Medical 3D printing with polyjet technology: effect of material type and printing orientation on printability, surface structure and cytotoxicity.

Due to its high printing resolution and ability to print multiple materials simultaneously, inkjet technology has found wide application in medicine. However, the biological safety of 3D-printed objects is not always guaranteed due to residues of uncured resins or support materials and must therefore be verified. The aim of this study was to evaluate the quality of standard assessment methods for determining the quality and properties of polyjet-printed scaffolds in terms of their dimensional accuracy, surface topography, and cytotoxic potential.Standardized 3D-printed samples were produced in two printing orientations (horizontal or vertical). Printing accuracy and surface roughness was assessed by size measurements, VR-5200 3D optical profilometer dimensional analysis, and scanning electron microscopy. Cytotoxicity tests were performed with a representative cell line (L929) in a comparative laboratory study. Individual experiments were performed with primary cells from clinically relevant tissues and with a Toxdent cytotoxicity assay.Dimensional measurements of printed discs indicated high print accuracy and reproducibility. Print accuracy was highest when specimens were printed in horizontal direction. In all cytotoxicity tests, the estimated mean cell viability was well above 70% (p < 0.0001) regardless of material and printing direction, confirming the low cytotoxicity of the final 3D-printed objects.

Metabolic rewiring controlled by c-Fos governs cartilage integrity in osteoarthritis.

OBJECTIVES: The activator protein-1 (AP-1) transcription factor component c-Fos regulates chondrocyte proliferation and differentiation, but its involvement in osteoarthritis (OA) has not been functionally assessed. METHODS: c-Fos expression was evaluated by immunohistochemistry on articular cartilage sections from patients with OA and mice subjected to the destabilisation of the medial meniscus (DMM) model of OA. Cartilage-specific c-Fos knockout (c-FosΔCh) mice were generated by crossing c-fosfl/fl to Col2a1-CreERT mice. Articular cartilage was evaluated by histology, immunohistochemistry, RNA sequencing (RNA-seq), quantitative reverse transcription PCR (qRT-PCR) and in situ metabolic enzyme assays. The effect of dichloroacetic acid (DCA), an inhibitor of pyruvate dehydrogenase kinase (Pdk), was assessed in c-FosΔCh mice subjected to DMM. RESULTS: FOS-positive chondrocytes were increased in human and murine OA cartilage during disease progression. Compared with c-FosWT mice, c-FosΔCh mice exhibited exacerbated DMM-induced cartilage destruction. Chondrocytes lacking c-Fos proliferate less, have shorter collagen fibres and reduced cartilage matrix. Comparative RNA-seq revealed a prominent anaerobic glycolysis gene expression signature. Consistently decreased pyruvate dehydrogenase (Pdh) and elevated lactate dehydrogenase (Ldh) enzymatic activities were measured in situ, which are likely due to higher expression of hypoxia-inducible factor-1α, Ldha, and Pdk1 in chondrocytes. In vivo treatment of c-FosΔCh mice with DCA restored Pdh/Ldh activity, chondrocyte proliferation, collagen biosynthesis and decreased cartilage damage after DMM, thereby reverting the deleterious effects of c-Fos inactivation. CONCLUSIONS: c-Fos modulates cellular bioenergetics in chondrocytes by balancing pyruvate flux between anaerobic glycolysis and the tricarboxylic acid cycle in response to OA signals. We identify a novel metabolic adaptation of chondrocytes controlled by c-Fos-containing AP-1 dimers that could be therapeutically relevant.

Activation of the Mechanosensitive Ion Channels Piezo1 and TRPV4 in Primary Human Healthy and Osteoarthritic Chondrocytes Exhibits Ion Channel Crosstalk and Modulates Gene Expression.

