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Proteoglycans are hierarchically organized structures that play an important role in the hydration and the compression resistance of cartilage matrix. In this study, the static and dynamic properties relevant to the biomechanical function of cartilage are determined at different levels of the hierarchical structure, using complementary osmotic pressure, neutron scattering (SANS) and light scattering (DLS) measurements. In cartilage proteoglycans (PGs), two levels of bottlebrush structures can be distinguished: the aggrecan monomer, which consists of a core protein to which are tethered charged glycosaminoglycan (GAG) chains, and complexes formed of the aggrecan monomers attached around a linear hyaluronic acid backbone. The principal component of GAG, chondroitin sulfate (CS), is used as a baseline in this comparison. The osmotic modulus, measured as a function of the proteoglycan concentration, follows the order CS < aggrecan < aggrecan-HA complex. This order underlines the benefit of the increasing complexity at each level of the molecular architecture. The hierarchical bottlebrush configuration, which prevents interpenetration among the bristles of the aggrecan monomers, enhances both the mechanical properties and the osmotic resistance. The osmotic pressure of the collagen solution is notably smaller than in the proteoglycan systems. This is consistent with its known primary role to provide tensile strength to the cartilage and to confine the aggrecan-HA complexes, as opposed to load bearing. The collective diffusion coefficient D governs the rate of recovery of biological tissue after compressive load. In CS solutions the diffusion process is fast, D ≈ 3 × 10 -6 cm 2 s -1 at concentrations comparable with that of the GAG chains inside the aggrecan molecule. In CS solutions D is a weakly decreasing function of calcium ion concentration, while in aggrecan and its complexes with HA, the relaxation rate is insensitive to the presence of calcium.

Articular cartilage is a hydrated macromolecular composite mainly composed of type II collagen fibrils and the large proteoglycan, aggrecan. Aggrecan is a key determinant of the load bearing and energy dissipation functions of cartilage. Previously, studies of cartilage biomechanics have been primarily focusing on the macroscopic, tissue-level properties, which failed to elucidate the molecular-level activities that govern cartilage development, function, and disease. This chapter provides a brief summary of Dr. Alan J. Grodzinsky's seminal contribution to the understanding of aggrecan molecular mechanics at the nanoscopic level. By developing and applying a series of atomic force microscopy (AFM)-based nanomechanical tools, Grodzinsky and colleagues revealed the unique structural and mechanical characteristics of aggrecan at unprecedented resolutions. In this body of work, the "bottle-brush"-like ultrastructure of aggrecan was directly visualized for the first time. Meanwhile, molecular mechanics of aggrecan was studied using a physiological-like 2D biomimetic assembly of aggrecan on multiple fronts, including compression, dynamic loading, shear, and adhesion. These studies not only generated new insights into the development, aging, and disease of cartilage, but established a foundation for designing and evaluating novel cartilage regeneration strategies. For example, building on the scientific foundation and methodology infrastructure established by Dr. Grodzinsky, recent studies have elucidated the roles of other proteoglycans in mediating cartilage integrity, such as decorin and perlecan, and evaluated the therapeutic potential of biomimetic proteoglycans in improving cartilage regeneration., (© 2023. The Author(s).)

It suggests that the poly (3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) scaffold can be used for cartilage tissue engineering, but PHBV is short of bioactivity that is required for cartilage regeneration. To fabricate a bioactive cartilage tissue engineering scaffold that promotes cartilage regeneration, quercetin (QUE) modified PHBV (PHBV-g-QUE) fibrous scaffolds were prepared by a two-step surface modification method. The PHBV-g-QUE fibrous scaffold facilitates the growth of chondrocytes and maintains chondrocytic phenotype resulting from the upregulation of SOX9, COL II, and ACAN. The PHBV-g-QUE fibrous scaffold inhibited apoptosis of chondrocyte and reduced oxidative stress of chondrocytes by regulating the transcription of related genes. Following PHBV-g-QUE fibrous scaffolds and PHBV fibrous scaffolds with adhered chondrocytes were implanted into nude mice for 4 weeks, it demonstrated that PHBV-g-QUE fibrous scaffolds significantly promoted cartilage regeneration compared with the PHBV fibrous scaffolds. Hence, it suggests that the PHBV-g-QUE fibrous scaffold can be potentially applied in the clinical treatment of cartilage defects in the future., (© 2021. The Author(s).)

Background: Cartilage injury and pathological degeneration are reported in millions of patients globally. Cartilages such as articular hyaline cartilage are characterized by poor self-regeneration ability due to lack of vascular tissue. Current treatment methods adopt foreign cartilage analogue implants or microfracture surgery to accelerate tissue repair and regeneration. These methods are invasive and are associated with the formation of fibrocartilage, which warrants further exploration of new cartilage repair materials. The present study aims to develop an injectable modified gelatin hydrogel., Method: The hydrogel effectively adsorbed proteoglycans secreted by chondrocytes adjacent to the cartilage tissue in situ, and rapidly formed suitable chondrocyte survival microenvironment modified by ε-poly-L-lysine (EPL). Besides, dynamic covalent bonds were introduced between glucose and phenylboronic acids (PBA). These bonds formed reversible covalent interactions between the cis-diol groups on polyols and the ionic boronate state of PBA. PBA-modified hydrogel induced significant stress relaxation, which improved chondrocyte viability and cartilage differentiation of stem cells. Further, we explored the ability of these hydrogels to promote chondrocyte viability and cartilage differentiation of stem cells through chemical and mechanical modifications., Results: In vivo and in vitro results demonstrated that the hydrogels exhibited efficient biocompatibility. EPL and PBA modified GelMA hydrogel (Gel-EPL/B) showed stronger activity on chondrocytes compared to the GelMA control group. The Gel-EPL/B group induced the secretion of more extracellular matrix and improved the chondrogenic differentiation potential of stem cells. Finally, thus hydrogel promoted the tissue repair of cartilage defects., Conclusion: Modified hydrogel is effective in cartilage tissue repair., (© 2021. The Author(s).)

Members of the mitochondrial carrier family [solute carrier family 25 (SLC25)] transport nucleotides, amino acids, carboxylic acids, fatty acids, inorganic ions, and vitamins across the mitochondrial inner membrane. They are important for many cellular processes, such as oxidative phosphorylation of lipids and sugars, amino acid metabolism, macromolecular synthesis, ion homeostasis, cellular regulation, and differentiation. Here, we describe the functional elements of the transport mechanism of mitochondrial carriers, consisting of one central substrate-binding site and two gates with salt-bridge networks on either side of the carrier. Binding of the substrate during import causes three gate elements to rotate inward, forming the cytoplasmic network and closing access to the substrate-binding site from the intermembrane space. Simultaneously, three core elements rock outward, disrupting the matrix network and opening the substrate-binding site to the matrix side of the membrane. During export, substrate binding triggers conformational changes involving the same elements but operating in reverse.

Cartilage is an avascular tissue with low cellularity and insufficient self-repair response. In clinical practice, a large articular cartilage defect is usually fixed by cartilage transplantation. Importantly, the fast repair process has been demanded postoperatively in the area between the host cartilage and the transplanted cartilage. In the past few years, magnetic nanoparticles have drawn great attention due to their biocompatible, biodegradable, and nontoxic properties. In addition, the nanoparticles can easily pass through the cell plasma membrane and increase the cellular uptake efficiency. Here, a therapeutic drug delivery strategy was proposed for cartilage repair. The prepared kartogenin (KGN)-conjugated magnetic nanocarriers (KGN@NCs) promoted the viability of chondrocytes in vitro. In a rat model of cartilage transplantation, intra-articularly delivered KGN@NCs generated cartilage with a flat surface and a high level of aggrecan in vivo. Notably, KGN@NCs were also capable of improving the pain-related motor functions. They promoted the motor functional parameters including the print area and intensity to restore to a normal level compared with the single KGN. Therefore, these therapeutic drug nanocarriers provided the potential for cartilage repair.

