrease in PRG4 staining uniformity and intensity. In contrast to early-timepoint in vitro RNA-seq analysis, which showed a high degree of concordance between protein- and gene therapy-treated chondrocytes, in vivo transcriptomic analysis, revealed few gene expression changes following protein treatment. On the other hand, the gene therapy treatment exhibited a high degree of durability and localization over the study period, upregulating several chondroanabolic genes while downregulating OA- and fibrocartilage-associated markers. FGF18 gene therapy treatment of OA joints can provide benefits to both cartilage and subchondral bone, with a high degree of localization and durability.

Triamcinolone acetonide has minimal effect on short- and long-term metabolic activities of cartilage.

Intra-articular corticosteroid injections, such as triamcinolone acetonide (TA), are commonly used by clinicians to manage joint synovial inflammation. However, due to conflicting evidence in literature, there is a fear among clinicians that the injections may be harmful to otherwise healthy cartilage in young patients. The purpose of this study was to evaluate the effects of TA on young, healthy chondrocytes. Articular cartilage samples were harvested from bovine knee joints (1-2 months old). In both healthy and inflammatory (interleukin-1β) challenged cartilage, samples were treated with TA at doses ranging from 1 nM to 200 μM. Following a short- (2 days) or long-term (10-14 days) treatment, chondrocyte viability, proliferation, and extracellular matrix (ECM) synthesis and degradation were evaluated with a click chemistry-based technique. Chondrocyte viability, proliferation, and anabolic activity were all minimally affected by short-term and long-term TA treatment. After both acute and sustained inflammatory challenges, TA reduced the catabolic activities in cartilage, reducing nascent glycosaminoglycan loss and maintaining cartilage mechanical properties. Overall, at physiologically relevant doses, TA had minimal negative impact on chondrocytes when maintained within their native ECM.Clinical significance: The findings provide new insight for current clinical practices concerning the use of TA in intra-articular injections, especially in young patients, and established a foundation for future investigations into the impact of corticosteroids on joint homeostasis.

Shedding light on the effects of blood on meniscus tissue: the role of mononuclear leukocytes in mediating meniscus catabolism

Traumatic meniscal injuries can cause acute pain, hemarthrosis (bleeding into the joint), joint immobility, and post-traumatic osteoarthritis (PTOA). However, the exact mechanism(s) by which PTOA develops following meniscal injuries is unknown. Since meniscus tears commonly coincide with hemarthrosis, investigating the direct effects of blood and its constituents on meniscus tissue is warranted. The goal of this study was to determine the direct effects of blood and blood components on meniscus tissue catabolism. Porcine meniscus explants or primary meniscus cells were exposed to whole blood or various fractions of blood for 3days to simulate blood exposure following injury. Explants were then washed and cultured for an additional 3days prior to collection for biochemical analyses. Whole blood increased matrix metalloproteinase (MMP) activity. Fractionation experiments revealed blood-derived red blood cells did not affect meniscus catabolism. Conversely, viable mononuclear leukocytes induced MMP activity, nitric oxide (NO) production, and loss of tissue sulfated glycosaminoglycan (sGAG) content, suggesting that these cells are mediating meniscus catabolism. These findings highlight the potential challenges of meniscus healing in the presence of hemarthrosis and the need for further research to elucidate the in vivo effects of blood and blood-derived mononuclear leukocytes due to both hemarthrosis and blood-derived therapeutics.

Conditional Knockout of Glycosaminoglycan Synthesis in Bladder Epithelium Has No Effect on Urinary Voiding in Mice

