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.

Bioprinting of human nasoseptal chondrocytes-laden collagen hydrogel for cartilage tissue engineering.

Skin cancer patients often have tumorigenic lesions on their noses. Surgical resection of the lesions often results in nasal cartilage removal. Cartilage grafts taken from other anatomical sites are used for the surgical reconstruction of the nasal cartilage, but donor-site morbidity is a common problem. Autologous tissue-engineered nasal cartilage grafts can mitigate the problem, but commercially available scaffolds define the shape and sizes of the engineered grafts during tissue fabrication. Moreover, the engineered grafts suffer from the inhomogeneous distribution of the functional matrix of cartilage. Advances in 3D bioprinting technology offer the opportunity to engineer cartilages with customizable dimensions and anatomically shaped configurations without the inhomogeneous distribution of cartilage matrix. Here, we report the fidelity of Freeform Reversible Embedding of Suspended Hydrogel (FRESH) bioprinting as a strategy to generate customizable and homogenously distributed functional cartilage matrix engineered nasal cartilage. Using FRESH and in vitro chondrogenesis, we have fabricated tissue-engineered nasal cartilage from combining bovine type I collagen hydrogel and human nasoseptal chondrocytes. The engineered nasal cartilage constructs displayed molecular, biochemical and histological characteristics akin to native human nasal cartilage.

Chondrosarcoma with Target-Like Chondrocytes: Update on Molecular Profiling and Specific Morphological Features.

This is the first histological and molecular analysis of two chondrosarcomas with target-like chondrocytes that were compared with a group of conventional chondrosarcomas and enchondromas. The unique histological feature of target-like chondrocytes is the presence of unusual hypertrophic eosinophilic APAS-positive perichondrocytic rings (baskets). In the sections stained with Safranin O/Fast green, the outer part of the ring was blue and the material in the lacunar space stained orange, similarly to intercellular regions. Immunohistochemical examination showed strong positivity for vimentin, factor XIIIa, cyclin D1, osteonectin, B-cell lymphoma 2 apoptosis regulator (Bcl-2), p53 and p16. The S-100 protein was positive in 25 % of neoplastic cells. Antibodies against GFAP, D2-40 (podoplanin), CD99, CKAE1.3 and CD10 exhibited weak focal positivity. Pericellular rings/baskets contained type VI collagen in their peripheral part, in contrast to the type II collagen in intercellular interterritorial spaces. Ultrastructural examination revealed that pericellular rings contained an intralacunar component composed of microfibrils with abundant admixture of aggregates of dense amorphous non-fibrillar material. The outer extralacunar zone was made up of a layer of condensed thin collagen fibrils with admixture of non-fibrillar dense material. NGS sequencing identified a fusion transcript involving fibronectin 1 (FN1) and fibroblast growth factor receptor 2 (FGFR2) at the RNA level. At the DNA level, no significant variant was revealed except for the presumably germline variant in the SPTA1 gene.

A competitive alphascreen assay for detection of hyaluronan.

A method for specific quantification of hyaluronan (HA) concentration using AlphaScreen® (Amplified Luminescent Proximity Homogeneous Assay) technology is described. Two types of hydrogel-coated and chromophore-loaded latex nanobeads are employed. The proximity of the beads in solution is detected by excitation of the donor bead leading to the production of singlet oxygen, and chemiluminescence from the acceptor bead upon exposure to singlet oxygen. In the HA assay, the donor bead is modified with streptavidin, and binds biotin-labeled HA. The acceptor bead is modified with Ni(II), and is used to bind a specific recombinant HA-binding protein (such as HABP; aggrecan G1-IGD-G2) with a His-tag. Competitive inhibition of the HA-HABP interaction by free unlabeled HA in solution is used for quantification. The assay is specific for HA, and not dependent on HA molecular mass above the decasaccharide. HA can be quantified over a concentration range of approximately 30-1600 ng/mL using 2.5 µL of sample, for a detectable mass range of approximately 0.08-4 ng HA. This sensitivity of the AlphaScreen assay is greater than existing ELISA-like methods, due to the small volume requirements. HA can be detected in biological fluids using the AlphaScreen assay, after removal of bound proteins from HA and dilution or removal of other interfering proteins and lipids.