Hyaluronan specifically binds to aggrecan globular domain 1, which is often referred to as just hyaluronan binding protein (HABP), however, the hyaluronan carbohydrate structure recognized by HABP had not been studied in detail. The aim of the present study was to investigate the important structure of hyaluronan for binding to HABP. We prepared hybrid oligosaccharides from hyaluronan and chondroitin, with or without modification of the reducing or non-reducing terminus, as tools to determine the preferred structure of hyaluronan for binding to the HABP by a competitive ELISA-like method. The non-reducing terminal structure was critical, especially, the glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) of the hyaluronan-unit are essential for complete HABP binding activity, and for any HABP binding activity, respectively. It is possible to replace GlcUAβ-1-3GlcNAc of the internal disaccharide units with GlcUAβ-1-3N-acetylgalactosamine (GalNAc), if the chain length is decasaccharide or larger., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This review examines aggrecan's roles in developmental embryonic tissues, in tissues undergoing morphogenetic transition and in mature weight-bearing tissues. Aggrecan is a remarkably versatile and capable proteoglycan (PG) with diverse tissue context-dependent functional attributes beyond its established role as a weight-bearing PG. The aggrecan core protein provides a template which can be variably decorated with a number of glycosaminoglycan (GAG) side chains including keratan sulphate (KS), human natural killer trisaccharide (HNK-1) and chondroitin sulphate (CS). These convey unique tissue-specific functional properties in water imbibition, space-filling, matrix stabilisation or embryonic cellular regulation. Aggrecan also interacts with morphogens and growth factors directing tissue morphogenesis, remodelling and metaplasia. HNK-1 aggrecan glycoforms direct neural crest cell migration in embryonic development and is neuroprotective in perineuronal nets in the brain. The ability of the aggrecan core protein to assemble CS and KS chains at high density equips cartilage aggrecan with its well-known water-imbibing and weight-bearing properties. The importance of specific arrangements of GAG chains on aggrecan in all its forms is also a primary morphogenetic functional determinant providing aggrecan with unique tissue context dependent regulatory properties. The versatility displayed by aggrecan in biodiverse contexts is a function of its GAG side chains.

B cells play a crucial role in the pathogenesis of rheumatoid arthritis. In Nkx2-3-deficient mice (Nkx2-3 -/- ) the spleen's histological structure is fundamentally changed; therefore, B cell homeostasis is seriously disturbed. Based on this, we were curious, whether autoimmune arthritis could be induced in Nkx2-3 -/- mice and how B cell activation and function were affected. We induced arthritis with immunization of recombinant human proteoglycan aggrecan G1 domain in Nkx2-3 -/- and control BALB/c mice. We followed the clinical picture, characterized the radiological changes, the immune response, and intracellular Ca 2+ signaling of B cells. Incidence of the autoimmune arthritis was lower, and the disease severity was milder in Nkx2-3 -/- mice than in control BALB/c mice. The radiological changes were in line with the clinical picture. In Nkx2-3 -/- mice, we measured decreased antigen-induced proliferation and cytokine production in spleen cell cultures; in the sera, we found less anti-CCP-IgG2a, IL-17 and IFNγ, but more IL-1β, IL-4 and IL-6. B cells isolated from the lymph nodes of Nkx2-3 -/- mice showed decreased intracellular Ca 2+ signaling compared to those isolated from BALB/c mice. Our findings show that the transcription factor Nkx2-3 might regulate the development of autoimmune arthritis most likely through modifying B cell activation.

Glycosaminoglycan (GAG) chemical exchange saturation transfer (gagCEST) is a potential method for cartilage quality assessment. The aim of this study was to investigate how the gagCEST effect depends on the types and molecular organization of GAG typically found in articular cartilage. gagCEST was performed on different concentrations of GAG in various forms: free chains of chondroitin sulfate (CS) of different types (-A and -C) and GAG bound to protein in aggregated and nonaggregated aggrecan extracted from calf articular cartilage. The measured magnetization transfer ratio asymmetry (MTR asym ) was compared with known GAG concentrations or GAG concentrations determined through biochemical analysis. The gagCEST effect was assessed through the linear regression coefficient with 95% confidence interval of MTR asym per GAG concentration. We observed a lower gagCEST effect in phantoms containing a mixture of CS-A and CS-C compared with phantoms containing mainly CS-A. The difference in response corresponds well to the difference in CS-A concentration. GAG bound in aggrecan from calf articular cartilage, where CS-A is assumed to be the major type of GAG, produed a similar gagCEST effect as that observed for free CS-A. The effect was also similar for aggregated (ie, bound to hyaluronic acid) and nonaggregated aggrecan. In conclusion, our results indicate that the aggrecan structure in itself does not impact the gagCEST effect, but that the effect is strongly dependent on GAG type. In phantoms, the current implementation of gagCEST is sensitive to CS-A while for CS-C, the main GAG component in mature human articular cartilage, the sensitivity is limited. This difference in gagCEST sensitivity between GAG types detected in phantoms is a strong motivation to also explore the possibility of a similar effect in vivo., (© 2020 John Wiley & Sons, Ltd.)

Aggrecan is a major matrix component of articular cartilage, and its dysregulated proteolysis is a crucial event in the pathogenesis of arthritis. Aggrecanases, members of ADAMTS family, play a pivotal role in aggrecan degradation with ADAMTS-4 and ADAMTS-5 being key enzymes. Cleavage events mediated by ADAMTSs are highly specific and very well characterized; therefore, it is possible to investigate aggrecanolysis by using antibodies reacting with the new N- and C-termini of the cleavage products (neoepitope antibodies). Here, we present a method for analyzing dynamic aggrecanolysis by Western blotting using neoepitope antibodies in combination with antibodies against total aggrecan fragments. The protocol is robust and has a broad application for investigation of aggrecanase activity in vitro and ex vivo.

Excessive degradation of the cartilage articular extracellular matrix (ECM) in chondrocytes has been considered as an important pathological characteristics of OA. In the present study, we demonstrate that the G protein-coupled receptor GPR39 is expressed on SW1353 chondrocytes and is significantly downregulated in response to advanced glycation end products (AGEs). Our findings show that agonism of GPR39 exerts significant protective effects against AGE-induced degradation of articular extracellular matrix. Agonism of GPR39 rescued degradation of type II collagen by decreasing expression of the collagen-degrading enzymes matrix metalloproteinase (MMP)-3 and MMP-13. Additionally, agonism of GPR39 rescued AGE-induced suppression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Agonism of GPR39 prevented degradation of aggrecan by downregulating AGE-induced expression of a disintegrin and metalloproteinase with type I thrombospondin motif (ADAMTS)-4 and ADAMTS-5. Finally, we demonstrate that the effects of GPR39 are mediated through the p38 mitogen activated protein kinase (MAPK)/nuclear factor-κB (NF-κB) cellular signaling pathway. Taken together, our findings show for the first time that targeted therapies involving GPR39 may provide a novel approach for the prevention and treatment of osteoarthritis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Joint biomechanical functions rely on the integrity of cartilage extracellular matrix. Understanding the molecular activities that govern cartilage matrix assembly is critical for developing effective cartilage regeneration strategies. This study elucidated the role of decorin, a small leucine-rich proteoglycan, in the structure and biomechanical functions of cartilage. In decorin-null cartilage, we discovered a substantial reduction of aggrecan content, the major proteoglycan of cartilage matrix, and mild changes in collagen fibril nanostructure. This loss of aggrecan resulted in significantly impaired biomechanical properties of cartilage, including decreased modulus, elevated hydraulic permeability, and reduced energy dissipation capabilities. At the cellular level, we found that decorin functions to increase the retention of aggrecan in the neo-matrix of chondrocytes, rather than to directly influence the biosynthesis of aggrecan. At the molecular level, we demonstrated that decorin significantly increases the adhesion between aggrecan and aggrecan molecules and between aggrecan molecules and collagen II fibrils. We hypothesize that decorin plays a crucial structural role in mediating the matrix integrity and biomechanical functions of cartilage by providing physical linkages to increase the adhesion and assembly of aggrecan molecules at the nanoscale.