Glycosaminoglycans (GAGs) on the luminal surface of the bladder epithelium have been suggested to play an important role in barrier function and in protection from infection. There is also a substantial literature that posits a breakdown in the GAG layer may underlie, or be associated with the development of painful bladder syndrome (PBS, aka interstitial cystitis) and indeed, GAG mimetics such as pentosan polysulphate provide symptomatic relief for some patients. However, opinions on the presence and role of GAGs vary, and incontrovertible evidence is lacking. We have taken a unique approach to answering this question by engineering a highly cell-specific genetic mouse model that lacks the obligatory initiating enzyme for GAG synthesis in urothelium. Uroplakin 2-Cre (UP2-Cre) mice were crossed with xylosyltransferase 2-floxed mice to knockout the xylt2 gene in urothelium. RT-PCR confirmed the tissue specificity of the knockout in urothelium but not in bladder smooth muscle. Further, lacZ reporter staining in cryosections demonstrated highly specific expression of the Cre construct in urothelium. Mammals express two xylosyltransferases, XylT1 and XylT2 in different proportions in different tissues. While functionally similar they appear to differ in their substrate specificity and kinetics. qPCR in dissected urothelium demonstrated that XylT2 was the dominant isoform, being expressed at approximately 80-fold higher level than XylT1. Void spot assays (VSA) on filter paper (4 hours) have proven to be a reliable indicator of voiding dysfunction in mice. We conducted VSAs on female Up2 cre/xylt2 fl/fl (n=12) and xylt2 fl/fl (n=11) littermate controls aged 8 – 16 weeks. There was no significant difference in void volumes, number of primary voids, volume/void or number of microvoids (<20 μl) between controls and knockouts, whereas transurethral instillation of protamine sulfate, which injures the apical cell layer, induced pain and a significantly greater number of microvoids, with reduced primary void volumes. We conclude preliminarily, that loss of GAGs in urothelium does not affect bladder function, suggesting that GAGs do not play a role in protecting the suburothelium and adjacent sensory afferents from noxious urine components. Future work will characterize and quantitate the distribution of GAGs within the urothelium to determine whether GAGs are deployed on the luminal surface and, whether they play a role in urothelial repair from injury. NIH grant P20DK097818. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Continuous Low-Intensity Ultrasound Improves Cartilage Repair in Rabbit Model of Subchondral Injury.

Subchondral drilling (SD), a bone marrow stimulation technique, is used to repair cartilage lesions that lack regenerative potential. Cartilage repair outcomes upon SD are typically fibrocartilaginous in nature with inferior functionality. The lack of cues to foster the chondrogenic differentiation of egressed mesenchymal stromal cells upon SD can be attributed for the poor outcomes. Continuous low-intensity ultrasound (cLIUS) at 3.8 MHz is proposed as a treatment modality for improving cartilage repair outcomes upon marrow stimulation. Bilateral defects were created by SD on the femoral medial condyle of female New Zealand white rabbits (n = 12), and the left joint received cLIUS treatment (3.8 MHz, 3.5 Vpp, 8 min/application/day) and the contralateral right joint served as the control. On day 7 postsurgery, synovial fluid was aspirated, and the cytokine levels were assessed by Quantibody™ assay. Rabbits were euthanized at 8 weeks and outcomes were assessed macroscopically and histologically. Defect areas in the right joints exhibited boundaries, incomplete fill, irregular cartilage surfaces, loss of glycosaminoglycan (GAG), and absence of chondrocytes. In contrast, the repaired defect area in the joints that received cLIUS showed complete fill, positive staining for GAG with rounded chondrocyte morphology, COL2A1 staining, and columnar organization. Synovial fluid collected from cLIUS-treated left knee joints had lower levels of IL1, TNFα, and IFNγ when compared to untreated right knee joints, alluding to the potential of cLIUS to mitigate early inflammation. Further at 8 weeks, left knee joints (n = 12) consistently scored higher on the O'Driscoll scale, with a higher percent hyaline cartilage score. No adverse impact on bone or change in the joint space was noted. Upon a single exposure of cLIUS to TNFα-treated cells, nuclear localization of pNFκB and SOX9 was visualized by double immunofluorescence and the expression of markers associated with the NFκB pathway was assayed by quantitative real-time polymerase chain reaction. cLIUS extends its chondroprotective effects by titrating pNFκB levels, preventing its nuclear translocation, while maintaining the expression of SOX9, the collagen II transcription factor. Our combined results demonstrate that healing of chondral defects treated with marrow stimulation by SD can be accelerated by employing cLIUS regimen that possesses chondroinductive and chondroprotective properties. Impact statement Repair of cartilage represents an unsolved biomedical burden. In vitro, continuous low-intensity ultrasound (cLIUS) has been demonstrated to possess chondroinductive and chondroprotective potential. To our best knowledge, the use of cLIUS to improve cartilage repair outcomes upon marrow stimulation, in vivo, has not been reported and our work reported here fills that gap. Our results demonstrated enhanced cartilage repair outcomes under cLIUS (3.8 MHz) in a rabbit model of subchondral injury by subchondral drilling. Enhanced repair stemmed from mesenchymal stem cell differentiation in vivo and the subsequent synthesis of articular cartilage-specific matrix.