A Complex Relationship between Visfatin and Resistin and microRNA: An In Vitro Study on Human Chondrocyte Cultures.

Growing evidence indicates the important role of adipokines and microRNA (miRNA) in osteoarthritis (OA) pathogenesis. The purpose of the present study was to investigate the effect of visfatin and resistin on some miRNA (34a, 140, 146a, 155, 181a, let-7e), metalloproteinases (MMPs), and collagen type II alpha 1 chain (Col2a1) in human OA chondrocytes and in the T/C-28a2 cell line. The implication of nuclear factor (NF)-κB in response to adipokines was also assessed. Chondrocytes were stimulated with visfatin (5 or 10 µg/mL) and resistin (50 or 100 ng/mL) with or without NF-κB inhibitor (BAY-11-7082, 1 µM) for 24 h. Viability and apoptosis were detected by MMT and cytometry, miRNA, MMP-1, MMP-13, and Col2a1 by qRT-PCR and NF-κB activation by immunofluorescence. Visfatin and resistin significantly reduced viability, induced apoptosis, increased miR-34a, miR-155, miR-181a, and miR-let7e, and reduced miR-140 and miR-146a gene expression in OA chondrocytes. MMP-1, MMP-13, and Col2a1 were significantly modulated by treatment of OA chondrocytes with adipokines. Visfatin and resistin significantly increased NF-κB activation, while the co-treatment with BAY11-7082 did not change MMPs or Col2a1 levels beyond that caused by single treatment. Visfatin and resistin regulate the expression levels of some miRNA involved in OA pathogenesis and exert catabolic functions in chondrocytes via the NF-κB pathway. These data confirm the complex relationship between adipokines and miRNA.

Oxygen-Dependent Lipid Profiles of Three-Dimensional Cultured Human Chondrocytes Revealed by MALDI-MSI.

Articular cartilage is exposed to a gradient of oxygen levels ranging from 5% at the surface to 1% in the deepest layers. While most cartilage research is performed in supraphysiological oxygen levels (19-21%), culturing chondrocytes under hypoxic oxygen levels (≤8%) promotes the chondrogenic phenotype. Exposure of cells to various oxygen levels alters their lipid metabolism, but detailed studies examining how hypoxia affects lipid metabolism in chondrocytes are lacking. To better understand the chondrocyte's behavior in response to oxygen, we cultured 3D pellets of human primary chondrocytes in normoxia (20% oxygen) and hypoxia (2.5% oxygen) and employed matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in order to characterize the lipid profiles and their spatial distribution. In this work we show that chondrocytes cultured in hypoxia and normoxia can be differentiated by their lipid profiles. Among other species, phosphatidylglycerol species were increased in normoxic pellets, whereas phosphatidylinositol species were the most prominent lipids in hypoxic pellets. Moreover, spatial mapping revealed that phospahtidylglyycerol species were less prominent in the center of pellets where the oxygen level is lower. Additional analysis revealed a higher abundance of the mitochondrial-specific lipids, cardiolipins, in normoxic conditions. In conclusion MALDI-MSI described specific lipid profiles that could be used as sensors of oxygen level changes and may especially be relevant for retaining the chondrogenic phenotype, which has important implications for the treatment of bone and cartilage diseases.

Differences in expression of Wnt antagonist Dkk1 in healthy versus pathological bone samples.

Wnt/ß-catenin signalling components was shown to affect bone cells function including chondrocytes.Secreted Dkk1, a potent osteogenesis inhibiting factor mediates bone loss in diseased bones by suppressing the biological actions of Wnt proteins. In addition, increased Dkk1 signalling inhibits chondrogenesis in new bone formation. Recent findings also show there exists a cross-talk between the chondrocytes and the cells of the osteoblast lineage, which are the most affected cell types in muskuloskeletal disorders. This study investigated whether spatial expression of Dkk1 is confined to only osteoblasts, osteocytes or chondrocytes. The second objective was to detect a difference in the Dkk1 expression pattern in healthy subjects when compared to pathological state. To elucidate the cell specificity of Dickkopf-1 (Dkk1) in healthy bones, samples from female Sprague-Dawley rats were tested against two different antibodies with the two most widely accepted visualization system (ABC and Envision). The findings show Dkk1 specificity predominantly for osteoblasts, chondrocytes and osteocytes depending upon the antibody used. In addition, Dkk1 expression was evaluated in different cells of human osteoarthritis (OA) and rheumatoid arthritis (OA) patients. Its overexpression in pathologic state also suggests the role of Dkk1 in bone formation. This is scientifically and clinically important in studying the effect of Dkk1 in bone healing and in designing treatments for patients with compromised bone status. Taking into consideration the paradigm that cartilage and subchondral bone behave as an interconnected functional unit, normalization of cell behaviour in one compartment may have benefits in both tissues.

