Cartilage Production Research Articles

The Role of Chondrocytes in Fracture Healing

Inflammation plays important roles in the early steps of fracture healing including, the recruitment, expansion, growth and differentiation of mesenchymal stem cells (MSCs), the coordinated interplay of many other cell types, growth factors, and extracellular matrix (ECM) components, as well as the production of cartilage and bone matrix in a temporally controlled manner.

Cartilage Resurfacing: Unresolved Enigma. Commentary on an article by L.R. Goodrich, DVM, PhD, et al.: "Addition of Mesenchymal Stem Cells to Autologous Platelet-Enhanced Fibrin Scaffolds in Chondral Defects. Does It Enhance Repair?".

Full-thickness cartilage defects present a substantial clinical challenge. People are remaining active longer, and orthopaedic surgeons need techniques that effectively produce repair tissue that can withstand the test of time. The challenge is to find a way to achieve filling of cartilage defects with sufficiently cartilage-like tissue that is able to withstand repetitive loads without degeneration and loss of function. To date, efforts have failed to achieve good long-term results, at least in part because of the inability to recapitulate the type-II-collagen and aggrecan content of normal hyaline cartilage. Recent efforts have focused on cellular therapies—using chondrocytes or mesenchymal stem cells—as the basis for regeneration of repair tissue and for gene therapy approaches to enhance healing. Chondrocytes are ideally suited for the production of hyaline cartilage but challenging to obtain without donor site morbidity or the potential for inciting an immune response to allogenic cells. Bone-marrow-derived mesenchymal stem cells (BMDMSCs) provide an appealing alternative cell source with the potential to differentiate into chondrocytes following implantation and ease of collection with acceptable morbidity. Platelet-rich plasma has also gained recent attention as a readily available source of anabolic growth factors and cytokines with the potential to drive chondrogenesis. The paper by Goodrich et al. describes the use of culture-expanded BMDMSCs combined with platelet-rich plasma in the form of an autologous platelet-enhanced fibrin (APEF) scaffold to enhance cartilage …

Chondrocytic ephrin B2 promotes cartilage destruction by osteoclasts in endochondral ossification.

The majority of the skeleton arises by endochondral ossification, whereby cartilaginous templates expand and are resorbed by osteoclasts then replaced by osteoblastic bone formation. Ephrin B2 is a receptor tyrosine kinase expressed by osteoblasts and growth plate chondrocytes that promotes osteoblast differentiation and inhibits osteoclast formation. We investigated the role of ephrin B2 in endochondral ossification using Osx1Cre-targeted gene deletion. Neonatal Osx1Cre.Efnb2(Δ/Δ) mice exhibited a transient osteopetrosis demonstrated by increased trabecular bone volume with a high content of growth plate cartilage remnants and increased cortical thickness, but normal osteoclast numbers within the primary spongiosa. Osteoclasts at the growth plate had an abnormal morphology and expressed low levels of tartrate-resistant acid phosphatase; this was not observed in more mature bone. Electron microscopy revealed a lack of sealing zones and poor attachment of Osx1Cre.Efnb2(Δ/Δ) osteoclasts to growth plate cartilage. Osteoblasts at the growth plate were also poorly attached and impaired in their ability to deposit osteoid. By 6 months of age, trabecular bone mass, osteoclast morphology and osteoid deposition by Osx1Cre.Efnb2(Δ/Δ) osteoblasts were normal. Cultured chondrocytes from Osx1Cre.Efnb2(Δ/Δ) neonates showed impaired support of osteoclastogenesis but no significant change in Rankl (Tnfsf11) levels, whereas Adamts4 levels were significantly reduced. A population of ADAMTS4(+) early hypertrophic chondrocytes seen in controls was absent from Osx1Cre.Efnb2(Δ/Δ) neonates. This suggests that Osx1Cre-expressing cells, including hypertrophic chondrocytes, are dependent on ephrin B2 for their production of cartilage-degrading enzymes, including ADAMTS4, and this might be required for attachment of osteoclasts and osteoblasts to the cartilage surface during endochondral ossification.