Mast cells represent a heterogeneous cell population that is well-known for the production of heparin and the release of histamine upon activation. Serglycin is a proteoglycan that within mast cell α-granules is predominantly decorated with the glycosaminoglycans heparin or chondroitin sulfate (CS) and has a known role in granule homeostasis. Heparanase is a heparin-degrading enzyme, is present within the α-granules, and contributes to granule homeostasis, but an equivalent CS-degrading enzyme has not been reported previously. In this study, using several approaches, including epitope-specific antibodies, immunohistochemistry, and EM analyses, we demonstrate that human HMC-1 mast cells produce the CS-degrading enzymes hyaluronidase-1 (HYAL1) and HYAL4. We observed that treating the two model CS proteoglycans aggrecan and serglycin with HYAL1 and HYAL4 in vitro cleaves the CS chains into lower molecular weight forms with nonreducing end oligosaccharide structures similar to CS stub neoepitopes generated after digestion with the bacterial lyase chondroitinase ABC. We found that these structures are associated with both the CS linkage region and with structures more distal toward the nonreducing end of the CS chain. Furthermore, we noted that HYAL4 cleaves CS chains into lower molecular weight forms that range in length from tetra- to dodecasaccharides. These results provide first evidence that mast cells produce HYAL4 and that this enzyme may play a specific role in maintaining α-granule homeostasis in these cells by cleaving CS glycosaminoglycan chains attached to serglycin., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2019 Farrugia et al.)

While articular cartilage defects affect millions of people worldwide from adolescents to adults, the repair of articular cartilage defects still remains challenging due to the limited endogenous regeneration of the tissue and poor integration with implants. In this study, we developed a 3D-printed scaffold functionalized with aggrecan that supports the cellular fraction of bone marrow released from microfracture, a widely used clinical procedure, and demonstrated tremendous improvement of regenerated cartilage tissue quality and joint function in a lapine model. Optical coherence tomography (OCT) revealed doubled thickness of the regenerated cartilage tissue in the group treated with our aggrecan functionalized scaffold compared to standard microfracture treatment. H&E staining showed 366 ± 95 chondrocytes present in the unit area of cartilage layer with the support of bioactive scaffold, while conventional microfracture group showed only 112 ± 26 chondrocytes. The expression of type II collagen appeared almost 10 times higher with our approach compared to normal microfracture, indicating the potential to overcome the fibro-cartilage formation associated with the current microfracture approach. The therapeutic effect was also evaluated at joint function level. The mobility was evaluated using a modified Basso, Beattie and Bresnahan (BBB) scale. While the defect control group showed no movement improvement over the course of study, all experimental groups showed a trend of increasing scores over time. The present work developed an effective method to regenerate critical articular defects by combining a 3D-printed therapeutic scaffold with the microfracture surgical procedure. This biofunctionalized acellular scaffold has great potential to be applied as a supplement for traditional microfracture to improve the quality of cartilage regeneration in a cost and labor effective way., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Biomimetic proteoglycans (BPGs) were designed to mimic the three-dimensional (3D) bottlebrush architecture of natural extracellular matrix (ECM) proteoglycans, such as aggrecan. BPGs were synthesized by grafting native chondroitin sulfate bristles onto a synthetic poly(acrylic acid) core to form BPGs at a molecular weight of approximately ∼1.6 MDa. The aggrecan mimics were characterized chemically, physically, and structurally, confirming the 3D bottlebrush architecture as well as a level of water uptake, which is greater than that of the natural proteoglycan, aggrecan. Aggrecan mimics were cytocompatible at physiological concentrations. Fluorescently labeled BPGs were injected into the nucleus pulposus of the intervertebral disc ex vivo and were retained in tissue before and after static loading and equilibrium conditioning. BPGs infiltrated the tissue, distributed and integrated with the ECM on a molecular scale, in the absence of a bolus, thus demonstrating a new molecular approach to tissue repair: molecular matrix engineering. Molecular matrix engineering may compliment or offer an acellular alternative to current regenerative medicine strategies., Statement of Significance: Aggrecan is a natural biomolecule that is essential for connective tissue hydration and mechanics. Aggrecan is composed of negatively charged chondroitin sulfate bristles attached to a protein core in a bottlebrush configuration. With age and degeneration, enzymatic degradation of aggrecan outpaces cellular synthesis resulting in a loss of this important molecule. We demonstrate a novel biomimetic molecule composed of natural chondroitin sulfate bristles grafted onto an enzymatically-resistant synthetic core. Our molecule mimics a 3D architecture and charge density of the natural aggrecan, can be delivered via a simple injection and is retained in tissue after equilibrium conditioning and loading. This novel material can serve as a platform for molecular repair, drug delivery and tissue engineering in regenerative medicine approaches., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Mutations in the aggrecan (ACAN) gene can cause short stature (with heterogeneous clinical phenotypes), impaired bone maturation, and large variations in response to growth hormone (GH) treatment. For such cases, long-term longitudinal therapy data from China are still scarce. We report that a previously unknown ACAN gene variant reduces adult height and we analyze the GH response in children from an affected large Chinese family., Methods: Two children initially diagnosed with idiopathic short stature (ISS) and a third mildly short child from a large Chinese family presented with poor GH response. Genetic etiology was identified by whole exome sequencing and confirmed via Sanger sequencing. Adult heights were analyzed, and the responses to GH treatment of the proband and two affected relatives are summarized and compared to other cases reported in the literature., Results: A novel ACAN gene variant c.7465 T > C (p. Gln2364Pro), predicted to be disease causing, was discovered in the children, without evident syndromic short stature; mild bone abnormity was present in these children, including cervical-vertebral clefts and apophyses in the upper and lower thoracic vertebrae. Among the variant carriers, the average adult male and female heights were reduced by - 5.2 and - 3.9 standard deviation scores (SDS), respectively. After GH treatment of the three children, first-year heights increased from 0.23 to 0.33 SDS (cases in the literature: - 0.5 to 0.8 SDS), and the average yearly height improvement was 0.0 to 0.26 SDS (cases in the literature: - 0.5 to 0.9 SDS)., Conclusions: We report a novel pathogenic ACAN variant in a large Chinese family which can cause severe adult nonsyndromic short stature without evident family history of bone disease. The evaluated cases and the reports from the literature reveal a general trend of gradually diminishing yearly height growth (measured in SDS) over the course of GH treatment in variant-carrying children, highlighting the need to develop novel management regimens.

Although the composition and structure of cartilaginous tissues is complex, collagen II fibrils and aggrecan are the most abundant assemblies in both articular cartilage (AC) and the nucleus pulposus (NP) of the intervertebral disc (IVD). Whilst structural heterogeneity of intact aggrecan ( containing three globular domains) is well characterised, the extent of aggrecan fragmentation in healthy tissues is poorly defined. Using young, yet skeletally mature (18-30 months), bovine AC and NP tissues, it was shown that, whilst the ultrastructure of intact aggrecan was tissue-dependent, most molecules (AC: 95 %; NP: 99.5 %) were fragmented (lacking one or more globular domains). Fragments were significantly smaller and more structurally heterogeneous in the NP compared with the AC (molecular area; AC: 8543 nm2; NP: 4625 nm2; p < 0.0001). In contrast, fibrillar collagen appeared structurally intact and tissue-invariant. Molecular fragmentation is considered indicative of a pathology; however, these young, skeletally mature tissues were histologically and mechanically (reduced modulus: AC: ≈ 500 kPa; NP: ≈ 80 kPa) comparable to healthy tissues and devoid of notable gelatinase activity (compared with rat dermis). As aggrecan fragmentation was prevalent in neonatal bovine AC (99.5 % fragmented, molecular area: 5137 nm2) as compared with mature AC (95.0 % fragmented, molecular area: 8667 nm2), it was hypothesised that targeted proteolysis might be an adaptive process that modified aggrecan packing (as simulated computationally) and, hence, tissue charge density, mechanical properties and porosity. These observations provided a baseline against which pathological and/or age-related fragmentation of aggrecan could be assessed and suggested that new strategies might be required to engineer constructs that mimic the mechanical properties of native cartilaginous tissues.