Osteoarthritis (OA) is the most common degenerative joint disease causing pain and functional limitations. Physical activity as a clinically relevant, effective intervention alleviates pain and promotes joint function. In chondrocytes, perception and transmission of mechanical signals are controlled by mechanosensitive ion channels, whose dysfunction in OA chondrocytes is leading to disease progression. Signaling of mechanosensitive ion channels Piezo/TRPV4 was analyzed by Yoda1/GSK1016790A application and calcium-imaging of Fura-2-loaded chondrocytes. Expression analysis was determined by qPCR and immunofluorescence in healthy vs. OA chondrocytes. Chondrocytes were mechanically stimulated using the Flexcell™ technique. Yoda1 and GSK1016790A caused an increase in intracellular calcium [Ca2+]i for Yoda1, depending on extracellularly available Ca2+. When used concomitantly, the agonist applied first inhibited the effect of subsequent agonist application, indicating mutual interference between Piezo/TRPV4. Yoda1 increased the expression of metalloproteinases, bone-morphogenic protein, and interleukins in healthy and OA chondrocytes to a different extent. Flexcell™-induced changes in the expression of MMPs and ILs differed from changes induced by Yoda1. We conclude that Piezo1/TRPV4 communicate with each other, an interference that may be impaired in OA chondrocytes. It is important to consider that mechanical stimulation may have different effects on OA depending on its intensity.

Targeting osteoarthritis-associated galectins and an induced effector class by a ditopic bifunctional reagent: Impact of its glycan part on binding measured in the tissue context.

Pairing glycans with tissue lectins controls multiple effector pathways in (patho)physiology. A clinically relevant example is the prodegradative activity of galectins-1 and -3 (Gal-1 and -3) in the progression of osteoarthritis (OA) via matrix metalloproteinases (MMPs), especially MMP-13. The design of heterobifunctional inhibitors that can block galectin binding and MMPs both directly and by preventing their galectin-dependent induction selectively offers a perspective to dissect the roles of lectins and proteolytic enzymes. We describe the synthesis of such a reagent with a bivalent galectin ligand connected to an MMP inhibitor and of two tetravalent glycoclusters with a subtle change in headgroup presentation for further elucidation of influence on ligand binding. Testing was performed on clinical material with mixtures of galectins as occurring in vivo, using sections of fixed tissue. Two-colour fluorescence microscopy monitored binding to the cellular glycome after optimization of experimental parameters. In the presence of the inhibitor, galectin binding to OA specimens was significantly reduced. These results open the perspective to examine the inhibitory capacity of custom-made ditopic compounds on binding of lectins in mixtures using sections of clinical material with known impact of galectins and MMPs on disease progression.

A Progress Report and Roadmap for Microphysiological Systems and Organ-On-A-Chip Technologies to Be More Predictive Models in Human (Knee) Osteoarthritis.

Osteoarthritis (OA), a chronic debilitating joint disease affecting hundreds of million people globally, is associated with significant pain and socioeconomic costs. Current treatment modalities are palliative and unable to stop the progressive degeneration of articular cartilage in OA. Scientific attention has shifted from the historical view of OA as a wear-and-tear cartilage disorder to its recognition as a whole-joint disease, highlighting the contribution of other knee joint tissues in OA pathogenesis. Despite much progress in the field of microfluidic systems/organs-on-a-chip in other research fields, current in vitro models in use do not yet accurately reflect the complexity of the OA pathophenotype. In this review, we provide: 1) a detailed overview of the most significant recent developments in the field of microsystems approaches for OA modeling, and 2) an OA-pathophysiology-based bioengineering roadmap for the requirements of the next generation of more predictive and authentic microscale systems fit for the purpose of not only disease modeling but also of drug screening to potentially allow OA animal model reduction and replacement in the near future.

Hydrolyzed Flavonoids from Cyrtosperma johnstonii with Superior Antioxidant, Antiproliferative, and Anti-Inflammatory Potential for Cancer Prevention.

Cyrtosperma johnstonii is one of the most interesting traditional medicines for cancer treatment. This study aimed to compare and combine the biological activities related to cancer prevention of the flavonoid glycosides rutin (RT) and isorhamnetin-3-o-rutinoside (IRR) and their hydrolysis products quercetin (QT) and isorhamnetin (IR) from C.johnstonii extract. ABTS and MTT assays were used to determine antioxidant activity and cytotoxicity against various cancer cells, as well as normal cells. Anti-inflammatory activities were measured by ELISA. The results showed that the antioxidant activities of the compounds decreased in the order of QT > IR > RT > IRR, while most leukemia cell lines were sensitive to QT and IR with low toxicity towards PBMCs. The reduction of IL-6 and IL-10 secretion by QT and IR was higher than that induced by RT and IRR. The combination of hydrolysis products (QT and IR) possessed a strong synergism in antioxidant, antiproliferative and anti-inflammatory effects, whereas the combination of flavonoid glycosides and their hydrolysis products revealed antagonism. These results suggest that the potential of the combination of hydrolyzed flavonoids from C. johnstonii can be considered as natural compounds for the prevention of cancer.