Sustained release effects of berberine-loaded chitosan microspheres on in vitro chondrocyte culture.

The low bioavailability and short biological half-life of berberine chloride (BBR) negatively affect the protective role of this compound against osteoarthritis (OA). The present study was performed to evaluate the effectiveness of sustained BBR release system. Novel BBR-loaded chitosan microspheres (BBR-loaded CMs) were successfully synthesized using an ionic cross-linking method for sustained release. The basic characteristics of the prepared microspheres were subsequently evaluated by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) techniques, encapsulation efficiency (EE), and in vitro release experiments. BBR-loaded CMs displayed spherical forms to encapsulate a considerable quantity of BBR (100.8 ± 2.7 mg/g); these microspheres also exhibited an ideal releasing profile. The FT-IR spectra and XRD results revealed that BBR-loaded CMs were successfully synthesized via electrostatic interaction. In vitro experiments further showed that BBR-loaded CMs significantly inhibited sodium nitroprusside (SNP)-stimulated chondrocyte apoptosis as well as cytoskeletal remodeling, and led to increasing mitochondrial membrane potential and maintaining the nuclear morphology. BBR-loaded CMs exerted markedly higher anti-apoptotic activity in the treatment of OA, and markedly inhibited the protein expression levels of caspase-3, a disintegrin, and metalloproteinase with thrombospondin motifs (ADAMTS)-5 and matrix metalloproteinase (MMP)-13 induced by SNP in rat articular chondrocytes, compared with free BBR at equivalent concentration. Therefore, novel BBR-loaded CMs may offer potential for application in the treatment of OA.

Evaluating the cartilage adjacent to the site of repair surgery with glycosaminoglycan-specific magnetic resonance imaging.

PURPOSE: The glycosaminoglycan (GAG) chemical exchange saturation transfer (CEST) imaging method (gagCEST) makes it possible to assess and quantify the GAG concentration in human cartilage. This biochemical imaging technique facilitates detection of the loss of GAG in the course of osteoarthritis. The gagCEST technique was used to analyse the perilesional zone (PLZ) adjacent to repair tissue after cartilage repair surgery, to determine whether there are biochemical changes present in the sense of degeneration. METHOD: Asymmetries in the PLZ of cartilage defects in 11 patients, who had been treated by microfracturing or matrix-associated autologous chondrocyte transplantation (MACT), were measured by gagCEST on a 7-T whole-body system. These results were correlated with gagCEST asymmetries of healthy reference cartilage (RC), measured anterior and posterior to the PLZ and to the repair tissue (RT). RESULTS: The mean gagCEST asymmetry for the anterior PLZ was 4.8% (±4.4), for the posterior PLZ 5.4% (±2.3), for the anterior RC 6.6% (±3.5) and 7.2% (±3.3) for the posterior RC and 4.5% (±2.3) for the RT. The difference between the anterior PLZ and the anterior RC (p = 0.019), the posterior PLZ and the posterior RC (p = 0.005), and the mean RC and the RT (p = 0.021) were all statistically significant. The measurements between RT and mean PLZ did not reveal significant results (p = 0.398). CONCLUSIONS: The gagCEST method provides a potentially useful biomarker for the loss of GAGs, indicating cartilage degeneration in the PLZ. Pre-operative and post-operative monitoring of the biomechanical state of the cartilage might influence intra-operative decision-making concerning the extent of cartilage resection or might give information of the success of the treatment post-operatively.

[Nrp-1 expression in healing process of traumatic brain injury combined with tibial fracture].