Lactoferrin from Camelus dromedarius inhibits nuclear transcription Factor-kappa B activation, cyclooxygenase-2 expression and prostaglandin E2 production in stimulated human chondrocytes

Background:Osteoarthritis (OA) is a progressive joint disorder, which remains the leading cause of chronic disability in aged people. Nuclear factor-kappa B (NF)-κB is a major cellular event in OA and its activation by interleukin-1β (IL-1β) plays a critical role in cartilage breakdown in these patients.Objective:In this study, we examined the effect of lactoferrin on NF-κB activation, cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production in stimulated human articular chondrocytes.Materials and Methods:Human chondrocytes were derived from OA articular cartilage and treated with camel lactoferrin and then stimulated with IL-1β. Gene expression was determined by TaqMan assays and protein expression was studied by Western immunoblotting. NF-κB activity and PGE2 levels were determined by ELISA based assays. NF-κB activity was also determined by treatment of chondrocytes with NF-κB specific inhibitor Bay 11–7082.Results:Lactoferrin inhibited IL-1β-induced activation and nuclear translocation of NF-κB p65 in human OA chondrocytes. Lactoferrin also inhibited mRNA/protein expression of COX-2 and production of PGE2. Moreover, Bay 11–7082 also inhibited IL-1β-induced expression of COX-2 and production of PGE2. The inhibitory effect of lactoferrin on the IL-1β induced expression of COX-2 or production of PGE2 was mediated at least in part via suppression of NF-κB activation.Conclusions:Our data determine camel lactoferrin as a novel inhibitor of IL-1β-induced activation of NF-κB signaling events and production of cartilage-degrading molecule PGE2 via inhibition of COX-2 expressions. These results may have important implications for the development of novel therapeutic strategies for the prevention/treatment of OA and other degenerative/inflammatory diseases.SUMMARY Lactoferrin shows anti-arthritic activity in IL-1β stimulated primary human chondrocytes.Lactoferrin inhibits IL-1β-induced NF-κB activation.Lactoferrin inhibits production of cartilage degrading PGE2 via inhibition of COX-2 expression. Abbreviations Used: OA: Osteoarthritis IL-1β: Interleukin-1 beta NF-κB: Nuclear factor-kappa B COX-2: cyclooxygenase-2 PGE2: prostaglandin E2

Biomechanical evaluation of intramedullary nail and bone plate for the fixation of distal metaphyseal fractures

Surgical treatment of distal metaphyseal fractures remains problematic, and whilst both intramedullary nailing and bone plate fixation are known as the acceptable methods for the internal fixation of this kind of fractures, neither technique demonstrated satisfactory clinical outcomes. In this research, a finite element based investigation was made to compare these two fixation techniques for the fixation of distal tibia fractures from the biomechanics point of view. For this purpose, a 3mm transverse fracture gap was created at the distal metaphyseal region of tibia and fixed by use of either a nail or a plate. The von Mises stress, interfragmentary movements, and the production of different tissue phenotypes at the fracture site were calculated. Results of this study showed that plating offers more advantageous biomechanical conditions at the fracture site, in which it provides sufficient amount of axial interfragmentary movement and considerable amount of cartilage production, while intramedullary nailing restricts axial movements but causes high magnitude of shear movements. However, nailing is superior to plating from the mechanical point of view and provides earlier weight bearing. In addition, it was shown that by using composite materials, biomechanical behavior of both fixation techniques will be improved through decreasing risk of failure and promoting cartilaginous tissue production.

Direct Cell-Cell Contact with Chondrocytes Is a Key Mechanism in Multipotent Mesenchymal Stromal Cell-Mediated Chondrogenesis.