Recent Advances in Additive Manufacturing and 3D Bioprinting for Organs-On-A-Chip and Microphysiological Systems.

The re-creation of physiological cellular microenvironments that truly resemble complex in vivo architectures is the key aspect in the development of advanced in vitro organotypic tissue constructs. Among others, organ-on-a-chip technology has been increasingly used in recent years to create improved models for organs and tissues in human health and disease, because of its ability to provide spatio-temporal control over soluble cues, biophysical signals and biomechanical forces necessary to maintain proper organotypic functions. While media supply and waste removal are controlled by microfluidic channel by a network the formation of tissue-like architectures in designated micro-structured hydrogel compartments is commonly achieved by cellular self-assembly and intrinsic biological reorganization mechanisms. The recent combination of organ-on-a-chip technology with three-dimensional (3D) bioprinting and additive manufacturing techniques allows for an unprecedented control over tissue structures with the ability to also generate anisotropic constructs as often seen in in vivo tissue architectures. This review highlights progress made in bioprinting applications for organ-on-a-chip technology, and discusses synergies and limitations between organ-on-a-chip technology and 3D bioprinting in the creation of next generation biomimetic in vitro tissue models.

What is the Sugar Code?

A code is defined by the nature of the symbols, which are used to generate information-storing combinations (e. g. oligo- and polymers). Like nucleic acids and proteins, oligo- and polysaccharides are ubiquitous, and they are a biochemical platform for establishing molecular messages. Of note, the letters of the sugar code system (third alphabet of life) excel in coding capacity by making an unsurpassed versatility for isomer (code word) formation possible by variability in anomery and linkage position of the glycosidic bond, ring size and branching. The enzymatic machinery for glycan biosynthesis (writers) realizes this enormous potential for building a large vocabulary. It includes possibilities for dynamic editing/erasing as known from nucleic acids and proteins. Matching the glycome diversity, a large panel of sugar receptors (lectins) has developed based on more than a dozen folds. Lectins 'read' the glycan-encoded information. Hydrogen/coordination bonding and ionic pairing together with stacking and C-H/π-interactions as well as modes of spatial glycan presentation underlie the selectivity and specificity of glycan-lectin recognition. Modular design of lectins together with glycan display and the nature of the cognate glycoconjugate account for the large number of post-binding events. They give an entry to the glycan vocabulary its functional, often context-dependent meaning(s), hereby building the dictionary of the sugar code.

Galectin network in osteoarthritis: galectin-4 programs a pathogenic signature of gene and effector expression in human chondrocytes in vitro.

Galectin-4 (Gal-4) is a member of the galectin family, which have been identified as galactose-binding proteins. Gal-4 possesses two tandem repeat carbohydrate recognition domains and acts as a cross-linking bridge in sulfatide-dependent glycoprotein routing. We herein document its upregulation in osteoarthritis (OA) in correlation with the extent of cartilage degradation in vivo. Primary human OA chondrocytes in vitro respond to carbohydrate-inhibitable Gal-4 binding with the upregulation of pro-degradative/-inflammatory proteins such as interleukin-1ß (IL-1ß) and matrix metalloproteinase-13 (MMP-13), as documented by RT-qPCR-based mRNA profiling and transcriptome data processing. Activation of p65 by phosphorylation of Ser536 within the NF-κB pathway and the effect of three p65 inhibitors on Gal-4 activity support downstream involvement of such signaling. In 3D (pellet) cultures, Gal-4 presence causes morphological and biochemical signs of degradation. Taken together, our findings strongly support the concept of galectins acting as a network in OA pathogenesis and suggest that blocking their activity in disease progression may become clinically relevant in the future.