Stomach engineering: region-specific characterization of the decellularized porcine stomach

PurposePatients affected by microgastria, severe gastroesophageal reflux, or those who have undergone subtotal gastrectomy, have commonly described reporting dumping syndromes or other symptoms that seriously impair the quality of their life. Gastric tissue engineering may offer an alternative approach to treating these pathologies. Decellularization protocols have great potential to generate novel biomaterials for large gastric defect repair. There is an urgency to define more reliable protocols to foster clinical applications of tissue-engineered decellularized gastric grafts.MethodsIn this work, we investigated the biochemical and mechanical properties of decellularized porcine stomach tissue compared to its native counterpart. Histological and immunofluorescence analyses were performed to screen the quality of decellularized samples. Quantitative analysis was also performed to assess extracellular matrix composition. At last, we investigated the mechanical properties and cytocompatibility of the decellularized tissue compared to the native.ResultsThe optimized decellularization protocol produced efficient cell removal, highlighted in the absence of native cellular nuclei. Decellularized scaffolds preserved collagen and elastin contents, with partial loss of sulfated glycosaminoglycans. Decellularized gastric tissue revealed increased elastic modulus and strain at break during mechanical tensile tests, while ultimate tensile strength was significantly reduced. HepG2 cells were seeded on the ECM, revealing matrix cytocompatibility and the ability to support cell proliferation.ConclusionOur work reports the successful generation of acellular porcine gastric tissue able to support cell viability and proliferation of human cells.

Chemonucleolysis combined with dynamic loading for inducing degeneration in bovine caudal intervertebral discs.

Chemonucleolysis has become an established method of producing whole organ culture models of intervertebral disc (IVD) degeneration. However, the field needs more side-by-side comparisons of the degenerative effects of the major enzymes used in chemonucleolysis towards gaining a greater understanding of how these organ culture models mimic the wide spectrum of characteristics observed in human degeneration. In the current work we induced chemonucleolysis in bovine coccygeal IVDs with 100µL of papain (65U/mL), chondroitinase ABC (chABC, 5U/mL), or collagenase II (col'ase, 0.5U/mL). Each enzyme was applied in a concentration projected to produce moderate levels of degeneration. After 7days of culture with daily dynamic physiological loading (0.02-0.2MPa, 0.2Hz, 2h), the cellular, biochemical and histological properties of the IVDs were evaluated in comparison to a PBS-injected control. Papain and collagenase, but not chABC, produced macroscopic voids in the tissues. Compared to day 0 intact IVDs, papain induced the greatest magnitude glycosaminoglycan (GAG) loss compared to chABC and col'ase. Papain also induced the greatest height loss (3%), compared to 0.7%, 1.2% and 0.4% for chABC, col'ase, and PBS, respectively. Cell viability in the region adjacent to papain and PBS-injection remained at nearly 100% over the 7-day culture period, whereas it was reduced to 60%-70% by chABC and col'ase. Generally, enzyme treatment tended to downregulate gene expression for major ECM markers, type I collagen (COL1), type II collagen (COL2), and aggrecan (ACAN) in the tissue adjacent to injection. However, chABC treatment induced an increase in COL2 gene expression, which was significant compared to the papain treated group. In general, papain and col'ase treatment tended to recapitulate aspects of advanced IVD degeneration, whereas chABC treatment captured aspects of early-stage degeneration. Chemonucleolysis of whole bovine IVDs is a useful tool providing researchers with a robust spectrum of degenerative changes and can be utilized for examination of therapeutic interventions.

An insight on the N-glycome of notochordal cell-rich porcine nucleus pulposus during maturation.