Human chondrocytes isolated from articular cartilage (AC) lesions as an alternative cell source to the standard nonweight-bearing notch biopsy site may hold clinical potential for cell-based therapies. The aim was to characterize human AC lesion site chondrocytes, compare them to notch chondrocytes, and evaluate their redifferentiation potential after monolayer expansion and subsequent three-dimensional (3D) alginate bead culture. Lesion chondrocytes from knee joints of 20 patients with International Cartilage Repair Society (ICRS) grade 3 and 4 cartilage defects were analyzed ex vivo or cultured in primary alginate bead culture, monolayer expansion, or redifferentiated in alginate culture following monolayer expansion. The mRNA expression of the types I, II, and X collagen, and the proteoglycan aggrecan was compared between the four groups. In addition, notch chondrocytes of nine patients were compared to lesion chondrocytes ex vivo. AC lesion chondrocytes displayed ex vivo a nondegenerative phenotype, characterized by a relatively high mRNA expression of aggrecan and type II and X collagen, but a low type I collagen expression and a low ratio of type I to II collagen mRNA expression. Compared to notch chondrocytes, the mRNA expression of aggrecan and type II collagen was comparable and the ratio of type I to II collagen mRNA expression was below 1 in both groups, indicating a functional chondrocyte phenotype. Dedifferentiation led to a significantly altered degenerative mRNA expression profile. Induced redifferentiation in alginate beads after monolayer expansion significantly improved the mRNA expression of aggrecan, the type I and II collagen, and the type I to II collagen ratio, compared to monolayer expansion only. These data suggested that redifferentiating lesion chondrocytes after monolayer expansion in alginate beads resulted in a pool of cells with greater chondrogenic potential, compared to expanded dedifferentiated chondrocytes. Collectively, these data suggest that ex vivo and redifferentiated lesion chondrocytes may hold nonutilized clinical potential for the tissue engineering of AC.

Although mutations in ACAN, FGFR3, NPR2, and SHOX typically lead to skeletal dysplasia, and mutations in GHRHR, GH1, GHR, STAT5B, IGF1, IGFALS, and IGF1R usually underlie hormonal defects of the growth hormone (GH)-insulin-like growth factor 1 (IGF1) axis, such mutations have also been identified in patients with idiopathic short stature (ISS). Of these, SHOX abnormalities are known to account for a certain percentage of ISS cases, whereas the frequency of mutations in the other 10 genes in ISS cohorts remains unknown. Here, we performed next-generation sequencing-based mutation screening of the 10 genes in 86 unrelated Japanese ISS patients without SHOX abnormalities. We searched for rare protein-altering variants. The functional significance of the identified variants was assessed by in silico analyses. Consequently, we identified 18 heterozygous rare variants in 19 patients, including four probable damaging variants in ACAN, six pathogenicity-unknown variants in FGFR3, GHRHR, GHR, and IGFALS, and eight possible benign variants. Pathogenic variants in NPR2, GH1, and IGF1 were absent from our cohort. Unlike previously reported patients with ACAN mutations, our four patients with ACAN variants manifested non-specific short stature with age-appropriate or mildly delayed bone ages, and had parents of normal stature. These results indicate that ACAN mutations can underlie ISS without characteristic skeletal features, and that such mutations are possibly associated with de novo occurrence or low penetrance. In addition, our data imply that mutations in FGFR3, NPR2, and GH-IGF1 axis genes play only limited roles in the etiology of ISS.

Articular cartilage is a connective tissue consisting of a specialized extracellular matrix (ECM) that dominates the bulk of its wet and dry weight. Type II collagen and aggrecan are the main ECM proteins in cartilage. However, little attention has been paid to less abundant molecular components, especially minor collagens, including type IV, VI, IX, X, XI, XII, XIII, and XIV, etc. Although accounting for only a small fraction of the mature matrix, these minor collagens not only play essential structural roles in the mechanical properties, organization, and shape of articular cartilage, but also fulfil specific biological functions. Genetic studies of these minor collagens have revealed that they are associated with multiple connective tissue diseases, especially degenerative joint disease. The progressive destruction of cartilage involves the degradation of matrix constituents including these minor collagens. The generation and release of fragmented molecules could generate novel biochemical markers with the capacity to monitor disease progression, facilitate drug development and add to the existing toolbox for in vitro studies, preclinical research and clinical trials.

A key event in arthritis pathogenesis is the degradation of aggrecan, the major component in articular cartilage. In this work, we investigate the effects of stimulated aggrecanolysis on the morphological and nanomechanical properties of cartilage harvested from wild-type mice and aggrecanase-resistant mutant mice named "Jaffa". The cartilages were native or were subjected to stimulated aggrecanolysis by interleukin-1[Formula: see text] (IL-1[Formula: see text]) treatment. The nanoscale morphological and mechanical properties of the sectioned cartilages were measured by using a sharp probe by atomic force microscopy (AFM). The IL-1[Formula: see text] treatment resulted in a higher nanoroughess and stiffness of the cartilage from wild-type mice. However, the same treatment did not lead to any measurable change in the nanoroughness or stiffness of the cartilage from mutant mice Jaffa. This suggests that blocking aggrecanolysis by genetic modification has created the stability in the structures and mechanical properties of the cartilage at nanoscale. The present study provides insight into the mechanism of aggrecan degradation, which can complement the examination by biochemical and histological techniques.

Aging and degeneration of human tissue come with the loss of tissue water retention and associated changes in physical properties partially due to degradation and subsequent loss of proteoglycans. We demonstrated a novel method of fabrication of biomimetic proteoglycans, which mimic the three-dimensional bottlebrush architecture and physical behavior of natural proteoglycans responsible for tissue hydration and structural integrity. Biomimetic proteoglycans are synthesized by an end-on attachment of natural chondroitin sulfate bristles to a synthetic poly(acryloyl chloride) backbone. Atomic force microscopy imaging suggested three-dimensional core-bristle architecture, and hydrodynamic size of biomimetic proteoglycans was estimated at 61.3 ± 12.3 nm using dynamic light scattering. Water uptake results indicated that biomimetic proteoglycans had a ∼50% increased water uptake compared to native aggrecan and chondroitin sulfate alone. The biomimetic proteoglycans are cytocompatible in the physiological ranges of concentrations and could be potentially used to repair damaged or diseased tissue with depleted proteoglycan content.

In a recent publication in Bioscience Reports "Contaminants in commercial preparations of 'purified' small leucine-rich proteoglycans may distort mechanistic studies", Brown et al. identified by mass spectrometry and immunoblotting that certain commercial preparations of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan, in fact, contained a mix of several proteoglycans that also included fibromodulin and aggrecan. The preparations were thus not suitable to study specific activities of decorin or biglycan. Decorin and biglycan are widely studied SLRPs that are considered to have highly multi-functional effects on cells. Decorin is of interest as a transforming growth factor-β antagonist and is also finding use in tissue engineering materials. This Commentary discusses Brown et al.'s findings and general issues raised for researchers who work with commercially sourced purified proteoglycans., (© 2017 The Author(s).)