Using a combination of articular chondrocytes (ACs) and mesenchymal stromal cells (MSCs) has shown to be a viable option for a single-stage cell-based treatment of focal cartilage defects. However, there is still considerable debate whether MSCs differentiate or have a chondroinductive role through trophic factors. In addition, it remains unclear whether direct cell-cell contact is necessary for chondrogenesis. Therefore, the aim of this study was to investigate whether direct or indirect cell-cell contact between ACs and MSCs is essential for increased cartilage production in different cellular environments and elucidate the mechanisms behind these cellular interactions. Human ACs and MSCs were cultured in a 10:90 ratio in alginate beads, fibrin scaffolds, and pellets. Cells were mixed in direct cocultures, separated by a Transwell filter (indirect cocultures), or cultured with conditioned medium. Short tandem repeat analysis revealed that the percentages of ACs increased during culture, while those of MSCs decreased, with the biggest change in fibrin glue scaffolds. For alginate, where the lack of cell-cell contact could be confirmed by histological analysis, no difference was found in matrix production between direct and indirect cocultures. For fibrin scaffolds and pellet cultures, an increased glycosaminoglycan production and type II collagen deposition were found in direct cocultures compared with indirect cocultures and conditioned medium. Positive connexin 43 staining and transfer of cytosolic calcein indicated communication through gap junctions in direct cocultures. Taken together, these results suggest that MSCs stimulate cartilage formation when placed in close proximity to chondrocytes and that direct cell-cell contact and communication through gap junctions are essential in this chondroinductive interplay.

Modeling and Validation of Multilayer Poly(Lactide-Co-Glycolide) Scaffolds for In Vitro Directed Differentiation of Juxtaposed Cartilage and Bone.

Polymeric scaffolds, which release growth factors in a temporally controlled manner, have successfully directed the differentiation of stem cells into monolithic tissues of a single lineage. However, engineering precise boundaries in multilineage functional tissues, such as the juxtaposed cartilaginous and osseous tissue present in articulated joints, often remains a challenge. This work demonstrates a precise materials system for in vitro reconstruction of the three-dimensional architecture of these types of human tissues. Multilayer poly(lactide-co-glycolide) (PLG) scaffolds were used to produce spatiotemporal gradients to direct the differentiation of an initially uniform population of mesenchymal stem cells (MSCs) into juxtaposed cartilage and bone. Specifically, growth factors (chondrogenic transforming growth factor-β3 and osteogenic bone morphogenetic protein-4) and their neutralizing antibodies were incorporated within distinct layers of the PLG scaffolds to create spatially segregated morphogen fields within the scaffold volume. The multilayer PLG scaffold designs were optimized by mathematical modeling, and generation of spatially segregated morphogen gradients was validated by assessing activity of luciferase reporter cell lines responsive to each growth factor. Scaffolds seeded with MSCs demonstrated production of juxtaposed cartilage and bone, as evaluated by biochemical staining and western blotting for tissue-specific matrix proteins. This work demonstrates a significant advance for the engineering of implantable constructs comprising tissues of multiple lineages, with potential applications in orthopedic regenerative medicine.

Transient Growth Factor Stimulation Improves Chondrogenesis in Static Culture and Under Dynamic Conditions in a Novel Shear and Perfusion Bioreactor

Tissue engineering emerges as a viable method to replace cartilage damaged by osteoarthritis or trauma. A key element for cartilage tissue engineering is to elucidate growth factor exposure regimes that enhance cartilage production from multipotent progenitor cells. To address this, we identify growth factor conditions that promote chondrogenesis of human mesenchymal stem cells cultured under static conditions. We identified transient exposure to bone morphogenic protein-2 in the presence of transforming growth factor beta-1 as a viable condition that promotes expression of chondrogenic phenotype for human mesenchymal stem cells seeded onto polycaprolactone scaffolds. We then use this growth factor regime as a proof of concept to test a new shear and perfusion bioreactor. This novel bioreactor design applies shear stress stimulation on the surface of constructs while allowing tissue perfusion. Constructs harvested from the bioreactor after 8 days showed cartilage development primarily near the construct surface exposed to shear stress. By day 14, cells, collagen deposition and proteoglycan deposition is observed throughout the scaffold, indicative of cartilage development.