OBJECTIVE: To examine the expression of neuropilin-1 (Nrp-1), semaphorin 3A (Sema3A) and vascular endothelial growth factor (VEGF) in the healing process of tibial fracture after traumatic brain injury and to explore the mechanism of Nrp-1 in the formation of pathological callus after nerve injury.
 Methods: A total of 192 Wister female rats, 8-10 weeks old and weighing 220-250 g, were randomly divided into a control group (Group C), a tibia fracture group (Group F), a traumatic brain injury group (Group TBI), a traumatic brain injury combined with the tibia fracture group (Group TBI+F) (n=48 in each group). Tissue samples were collected at 3, 5, 7, 14, 21 and 28 days, respectively (n=8 for each time point). The expression of Nrp-1 in callus tissues were examined by immunohistochemistry and Western blot, while the expression of Sema3A and VEGF were detected by Western blot.
 Results: Compared with the Group F , the expression of Nrp-1 in chondrocytes of bone formation area and cartilage area was higher in the Group TBI+F, particularly at the 7th , 14th and 21st day (P<0.05), while the expression of Nrp-1 in osteoblast cells of fresh bone trabecula region was lower in the Group TBI+F, particularly at the 14th and 21st day (both P<0.05). Western blot showed that the expression of Nrp-1, Sema 3A and VEGF had the same trends in the Group TBI+F and the Group F. However, at each time point, the expression of Nrp-1 in the Group TBI+F was significantly higher and slowly decreased, particularly at the 14th, 21st and 28th day (all P<0.05). Meanwhile, the ratio of Sema 3A/VEGF in the Group TBI+F was significantly higher than that in the Group F, with statistical difference (P<0.05).
 Conclusion: The Nrp-1 is expressed abnormally in the process of fracture healing after nerve injury. It may play a role in the formation of pathological callus after nerve injury by promoting the preliminary and proliferation of chondrocytes, and inhibiting the growth of nerve fibers in the soft callus as well as the differentiation of osteoblast cell.

Development of a Rat Model of Mechanically Induced Tunable Pain and Associated Temporomandibular Joint Responses.

PURPOSE: Although mechanical overloading of the temporomandibular joint (TMJ) is implicated in TMJ osteoarthritis (OA) and orofacial pain, most experimental models of TMJ-OA induce only acute and resolving pain, which do not meaningfully simulate the pathomechanisms of TMJ-OA in patients with chronic pain. The aim of this study was to adapt an existing rat model of mechanically induced TMJ-OA, to induce persistent orofacial pain by altering only the jaw-opening force, and to measure the expression of common proxies of TMJ-OA, including degradation and inflammatory proteins, in the joint. MATERIALS AND METHODS: TMJ-OA was mechanically induced in a randomized, prospective study using 2 magnitudes of opening loads in separate groups (ie.,. 2-N, 3.5-N and sham control [no load]). Steady mouth opening was imposed daily (60 minutes/day for 7 days) in female Holtzman rats, followed by 7 days of rest, and orofacial sensitivity was measured throughout the loading and rest periods. Joint structure and extent of degeneration were assessed at day 14 and expression of matrix metalloproteinase-13 (MMP-13), hypoxia-inducible factor-1α (HIF-1α), and tumor necrosis factor-α (TNF-α) in articular cartilage was evaluated by immunohistochemistry and quantitative densitometry methods at day 7 between the 2 loading and control groups. Statistical differences of orofacial sensitivity and chondrocyte expression between loading groups were computed and significance was set at a P value less than .05. RESULTS: Head-withdrawal thresholds for the 2 loading groups were significantly decreased during loading (P < .0001), but that decrease remained through day 14 only for the 3.5-N group (P < .00001). At day 14, TMJs from the 2-N and 3.5-N groups exhibited truncation of the condylar cartilage, typical of TMJ-OA. In addition, a 3.5-N loading force significantly upregulated MMP-13 (P < .0074), with nearly a 2-fold increase in HIF-1α (P < .001) and TNF-α (P < .0001) at day 7, in 3.5-N loaded joints over those loaded by 2 N. CONCLUSION: Unlike a 2-N loading force, mechanical overloading of the TMJ using a 3.5-N loading force induced constant and nonresolving pain and the upregulation of inflammatory markers only in the 3.5-N group, suggesting that these markers could predict the maintenance of persistent orofacial pain. As such, the development of a tunable experimental TMJ-OA model that can separately induce acute or persistent orofacial pain using similar approaches provides a platform to better understand the pathomechanisms involved and possibly to evaluate potential treatment strategies for patients with painful TMJ-OA.

