Journal: Cells

Article Title: Heat-Treated Lacticaseibacillus rhamnosus Strains Modulate Inflammatory and Metabolic Processes in In Vitro Systems Relevant to Canine Osteoarthritis

doi: 10.3390/cells15040336

Figure Lengend Snippet: Screening of the anti-inflammatory effect of different heat-treated strains in canine chondrocytes. IL-6 secretion by primary canine chondrocytes stimulated with IL-1β was measured in the presence of a collection of candidates. Data are represented as the percentage of IL-6 secretion relative to IL-1β stimulated condition without inactivated cells. Control condition refers to chondrocytes stimulated with IL-1β, whereas negative control (NC) corresponds to chondrocytes without stimulation. Data are presented as the mean ± SD of three independent experiments. Statistical test: One-way ANOVA with Dunnett’s multiple comparisons test (Postbiotic vs. Control). Significance levels were indicated as follows: p -value > 0.05 (ns: non-significant), p -value < 0.01(**), p -value < 0.0001 (****).

Article Snippet: Alginate beads were washed 5 times with NaCl solution (0.9%) and finally, one more time in chondrocyte differentiation medium (Cell Applications Inc., San Diego, CA, USA).

Techniques: Control, Negative Control

Journal: Cells

Article Title: Heat-Treated Lacticaseibacillus rhamnosus Strains Modulate Inflammatory and Metabolic Processes in In Vitro Systems Relevant to Canine Osteoarthritis

doi: 10.3390/cells15040336

Figure Lengend Snippet: Insoluble type II collagen production by primary canine chondrocytes cultured in spheroids structure. Data are presented as the mean ± SD of three independent experiments and expressed as the percentage of insoluble type II collagen production relative to the control condition. In the control condition, canine chondrocytes were cultured without supplementation of heat-treated strains. Statistical test: Kruskal-Wallis test, followed by Dunn’s multiple comparison test (Control vs. Postbiotic). Significance levels were indicated as follows: p -value < 0.05 (*).

Article Snippet: Alginate beads were washed 5 times with NaCl solution (0.9%) and finally, one more time in chondrocyte differentiation medium (Cell Applications Inc., San Diego, CA, USA).

Techniques: Cell Culture, Control, Comparison

Journal: Open Veterinary Journal

Article Title: Generation and characterization of mesenchymal stem cells from the affected femoral heads of dogs with Legg Calvé Perthes disease

doi: 10.5455/OVJ.2024.v14.i5.12

Figure Lengend Snippet: Trilineage differentiation potency. Canine LCPD-affected femoral head-derived adherent cells (A-C) and BMMSCs (D-F) differentiated into adipocytes (A and D), osteoblasts (B and E), and chondrocytes (C and F). (A and D): Sudan III staining, (B and E): Alizarin Red staining, and (C and F): Alcian blue staining. Scale bars = 100 μm or 1 mm.

Article Snippet: To induce differentiation into chondrocytes, 5 × 10 5 cells were placed in 15 ml polypropylene tubes, centrifuged to form pellets, and cultured with canine chondrocyte differentiation medium (Cell Applications, Inc.) for 3 weeks.

Techniques: Derivative Assay, Staining

Journal: BMC Musculoskeletal Disorders

Article Title: ARG098, a novel anti-human Fas antibody, suppresses synovial hyperplasia and prevents cartilage destruction in a severe combined immunodeficient-HuRAg mouse model

doi: 10.1186/1471-2474-11-221

Figure Lengend Snippet: ARG098 cytotoxicity to human chondrocytes . (A) ARG098 binding activity to human chondrocytes was assessed by flow cytometory. The white area is stained with human IgM and the black area is stained with ARG098. (B) RA synoviocytes pretreated with anti-Fas neutralizing antibody clone SM1/23. (C) Representative cell viability of human chondrocytes following ARG098 treatment (Experiment 1: from 0.1 to 1000 ng/mL, Experiment 2: from 1 to 100 μg/mL) versus untreated condition, as measured by the WST assay. Black circles and black triangles, ARG098; white circles and white triangles, human IgM. Each point represents mean ± SEM (n = 5).

Article Snippet: The sources of the other materials used in this study are as follows: human IgM was obtained from ICN Biomedicals Inc. (Aliso Viejo, CA, USA), chondrocyte basal medium supplemented with chondrocyte growth supplement was obtained from Cell Applications, Inc. (San Diego, CA, USA), and the neutralizing antibody anti-human APO-1/Fas (SM1/23) was obtained from Bender Medsystems GmbH (Vienna, Austria).

