Diterbutyl phthalate attenuates osteoarthritis in ACLT mice via suppressing ERK/c-fos/NFATc1 pathway, and subsequently inhibiting subchondral osteoclast fusion.

Osteoarthritis (OA) is the most common arthritis with a rapidly increasing prevalence. Disease progression is irreversible, and there is no curative therapy available. During OA onset, abnormal mechanical loading leads to excessive osteoclastogenesis and bone resorption in subchondral bone, causing a rapid subchondral bone turnover, cyst formation, sclerosis, and finally, articular cartilage degeneration. Moreover, osteoclast-mediated angiogenesis and sensory innervation in subchondral bone result in abnormal vascularization and OA pain. The traditional Chinese medicine Panax notoginseng (PN; Sanqi) has long been used in treatment of bone diseases including osteoporosis, bone fracture, and OA. In this study we established two-dimensional/bone marrow mononuclear cell/cell membrane chromatography/time of flight mass spectrometry (2D/BMMC/CMC/TOFMS) technique and discovered that diterbutyl phthalate (DP) was the active constituent in PN inhibiting osteoclastogenesis. Then we explored the therapeutic effect of DP in an OA mouse model with anterior cruciate ligament transaction (ACLT). After ACLT was conducted, the mice received DP (5 mg·kg-1·d-1, ip) for 8 weeks. Whole knee joint tissues of the right limb were harvested at weeks 2, 4, and 8 for analysis. We showed that DP administration impeded overactivated osteoclastogenesis in subchondral bone and ameliorated articular cartilage deterioration. DP administration blunted aberrant H-type vessel formation in subchondral bone marrow and alleviated OA pain assessed in Von Frey test and thermal plantar test. In RANKL-induced RAW264.7 cells in vitro, DP (20 µM) retarded osteoclastogenesis by suppressing osteoclast fusion through inhibition of the ERK/c-fos/NFATc1 pathway. DP treatment also downregulated the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and d2 isoform of the vacuolar (H+) ATPase V0 domain (Atp6v0d2) in the cells. In conclusion, we demonstrate that DP prevents OA progression by inhibiting abnormal osteoclastogenesis and associated angiogenesis and neurogenesis in subchondral bone.

Positive effects of platelet-rich plasma (PRP) and a Sanguisorba officinalis polysaccharide on the proliferation and differentiation of anterior cruciate ligament (ACL) fibroblasts in vitro.

Context: Sanguisorba officinalis L. (Rosaceae), a famous traditional Chinese medicine. It was recently reported that its polysaccharide could facilitate collagen production.Objectives: We investigated the mechanism by which S. officinalis polysaccharide (SOWPa) and/or platelet-rich plasma (PRP) promote regenerative potential of anterior cruciate ligament (ACL) in vitro.Materials and methods: ACL fibroblasts were treated with SOWPa (25 and 100 mg/kg), PRP, PRP + SOWPa (25 and 100 mg/kg) or vehicle alone for 24, 48, or 72 h. Cell viability, migration ability and apoptosis were evaluated by MTT, transwell and flow cytometry, respectively. Western blot analysis was performed to assess associated protein expression.Results: PRP, SOWPa (100 mg/kg) or PRP + SOWPa (100 mg/kg) treatment for 72 h significantly improved the cell viability of ACL fibroblasts from 100 ± 7.5% (control) to 156.85 ± 12.82%, 188.08 ± 15.92%, and 223.67 ± 18.82%, respectively, which was evidenced by individual decreased apoptosis rate from 31.26 ± 2.35% (control) to 20.80 ± 1.89%, 18.01 ± 1.55% and 9.33 ± 0.78%. Furthermore, the motility of ACL fibroblasts was significantly improved with increased migrated cell number per field from 5 for control to 26 for PRP, 36 for SOWPa and 44 for PRP + SOWPa, respectively. Moreover, the protein expression of differentiation markers (RUNX2, ALP, BMP2 and Col I) and TLR-4 and phosphorylated p65 (p-p65) was inhibited by the above treatment.Discussion and conclusions: Data suggested that the addition of SOWPa to PRP increased the regenerative ability of ACL fibroblasts by blocking the TLR-4/NF-κB pathway.

Evaluation of Chondroprotective Activity of Channa striatus in Rabbit Osteoarthritis Model.