Degeneration of the intervertebral disc is an age-related condition. It also accompanies the disappearance of the notochordal cells, which are remnants of the developmental stages of the nucleus pulposus (NP). Molecular changes such as extracellular matrix catabolism, cellular phenotype, and glycosaminoglycan loss in the NP have been extensively studied. However, as one of the most significant co- and posttranslational modifications, glycosylation has been overlooked in cells in degeneration. Here, we aim to characterize the N-glycome of young and mature NP and identify patterns related to aging. Accordingly, we isolated N-glycans from notochordal cell-rich NP from porcine discs, characterized them using a combined approach of exoglycosidase digestions and analysis with hydrophilic interaction ultra-performance liquid chromatography and mass spectrometry. We have assigned over 300 individual N-glycans for each age group. Moreover, we observed a notable abundance of antennary structures, galactosylation, fucosylation, and sialylation in both age groups. In addition, as indicated from our results, increasing outer arm fucosylation and decreasing α(2,3)-linked sialylation with aging suggest that these traits are age-dependent. Lastly, we have focused on an extensive characterization of the N-glycome of the notochordal cell-rich NP in aging without inferred degeneration, describing glycosylation changes specific for aging only. Our findings in combination with those of other studies, suggest that the degeneration of the NP does not involve identical processes as aging.

Incompletely Decellularized Tracheal Matrix Scaffold for Tissue Engineering.

Dense cartilaginous extracellular matrix makes decellularization and repopulation of tracheal cartilage difficult. However, the dense matrix isolates cartilaginous antigens from the recipient's immune system. Therefore, allorejection may be avoided by removing antigens from noncartilaginous tissues. In this study, incompletely decellularized tracheal matrix scaffolds were developed for tracheal tissue engineering. Brown Norway rat tracheae were decellularized with 4% sodium deoxycholate treatment. The cell and antigen removal efficacy, histoarchitecture, surface ultrastructure, glycosaminoglycan, collagen contents, mechanical properties, and chondrocyte viability of the scaffold were evaluated in vitro. Brown Norway rat tracheal matrix scaffolds ( n = 6) were implanted subcutaneously into Lewis rats and observed for 4 weeks. Brown Norway rat tracheae ( n = 6) and Lewis rat scaffolds ( n = 6) were implanted as controls. Histologic analysis of macrophage and lymphocyte infiltration was performed. One decellularization cycle removed all cells and antigens from noncartilaginous tissue. Incomplete decellularization preserved the structural integrity of the tracheal matrix and chondrocyte viability. Except for 31% glycosaminoglycan loss, the scaffold had comparable collagen content and tensile and compressive mechanical properties to those of the native trachea. The allogeneic scaffold showed remarkably reduced CD68 + , CD8 + , and CD4 + cell infiltration compared with the allografts and demonstrated similar cell infiltration to the syngeneic scaffold. It also maintained the three-dimensional tracheal structure and cartilage viability in vivo. Incompletely decellularized trachea did not induce immunorejection and maintained the integrity and viability of cartilage in vivo. Tracheal decellularization and repopulation can be simplified for urgent tracheal replacement. The present study describes the development of an incomplete decellularization protocol that creates a decellularized matrix scaffold for tracheal tissue engineering, aiming to provide preliminary data that this method may generate suitable tracheal scaffolds for use in tracheal replacement.

In Vitro Mineralisation of Tissue-Engineered Cartilage Reduces Endothelial Cell Migration, Proliferation and Tube Formation.

Tissue engineering bone via endochondral ossification requires the generation of a cartilage template which undergoes vascularisation and remodelling. While this is a promising route for bone repair, achieving effective cartilage vascularisation remains a challenge. Here, we investigated how mineralisation of tissue-engineered cartilage affects its pro-angiogenic potential. To generate in vitro mineralised cartilage, human mesenchymal stromal cell (hMSC)-derived chondrogenic pellets were treated with β-glycerophosphate (BGP). After optimising this approach, we characterised the changes in matrix components and pro-angiogenic factors by gene expression analysis, histology and ELISA. Human umbilical vein endothelial cells (HUVECs) were exposed to pellet-derived conditioned media, and migration, proliferation and tube formation were assessed. We established a reliable strategy to induce in vitro cartilage mineralisation, whereby hMSC pellets are chondrogenically primed with TGF-β for 2 weeks and BGP is added from week 2 of culture. Cartilage mineralisation determines loss of glycosaminoglycans, reduced expression but not protein abundance of collagen II and X, and decreased VEGFA production. Finally, the conditioned medium from mineralised pellets showed a reduced ability to stimulate endothelial cell migration, proliferation and tube formation. The pro-angiogenic potential of transient cartilage is thus stage-dependent, and this aspect must be carefully considered in the design of bone tissue engineering strategies.