Chondrocyte-seeded, photo-cross-linked hydrogels prepared from solutions containing 50% mass fractions of methacrylated glycol chitosan or methacrylated hyaluronic acid (MHA) with methacrylated chondroitin sulfate (MCS) are cultured in vitro under static conditions over 35 d to assess their suitability for load-bearing soft tissue repair. The photo-cross-linked hydrogels have initial equilibrium moduli between 100 and 300 kPa, but only the MHAMCS hydrogels retain an approximately constant modulus (264 ± 5 kPa) throughout the culture period. Visually, the seeded chondrocytes in the MHAMCS hydrogels are well distributed with an apparent constant viability in culture. Multicellular aggregates are surrounded by cartilaginous matrix, which contain aggrecan and collagen II. Thus, co-cross-linked MCS and MHA hydrogels may be suited for use in an articular cartilage or nucleus pulposus repair applications., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Rheumatoid arthritis (RA) is an autoimmune disease of the synovial joints. The autoimmune character of RA is underscored by prominent production of autoantibodies such as those against IgG (rheumatoid factor), and a broad array of joint tissue-specific and other endogenous citrullinated proteins. Anti-citrullinated protein antibodies (ACPA) can be detected in the sera and synovial fluids of RA patients and ACPA seropositivity is one of the diagnostic criteria of RA. Studies have demonstrated that RA T cells respond to citrullinated peptides (epitopes) of proteoglycan (PG) aggrecan, which is one of the most abundant macromolecules of articular cartilage. However, it is not known if the PG molecule is citrullinated in vivo in human cartilage, and if so, whether citrulline-containing neoepitopes of PG (CitPG) can contribute to autoimmunity in RA., Methods: CitPG was detected in human cartilage extracts using ACPA+ RA sera in dot blot and Western blot. Citrullination status of in vitro citrullinated recombinant G1 domain of human PG (rhG1) was confirmed by antibody-based and chemical methods, and potential sites of citrullination in rhG1 were explored by molecular modeling. CitPG-specific serum autoantibodies were quantified by enzyme-linked immunosorbent assays, and CitPG was localized in osteoarthritic (OA) and RA cartilage using immunohistochemistry., Findings: Sera from ACPA+ RA patients reacted with PG purified from normal human cartilage specimens. PG fragments (mainly those containing the G1 domain) from OA or RA cartilage extracts were recognized by ACPA+ sera but not by serum from ACPA- individuals. ACPA+ sera also reacted with in vitro citrullinated rhG1 and G3 domain-containing fragment(s) of PG. Molecular modeling suggested multiple sites of potential citrullination within the G1 domain. The immunohistochemical localization of CitPG was different in OA and RA cartilage., Conclusions: CitPG is a new member of citrullinated proteins identified in human joints. CitPG could be found in both normal and diseased cartilage specimens. Antibodies against CitPG may trigger or augment arthritis by forming immune complexes with this autoantigen in the joints of ACPA+ RA patients.

Severe knee trauma, such as an ACL disruption, produces aggrecan degradation as evidenced by elevated synovial fluid (SF) N-terminal (393) Alanine-Arginine-Glycine-Serine (ARGS) neoepitope (or ARGS-aggrecan) and is associated with inflammatory activity soon after injury. However, it is not known if this process persists for a substantial time interval following the initial trauma. The purpose of this study was to evaluate relationships between SF ARGS concentrations and an array of cytokines, matrix metalloproteases (MMPs), and tissue inhibitor of metalloproteases (TIMPs) during the initial 6 months following ACL rupture. SF samples from 67 ACL-injured subjects (29 women) were analyzed within 6 months of injury (18-155 days), immediately prior to surgical ACL reconstruction. Relationships between ARGS and individual analyte concentrations, as well as MMP/TIMP ratios were evaluated. Statistically significant relationships were found between ARGS and basic fibroblast growth factor (FGF2) (p=0.03) and TIMP-3 (p=0.01). Our findings suggest that FGF2, considered to be primarily catabolic in articular cartilage, is not downregulated as ARGS concentration declines over time since injury. In addition, these results support the hypothesis that an upregulation of TIMP-3, the primary aggrecanase inhibitor, is elicited in response to increased aggrecan degradation, which may inhibit further cleavage., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

In many cells hyaluronan receptor CD44 mediates the endocytosis of hyaluronan and its delivery to endosomes/lysosomes. The regulation of this process remains largely unknown. In most extracellular matrices hyaluronan is not present as a free polysaccharide but often is found in complex with other small proteins and macromolecules such as proteoglycans. This is especially true in cartilage, where hyaluronan assembles into an aggregate structure with the large proteoglycan termed aggrecan. In this study when purified aggrecan was added to FITC-conjugated hyaluronan, no internalization of hyaluronan was detected. This suggested that the overall size of the aggregate prevented hyaluronan endocytosis and furthermore that proteolysis of the aggrecan was a required prerequisite for local, cell-based turnover of hyaluronan. To test this hypothesis, limited C-terminal digestion of aggrecan was performed to determine whether a size range of aggrecan exists that permits hyaluronan endocytosis. Our data demonstrate that only limited degradation of the aggrecan monomer was required to allow for hyaluronan internalization. When hyaluronan was combined with partially degraded, dansyl chloride-labeled aggrecan, blue fluorescent aggrecan was also visualized within intracellular vesicles. It was also determined that sonicated hyaluronan of smaller molecular size was internalized more readily than high molecular mass hyaluronan. However, the addition of intact aggrecan to hyaluronan chains sonicated for 5 and 10 s reblocked their endocytosis, whereas aggregates containing 15-s sonicated hyaluronan were internalized. These data suggest that hyaluronan endocytosis is regulated in large part by the extracellular proteolytic processing of hyaluronan-bound proteoglycan., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Poroelastic interactions between interstitial fluid and the extracellular matrix of connective tissues are critical to biological and pathophysiological functions involving solute transport, energy dissipation, self-stiffening and lubrication. However, the molecular origins of poroelasticity at the nanoscale are largely unknown. Here, the broad-spectrum dynamic nanomechanical behavior of cartilage aggrecan monolayer is revealed for the first time, including the equilibrium and instantaneous moduli and the peak in the phase angle of the complex modulus. By performing a length scale study and comparing the experimental results to theoretical predictions, we confirm that the mechanism underlying the observed dynamic nanomechanics is due to solid-fluid interactions (poroelasticity) at the molecular scale. Utilizing finite element modeling, the molecular-scale hydraulic permeability of the aggrecan assembly was quantified (kaggrecan = (4.8 ± 2.8) × 10(-15) m(4)/N·s) and found to be similar to the nanoscale hydraulic permeability of intact normal cartilage tissue but much lower than that of early diseased tissue. The mechanisms underlying aggrecan poroelasticity were further investigated by altering electrostatic interactions between the molecule's constituent glycosaminoglycan chains: electrostatic interactions dominated steric interactions in governing molecular behavior. While the hydraulic permeability of aggrecan layers does not change across species and age, aggrecan from adult human cartilage is stiffer than the aggrecan from newborn human tissue.

