TGF-β1 upregulates the expression of hyaluronan synthase 2 and hyaluronan synthesis in culture models of equine articular chondrocytes

We investigated the effect of transforming growth factor beta 1 (TGF-β1) on equine hyaluronan synthase 2 (HAS2) gene expression and hyaluronan (HA) synthesis in culture models of articular chondrocytes. Equine chondrocytes were treated with TGF-β1 at different concentrations and times in monolayer cultures. In three-dimensional cultures, chondrocyte-seeded gelatin scaffolds were cultured in chondrogenic media containing 10 ng/mL of TGF-β1. The amounts of HA in conditioned media and in scaffolds were determined by enzyme-linked immunosorbent assays. HAS2 mRNA expression was analyzed by semi-quantitative reverse transcription polymerase chain reaction. The uronic acid content and DNA content of the scaffolds were measured by using colorimetric and Hoechst 33258 assays, respectively. Cell proliferation was evaluated by using the alamarBlue assay. Scanning electron microscopy (SEM), histology, and immunohistochemistry were used for microscopic analysis of the samples. The upregulation of HAS2 mRNA levels by TGF-β1 stimulation was dose and time dependent. TGF-β1 was shown to enhance HA and uronic acid content in the scaffolds. Cell proliferation and DNA content were significantly lower in TGF-β1 treatments. SEM and histological results revealed the formation of a cartilaginous-like extracellular matrix in the TGF-β1-treated scaffolds. Together, our results suggest that TGF-β1 has a stimulatory effect on equine chondrocytes, enhancing HA synthesis and promoting cartilage matrix generation.

Raised Chondroitin Sulphate (WF6 Epitope) Levels around Maxillary Molars and Miniscrew Implants during Orthodontic Molar Intrusion

This study aimed to monitor chondroitin sulphate (CS; WF6 epitope) levels in crevicular fluid around maxillary molars and miniscrew implants during orthodontic molar intrusion.              One miniscrew implant was placed in the midpalatal area of each of ten patients with open skeletal configurations, who required orthodontic molar intrusion, and two Sentalloy® closed-coil springs (100 g) were used for molar intrusion.  Gingival crevicular fluid (GCF) around experimental and control molars, and peri-miniscrew implant crevicular fluid (PMICF) were collected before and during load application. Competitive ELISA with monoclonal antibody WF6 and colorimetric protein assay were used to detect CS (WF6 epitope), and total protein concentration, respectively.The results showed that the median CS (WF6 epitope) levels around experimental molars during the loaded period (12 weeks) (2.099 ng/µg of total protein) and those during each two-week interval of the loaded period (1.952, 1.854, 2.604, 2.414, 1.844, 1.44 ng/ µg of total protein respectively) were significantly greater than those during the unloaded period (2 weeks) (0.832 ng/ µg of total protein) (P

ZINGIBER CASSUMUNAR ROXB. INHIBITS HYALURONAN PRODUCTION IN HUMAN ORAL FIBROBLASTS

Zingiber cassumunar Roxb. was previously shown to possess potent anti-inflammatory activity. We investigated the effect of its ethanol extract on hyaluronan (HA), an extracellular matrix compound with proinflammatory activity synthesis in human oral fibroblasts. Cultured fi broblasts were treated at various times with different concentrations of the extract (0.1-50 μg/mL) with or without retinoic acid (RA; 10 μM), or left untreated as a control. Culture medium was analyzed for HA quantities by the ELISA-based assay. Total RNA was harvested and RT-PCR analyses were performed to determine mRNA expression of hyaluronan synthase (HAS) -1, -2, and –3. Z. cassumunar extract, at 25 and 50 μg/mL, with or without 10 μM of RA, significantly decreased HA levels (p \< 0.05). Consistent with decreased HA levels, mRNA expression of HAS-2, but not HAS-1 or HAS-3, was selectively down-regulated by the extract. Collectively, these data indicate that the ethanol extract of Z. cassumunar inhibits HA synthesis in human oral fibroblasts, which may be involved in chronic inflammatory disorders, particularly in the oral cavity.

The effect of doxycycline on canine hip osteoarthritis: design of a 6-months clinical trial

Twenty-five dogs were included in a randomized, double-blind trial to assess the efficacy of doxycycline (DOX) orally administered twice a day at 4 mg/kg/day (n = 12) for the treatment of osteoarthritis of the hip. Chondroitin sulfate (CS; 525 mg/day) was used as a positive control (n = 13). Dogs were re-examined monthly for 6 months after initiation of treatment. The assessment protocol included clinical score, radiographic findings and serum osteoarthritis biomarkers. Dogs treated with DOX showed statistically significant improvements (p < 0.05) in lameness, joint mobility, pain on palpation, weight-bearing and overall score at 2, 6, 4, 4 and 4 months, respectively, after treatment. Biomarker levels of CS-WF6 epitope and hyaluronan were significantly increased and decreased (p < 0.05) at 2 and 3 months after treatment compared to pretreatment. These results showed that DOX had a positive therapeutic effect in dogs with osteoarthritis.

Evaluation of serum chondroitin sulfate and hyaluronan: biomarkers for osteoarthritis in canine hip dysplasia

Hip dysplasia (HD) is one of the most important bone and joint diseases in dogs. Making the radiographic diagnosis is sometime possible when the disease has markedly progressed. Chondroitin sulfate (CS) and hyaluronan (HA) are the most important cartilage biomolecules that are elevated in the serum taken from dogs with osteoarthritis. The serum CS and HA can be detected by an ELISA technique, with using monoclonal antibodies against CS epitope 3B3 and WF6 and the HA chain as the primary antibodies. The aim of this study was to compare the levels of serum CS (both epitopes) and HA in non-HD and HD dogs. All 123 dogs were categorized into 2 groups. The non-HD group was composed of 98 healthy dogs, while the HD group was comprised of 25 HD dogs. Blood samples were collected for analyzing the serum CS and HA levels with using the ELISA technique. The results showed that the average serum level of the CS epitope WF6 in the HD group (2,594 +/- 3,036.10 ng/ml) was significantly higher than that in the non-HD group (465 +/- 208.97 ng/ml) (p < 0.01) while the epitope 3B3 in the HD group (105 +/- 100.05 ng/ml) was significantly lower than that in the non-HD group (136 +/- 142.03 ng/ml) (p < 0.05). The amount of serum HA in the HD group (134.74 +/- 59.71 ng/ml) was lower than that in the non HD group (245.45 +/- 97.84 ng/ml) (p < 0.05). The results indicate that the serum CS and HA levels might be used as biomarkers for osteoarthritis in HD dogs.