CHARACTERIZATION OF NATIVE AND DECELLULARIZED PORCINE TENDON UNDER TENSION AND COMPRESSION: A CLOSER LOOK AT GLYCOSAMINOGLYCAN CONTRIBUTION TO TENDON MECHANICS

Decellularised porcine superflexor tendon (pSFT) has been demonstrated to be a suitable scaffold for anterior cruciate ligament reconstruction[1]. While the role of collagen in tendons is well known, the mechanical role of glycosaminoglycans (GAGs) is less clear and may be altered by the decellularisation process.To determine the effects of decellularisation on pSFT GAG content and mechanical function and to investigate the consequences of GAG loss in tensile and compressive loading.pSFTs were decellularised following previous techniques [2]. For GAG removal, native pSFTs were treated with chondroitinase ABC (ChABC; 0.1U/mL, 72h). Cell and GAG removal was validated using histology and quantitative assays. Native, decellularised and ChABC treated groups (n=6) were biomechanically characterised. In tension, specimens underwent stress relaxation and strength testing using previous protocols [1]. Stress relaxation data was fitted to a modified Maxwell-Weichert model to determine time-dependent (E1 & E2) and time-independent moduli (E0). The toe and linear region moduli (Etoe, Elinear), in addition to tensile strength (UTS) and failure strain were determined from strength testing. In compression, specimens underwent confined loading conditions (ramp at 10 s-1 to 10% strain and hold). The aggregate modulus (HA) and zero-strain permeability (k0) were determined using previous techniques [3]. Data was analysed by one-way ANOVA with Tukey post-hoc test to determine significant differences between test groups (p<0.05).Quantitative assays showed no GAG reduction post-decellularisation, but a significant reduction after ChABC treatment. HA was only significantly reduced in the ChABC group. k0 was significantly higher for the ChABC group compared to decellularised. E0 was significantly reduced in the decellularised group compared to native and ChABC groups, while E1 and E2 were not different between groups. Etoe, Elinear, UTS and failure strain were not different between groups.Decellularisation does not affect GAG content or impair mechanical function in pSFT. GAG loss adversely affects pSFT compressive properties, revealing major mechanical contribution under compression, but no significant role under tension.

Effects of dexamethasone and dynamic loading on cartilage of human osteochondral explants challenged with inflammatory cytokines

Post-traumatic osteoarthritis (PTOA), characterized by articular cartilage degradation initiated in an inflammatory environment after traumatic joint injury, can lead to alterations in cartilage biomechanical properties. Low dose dexamethasone (Dex) shows chondroprotection in cartilage challenged with inflammatory cytokines, but little is known about the structural biomechanical response of human cartilage to Dex in such a diseased state. This study examined changes in the biomechanical properties and biochemical composition of the cartilage within human osteochondral explants in response to treatment with exogenous cytokines, Dex, and a regimen of cyclic loading at the start and end of culture. Osteochondral explants were harvested from five pairs of human ankle talocrural joints (Collins grade 0–1) and cultured for 10 days with/without exogenous cytokines (100 ng/mL TNFα, 50 ng/mL IL-6, 250 ng/mL sIL-6R) ± Dex (100 nM). Biomechanical testing on day-0 and day-10 enabled estimation of the unconfined compression equilibrium modulus (Ey), dynamic stiffness (Ed) and hydraulic permeability (kp) of cartilage excised from bone, accompanied by biochemical assessment of media and cartilage tissue. Dex preserved chondrocyte cell viability and decreased sulfated glycosaminoglycan (sGAG) loss and nitric oxide release, but did not alter Ey, Ed and kp (before or after loading) on day-10. In the cytokine/cytokine+Dex treated groups, sGAG content exhibited a weaker correlation with Ey and Ed than at baseline, suggesting an important role for structural rather than biochemical changes in producing biomechanical alterations in response to cytokines and Dex. These findings aid in forming a more complete profile of potential clinical effects of Dex for use in OA/PTOA treatment regimens.