Influence of the scaffold geometry on the spatial and temporal evolution of the mechanical properties of tissue-engineered cartilage: insights from a mathematical model.

The production of tissue-engineered cartilage in vitro with inhomogeneous mechanical properties is a problem yet to be solved. Different geometries have been studied to overcome this caveat; however, the reported measurements are limited to average values of some properties and qualitative measures of spatial distributions. We will apply a coupled model to extend knowledge about the introduction of a macrochannel in a scaffold by calculating spatiotemporal patterns for several interest variables related to the remodeling of the mechanical properties. Model parameters were estimated based on experimental data on the temporal patterns of glycosaminoglycans, collagen and compressive Young's modulus for channel-free constructs. The model reproduced the experimental data trends in both geometries, with experimental-numerical correlations between 0.84 and 0.97. The channel had a higher impact on the reduction in spatial heterogeneities and delay of saturation of core properties than in the improvement of average properties. Despite the possible improvement of cell densities for longer periods than 56 days, it is estimated that it will not cause further significant improvements of the mechanical properties. The degrees of spatial heterogeneity of the Young's modulus and permeability in the channeled geometry are 23 and 27% of the channel-free values. While the average Young's modulus values are in the range of native cartilage, the permeabilities are one to three degrees of magnitude higher than the native cartilage, suggesting that limiting factors such as scaffold porosity and initial permeability are more relevant than scaffold geometry to effectively decrease the tissue permeability.

TGF-β1, GDF-5, and BMP-2 stimulation induces chondrogenesis in expanded human articular chondrocytes and marrow-derived stromal cells.

Replacement of degenerated cartilage with cell-based cartilage products may offer a long-term solution to halt arthritis' degenerative progression. Chondrocytes are frequently used in cell-based FDA-approved cartilage products; yet human marrow-derived stromal cells (hMSCs) show significant translational potential, reducing donor site morbidity and maintaining their undifferentiated phenotype with expansion. This study sought to investigate the effects of transforming growth factor β1 (TGF-β1), growth/differentiation factor 5 (GDF-5), and bone morphogenetic protein 2 (BMP-2) during postexpansion chondrogenesis in human articular chondrocytes (hACs) and to compare chondrogenesis in passaged hACs with that of passaged hMSCs. Through serial expansion, chondrocytes dedifferentiated, decreasing expression of chondrogenic genes while increasing expression of fibroblastic genes. However, following expansion, 10 ng/mL TGF-β1, 100 ng/mL GDF-5, or 100 ng/mL BMP-2 supplementation during three-dimensional aggregate culture each upregulated one or more markers of chondrogenic gene expression in both hACs and hMSCs. Additionally, in both cell types, the combination of TGF-β1, GDF-5, and BMP-2 induced the greatest upregulation of chondrogenic genes, that is, Col2A1, Col2A1/Col1A1 ratio, SOX9, and ACAN, and synthesis of cartilage-specific matrix, that is, glycosaminoglycans (GAGs) and ratio of collagen II/I. Finally, TGF-β1, GDF-5, and BMP-2 stimulation yielded mechanically robust cartilage rich in collagen II and GAGs in both cell types, following 4 weeks maturation. This study illustrates notable success in using the self-assembling method to generate robust, scaffold-free neocartilage constructs using expanded hACs and hMSCs.