Proteoglycans (PGs) are involved in various cellular functions including cell growth, adhesion, and differentiation; however, their physiological roles are not fully understood. In this study, we examined the effect of PG purified from salmon nasal cartilage (SNC-PG) on wound closure using tissue-cultured cell monolayers, an in vitro wound-healing assay. The results indicated that SNC-PG significantly promoted wound closure in NIH/3T3 cell monolayers by stimulating both cell proliferation and cell migration. SNC-PG was effective in concentrations from 0.1 to 10μg/ml, but showed much less effect at higher concentrations (100-1000μg/ml). The effect of SNC-PG was abolished by chondroitinase ABC, indicating that chondroitin sulfates (CSs), a major component of glycosaminoglycans (GAGs) in SNC-PG, are crucial for the SNC-PG effect. Furthermore, chondroitin 6-sulfate (C-6-S), a major CS of SNC-PG GAGs, could partially reproduce the SNC-PG effect and partially inhibit the binding of SNC-PG to cells, suggesting that SNC-PG exerts its effect through an interaction between the GAGs in SNC-PG and the cell surface. Neutralization by anti-CD44 antibodies or CD44 knockdown abolished SNC-PG binding to the cells and the SNC-PG effect on wound closure. These results suggest that interactions between CS-rich GAG-chains of SNC-PG and CD44 on the cell surface are responsible for the SNC-PG effect on wound closure., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In healthy cartilage, mechano-electrochemical phenomena act together to maintain tissue homeostasis. Osteoarthritis (OA) and degenerative diseases disrupt this biological equilibrium by causing structural deterioration and subsequent dysfunction of the tissue. Swelling and ion flux alteration as well as abnormal ion distribution are proposed as primary indicators of tissue degradation. In this paper, we present an extension of a previous three-dimensional computational model of the cartilage behaviour developed by the authors to simulate the contribution of the main tissue components in its behaviour. The model considers the mechano-electrochemical events as concurrent phenomena in a three-dimensional environment. This model has been extended here to include the effect of repulsion of negative charges attached to proteoglycans. Moreover, we have studied the fluctuation of these charges owning to proteoglycan variations in healthy and pathological articular cartilage. In this sense, standard patterns of healthy and degraded tissue behaviour can be obtained which could be a helpful diagnostic tool. By introducing measured properties of unhealthy cartilage into the computational model, the severity of tissue degeneration can be predicted avoiding complex tissue extraction and subsequent in vitro analysis. In this work, the model has been applied to monitor and analyse cartilage behaviour at different stages of OA and in both short (four, six and eight weeks) and long-term (11 weeks) fully immobilized joints. Simulation results showed marked differences in the corresponding swelling phenomena, in outgoing cation fluxes and in cation distributions. Furthermore, long-term immobilized patients display similar swelling as well as fluxes and distribution of cations to patients in the early stages of OA, thus, preventive treatments are highly recommended to avoid tissue deterioration., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Objectives: To fully sequence and characterize equine aggrecan and confirm conservation of major aggrecanase, calpain and matrix metalloproteinase (MMP) cleavage sites., Methods: Reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends were used to generate clones that encompassed the complete equine aggrecan sequence. Clones were sequenced and compared with the equine genome database to determine intron-exon boundaries., Results: The aggrecan gene spans over 61 kb on chromosome 1 and is encoded by 17 exons. Two major variants of aggrecan were cloned; one containing 8187 bp (2728 amino acids) and a second sequence of 8061 nucleotides (2686 amino acids). The variation was due to a CS1 domain polymorphism. Both sequences are substantially larger than predicted by the genomic database; 11 CS1 repeat elements are absent in the database sequence. The equine amino acid sequence was compared with human, bovine and murine sequences. Globular domains 1, 2 and 3 are highly conserved (overall identity over 80%). Equine CS1 is considerably larger than in other species and, therefore, is the least conserved domain (an overall amino acid identity of 22%). Previously defined aggrecanase, calpain and MMP cleavage sites were identified. Western blotting of chondrocyte culture samples showed complex post-secretion processing., Clinical Significance: The complete equine aggrecan sequence will support more in-depth research on aggrecan processing and degradation in equine articular cartilage and other musculoskeletal tissues.

Aggrecan, the most abundant extracellular proteoglycan in cartilage (~35 % by dry weight), plays a key role in the biophysical and biomechanical properties of cartilage. Here, we review several approaches based on atomic force microscopy (AFM) to probe the physical, mechanical, and structural properties of aggrecan at the molecular level. These approaches probe the response of aggrecan over a wide time (frequency) scale, ranging from equilibrium to impact dynamic loading. Experimental and theoretical methods are described for the investigation of electrostatic and fluid-solid interactions that are key mechanisms underlying the biomechanical and physicochemical functions of aggrecan. Using AFM-based imaging and nanoindentation, ultrastructural features of aggrecan are related to its mechanical properties, based on aggrecans harvested from human vs. bovine, immature vs. mature, and healthy vs. osteoarthritic cartilage.

T-cell recognition of MHC–peptide complexes shows a high degree of polyspecificity extending to recognition of a large number of structurally unrelated peptides. Examples of polyspecificity reported to date are confined to recognition of epitopes from distinct proteins or synthetic peptide libraries. Here we describe intramolecular polyspecificity of CD4 T cells specific for several epitopes within proteoglycan aggrecan, a structural glycoprotein of cartilage and candidate autoantigen in rheumatoid arthritis. T-cell hybridomas from aggrecan-immunized mice recognized four structurally unrelated epitopes from the G1 domain of aggrecan, but not other aggrecan epitopes or a variety of other peptide epitopes restricted by the same MHC class II allele. We also showed that the hierarchy of cross-reactivity broadly correlated with the strength of peptide binding to MHC class II. Similar polyspecificity was observed in responses of lymph node cells from peptide-immunized mice, suggesting polyspecificity of a significant proportion of the in vivo aggrecan specific T-cell repertoire. Polyspecific recognition of several epitopes within the same autoantigen may provide a novel mechanism to reach the activation threshold of low-affinity autoreactive T cells in the initiation of autoimmune diseases.

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.

The direct delivery of growth factors to sites of tissue healing is complicated by their relative instability. In many tissues, the glycosaminoglycan (GAG) side chains of proteoglycans like aggrecan stabilize growth factors in the pericellular and extracellular space, creating a local reservoir that can be accessed during a wound healing response. GAGs also regulate growth factor-receptor interactions at the cell surface. Here we report the development of nanoparticles for growth factor delivery that mimic the size, GAG composition, and growth factor binding and stabilization of aggrecan. The aggrecan-mimetic nanoparticles are easy to assemble, and their structure and composition can be readily tuned to alter their physical and biological properties. We use basic fibroblast growth factor (FGF-2) as a model heparin-binding growth factor, demonstrating that aggrecan-mimetic nanoparticles can preserve its activity for more than three weeks. We evaluate FGF-2 activity by measuring both the proliferation and metabolic activity of bone marrow stromal cells to demonstrate that chondroitin sulfate-based aggrecan mimics are as effective as aggrecan, and heparin-based aggrecan mimics are superior to aggrecan as delivery vehicles for FGF-2.

Proteoglycans consist of a protein core to which at least one glycosaminoglycan chain is attached. They play important roles in the physiology and biomechanical function of tendons, ligaments and cardiovascular system through their involvement in regulation of assembly and maintenance of extracellular matrix, and as they participate in cell proliferation through their interactions with growth factors. They can be divided into two main groups of small and large proteoglycans. The small proteoglycans are also known as small leucine-rich proteoglycans (or SLRPs) which are encoded by 17 genes and are further subclassified into Classes I-V. Several members of Class I and II, such as decorin and biglycan from Class I, and Class II fibromodulin and lumican, are known to regulate collagen fibrillogenesis. Decorin limits the diameter of collagen fibrils during fibrillogenesis. The function of biglycan in fibrillogenesis is similar to that of decorin. Though biomechanical function of tendon is compromised in decorin-deficient mice, decorin can substitute for lack of biglycan in biglycan-deficient mice. New data also indicate an important role for biglycan in disorders of the cardiovascular system, including aortic valve stenosis and aortic dissection. Two members of the Class II of SLRPs, fibromodulin and lumican bind to the same site within the collagen molecule and can substitute for each other in fibromodulin- or lumican-deficient mice.Aggrecan and versican are the major representatives of the large proteoglycans. Though they are mainly found in the cartilage where they provide resilience and toughness, they are also present in tensile portions of tendons and, in slightly different biochemical form in fibrocartilage. Degradation with aggrecanase is responsible for the appearance of different forms of aggrecan and versican in different parts of the tendon where these cleaved forms play different roles. In addition, they are important components of the ventricularis of cardiac valves. Mutations in the gene for versican or in the gene for elastin (which binds to versican) lead to severe disruptions of normal developmental of the heart at least in mice.