Teriparatide ameliorates articular cartilage degradation and aberrant subchondral bone remodeling in DMM mice.

Knee osteoarthritis (KOA) is a highly prevalent musculoskeletal disorder characterized by degeneration of cartilage and abnormal remodeling of subchondral bone (SCB). Teriparatide (PTH (1-34)) is an effective anabolic drug for osteoporosis (OP) and regulates osteoprotegerin (OPG)/receptor activator of nuclear factor ligand (RANKL)/RANK signaling, which also has a therapeutic effect on KOA by ameliorating cartilage degradation and inhibiting aberrant remodeling of SCB. However, the mechanisms of PTH (1-34) in treating KOA are still uncertain and remain to be explored. Therefore, we compared the effect of PTH (1-34) on the post-traumatic KOA mouse model to explore the potential therapeutic effect and mechanisms. In vivo study, eight-week-old male mice including wild-type (WT) (n​=​54) and OPG-/- (n​=​54) were investigated and compared. Post-traumatic KOA model was created by destabilization of medial meniscus (DMM). WT mice were randomly assigned into three groups: the sham group (WT-sham; n​=​18), the DMM group (WT-DMM; n​=​18), and the PTH (1-34)-treated group (WT-DMM​+​PTH (1-34); n​=​18). Similarly, the OPG-/- mice were randomly allocated into three groups as well. The designed mice were executed at the 4th, 8th, and 12th weeks to evaluate KOA progression. To further explore the chondro-protective of PTH (1-34), the ATDC5 chondrocytes were stimulated with different concentrations of PTH (1-34) in vitro. Compared with the WT-sham mice, significant wear of cartilage in terms of reduced cartilage thickness and glycosaminoglycan (GAG) loss was detected in the WT-DMM mice. PTH (1-34) exhibited cartilage-protective by alleviating wear, retaining the thickness and GAG contents. Moreover, the deterioration of the SCB was alleviated and the expression of PTH1R/OPG/RANKL/RANK were found to increase after PTH (1-34) treatment. Among the OPG-/- mice, the cartilage of the DMM mice displayed typical KOA change with higher OARSI score and thinner cartilage. The damage of the cartilage was alleviated but the abnormal remodeling of SCB didn't show any response to the PTH (1-34) treatment. Compared with the WT-DMM mice, the OPG-/--DMM mice caught more aggressive KOA with thinner cartilage, sever cartilage damage, and more abnormal remodeling of SCB. Moreover, both the damaged cartilage from the WT-DMM mice and the OPG-/--DMM mice were alleviated but only the deterioration of SCB in WT-DMM mice was alleviated after the administration of PTH (1-34). In vitro study, PTH (1-34) could promote the viability of chondrocytes, enhance the synthesis of extracellular matrix (ECM) (AGC, COLII, and SOX9) at the mRNA and protein level, but inhibit the secretion of inflammatory cytokines (TNF-α and IL-6). Both wear of the cartilage was alleviated and aberrant remodeling of the SCB was inhibited in the WT mice, but only the cartilage-protective effect was observed in the OPG-/- mice. PTH (1-34) exhibited chondro-protective effect by decelerating cartilage degeneration in vivo as well as by promoting the proliferation and enhancing ECM synthesis of chondrocytes in vitro. The current investigation implied that the rescue of the disturbed SCB is dependent on the regulation of OPG while the chondro-protective effect is independent of modulation of OPG, which provides proof for the treatment of KOA. Systemic administration of PTH (1-34) could exert a therapeutic effect on both cartilage and SCB in different mechanisms to alleviate KOA progression, which might be a novel therapy for KOA.