A4.2 IL4–10 synerkine for the treatment of osteoarthritis

Background and objectives Osteoarthritis (OA) is a degenerative joint disease, clinically characterised by joint pain and disability. Underlying structural changes are degeneration of articular cartilage, intra-articular inflammation with synovitis and changes in peri-articular and subchondral bone. The ideal drug to treat OA should have analgesic, anti-inflammatory and cartilage protective effects. To date, no such drug has been described. In human and animal in and ex-vivo models, the immunoregulatory cytokines interleukin 4 (IL-4) and interleukin 10 (IL-10), especially in combination therapy, can prevent joint degeneration effectively. To use the combined activities of both IL-4 and IL-10, we developed IL4–10 synerkine, preserving the functional activity of each cytokine. In several well-established animal models, we have shown the potential of IL4–10 synerkine to inhibit inflammatory pain in animal models. In this study, we evaluated the potential of the synerkine to reduce inflammation and to provide cartilage protection in an ex vivo model for human OA. Materials and methods Osteoarthritic cartilage (n = 8) and synovium (n = 6) was obtained at joint replacement surgery and was cultured for 72 h in conditioned medium, in the presence or absence of equimolar concentrations of IL-4 (10 ng/ml), IL-10 (10 ng/ml), the combination of IL-4 and IL-10 (10 ng/ml + 10 ng/ml) and IL4-10 synerkine (20 ng/ml). After culture, cartilage was selected to study changes in proteoglycan (PG) synthesis and release. Supernatants of both cartilage and synovium cultures were collected for measurement of a broad spectrum of inflammatory mediators. Results In both synovium and cartilage cultures, elevated levels of pro-inflammatory mediators like IL-6 (resp. 7.5 µg/mg and 531 µg/mg) and IL-8 (resp. 2.6 µg/mg and 142 µg/mg) were measured. These levels were strongly inhibited by the IL4-10 synerkine (about 80%; all p Conclusions IL4-10 synerkine directly affects proteoglycan turnover and cytokine production of OA cartilage, combined with reduced secretion of inflammatory cytokines and proteases by OA synovial tissue. The immunoregulatory and cartilage protective effects in combination with it’s analgesic effects, suggest that the synerkine is an effective disease modifying drug in the treatment of osteoarthritis.

Cartilage Regeneration in the Head and Neck Area: Combination of Ear or Nasal Chondrocytes and Mesenchymal Stem Cells Improves Cartilage Production.

Cartilage tissue engineering can offer promising solutions for restoring cartilage defects in the head and neck area and has the potential to overcome limitations of current treatments. However, to generate a construct of reasonable size, large numbers of chondrocytes are required, which limits its current applicability. Therefore, the authors evaluate the suitability of a combination of cells for cartilage regeneration: bone marrow-derived mesenchymal stem cells and ear or nasal chondrocytes. Human bone marrow-derived mesenchymal stem cells were encapsulated in alginate hydrogel as single-cell-type populations or in combination with bovine ear chondrocytes or nasal chondrocytes at an 80:20 ratio. Constructs were either cultured in vitro or implanted directly subcutaneously into mice. Cartilage formation was evaluated with biochemical and biomechanical analyses. The use of a xenogeneic coculture system enabled the analyses of the contribution of the individual cell types using species-specific gene-expression analyses. In vivo, human bone marrow-derived mesenchymal stem cells/bovine ear chondrocytes or human bone marrow-derived mesenchymal stem cells/bovine nasal chondrocytes contained amounts of cartilage components similar to those of constructs containing chondrocytes only (i.e., bovine ear and nasal chondrocytes). In vitro, species-specific gene-expression analyses demonstrated that aggrecan was expressed by the chondrocytes only, which suggests a more trophic role for human bone marrow-derived mesenchymal stem cells. Furthermore, the additional effect of human bone marrow-derived mesenchymal stem cells was more pronounced in combination with bovine nasal chondrocytes. By supplementing low numbers of bovine ear or nasal chondrocytes with human bone marrow-derived mesenchymal stem cells, the authors were able to engineer cartilage constructs with properties similar to those of constructs containing chondrocytes only. This makes the procedure more feasible for future applicability in the reconstruction of cartilage defects in the head and neck area because fewer chondrocytes are required.

Cartilage synthesis in hyaluronic acid-tyramine constructs.