Objective: We previously described a messenger RNA variant of ADAMTS4 (ADAMTS4&#95;v1) in human synovial cell cocultures obtained from patients with osteoarthritis (OA). This RNA message has been found only in OA synovium and, if translated, would result in a protein identical to ADAMTS-4, except that the C-terminal spacer domain would be different. The purpose of this study was to determine whether ADAMTS4&#95;v1 is translated into a protein, is expressed in vivo, and acts as a functional aggrecanase., Methods: Polyclonal antibodies were raised against unique C-terminal sequences of ADAMTS-4&#95;v1. An immunohistochemical study of human OA synovium was performed. A mammalian expression vector coding for FLAG-tagged human ADAMTS4 was mutated to contain the different sequences of ADAMTS4&#95;v1, and the resultant plasmid was used to transfect HEK 293 cells. ADAMTS-4&#95;v1 produced by these cells was purified via the FLAG epitope, and the ability of this recombinant protein to cleave aggrecan, biglycan, and decorin was investigated., Results: An antibody specific for ADAMTS-4&#95;v1 was found to bind to the synovial membrane surface on cryosections, and the protein was detected in cell lysates from synovium obtained from OA patients. The recombinant ADAMTS-4&#95;v1 demonstrated enzyme activity toward the target substrate in a commercial aggrecanase 1 enzyme-linked immunosorbent assay and was also found to cleave aggrecan at the pathologically important Glu(373↓374) Ala aggrecanase site., Conclusion: ADAMTS-4&#95;v1 is expressed as a protein in vivo in human OA synovium, functions as an aggrecanase, and cleaves other proteoglycan substrates. This splice variant may be a major contributor to loss of aggrecan from the superficial zone of OA cartilage., (© The Authors. Arthritis & Rheumatism is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

Articular cartilage defects are a significant source of pain, have limited ability to heal, and can lead to the development of osteoarthritis. However, a surgical solution is not available. To tackle this clinical problem, non-degradable implants capable of carrying mechanical load immediately after implantation and for the duration of implantation, while integrating with the host tissue, may be viable option. But integration between articular cartilage and non-degradable implants is not well studied. Our objective was to assess the in vivo performance of a novel macroporous, nondegradable, polyvinyl alcohol construct. We hypothesized that matrix generation within the implant would be enhanced with partial digestion of the edges of articular cartilage. Our hypothesis was tested by randomizing an osteochondral defect created in the trochlea of 14 New Zealand white rabbits to treatment with: (i) collagenase or (ii) saline, prior to insertion of the implant. At 1 and 3-month post-operatively, the gross morphology and histologic appearance of the implants and the surrounding tissue were assessed. At 3 months, the mechanical properties of the implant were also quantified. Overall, the hydrogel implants performed favorably; at all time-points and in all groups the implants remained well fixed, did not cause inflammation or synovitis, and did not cause extensive damage to the opposing articular cartilage. Regardless of treatment with saline or collagenase, at 1 month post-operatively implants from both groups had a contiguous interface with adjacent cartilage and were populated with chondrocyte-like cells. At 3 months fibrous encapsulation of all implants was evident, there was no difference between area of aggrecan staining in the collagenase versus saline groups, and implant modulus was similar in both groups; leading us to reject our hypothesis. In summary, a porous PVA osteochondral implant remained well fixed in a short term in vivo osteochondral defect model; however, matrix generation within the implant was not enhanced with partial digestion of adjacent articular cartilage.

Polyvinyl alcohol (PVA) hydrogel chains were crosslinked by urethane pre-polymer (PPU) in order to fabricate a new substitute for cartilage lesions. The microscopy images showed that the cultured chondrocytes had spherical morphology on PVA-PPU sample after 4 weeks of isolation in vitro. The alcian blue and safranin O staining proved the presence of proteoglycan on the surface of PVA-PPU sample secreted by cultured chondrocytes. This was confirmed by the detection of sulfate ions in the wavelength dispersive X-ray (WDX) analysis. In addition, the expression of collagen type II and aggrecan were observed in chondrocytes cultured on PVA-PPU by RT-PCR. Moreover, the implantation of the PVA-PPU sample with autologous cultured chondrocytes revealed the formation of neocartilage tissue in a rabbit model during 12 weeks follow up. In conclusion, the results verified that isolated chondrocytes cultured on PVA-PPU retain their original phenotype and this composition can be considered as promising substrate for cartilage tissue engineering.

Composite agarose (1.2 %) polyacrylamide (0.6 %) gel electrophoresis was used to separate discrete populations of native aggrecan and perlecan in newborn to 10 year old ovine intervertebral discs (IVDs). Semi-dry immunoblotting using core-protein and glycosaminoglycan (GAG) side chain specific monoclonal antibodies in combination with chondroitin ABC lyase demonstrated intra-chain native 7-D-4 chondroitin sulphate (CS) sulphation motifs and variable proportions of non-reducing terminal Δ4,5-unsaturated uronate-N-acetylgalactosamine-4-sulphate [2B6(+)] and Δ4,5-unsaturated glucuronate-N-acetylgalactosamine-6-sulphate [3B3(+)] disaccharides. The relative abundance of 2-B-6(+) aggrecan increased with advancing age of the IVD samples while the converse was true for the 3-B-3(+) aggrecan population. Relative 7D4 levels in aggrecan and perlecan were highest in the newborn IVD and significantly lower in the older IVD and other cartilage samples. Quantitation of 7D4 proteoglycan by enzyme linked immunosorbent inhibition assay confirmed the newborn ovine nucleus pulposus (NP) and inner annulus fibrosus (AF) contained higher levels (1.2-1.32 μg 7-D-4-proteoglycan/mg tissue wet weight) than the 2 (0.35-0.42 μg/mg wet weight tissue) and 10 year old IVD samples (0.16-0.22 μg/mg tissue wet weight) with the outer AF zones consistently containing lower levels of 7-D-4 epitope in all cases (P < 0.001). Cell populations on the margins of the AF and cartilaginous vertebral rudiments in newborn ovine and human foetal IVD strongly expressed 7-D-4 CS epitope and perlecan, This was co-distributed with Notch-1 expression in human foetal IVDs consistent with the 7-D-4 CS sulphation motif representing a marker of tissue development expressed by disc progenitor cell populations.

Loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier to the application of chondrocytes for tissue engineering. In previous study, we showed that dedifferentiation of chondrocytes during the passage culture was delayed by matrices formed by primary chondrocytes (P0-ECM). In this study, we investigated bovine chondrocyte functions when being cultured on isolated extracellular matrix (ECM) protein-coated substrata and P0-ECM. Low chondrocyte attachment was observed on aggrecan-coated substratum and P0-ECM. Cell proliferation on aggrecan- and type II collagen/aggrecan-coated substrata and P0-ECM was lower than that on the other ECM protein (type I collagen and type II collagen)-coated substrata. When chondrocytes were subcultured on aggrecan-coated substratum, decline of cartilaginous gene expression was delayed, which was similar to the cells subcultured on P0-ECM. These results indicate that aggrecan plays an important role in the regulation of chondrocyte functions and P0-ECM may be a good experimental control for investigating the role of each ECM protein in cartilage ECM., (© 2013 American Institute of Chemical Engineers.)

Molecular interactions involving glycosaminoglycans (GAGs) are important for biological processes in the extracellular matrix (ECM) and at cell surfaces, and also in biotechnological applications. Enzymes in the ECM constantly modulate the molecular structure and the amount of GAGs in our tissues. Specifically, the changeable sulfation patterns of many GAGs are expected to be important in interactions with proteins. Biotinylation is a convenient method for immobilizing molecules to surfaces. When studying interactions at the molecular, cell and tissue level, the native properties of the immobilized molecule, i.e. its biofunctionality, need to be retained upon immobilization. Here, the GAGs hyaluronan (HA) and chondroitin sulfate (CS), and synthetically sulfated derivatives of the two, were immobilized using biotin-streptavidin binding. The degree of biotinylation and the placement of biotin groups (end-on/side-on) were varied. The introduction of biotin groups could have unwanted effects on the studied molecule, but this aspect that is not always straightforward to evaluate. Hyaluronidase, an enzyme that degrades HA and CS in the ECM, was investigated as a probe to evaluate the biofunctionality of the immobilized GAGs, using both quartz crystal microbalance and high-performance liquid chromatography. Our results showed that end-on biotinylated HA was efficiently degraded by hyaluronidase, whereas already a low degree of side-on biotinylation destroyed the degrading ability of the enzyme. Synthetically introduced sulfate groups also had this effect. Hence hyaluronidase degradation is a cheap and easy way to investigate how molecular function is influenced by the introduced functional groups. Binding experiments with the proteoglycan aggrecan emphasized the influence of protein size and surface orientation of the GAGs for in-depth studies of GAG behavior., (Copyright © 2013. Published by Elsevier Ltd.)