A bio-inspired nano-material recapitulating the composition, ultra-structure, and function of the glycosaminoglycan-rich extracellular matrix of nucleus pulposus

The nucleus pulposus (NP) of intervertebral disc represents a soft gel consisting of glycosaminoglycans (GAGs)-rich extracellular matrix (ECM). Significant loss of GAGs and normal functions are the most prevalent changes in degenerated disc. Attempts targeted to incorporate GAGs into collagen fibrous matrices have been made but the efficiency is very low, and the resulting structures showed no similarity with native NP. Inspired by the characteristic composition and structures of the ECM of native NP, here, we hypothesize that by chemically modifying the collagen (Col) and hyaluronic acid (HA) and co-precipitating with GAGs, a bio-inspired nano-material recapitulating the composition, ultra-structure and function of the GAG-rich ECM will be fabricated. Compositionally, the bio-inspired nano-material namely Aminated Collagen-Aminated Hyaluronic Acid-GAG (aCol-aHA-GAG) shows a record high GAG/hydroxyproline ratio up to 39.1:1 in a controllable manner, out-performing that of the native NP. Ultra-structurally, the nano-material recapitulates the characteristic ‘nano-beads’ (25 nm) and ‘bottle-brushes’ (133 nm) features as those found in native NP. Functionally, the nano-material supports the viability and maintains the morphological and phenotypic markers of bovine NP cells, and shows comparable mechanical properties of native NP. This work contributes to the development of a compositionally, structurally, and functionally biomimetic nano-material for NP tissue engineering.

Maltol ameliorates intervertebral disc degeneration through inhibiting PI3K/AKT/NF-κB pathway and regulating NLRP3 inflammasome-mediated pyroptosis

ObjectivesAs one of the major causes of low back pain, intervertebral disc degeneration (IDD) has caused a huge problem for humans. Increasing evidence indicates that NLRP3 inflammasome-mediated pyroptosis of NP cells displays an important role in the progression of IDD. Maltol (MA) is a flavoring agent extracted from red ginseng. Due to its anti-inflammatory and antioxidant effects, MA has been widely considered by researchers. Therefore, we hypothesized that MA may be a potential IVD protective agent by regulating NP cells and their surrounding microenvironment.MethodsIn vitro, qRT-PCR, and Western blot were used to explore the effect of MA on the transcription and protein expression of the anabolic protein (ADAMTS5, MMP3, MMP9) catabolic protein (Aggrecan), and pro-inflammatory factor (iNOS COX-2). Next, the effects of MA on PI3K/AKT/NF-κB pathway and pyroptosis pathway were analyzed by Western blot and immunofluorescence. Molecular docking was used to investigate the relationship between PI3K and MA. Moreover, ELISA was also used to detect the effects of MA on inflammatory factors (TNF-α, PGE2, IL-1β, and IL-18). In vivo, the effects of MA on the vertebral structure of IDD mice were studied by HE and SO staining and the effects of MA on ECM and PI3K/AKT/NF-κB and pyroptosis pathway of IDD mice were studied by immunohistochemical staining.ResultsMA can ameliorate intervertebral disc degeneration in vivo and in vitro. Specifically, the molecular docking results showed that the binding degree of MA and PI3K was significant. Second, in vitro studies showed that MA inhibited the degradation of ECM and inflammatory response by inhibiting the PI3K/AKT/NF-κB pathway and the pyroptosis mediated by NLRP3 inflammasome, which increased the expression of anabolic proteins, decreased the expression of catabolic proteins, and decreased the secretion of inflammatory mediators such as IL-18 and IL-1β. In addition, according to the study results of the mouse lumbar instability model, MA also improved the tissue disorder and degradation of the intervertebral disc, reduced the loss of proteoglycan and glycosaminoglycan, and inhibited intervertebral disc inflammation, indicating that MA has a protective effect on the intervertebral disc to intervertebral disc in mice.ConclusionsOur results suggest that MA slowed IDD development through the PI3K/AKT/NF-κB signaling pathway and NLRP3 inflammasome-mediated pyroptosis, indicating that MA appeared to be a viable medication for IDD treatment.