The objective of this study was to determine the potential for cartilage production within a hyaluronic acid-tyramine (HA-Tyr) hydrogel scaffold. Chondrocytes were encapsulated within HA-Tyr hydrogels and subcutaneously implanted in mice. The HA-Tyr hydrogels were formed by the oxidative coupling of Tyr moieties catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). Harvested constructs were shown to achieve a glycosaminoglycan (GAG) content of 1.2 wt%, and they demonstrated 40% of the collagen content of normal articular cartilage, including the presence of Type II collagen, which is the characteristic of articular cartilage. Matrix production was found to be influenced by the initial cell density, scaffold degradation rate and Type II collagen concentration. Injectability was also imparted to the system by delivering HRP through thermoresponsive liposomes. The method of HRP delivery, either by simple addition or through thermoresponsive liposomes, was not shown to have an effect on matrix production.

A survey of self-medication practices and related factors in the general population: the Ohasama study

Encouraging self-medication is expected to reduce healthcare costs. To assess the current situation of self-medication practices in the general population, we conducted a questionnaire survey regarding the use of over-the-counter (OTC) medications or dietary supplements in 1008 participants (37% men; mean age, 64±13 years) from Ohasama, a rural Japanese community. A total of 519 (52%) participants used OTC medications or dietary supplements, with common cold medication (36%) and supplements (28%) such as shark cartilage products representing the most common choices. Stepwise logistic regression showed female gender, a higher frequency of visits from a household medicine kit distributor, dyslipidemia, and lower home systolic blood pressure levels as predictors for the use of such materials (chi-square values: 25.3, 12.6, 7.0, and 4.6, respectively; all p<0.03). Stratifying the participants according to the use of antihypertensive treatment showed a negative association between systolic blood pressure and the use of OTC medications or supplements only in participants being treated for hypertension. These results suggest that although the adoption rate of self-medication in Japan can be increased in rural areas, it may remain lower in urban areas. The present study clarifies the factors associated with the use of OTC medications or dietary supplements and indicates that appropriate self-medication practices might improve the control of hypertension, particularly in patients undergoing antihypertensive treatment.

Dynamic nanomechanics of individual bone marrow stromal cells and cell-matrix composites during chondrogenic differentiation

Dynamic nanomechanical properties of bovine bone marrow stromal cells (BMSCs) and their newly synthesized cartilage-like matrices were studied at nanometer scale deformation amplitudes. The increase in their dynamic modulus, |E⁎| (e.g., 2.4±0.4kPa at 1Hz to 9.7±0.2kPa at 316Hz at day 21, mean±SEM), and phase angle, δ, (e.g., 15±2° at 1Hz to 74±1° at 316Hz at day 21) with increasing frequency were attributed to the fluid flow induced poroelasticity, governed by both the newly synthesized matrix and the intracellular structures. The absence of culture duration dependence suggested that chondrogenesis of BMSCs had not yet resulted in the formation of a well-organized matrix with a hierarchical structure similar to cartilage. BMSC-matrix composites demonstrated different poro-viscoelastic frequency-dependent mechanical behavior and energy dissipation compared to chondrocyte-matrix composites due to differences in matrix molecular constituents, structure and cell properties. This study provides important insights into the design of optimal protocols for tissue-engineered cartilage products using chondrocytes and BMSCs.

Enhanced cell-induced articular cartilage regeneration by chondrons; the influence of joint damage and harvest site

Interactions between chondrocytes and their native pericellular matrix provide optimal circumstances for regeneration of cartilage. However, cartilage diseases such as osteoarthritis change the pericellular matrix, causing doubt to them as a cell source for autologous cell therapy. Chondrons and chondrocytes were isolated from stifle joints of goats in which cartilage damage was surgically induced in the right knee. After 4 weeks of regeneration culture, DNA content and proteoglycan and collagen content and release were determined. The cartilage regenerated by chondrons isolated from the damaged joint contained less proteoglycans and collagen compared to chondrons from the same harvest site in the nonoperated knee (P<0.01). Besides, chondrons still reflected whether they were isolated from a damaged joint, even if they where isolated from the opposing or adjacent condyle. Although chondrocytes did not reflect this diseased status of the joint, chondrons always outperformed chondrocytes, even when isolated from the damaged joints (P<0.0001). Besides increased cartilage production, the chondrons showed less collagenase activity compared to the chondrocytes. Chondrons still outperform chondrocytes when they were isolated from a damaged joint and they might be a superior cell source for articular cartilage repair and cell-induced cartilage formation.