Engineering zonal cartilage through bioprinting collagen type II hydrogel constructs with biomimetic chondrocyte density gradient.

BACKGROUND: Cartilage tissue engineering is a promising approach for repairing and regenerating cartilage tissue. To date, attempts have been made to construct zonal cartilage that mimics the cartilaginous matrix in different zones. However, little attention has been paid to the chondrocyte density gradient within the articular cartilage. We hypothesized that the chondrocyte density gradient plays an important role in forming the zonal distribution of extracellular matrix (ECM). METHODS: In this study, collagen type II hydrogel/chondrocyte constructs were fabricated using a bioprinter. Three groups were created according to the total cell seeding density in collagen type II pre-gel: Group A, 2 × 10(7) cells/mL; Group B, 1 × 10(7) cells/mL; and Group C, 0.5 × 10(7) cells/mL. Each group included two types of construct: one with a biomimetic chondrocyte density gradient and the other with a single cell density. The constructs were cultured in vitro and harvested at 0, 1, 2, and 3 weeks for cell viability testing, reverse-transcription quantitative PCR (RT-qPCR), biochemical assays, and histological analysis. RESULTS: We found that total ECM production was positively correlated with the total cell density in the early culture stage, that the cell density gradient distribution resulted in a gradient distribution of ECM, and that the chondrocytes' biosynthetic ability was affected by both the total cell density and the cell distribution pattern. CONCLUSIONS: Our results suggested that zonal engineered cartilage could be fabricated by bioprinting collagen type II hydrogel constructs with a biomimetic cell density gradient. Both the total cell density and the cell distribution pattern should be optimized to achieve synergistic biological effects.

Finite difference time domain model of ultrasound propagation in agarose scaffold containing collagen or chondrocytes.

Measurement of ultrasound backscattering is a promising diagnostic technique for arthroscopic evaluation of articular cartilage. However, contribution of collagen and chondrocytes on ultrasound backscattering and speed of sound in cartilage is not fully understood and is experimentally difficult to study. Agarose hydrogels have been used in tissue engineering applications of cartilage. Therefore, the aim of this study was to simulate the propagation of high frequency ultrasound (40 MHz) in agarose scaffolds with varying concentrations of chondrocytes (1 to 32 × 10(6) cells/ml) and collagen (1.56-200 mg/ml) using transversely isotropic two-dimensional finite difference time domain method (FDTD). Backscatter and speed of sound were evaluated from the simulated pulse-echo and through transmission measurements, respectively. Ultrasound backscatter increased with increasing collagen and chondrocyte concentrations. Furthermore, speed of sound increased with increasing collagen concentration. However, this was not observed with increasing chondrocyte concentrations. The present study suggests that the FDTD method may have some applicability in simulations of ultrasound scattering and propagation in constructs containing collagen and chondrocytes. Findings of this study indicate the significant role of collagen and chondrocytes as ultrasound scatterers and can aid in development of modeling approaches for understanding how cartilage architecture affects to the propagation of high frequency ultrasound.

[Hydrogen sulfide in cartilage and its inhibitory effect on matrix metalloproteinase 13 expression in chondrocytes induced by interlukin-1ß].