Excessive complement activation contributes to joint diseases such as rheumatoid arthritis and osteoarthritis during which cartilage proteins are fragmented and released into the synovial fluid. Some of these proteins and fragments activate complement, which may sustain inflammation. The G3 domain of large cartilage proteoglycan aggrecan interacts with other extracellular matrix proteins, fibulins and tenascins, via its C-type lectin domain (CLD) and has important functions in matrix organization. Fragments containing G3 domain are released during normal aggrecan turnover, but increasingly so in disease. We now show that the aggrecan CLD part of the G3 domain activates the classical and to a lesser extent the alternative pathway of complement, via binding of C1q and C3, respectively. The complement control protein (CCP) domain adjacent to the CLD showed no effect on complement initiation. The binding of C1q to G3 depended on ionic interactions and was decreased in D2267N mutant G3. However, the observed complement activation was attenuated due to binding of complement inhibitor factor H to CLD and CCP domains. This was most apparent at the level of deposition of terminal complement components. Taken together our observations indicate aggrecan CLD as one factor involved in the sustained inflammation of the joint.

The P70-84 peptide (also called 5/4E8 epitope) of the human cartilage proteoglycan (PG) aggrecan is the dominant/arthritogenic epitope in both humans and arthritis-prone BALB/c mice (PG-induced arthritis, PGIA). An elevated T cell reactivity was demonstrated to a citrullinated version of the P70-84 epitope in most of the patients with rheumatoid arthritis (RA). The goal of this study was to understand better how a T cell epitope, if citrullinated, may affect antigenicity/arthritogenicity in PGIA, a murine model of RA. T cell reactivity to differentially citrullinated versions of either the human PG aggrecan P70-84 peptide or the corresponding mouse sequence was assessed in peptide or aggrecan-immunized and arthritic BALB/c mice as well as in T cell receptor transgenic mice specific for peptide P70-84 sequence. Peripheral T cell responses were induced by priming BALB/c mice with either the human wild-type or its citrullinated versions. Unexpectedly, priming with the citrullinated self-peptide induced a higher T cell response compared to the wild-type sequence (p<0.001), and the citrullination of the human peptide abolished T cell reactivity in PGIA. Our data suggest that T cells reactive to the citrullinated P70-84 peptide escaped thymic selection and are present in the peripheral T cell repertoire. Results of this study provide evidence that citrullination of an immunodominant T cell epitope may substantially alter, either increase or abolish, T cell recognition at the periphery in an experimental model of arthritis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Biomimetic scaffolds that promote regeneration and resist proteolysis are required as a tissue engineering solution to repair or replace a broad range of diseased tissues. Native corrosive environments, such as the richly enzymatic milieu of diseased articular cartilage, degrade the local extracellular matrix structure, so an implantable replacement must both replicate the healthy structure and demonstrate substantial proteolytic immunity, yet promote regeneration, if long-term functional success is to be achieved. Here, we combine magnetically aligned collagen with peptidoglycans, biosynthetic molecules that mimic proteoglycan activity but lack core proteins susceptible to proteases, to develop cartilage scaffold analogs with tailored functionality. With the incorporation of the aggrecan mimic, we demonstrate an ability to enhance bulk mechanical properties and prevent cytokine-induced degradation. Furthermore, fiber alignment in collagen scaffolds enhanced the gene expression of aggrecan, indicating cell responsiveness to anisotropy that also better replicates the natural environment of cartilage. Finally, the expression of type II collagen is enhanced with both alignment and incorporation of the aggrecan mimic, showing synergism between fiber alignment and incorporation of the aggrecan mimic. The work presented here identified a mechanistic synergy of matrix molecules and organization to prevent proteolysis while simultaneously upregulating protein expression., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Aggrecan is the prominent proteoglycan in cartilage and is modified with approximately 100 chondroitin sulfate (CS) chains through a tetrasaccharide linkage structure. In osteoarthritis (OA), the viscoelastic properties of cartilage are compromised on both the quantity and integrity of aggrecan core protein expressed as well as reduced overall CS chain length. Herein, we postulated that chronic low-level inflammation may also contribute to OA progression by promoting regulatory mechanisms in early CS biosynthesis that yield incomplete linkage structures on aggrecan. To test this idea, chondrocytes extracted from human tali were cultured in alginate beads and challenged with 5 ng/mL IL-1β as a model for chronic inflammation leading to OA progression. Novel mass spectrometry-based methods were devised to detect and quantify partially elongated linkage structures relative to control cultures. The total mole fraction of unelongated xylose residues per aggrecan was significantly less (p = 0.03) after IL-1β treatment compared to control cultures, with unelongated xylose residues constituting between 6% and 12% of the fraction of total CS measured. A portion (<1%) of the partially elongated linkage structures was found to be either phosphorylated or sulfated. These results establish quantitative mass spectrometry as a very sensitive and effective platform for evaluating truncated proteoglycan linkage structures. Our observations using this method suggest a possible role for aberrant linkage structure elongation in OA progression.

The aggregating proteoglycans of the lectican family are important components of extracellular matrices. Aggrecan is the most well studied of these and is central to cartilage biomechanical properties and skeletal development. Key to its biological function is the fixed charge of the many glycosaminoglycan chains, that provide the basis for the viscoelastic properties necessary for load distribution over the articular surface. This review is focused on the globular domains of aggrecan and their role in anchoring the proteoglycans to other extracellular matrix components. The N-terminal G1 domain is vital in that it binds the proteoglycan to hyaluronan in ternary complex with link protein, retaining the proteoglycan in the tissue. The importance of the C-terminal G3 domain interactions has recently been emphasized by two different human hereditary disorders: autosomal recessive aggrecan-type spondyloepimetaphyseal dysplasia and autosomal dominant familial osteochondritis dissecans. In these two conditions, different missense mutations in the aggrecan C-type lectin repeat have been described. The resulting amino acid replacements affect the ligand interactions of the G3 domain, albeit with widely different phenotypic outcomes.

Although it has been shown that aggrecanases are involved in aggrecan degradation, the role of MMP (matrix metalloproteinase) aggrecanolysis is less well studied. To investigate MMP proteolysis of human aggrecan, in the present study we used neoepitope antibodies against MMP cleavage sites and Western blot analysis to identify MMP-generated fragments in normal and OA (osteoarthritis/osteoarthritic) cartilage, and in normal, knee injury and OA and SF (synovial fluid) samples. MMP-3 in vitro digestion showed that aggrecan contains six MMP cleavage sites, in the IGD (interglobular domain), the KS (keratan sulfate) region, the border between the KS region and CS (chondroitin sulfate) region 1, the CS1 region, and the border between the CS2 and the G3 domain, and kinetic studies showed a specific order of digestion where the cleavage between CS2 and the G3 domain was the most preferred. In vivo studies showed that OA cartilage contained (per dry weight) 3.4-fold more MMP-generated FFGV fragments compared with normal cartilage, and although aggrecanase-generated SF-ARGS concentrations were increased 14-fold in OA and knee-injured patients compared with levels in knee-healthy reference subjects, the SF-FFGV concentrations did not notably change. The results of the present study suggest that MMPs are mainly involved in normal aggrecan turnover and might have a less-active role in aggrecan degradation during knee injury and OA.

Many cells express a membrane-coupled external mechanical layer, the pericellular matrix (PCM), which often contains long-chain polymers. Its role and properties are not entirely known, but its functions are believed to include physical protection, mechanosensing, chemical signalling or lubrication. The viscoelastic response of the PCM, with polysaccharides as the main structural components, is therefore crucial for the understanding of its function. We have here applied microrheology, based on optically trapped micrometre-sized colloids, to the PCM of cultured PC3 prostate cancer cells. This technology allowed us to measure the extremely soft response of the PCM, with approximately 1 µm height resolution. Exogenously added aggrecan, a hyaluronan-binding proteoglycan, caused a remarkable increase in thickness of the viscoelastic layer and also triggered filopodia-like protrusions. The viscoelastic response of the PCM, however, did not change significantly.