Techniques: Binding Assay, Activity Assay, Staining, WST Assay

Journal: Arthritis Research & Therapy

Article Title: Anabolic role of lysyl oxidase like-2 in cartilage of knee and temporomandibular joints with osteoarthritis

doi: 10.1186/s13075-017-1388-8

Figure Lengend Snippet: Regulation of LOXL2 gene expression by osteoarthritis (OA)-related mediators: Human articular OA chondrocytes (HAC-OA) were treated with TNF-α, IL-1β, or transforming growth factor-β1 ( TGF-β1 ) at 10 or 30 ng/mL for 24 h, and LOXL2 mRNA levels were assessed by RTqPCR. Data are represented as mean ± SEM of three independent experiments with 95% confidence interval (CI) (* P < 0.001; Student’s t test). LOXL2 gene expression was significantly upregulated by TGF-β1 ( P < 0.001) and downregulated by TNF-α ( P < 0.001) and IL-1β ( P < 0.001).

Article Snippet: Cell lines were grown in chondrocyte growth medium (CGMTM BulletKitTM, CC-3216, Lonza Inc.) or in chondrocyte differentiation medium (CDM BulletKit, CC-3225, Lonza), both of which contain 5% fetal bovine serum (FBS), R3-insulin-like growth factor-1, human recombinant fibroblast growth factor-beta; insulin-transferrin-selenium (ITS), and gentamycin/amphotericin-B for in vitro studies.

Techniques: Gene Expression

Journal: Arthritis Research & Therapy

Article Title: Anabolic role of lysyl oxidase like-2 in cartilage of knee and temporomandibular joints with osteoarthritis

doi: 10.1186/s13075-017-1388-8

Figure Lengend Snippet: LOXL2 promotes a differentiated phenotype and inhibits apoptosis in human articular chondrocytes from cartilage affected by osteoarthritis ( OA ). a Human articular OA chondrocytes ( HAC-OA ) transduced with adenoviruses for transient expression of LOXL2 (Ad-RFP-LOXL2) compared to cells transduced with its empty vector ( EV ) control (Ad-FP-EV) did not affect chondrocyte proliferation after 24 h. b Lentiviral cytomegalovirus (CMV)-LOXL2 transduction of HAC-OA compared to CMV-EV increased the mRNA levels of collagen type II α1 ( COL2A1 ) ( P = 0.043), sex determining region Y-box containing gene 9 ( SOX9 ) (* P < 0.001), and aggrecan ( ACAN ) ( P = 0.002). c Ad-RFP-LOXL2 transduction inhibited caspase 3/7 activity induced by TNF-α or IL-1β (* P = 0.006) compared to Ad-RFP-EV in HAC-normal ( HAC-N ) and HAC-OA cells in serum-free medium after 24 h. d Adenoviral transduction (Ad-RFP-LOXL2) HAC-OA in serum-free medium increased COL2A1 mRNA levels (* P < 0.001) compared to control (Ad-RFP-EV) in the presence of TNF-α (* P = 0.001)

Article Snippet: Cell lines were grown in chondrocyte growth medium (CGMTM BulletKitTM, CC-3216, Lonza Inc.) or in chondrocyte differentiation medium (CDM BulletKit, CC-3225, Lonza), both of which contain 5% fetal bovine serum (FBS), R3-insulin-like growth factor-1, human recombinant fibroblast growth factor-beta; insulin-transferrin-selenium (ITS), and gentamycin/amphotericin-B for in vitro studies.

Techniques: Transduction, Expressing, Plasmid Preparation, Control, Activity Assay