OBJECTIVE: To investigate whether endogenous hydrogen sulfide (H2S) was involved in the pathogenesis of osteoarthritis (OA) and its underlying mechanism, to detect H2S and its synthases expression in knee cartilage in patients diagnosed with different severity of OA, and to explore the transcription and expression of gene MMP-13 in chondrocytes treated with IL-1ß or H2S. METHODS: Synovial fluids of the in-patients with different severity of OA hospitalized in Peking University First Hospital were collected for measurement of H2S content using methylene blue assay. Articular cartilages of the patients who underwent knee arthroplasty were collected for the cell culture of relatively normal chondrocytes. The chondrocytes were cultured to the P3 generation and H2S molecular probes were used for detection of endogenous H2S generation in the chondrocytes. Immunocytochemistry was used to detect the localization of H2S synthases including cystathionine ß-synthase (CBS), cystathionine-γ-lyase (CSE), and mercaptopyruvate sulfurtransferase (MPST) in OA chondrocytes. Western blot was used to quantify the protein expressions of CSE, MPST, and CBS in cartilage tissues of the patients who were diagnosed with OA and underwent knee arthroplasty. The relatively normal human chondrocytes were cultured to passage 3 and then divided into 4 groups for different treatments: (1)the normal control group, no reagent was added; (2)the IL-1ß group, 5 µg/L of IL-1ß was added; (3)the IL-1ß+H2S group, 200 µmol/L of NaHS was added 30 min before adding 5 µg/L of IL-1ß;(4)the H2S group, 200 µmol/L of NaHS was added. The transcription and expression of gene MMP-13 in chondrocytes of each group were determined with Real-time PCR and Western blot, respectively. And the total NF-κB p65 and phosphorylated NF-κB p65 in chondrocytes were detected with Western blot. RESULTS: The content of H2S in the synovial fluid of degenerative knee was (14.3±3.3) µmol/L. Expressions of endogenous H2S and its synthases including CBS, CSE and MPST were present in the cytoplasm of chondrocytes.CSE protein expression in Grade 3 (defined by outerbridge grading) cartilage tissues was significantly increased as compared with that of Grade 1 cartilage tissues (1.67±0.09 vs. 1.26±0.11, P< 0.05). However, no significant difference of CBS or MPST expression among the different groups was observed. The expression of MMP-13 protein in the IL-1ßgroup was significantly higher than that in the normal chondrocytes (1.87±0.67 vs. 0.22±0.10, P<0.05), and that in the IL-1ß+H2S group was significantly decreased than that in the IL-1ß group (0.55±0.11 vs. 1.87±0.67, P< 0.05), and that in the H2S group had no significant difference compared with that in the normal control group. The transcription of MMP-13 protein in the IL-1ß group was significantly higher than that in the normal chondrocytes (31.40±0.31 vs. 1.00±0.00, P<0.05), and that in the IL-1ß+H2S group was significantly decreased than that in the IL-1ß group (24.41±1.28 vs. 31.40±0.31, P<0.05), and that in the H2S group had no significant difference compared with that in the normal control group. The total NF-κB p65 in the IL-1ß group was significantly higher than that in the normal chondrocytes (2.13±0.08 vs. 0.73±0.08, P< 0.05), and that in the IL-1ß+H2S group was significantly decreased than that in the IL-1ß group (1.24±0.13 vs. 2.13±0.08, P<0.05), and that in the H2S group had no significant difference compared with that in the normal control group. The phosphorylated NF-κB p65 in IL-1ß group was significantly higher than that in the normal chondrocytes (1.30±0.13 vs. 0.19±0.04, P<0.05), and that in IL-1ß+H2S group was significantly decreased than that in the IL-1ß group (0.92±0.26 vs. 1.30±0.13, P<0.05), and that in the H2S group had no significant difference compared with that in the normal control group. CONCLUSION: H2S affected the cartilage degeneration by partly inhibiting the degradation of extracellular matrix.

[Protective effect of diosgenin on chondrocytes mediated by JAK2/STAT3 signaling pathway in mice with osteoarthritis].