Establishment of a human three-dimensional chip-based chondro-synovial coculture joint model for reciprocal cross talk studies in arthritis research.

Rheumatoid arthritis is characterised by a progressive, intermittent inflammation at the synovial membrane, which ultimately leads to the destruction of the synovial joint. The synovial membrane as the joint capsule's inner layer is lined with fibroblast-like synoviocytes that are the key player supporting persistent arthritis leading to bone erosion and cartilage destruction. While microfluidic models that model molecular aspects of bone erosion between bone-derived cells and synoviocytes have been established, RA's synovial-chondral axis has not yet been realised using a microfluidic 3D model based on human patient in vitro cultures. Consequently, we established a chip-based three-dimensional tissue coculture model that simulates the reciprocal cross talk between individual synovial and chondral organoids. When co-cultivated with synovial organoids, we could demonstrate that chondral organoids induce a higher degree of cartilage physiology and architecture and show differential cytokine response compared to their respective monocultures highlighting the importance of reciprocal tissue-level cross talk in the modelling of arthritic diseases.

Characterization of the Inducible and Slow-Releasing Hydrogen Sulfide and Persulfide Donor P*: Insights into Hydrogen Sulfide Signaling.

Hydrogen sulfide (H2S) is an important mediator of inflammatory processes. However, controversial findings also exist, and its underlying molecular mechanisms are largely unknown. Recently, the byproducts of H2S, per-/polysulfides, emerged as biological mediators themselves, highlighting the complex chemistry of H2S. In this study, we characterized the biological effects of P*, a slow-releasing H2S and persulfide donor. To differentiate between H2S and polysulfide-derived effects, we decomposed P* into polysulfides. P* was further compared to the commonly used fast-releasing H2S donor sodium hydrogen sulfide (NaHS). The effects on oxidative stress and interleukin-6 (IL-6) expression were assessed in ATDC5 cells using superoxide measurement, qPCR, ELISA, and Western blotting. The findings on IL-6 expression were corroborated in primary chondrocytes from osteoarthritis patients. In ATDC5 cells, P* not only induced the expression of the antioxidant enzyme heme oxygenase-1 via per-/polysulfides, but also induced activation of Akt and p38 MAPK. NaHS and P* significantly impaired menadione-induced superoxide production. P* reduced IL-6 levels in both ATDC5 cells and primary chondrocytes dependent on H2S release. Taken together, P* provides a valuable research tool for the investigation of H2S and per-/polysulfide signaling. These data demonstrate the importance of not only H2S, but also per-/polysulfides as bioactive signaling molecules with potent anti-inflammatory and, in particular, antioxidant properties.

N-Glycan profiling of chondrocytes and fibroblast-like synoviocytes: Towards functional glycomics in osteoarthritis.

PURPOSE: N-Glycan profiling provides an indicator of the cellular potential for functional pairing with tissue lectins. Following the discovery of galectin expression by chondrocytes as a factor in osteoarthritis pathobiology, mapping of N-glycans upon their phenotypic dedifferentiation in culture and in fibroblast-like synoviocytes is a step to better understand glycobiological contributions to disease progression. EXPERIMENTAL DESIGN: The profiles of cellular N-glycans of human osteoarthritic chondrocytes and fibroblast-like synoviocytes were characterized by mass spectrometry. RT-qPCR experiments determined mRNA levels of 16 glycosyltransferases. Responsiveness of cells to galectins was quantified by measuring the mRNA level for interleukin-1ß. RESULTS: The shift of chondrocytes to a fibroblastic phenotype (dedifferentiation) is associated with changes in N-glycosylation. The N-glycan profile of chondrocytes at passage 4 reflects characteristics of synoviocytes. Galectins-1 and -3 enhance expression of interleukin-1ß mRNA in both cell types, most pronounced in primary culture. Presence of interleukin-1ß leads to changes in sialylation in synoviocytes that favor galectin binding. CONCLUSIONS AND CLINICAL RELEVANCE: N-Glycosylation reflects phenotypic changes of osteoarthritic cells in vitro. Like chondrocytes, fibroblast-like synoviocytes express N-glycans that are suited to bind galectins, and these proteins serve as inducers of pro-inflammatory markers in these cells. Synoviocytes can thus contribute to disease progression in osteoarthritis in situ.