Journal: Arthritis Research & Therapy

Article Title: Anabolic role of lysyl oxidase like-2 in cartilage of knee and temporomandibular joints with osteoarthritis

doi: 10.1186/s13075-017-1388-8

Figure Lengend Snippet: LOXL2 overexpression differentially regulates genes in osteoarthritis (OA) chondrocytes. Human articular chondrocytes from OA cartilage (HAC-OA) were transduced with adenoviruses for transient expression of LOXL2 (Ad-RFP-LOXL2) or Ad-RFP-empty vector ( EV ), and total RNA extracts were subjected to global gene expression analysis. LOXL2 overexpression in HAC-OA induced the upregulated gene signature ( a ) and the downregulated gene signature ( b ). c Statistical significance for differential gene expression. d Exogenous LOXL2 expression increased anabolic markers in HAC-OA chondrocytes, whereas genes related to chondrocyte hypertrophy and catabolic markers were not changed as validated by RT-qPCR ( e ) (* P < 0.001; Student’s t test). LOXL2 transduction increased the expression of collagen type II α1 ( COL2A1 ) ( P = 0.063), Sex determining region Y-box containing gene 9 ( SOX9 ) ( P = 0.002), and aggrecan ( ACAN ) ( P = 0.003). NPPC natriuretic peptide C, CSPG chondroitin sulfate proteoglycan, GDF5 growth differentiation factor 5, TGFB3 transforming growth factor B3, IGFBP3 insulin-like growth factor binding protein, CABP5 calcium binding protein 5, SFRP1 secreted frizzled-related protein 1, TOBP1 topoisomerase II binding protein 1, VCAM1 vascular cell adhesion molecule 1, E2F8 E2F transcription factor 8, DKK1 Dickkopf WNT signaling inhibitor 1, CCNE2 cyclin E2, TPX microtubule-associated homolog, NAA N(alpha)-acetyltransferase, FDR false discovery rate, MMP matrix metalloproteinase

Article Snippet: Cell lines were grown in chondrocyte growth medium (CGMTM BulletKitTM, CC-3216, Lonza Inc.) or in chondrocyte differentiation medium (CDM BulletKit, CC-3225, Lonza), both of which contain 5% fetal bovine serum (FBS), R3-insulin-like growth factor-1, human recombinant fibroblast growth factor-beta; insulin-transferrin-selenium (ITS), and gentamycin/amphotericin-B for in vitro studies.

Techniques: Over Expression, Transduction, Expressing, Plasmid Preparation, Gene Expression, Quantitative RT-PCR, Binding Assay

Journal: Arthritis Research & Therapy

Article Title: Anabolic role of lysyl oxidase like-2 in cartilage of knee and temporomandibular joints with osteoarthritis

doi: 10.1186/s13075-017-1388-8

Figure Lengend Snippet: LOXL2 overexpression differentially regulates signaling pathways in osteoarthritis chondrocytes. Gene set enrichment analysis data shown are reactome signaling by transforming growth factor -β1 ( TGF-β1 ) receptor complex ( a ), Biocarta TNF-α receptor 1 pathway ( b ), reactome activation genes by ATF4 ( c ), reactome mitogen-activated protein kinase ( MAPK ) targets nuclear events mediated by MAPK ( d ), negative regulation of cell proliferation ( e ), regulation of angiogenesis ( f ), regulation of apoptosis ( g ), regulation of IkB kinase NF-ĸB cascade ( h ), and signaling by MiR-191 i compared to control (EV). j LOXL2 inhibits IL-1β-induced phospho-NF-ĸB and TGF-β1-induced phospho-ERK1/2 shown by western blot analysis, whereas TNF-α induced phospho-NF-ĸB and phospho-ERK1/2 are not affected. k Quantification is shown as fold-change normalized to β-actin (* P < 0.001; Student’s t test; n = 3)

Article Snippet: Cell lines were grown in chondrocyte growth medium (CGMTM BulletKitTM, CC-3216, Lonza Inc.) or in chondrocyte differentiation medium (CDM BulletKit, CC-3225, Lonza), both of which contain 5% fetal bovine serum (FBS), R3-insulin-like growth factor-1, human recombinant fibroblast growth factor-beta; insulin-transferrin-selenium (ITS), and gentamycin/amphotericin-B for in vitro studies.