Hypertonic conditions enhance cartilage formation in scaffold-free primary chondrocyte cultures.

The potential of hypertonic conditions at in vivo levels to promote cartilage extracellular matrix accumulation in scaffold-free primary chondrocyte cultures was investigated. Six million bovine primary chondrocytes were cultured in transwell inserts in low glucose (LG), high glucose (HG), or hypertonic high glucose (HHG) DMEM supplemented with fetal bovine serum, antibiotics, and ascorbate under 5 % or 20 % O2 tension with and without transforming growth factor (TGF)-β3 for 6 weeks. Samples were collected for histological staining of proteoglycans (PGs) and type II collagen, analysis by quantitative reverse transcription plus the polymerase chain reaction (RT-PCR) of mRNA expression of aggrecan and procollagen α1 (II) and of Sox9 and procollagen α2 (I), and quantitation of PGs and PG separation in agarose gels. Cartilage tissues produced at 20 % O2 tension were larger than those formed at 5 % O2 tension. Compared with LG, the tissues grew to larger sizes in HG or HHG medium. Histological staining showed the strongest PG and type II collagen staining in cartilage generated in HG or HHG medium at 20 % O2 tension. Quantitative RT-PCR results indicated significantly higher expression of procollagen α1 (II) mRNA in cartilage generated in HHG medium at 20 % O2 tension compared with that in the other samples. TGF-β3 supplements in the culture medium provided no advantage for cartilage formation. Thus, HHG medium used at 20 % O2 tension is the most beneficial combination of the tested culture conditions for scaffold-free cartilage production in vitro and should improve cell culture for research into cartilage repair or tissue engineering.

Genomic Analysis and Differential Expression of HMG and S100A Family in Human Arthritis: Upregulated Expression of Chemokines, IL-8 and Nitric Oxide by HMGB1

We applied global gene expression arrays, quantitative real-time PCR, immunostaining, and functional assays to untangle the role of High Mobility Groups proteins (HMGs) in human osteoarthritis (OA)-affected cartilage. Bioinformatics analysis showed increased mRNA expression of Damage-Associated Molecular Patterns (DAMPs): HMGA, HMGB, HMGN, SRY, LEF1, HMGB1, MMPs, and HMG/RAGE-interacting molecules (spondins and S100A4, S100A10, and S100A11) in human OA-affected cartilage as compared with normal cartilage. HMGB2 was down-regulated in human OA-affected cartilage. Immunohistological staining identified HMGB1 in chondrocytes in the superficial cartilage. Cells of the deep cartilage and subchondral bone showed increased expression of HMGB1 in OA-affected cartilage. HMGB1 was expressed in the nucleus, cytosol, and extracellular milieu of chondrocytes in cartilage. Furthermore, HMGB1 was spontaneously released from human OA-affected cartilage in ex vivo conditions. The effects of recombinant HMGB1 was tested on human cartilage and chondrocytes in vitro. HMGB1 stimulated mRNA of 2 NFκB gene enhancers (NFκB1 and NFκB2), 16 CC and CXC chemokines (IL-8, CCL2, CCL20, CCL3, CCL3L1, CCL3L3, CCL4, CCL4L1, CCL4L2, CCL5, CCL8, CXCL1, CXCL10, CXCL2, CXCL3, and CXCL6) by ≥10-fold. Furthermore, HMGB1 and IL-1β and/or tumor necrosis factor α (but not HMGI/Y) also significantly induced inducible nitric oxide synthase, NO, and interleukin (IL)-8 production in human cartilage and chondrocytes. The recombinant HMGB1 utilized in this study shows properties that are similar to disulfide-HMGB1. The differential, stage and/or tissue-specific expression of HMGB1, HMGB2, and S100A in cartilage was associated with regions of pathology and/or cartilage homeostasis in human OA-affected cartilage. Noteworthy similarities in the expression of mouse and human HMGB1 and HMGB2 were conserved in normal and arthritis-affected cartilage. The multifunctional forms of HMGB1 and S100A could perpetuate damage-induced cartilage inflammation in late-stage OA-affected joints similar to sterile inflammation. The paracrine effects of HMGB1 can induce chemokines and NO that are perceived to change cartilage homeostasis in human OA-affected cartilage.