Objective: To investigate the effect of diosgenin (Dgn) on chondrocytes and its relation to JAK2/STAT3 signaling pathway in mice with osteoarthritis (OA).Methods: Fifteen male C57BL/6 mice were randomly divided into three groups:control group, OA group and OA+Dgn group. After 4 weeks of treatment, the histopathological changes of cartilage tissue were observed by toluidine blue staining under light microscopy and the ultrastructure of chondrocytes was observed under electron microscopy. The primarily cultured chondrocytes of OA mice were randomly divided into 4 groups:(1) OA group, (2) Dgn group, (3) Dgn+AG490 group, (4) AG490 group. The expression of p-JAK2, p-STAT3, Bax, succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) were detected by Western blotting, and superoxide dismutase (SOD) was detected using colorimetric method. Results: The morphological observation showed that the chondrocytes of OA group presented considerable pathological changes, while the chondrocytes in OA+Dgn group maintained intact membrane. Electron microscopy observation found obvious injury in cartilage tissues of OA group, while that in OA+Dgn group remained smooth. Compared with OA group, the expressions of p-JAK2 and p-STAT3 in chondrocytes of Dgn group were increased (all P<0.05), and the expressions of Bax protein, SDH, COX and SOD were decreased (all P<0.05). While compared with Dgn group, the expressions of p-JAK2, p-STAT3, SDH, COX and SOD in chondrocytes of Dgn+AG490 group were decreased (all P<0.05), and the expression of Bax protein was increased (P<0.05). Conclusion: Diosgenin can inhibit apoptosis and increase mitochondrial oxidative stress capacity of chondrocytes in mice with osteoarthritis, which is closely related to the activation of JAK2/STAT3 signaling pathway.

Modulation of ten-eleven translocation 1 (TET1), Isocitrate Dehydrogenase (IDH) expression, α-Ketoglutarate (α-KG), and DNA hydroxymethylation levels by interleukin-1ß in primary human chondrocytes.

5-Hydroxymethylcytosine (5-hmC) generated by ten-eleven translocation 1-3 (TET1-3) enzymes is an epigenetic mark present in many tissues with different degrees of abundance. IL-1ß and TNF-α are the two major cytokines present in arthritic joints that modulate the expression of many genes associated with cartilage degradation in osteoarthritis. In the present study, we investigated the global 5-hmC content, the effects of IL-1ß and TNF-α on 5-hmC content, and the expression and activity of TETs and isocitrate dehydrogenases in primary human chondrocytes. The global 5-hmC content was found to be ∼0.1% of the total genome. There was a significant decrease in the levels of 5-hmC and the TET enzyme activity upon treatment of chondrocytes with IL-1ß alone or in combination with TNF-α. We observed a dramatic (10-20-fold) decrease in the levels of TET1 mRNA expression and a small increase (2-3-fold) in TET3 expression in chondrocytes stimulated with IL-1ß and TNF-α. IL-1ß and TNF-α significantly suppressed the activity and expression of IDHs, which correlated with the reduced α-ketoglutarate levels. Whole genome profiling showed an erasure effect of IL-1ß and TNF-α, resulting in a significant decrease in hydroxymethylation in a myriad of genes including many genes that are important in chondrocyte physiology. Our data demonstrate that DNA hydroxymethylation is modulated by pro-inflammatory cytokines via suppression of the cytosine hydroxymethylation machinery. These data point to new mechanisms of epigenetic control of gene expression by pro-inflammatory cytokines in human chondrocytes.

Effects of chondrocyte-derived extracellular matrix in a dry eye mouse model.

PURPOSE: The occurrence of repetitive dry eye is accompanied by inflammation. This study investigated the anti-inflammatory effects of chondrocyte-derived extracellular matrix (CDECM) on the cornea and conjunctiva in a dry eye mouse model. METHODS: Dry eyes were experimentally induced in 12- to 16-week-old NOD.B10.H2(b) mice (Control) via subcutaneous injections of scopolamine (muscarinic receptor blocker) and exposure to an air draft for 10 days (desiccation stress [DS] 10D group). Tear volume and corneal smoothness were measured at 3, 5, 7, and 10 days after the instillation of PBS (PBS group) or CDECM (CDECM group). The corneas and conjunctivas were sectioned and stained with hematoxylin and eosin (H&E) and periodic acid Schiff (PAS). The expression of inflammatory markers (i.e., tumor necrosis factor-α [TNF-α], matrix metalloproteinase-2 [MMP-2], MMP-9, intercellular adhesion molecule-1 [ICAM-1], and vascular cell adhesion molecule-1 [VCAM-1]) was detected by quantitative real-time (qRT)-PCR and western blotting. All data were statistically processed using SPSS version 18.0. RESULTS: The instillation of CDECM after the removal of the DS increased tear production by up to 3.0-fold, and corneal smoothness improved to 80% compared to the PBS group (p<0.05). In the CDECM group, the detachment of the corneal epithelial cells was reduced by 73.3% compared to the PBS group, and the conjunctival goblet cell density was significantly recovered to the control levels (p<0.05). The expression of inflammatory factors was decreased in the cornea and conjunctiva of the CDECM group compared to the PBS group. CONCLUSIONS: These observations suggest that CDECM induced effective anti-inflammatory improvements in the cornea and conjunctiva in this experimental model of dry eye.