A 3-Dimensional In Vitro Model of Zonally Organized Extracellular Matrix.

OBJECTIVE: Functional cartilage repair requires the new formation of organized hyaline cartilaginous matrix to avoid the generation of fibrous repair tissue. The potential of mesenchymal progenitors was used to assemble a 3-dimensional structure in vitro, reflecting the zonation of collagen matrix in hyaline articular cartilage. DESIGN: The 3-dimensional architecture of collagen alignment in pellet cultures of chondroprogenitors (CPs) was assessed with Picrosirius red staining analyzed under polarized light. In parallel assays, the trilineage capability was confirmed by calcium deposition during osteogenesis by alizarin S staining and alkaline phosphatase staining. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), mRNA levels of ALP, RUNX2, and BGLAP were assessed after 21 days of osteoinduction. Lipid droplets were stained with oil red O and adipogenic differentiation was confirmed by RT-qPCR analysis of PPARG and LPL gene expression. RESULTS: Under conditions promoting the chondrogenic signature in self-assembling constructs, CPs formed an aligned extracellular matrix, positive for glycosaminoglycans and collagen type II, showing developing zonation of birefringent collagen fibers along the cross section of pellets, which reflect the distribution of collagen fibers in hyaline cartilage. Induced osteogenic and adipogenic differentiation confirmed the trilineage potential of CPs. CONCLUSION: This model promotes the differentiation and self-organization of postnatal chondroprogenitors, resulting in the formation of zonally organized engineered hyaline cartilage comparable to the 3 zones of native cartilage.

Long-term impact of sagittal malalignment on hardware after posterior fixation of the thoracolumbar spine: a retrospective study.

BACKGROUND: The importance of sagittal alignment in healthy individuals and in reconstructive spinal surgery has been studied over the last 15 years. The aim of the present study was to assess the long-term effects of abnormal sagittal alignment on hardware after posterior thoracolumbar spinal fusion. METHODS: Patients who had undergone revision surgery (revision cohort, n = 34) due to breakage of their implants were compared retrospectively with patients who had intact implants at the final follow-up investigation after a long posterior thoracolumbar and/or lumbar spinal fusion (control cohort, n = 22). Clinical data and radiological parameters including the sagittal vertical axis (SVA), pelvic incidence (PI), lordosis gap (LG), pelvic tilt (PT), sacral slope (SS), lumbar lordosis (LL), thoracic kyphosis (TK), and the femoral obliquity angle (FOA) were assessed on full-spine lateral radiographs obtained in regular standing position. Data were analysed using descriptive statistics, parametric and non-parametric inferential statistics. RESULTS: Patients in the breakage group (female n = 21, male n = 9, mean age 60.9 ± 15.6 years) had a higher anterior shift of the C7 plumb line (SVA) (p = 0.02), retroversion of the pelvis (PT) (p < 0.001), PI-LL mismatch (LG) (p = 0.001), and PI (p = 0.002) than the intact group (female n = 10, male n = 12, mean age 65.7 ± 12.4 years). No significant difference was registered between groups in regard of SS, LL, TK, FOA, and the mean number of comorbidities. CONCLUSION: Failure of restoration of the SVA and the LG to the acceptable ranges, especially in patients with a high PI, may be regarded as a risk factor for the long-term failure of implants after posterior thoracolumbar spinal fusion.

Biological Regeneration of Articular Cartilage in an Early Stage of Compartmentalized Osteoarthritis: 12-Month Results.