New strategies for cartilage regeneration exploiting selected glycosaminoglycans to enhance cell fate determination.

Most research strategies for cartilage tissue engineering use extended culture with complex media loaded with costly GFs (growth factors) to drive tissue assembly and yet they result in the production of cartilage with inferior mechanical and structural properties compared with the natural tissue. Recent evidence suggests that GAGs (glycosaminoglycans) incorporated into tissue engineering scaffolds can sequester and/or activate GFs and thereby more effectively mimic the natural ECM (extracellular matrix). Such approaches may have potential for the improvement of cartilage engineering. However, natural GAGs are structurally complex and heterogeneous, making structure-function relationships hard to determine and clinical translation difficult. Importantly, subfractions of GAGs with specific chain lengths and sulfation patterns have been shown to activate key signalling processes during stem cell differentiation. In addition, recently, GAGs have been bound to synthetic biomaterials, such as electrospun scaffolds and hydrogels, in biologically active conformations, and methods to purify and select affinity-matched GAGs for specific GFs have also been developed. The identification and use of specific GAG moieties to promote chondrogenesis is therefore an exciting new avenue of research. Combining these with synthetic biomaterials may allow a more effective mimicry of the natural ECM, reduction in the need for expensive GFs, and perhaps the deposition of an articular cartilage-like matrix in a clinically relevant manner.

Environmental neurotoxins β-N-methylamino-l-alanine (BMAA) and mercury in shark cartilage dietary supplements

Shark cartilage products are marketed as dietary supplements with claimed health benefits for animal and human use. Shark fin and cartilage products sold as extracts, dry powders and in capsules are marketed based on traditional Chinese medicine claims that it nourishes the blood, enhances appetite, and energizes multiple internal organs. Shark cartilage contains a mixture of chondroitin and glucosamine, a popular nutritional supplement ingested to improve cartilage function. Sharks are long-lived apex predators, that bioaccumulate environmental marine toxins and methylmercury from dietary exposures. We recently reported detection of the cyanobacterial toxin β-N-methylamino-l-alanine (BMAA) in the fins of seven different species of sharks from South Florida coastal waters. Since BMAA has been linked to degenerative brain diseases, the consumption of shark products may pose a human risk for BMAA exposures. In this report, we tested sixteen commercial shark cartilage supplements for BMAA by high performance liquid chromatography (HPLC-FD) with fluorescence detection and ultra performance liquid chromatography/mass spectrometry/mass spectrometry (UPLC–MS/MS). Total mercury (Hg) levels were measured in the same shark cartilage products by cold vapor atomic fluorescence spectrometry (CVAFS). We report here that BMAA was detected in fifteen out of sixteen products with concentrations ranging from 86 to 265μg/g (dry weight). All of the shark fin products contained low concentrations of Hg. While Hg contamination is a known risk, the results of the present study demonstrate that shark cartilage products also may contain the neurotoxin BMAA. Although the neurotoxic potential of dietary exposure to BMAA is currently unknown, the results demonstrate that shark cartilage products may contain two environmental neurotoxins that have synergistic toxicities.