Techniques: Over Expression, Protein-Protein interactions, Activation Assay, Control, Western Blot

Journal: Arthritis Research & Therapy

Article Title: Anabolic role of lysyl oxidase like-2 in cartilage of knee and temporomandibular joints with osteoarthritis

doi: 10.1186/s13075-017-1388-8

Figure Lengend Snippet: LOXL2 promotes anabolic responses in implants of the human knee and osteoarthritic temporomandibular joint ( TMJ-OA ) chondrocytes in vivo. a In vitro imaging systems ( IVIS ) imaging of human articular chondrocytes (HAC)/Matrigel implants in nude mice after local injection every week for 6 weeks with adenoviruses for transient expression of LOXL2 ( Ad-RFP-LOXL2 ) or Ad-RFP-empty vector ( EV ) ( Ad-RFP-empty ). Knee HAC-OA/Matrigel implants were injected weekly with Ad-RFP-LOXL2 or Ad-RFP-EV, harvested after 6 weeks, and analyzed by Safranin O/Fast Green, Alcian blue, and Masson’s trichrome staining ( b ) with relative quantification ( c ), and RT-qPCR using human primers ( d ). TMJ-OA/Matrigel implants were injected weekly with Ad-RFP-LOXL2 or Ad-RFP-EV, harvested after 6 weeks, and analyzed by Safranin O/Fast Green, Alcian blue, and Masson’s Trichrome staining ( e ) with relative quantification ( f ), and RT-qPCR ( g ) using human primers (n = 5/condition; * P < 0.05 and ** P < 0.01; Student’s t test). SOX 9 sex determining region Y-box containing gene 9, ACAN aggrecan, COL2A1 collagen type II α1, MMP matrix metalloproteinase

Article Snippet: Cell lines were grown in chondrocyte growth medium (CGMTM BulletKitTM, CC-3216, Lonza Inc.) or in chondrocyte differentiation medium (CDM BulletKit, CC-3225, Lonza), both of which contain 5% fetal bovine serum (FBS), R3-insulin-like growth factor-1, human recombinant fibroblast growth factor-beta; insulin-transferrin-selenium (ITS), and gentamycin/amphotericin-B for in vitro studies.

Techniques: In Vivo, In Vitro, Imaging, Injection, Expressing, Plasmid Preparation, Staining, Quantitative Proteomics, Quantitative RT-PCR

Journal: Arthritis Research & Therapy

Article Title: Anabolic role of lysyl oxidase like-2 in cartilage of knee and temporomandibular joints with osteoarthritis

doi: 10.1186/s13075-017-1388-8

Figure Lengend Snippet: LOXL2 co-expresses with sex determining region Y-box containing gene 9 ( SOX9 ) in the knee joint and osteoarthritic temporomandibular joint ( TMJ-OA ) chondrocytes implants in nude mice. a Human knee articular chondrocytes in osteoarthritis ( HAC-OA )/Matrigel implants or TMJ-OA/implants ( b ) from nude mice after treatments as described in Fig. . In the image , localization of proteins is shown with different colors such as LOXL2 ( red ), 4′,6-diamidino-2-phenylindole ( DAP I) ( blue ), and LOXL2 and SOX9 co-localization ( yellow ). EV empty vector

Article Snippet: Cell lines were grown in chondrocyte growth medium (CGMTM BulletKitTM, CC-3216, Lonza Inc.) or in chondrocyte differentiation medium (CDM BulletKit, CC-3225, Lonza), both of which contain 5% fetal bovine serum (FBS), R3-insulin-like growth factor-1, human recombinant fibroblast growth factor-beta; insulin-transferrin-selenium (ITS), and gentamycin/amphotericin-B for in vitro studies.

Techniques: Plasmid Preparation

Journal: Arthritis Research & Therapy

Article Title: Anabolic role of lysyl oxidase like-2 in cartilage of knee and temporomandibular joints with osteoarthritis

doi: 10.1186/s13075-017-1388-8

Figure Lengend Snippet: LOXL2 transduction inhibits phospho-SMAD2/3 and increases type II collagen ( COL2A1 ). a Human knee articular chondrocytes in osteoarthritis ( HAC-OA )/Matrigel implants and osteoarthritic temporomandibular joint ( TMJ-OA )/Matrigel implants b were stained with antibodies against RFP, LOXL2, phspho-SMAD2/3, or COL2A1 with respective 4′,6-diamidino-2-phenylindole ( DAPI ) staining

Article Snippet: Cell lines were grown in chondrocyte growth medium (CGMTM BulletKitTM, CC-3216, Lonza Inc.) or in chondrocyte differentiation medium (CDM BulletKit, CC-3225, Lonza), both of which contain 5% fetal bovine serum (FBS), R3-insulin-like growth factor-1, human recombinant fibroblast growth factor-beta; insulin-transferrin-selenium (ITS), and gentamycin/amphotericin-B for in vitro studies.

Techniques: Transduction, Staining