3D Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications.

The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine. The 3D bioprinter is able to dispense materials while moving in X, Y, and Z directions, which enables the engineering of complex structures from the bottom up. In this study, a bioink that combines the outstanding shear thinning properties of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate was formulated for the 3D bioprinting of living soft tissue with cells. Printability was evaluated with concern to printer parameters and shape fidelity. The shear thinning behavior of the tested bioinks enabled printing of both 2D gridlike structures as well as 3D constructs. Furthermore, anatomically shaped cartilage structures, such as a human ear and sheep meniscus, were 3D printed using MRI and CT images as blueprints. Human chondrocytes bioprinted in the noncytotoxic, nanocellulose-based bioink exhibited a cell viability of 73% and 86% after 1 and 7 days of 3D culture, respectively. On the basis of these results, we can conclude that the nanocellulose-based bioink is a suitable hydrogel for 3D bioprinting with living cells. This study demonstrates the potential use of nanocellulose for 3D bioprinting of living tissues and organs.

Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues.

Much of our understanding of the biological mechanisms that underlie cellular functions, such as migration, differentiation and force-sensing has been garnered from studying cells cultured on two-dimensional (2D) glass or plastic surfaces. However, more recently the cell biology field has come to appreciate the dissimilarity between these flat surfaces and the topographically complex, three-dimensional (3D) extracellular environments in which cells routinely operate in vivo. This has spurred substantial efforts towards the development of in vitro 3D biomimetic environments and has encouraged much cross-disciplinary work among biologists, material scientists and tissue engineers. As we move towards more-physiological culture systems for studying fundamental cellular processes, it is crucial to define exactly which factors are operative in 3D microenvironments. Thus, the focus of this Commentary will be on identifying and describing the fundamental features of 3D cell culture systems that influence cell structure, adhesion, mechanotransduction and signaling in response to soluble factors, which - in turn - regulate overall cellular function in ways that depart dramatically from traditional 2D culture formats. Additionally, we will describe experimental scenarios in which 3D culture is particularly relevant, highlight recent advances in materials engineering for studying cell biology, and discuss examples where studying cells in a 3D context provided insights that would not have been observed in traditional 2D systems.

Modeling of signaling pathways in chondrocytes based on phosphoproteomic and cytokine release data.

OBJECTIVE: Chondrocyte signaling is widely identified as a key component in cartilage homeostasis. Dysregulations of the signaling processes in chondrocytes often result in degenerative diseases of the tissue. Traditionally, the literature has focused on the study of major players in chondrocyte signaling, but without considering the cross-talks between them. In this paper, we systematically interrogate the signal transduction pathways in chondrocytes, on both the phosphoproteomic and cytokine release levels. METHODS: The signaling pathways downstream 78 receptors of interest are interrogated. On the phosphoproteomic level, 17 key phosphoproteins are measured upon stimulation with single treatments of 78 ligands. On the cytokine release level, 55 cytokines are measured in the supernatant upon stimulation with the same treatments. Using an Integer Linear Programming (ILP) formulation, the proteomic data is combined with a priori knowledge of proteins' connectivity to construct a mechanistic model, predictive of signal transduction in chondrocytes. RESULTS: We were able to validate previous findings regarding major players of cartilage homeostasis and inflammation (e.g., IL1B, TNF, EGF, TGFA, INS, IGF1 and IL6). Moreover, we studied pro-inflammatory mediators (IL1B and TNF) together with pro-growth signals for investigating their role in chondrocytes hypertrophy and highlighted the role of underreported players such as Inhibin beta A (INHBA), Defensin beta 1 (DEFB1), CXCL1 and Flagellin, and uncovered the way they cross-react in the phosphoproteomic level. CONCLUSIONS: The analysis presented herein, leveraged high throughput proteomic data via an ILP formulation to gain new insight into chondrocytes signaling and the pathophysiology of degenerative diseases in articular cartilage.