BACKGROUND: Biological regeneration in an early stage of osteoarthritis (OA) is an important clinical challenge. An early-stage compartmentalized OA model was used to evaluate different biological regeneration techniques. HYPOTHESIS: Biological regeneration in an early stage of compartmentalized OA is possible. STUDY DESIGN: Controlled laboratory study. METHODS: A 7-mm cartilage defect was surgically created in 24 sheep. After 3 months, by which time early OA had set in, the sheep were randomized into 4 different treatment groups and operated for the second time. One group (CONTROL) served as a long-term follow-up group for the further development of OA. The other 3 groups (regeneration groups) each underwent a different regeneration procedure after abrasion of the subchondral bone (defect size: 20 × 10 mm with a depth of 2.5 mm): spongialization alone (SPONGIO), spongialization followed by implantation of an unseeded hyaluronan matrix (MATRIX), or spongialization followed by implantation of a hyaluronan matrix seeded with autologous chondrocytes (MACT). Then, 12 months after the second operative procedure, the animals were euthanized and the defects subjected to macroscopic and histological grading. Historical 4-month data were compared with the 12-month results. RESULTS: After 12 months of follow-up, advanced cartilage degeneration was observed in the CONTROL group. On the other hand, all regeneration groups improved significantly compared with the 4-month results using the Mankin score. Cartilage quality in the MACT group was significantly better than in the MATRIX group, as determined by the Mankin and the O'Driscoll scores. CONCLUSION: There are no existing clinical options for preventing early OA from progressing to a severe disease. This study provides important information on how a surgical intervention can forestall the development of OA. CLINICAL RELEVANCE: OA of the knee is very common. Total joint replacement is not an acceptable option for active patients. Biological regeneration in OA is successful for focal cartilage defects; however, a long-term follow-up for biological regeneration in OA is missing. It is essential to have long-term results for a regenerative procedure involving cartilage, which is a tissue with a very slow turnover.

Galectins-1 and -3 in Human Intervertebral Disc Degeneration: Non-Uniform Distribution Profiles and Activation of Disease Markers Involving NF-κB by Galectin-1.

Degeneration of the human intervertebral disc (IVD) is assumed to underlie severe clinical symptoms, in particular chronic back pain. Since adhesion/growth-regulatory galectins are linked to arthritis/osteoarthritis pathogenesis by activating a pro-degradative/-inflammatory gene expression signature, we hypothesized a similar functional involvement of galectins in IVD degeneration. Immunohistochemical evidence for the presence of galectins-1 and -3 in IVD is provided comparatively for specimens of spondylochondrosis, spondylolisthesis, and spinal deformity. Immunopositivity was detected in sections of fixed IVD specimens in each cellular compartment with age-, disease-, and galectin-type-related differences. Of note, presence of both galectins correlated with IVD degeneration, whereas correlation with age was seen only for galectin-3. In addition, staining profiles for these two galectins showed different distribution patterns in serial sections, an indication for non-redundant functionalities. In vitro, both galectins bound to IVD cells in a glycan-dependent manner. However, exclusively galectin-1 binding triggered a significant induction of functional disease markers (i.e., IL6, CXCL8, and MMP1/3/13) with involvement of the nuclear factor-kB pathway. This study thus gives direction to further network analyses and functional studies on galectins in IVD degeneration. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2204-2216, 2019.

Galectin-8 induces functional disease markers in human osteoarthritis and cooperates with galectins-1 and -3.

The reading of glycan-encoded signals by tissue lectins is considered a major route of the flow of biological information in many (patho)physiological processes. The arising challenge for current research is to proceed from work on a distinct protein to family-wide testing of lectin function. Having previously identified homodimeric galectin-1 and chimera-type galectin-3 as molecular switches in osteoarthritis progression, we here provide proof-of-principle evidence for an intra-network cooperation of galectins with three types of modular architecture. We show that the presence of tandem-repeat-type galectin-8 significantly correlated with cartilage degeneration and that it is secreted by osteoarthritic chondrocytes. Glycan-inhibitable surface binding of galectin-8 to these cells increased gene transcription and the secretion of functional disease markers. The natural variant galectin-8 (F19Y) was less active than the prevalent form. Genome-wide array analysis revealed induction of a pro-degradative/inflammatory gene signature, largely under control of NF-κB signaling. This signature overlapped with respective gene-expression patterns elicited by galectins-1 and -3, but also presented supplementary features. Functional assays with mixtures of galectins that mimic the pathophysiological status unveiled cooperation between the three galectins. Our findings shape the novel concept to consider individual galectins as part of a so far not realized teamwork in osteoarthritis pathogenesis, with relevance beyond this disease.

Brazilin blocks catabolic processes in human osteoarthritic chondrocytes via inhibition of NFKB1/p50.