A novel whole “Joint-in-Motion” device reveals a permissive effect of high glucose levels and mechanical stress on joint destruction

Osteoarthritis (OA) has recently been suggested to be associated with diabetes. However, this association often disappears when accounting for body mass index (BMI), suggesting that mechanical stress may be a confounding factor. We investigated the combined influence of glucose level and loading stress on OA progression using a novel whole joint-in-motion (JM) culture system. Whole mouse knee joints were placed in an enclosed chamber with culture media and actuated to recapitulate leg movement, with a dynamic stress regimen of 0.5Hz, 8h/day for 7 days. These joints were treated with varying levels of glucose and controlled for osmolarity and diffusion. Joint movement and joint space were examined by X-ray fluoroscopy and microCT. Cartilage matrix levels were quantified by blinded Mankin scoring and immunohistochemistry. Culturing in the JM device facilitated proper leg extension and flexion movements, and adequate mass transport for analyzing the effect of glucose on cartilage. Treatment with higher levels of glucose either via media supplementation or intra-articular injection caused a significant decrease in levels of glycosaminoglycan (GAG) and an increase in aggrecan neoepitope in articular cartilage, but only under dynamic stress. Additionally, collagen II level was slightly reduced by high glucose levels. High levels of glucose and dynamic stress have permissive effects on articular cartilage GAG loss and aggrecan degradation, implicating that mechanical stress confounds the association of diabetes with OA. The JM device supports novel investigation of mechanical stress on the integrity of an intact living mouse joint to provide insights into OA pathogenesis.

Detergent-Free Decellularization of Notochordal Cell-Derived Matrix Yields a Regenerative, Injectable, and Swellable Biomaterial

Porcine notochordal cell-derived matrix (NCM) has anti-inflammatory and regenerative effects on degenerated intervertebral discs. For its clinical use, safety must be assured. The porcine DNA is concerning because of (1) the transmission of endogenous retroviruses and (2) the inflammatory potential of cell-free DNA. Here, we present a simple, detergent-free protocol: tissue lyophilization lyses cells, and matrix integrity is preserved by limiting swelling during decellularization. DNA is digested quickly by a high nuclease concentration, followed by a short washout. Ninety-four percent of DNA was removed, and there was no loss of glycosaminoglycans or collagen. Forty-three percent of the total proteins remained in the decellularized NCM (dNCM). dNCM stimulated as much GAG production as NCM in nucleus pulposus cells but lost some anti-inflammatory effects. Reconstituted pulverized dNCM yielded a soft, shear-thinning biomaterial with a swelling ratio of 350% that also acted as an injectable cell carrier (cell viability >70%). dNCM can therefore be used as the basis for future biomaterials aimed at disc regeneration on a biological level and may restore joint mechanics by creating swelling pressure within the intervertebral disc.

Osteoarthritis: management strategies depending on the location of lesions

Osteoarthritis (OA) is a multifactorial disease that can be caused by genetic factors, obesity, joint microtrauma, and excessive physical exercises. The key features of OA include gradual loss of articular cartilage, bone tissue remodeling, development of osteophytes, and subchondral sclerosis. The pathogenesis of OA is based on the loss of glycosaminoglycans, collagen disorganization, and inflammation mediated by a cytokine cascade. OA is a complex disease affecting not only cartilage, but also intraarticular and periarticular tissues. Together, these changes compromise joint function and cause chronic pain. Considerable attention has beer recently paid to OA phenotyping in order to introduce a tailored approach into patient management. Phenotypes are applicable to the three most common peripheral OA locations: gonarthrosis, coxarthrosis, and hand OA. An international group of experts of primary care has developed algorithms for OA patient management, including diagnostic criteria, treatment options, and criteria for referring the patient to a specialist (rheumatologist). Current guidelines recommend non-drug therapy, pharmacotherapy, and surgery. There is a growing interest in phytopharmaceuticals, a heterogeneous group of molecules with a high capacity of suppressing inflammation, oxidative stress, and pain, as well as improving joint function. Mucosate® in capsules (DIAMED-pharma, Russia) is one of currently available complex products with phytocompounds. It contains the NEM® complex (natural eggshell membrane), Harpagophytum root extract, glucosamine sulfate, chondroitin sulfate, and manganese sulfate monohydrate.