This study aimed to evaluate the chondroprotective and anti-inflammatory activity of brazilin in human osteoarthritic (OA) cartilage and chondrocytes with particular focus on the nuclear factor-kappa B (NF-κB) pathway. Therefore, brazilin was isolated from Caesalpinia sappan and identified using high performance liquid chromatography (HPLC). The effect of brazilin was assessed in cartilage explants treated with 10 ng/ml interleukin (IL)-1ß and 10 ng/ml tumor necrosis factor (TNF)-α using histological and biochemical glycosaminoglycan (GAG) analyses and in primary chondrocytes treated with 10 ng/ml IL-1ß using RT-qPCR, ELISA, and Western blot. The involvement of NF-κB signaling was examined using a human NF-κB signaling array and in silico pathway analysis. Brazilin was found to reduce the GAG loss from cartilage explants stimulated with IL-1ß and TNF-α. NF-κB pathway analysis in chondrocytes revealed NFKB1/p50 as a central player regulating the anti-inflammatory activities of brazilin. Brazilin suppressed the IL-1ß-mediated up-regulation of OA markers and the induction of NFKB1/p50 in chondrocytes. In conclusion, brazilin effectively attenuates catabolic processes in human OA cartilage and chondrocytes-at least in part due to the inhibition of NFKB1/p50-which indicates a chondroprotective potential of brazilin in OA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2431-2438, 2018.

Galectins: their network and roles in immunity/tumor growth control.

One route of realizing the information of glycans involves endogenous receptors (lectins). Occurrence at branch ends renders galactosides particularly accessible. Thus, they are suited for such a recognition process. Fittingly, these epitopes serve as physiological ligands. The ga(lactoside-binding) lectins share the ß-sandwich fold with a sequence signature around a central tryptophan residue besides this specificity. Three modes of presentation of the carbohydrate recognition domain are known for galectins, and genome monitoring from fungi to mammals discloses that galectins form a network. The extent of its complexity varies considerably between organisms, for chicken reaching seven proteins, more for mammals. The current status of network analysis reveals overlapping and distinct expression profiles. Matching intra- and extracellular galectin presence, they have a broad range of functions at each site depending on their specific counterreceptor(s), with the possibility even for functional antagonism between family members. Orchestration of expression of galectin, the cognate glycan, its scaffold (protein or sphingolipid) and spatial aspects of glycoconjugate presentation has been detected to lead to growth regulation of immune and tumor cells. To delineate the factors that underlie the specificity of a galectin for its counterreceptor(s) in the cellular context and the details of structure-activity relationships by comparatively analyzing natural and rationally engineered proteins is the main challenge for ongoing research.

Galectin-3 Induces a Pro-degradative/inflammatory Gene Signature in Human Chondrocytes, Teaming Up with Galectin-1 in Osteoarthritis Pathogenesis.

Inflammatory chemo- and cytokines and matrix-degrading proteases underlie the progression of osteoarthritis (OA). Aiming to define upstream regulators for these disease markers, we pursued initial evidence for an upregulation of members of the adhesion/growth-regulatory galectin family. Immunohistochemical localization of galectin-3 (Gal-3) in sections of human cartilage with increasing levels of degeneration revealed a linear correlation reaching a chondrocyte positivity of 60%. Presence in situ was cytoplasmic, the lectin was secreted from OA chondrocytes in culture and binding of Gal-3 yielded lactose-inhibitable surface staining. Exposure of cells to the lectin led to enhanced gene expression and secretion of functional disease markers. Genome-wide transcriptomic analysis broadened this result to reveal a pro-degradative/inflammatory gene signature under the control of NF-κB. Fittingly, targeting this route of activation by inhibitors impaired the unfavourable response to Gal-3 binding, as also seen by shortening the lectin's collagen-like repeat region. Gal-3's activation profile overlaps with that of homodimeric galectin-1 (Gal-1) and also has distinctive (supplementing) features. Tested at subsaturating concentrations in a mixture, we found cooperation between the two galectins, apparently able to team up to promote OA pathogenesis. In summary, our results suggest that a network of endogenous lectins is relevant for initiating this process cascade.