OBJECTIVES: The objective of the study is to evaluate the chondroprotective activity of Channa striatus (Channa) and glucosamine sulphate (glucosamine) on histomorphometric examinations, serum biomarker, and inflammatory mediators in experimental osteoarthritis (OA) rabbit model. DESIGN: Anterior cruciate ligament transection (ACLT) was performed to induce OA in thirty-three male New Zealand white rabbits and were randomly divided into three groups: Channa, glucosamine, and control group. The control group received drinking water and the Channa and glucosamine groups were orally administered with 51.4 mg/kg of Channa extract and 77.5 mg/kg of glucosamine sulphate in drinking water, respectively, for eight weeks and then sacrificed. The articular cartilage was evaluated macroscopically and histologically using semiquantitative and quantitative methods. Serum cartilage oligomeric matric protein (COMP), cyclooxygenase 2 (COX-2) enzyme, and prostaglandin E2 (PGE2) were also determined. RESULTS: Macroscopic analysis revealed that Channa group have a significantly lower severity grade of total macroscopic score compared to the control (p < 0.001) and glucosamine (p < 0.05) groups. Semiquantitative histology scoring showed that both Channa and glucosamine groups had lower severity grading of total histology score compared to the control group (p < 0.001). In comparison with the control, Channa group had lower histopathological changes in three compartments of the joint compared to glucosamine group which had lower histological scoring in two compartments only. The cartilage thickness, area, and roughness of both Channa (p < 0.05) and glucosamine (p < 0.05) groups were superior compared to the control group. However, the Channa group demonstrated significantly less cartilage roughness compared to the glucosamine group (p < 0.05). Serum COMP levels were lower in both Channa (p < 0.05) and glucosamine (p < 0.05) groups compared to the control group. CONCLUSION: Both oral administration of Channa extract and glucosamine exhibited chondroprotective action on an ACLT OA-induced rabbit model. However, Channa was superior to glucosamine in maintaining the structure of the cartilage.

Mechanism of Activating the Proprioceptive NT-3/TrkC Signalling Pathway by Reverse Intervention for the Anterior Cruciate Ligament-Hamstring Reflex Arc with Electroacupuncture.

The anterior cruciate ligament (ACL) is an important structure maintaining stability of the knee joints. Deficits in physical stability and the proprioceptive capabilities of the knee joints are observed, when the ACL is damaged. Additionally, a unilateral ACL injury can affect bilateral knee proprioception; therefore, proprioception of the ACL may play a key role in stability. Electroacupuncture therapy has a definite effect nerve regeneration. In this study, cynomolgus monkeys were randomly divided into 4 groups: the model control group, intervention of the injured knee with electroacupuncture (IIKE) group, intervention of the bilateral knees with electroacupuncture (IBKE) group, and the blank control group. The unilateral ACL injury model was developed in IIKE and IBKE groups; acupuncture points around the knees underwent intervention similarly in the IIKE and IBKE groups. Then, mRNA and protein expressions of NT-3 and TrkC in the dorsal root ganglion and of growth-associated protein-43 in the ACL increased according to reverse-transcription quantitative polymerase chain reaction and Western blotting results. Decreased incubations and increased amplitudes were found for somatosensory-evoked potentials and motor nerve conduction velocity. The finding indicates that electroacupuncture may play an important role in the recovery of proprioception in the ACL by activating the NT-3/TrkC signalling pathway.

Effects of biological factors and health condition on mercury and selenium concentrations in the cartilage, meniscus and anterior cruciate ligament.

Due the long-term nature of joint tissue remodeling processes, knee structures including meniscus and anterior cruciate ligament (ACL) can be a model for studying the bioaccumulation of mercury (Hg) and selenium (Se). The accumulation and retention of Hg in human tissues can have a negative effect on the proper functioning of homeostasis-affecting organisms. A factor of chronic poisoning with Hg forms is probably the Se:Hg ratio in tissues. Se:Hg molar ratios below one may increase Hg toxicity potentials, while molar ratios that approach or exceed one effectively may protect against Hg toxicity. Therefore, the aim of the study was to determine total mercury (THg), Se, and Se:THg molar ratios in the cartilage, meniscus and ACL of patients with osteoarthritis (OA) from northwestern Poland. In all studied samples (n=95), we observed higher Se than THg concentration. Taking into consideration the biological factors, we found significantly higher THg levels in the cartilage of women, patients under 65 years of age, patients without hypertension and in the ACL of patients with spinal degenerative disease. We found higher Se levels in the meniscus in women than in men. In all studied parts of the knee joint, we found the Se:THg molar ratio higher than one, which suggests that the joint forming structures are not much exposed to THg. Moreover the results reported here may provide a basis for establishing reference values for the meniscus and ACL in patients with OA who had undergone knee replacement surgery.

Inhibition of cartilage degradation and suppression of PGE2 and MMPs expression by pomegranate fruit extract in a model of posttraumatic osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is characterized by cartilage degradation in the affected joints. Pomegranate fruit extract (PFE) inhibits cartilage degradation in vitro. The aim of this study was to determine whether oral consumption of PFE inhibits disease progression in rabbits with surgically induced OA. METHODS: OA was surgically induced in the tibiofemoral joints of adult New Zealand White rabbits. In one group, animals were fed PFE in water for 8 wk postsurgery. In the second group, animals were fed PFE for 2 wk before surgery and for 8 wk postsurgery. Histologic assessment and scoring of the cartilage was per Osteoarthritis Research Society International guidelines. Gene expression and matrix metalloproteinases (MMP) activity were determined using quantitative reverse transcriptase polymerase chain reaction and fluorometric assay, respectively. Interleukin (IL)-1 ß, MMP-13, IL-6, prostaglandin (PG)E2, and type II collagen (COL2A1) levels in synovial fluid/plasma/culture media were quantified using enzyme-linked immunosorbent assay. Expression of active caspase-3 and poly (ADP-ribose) polymerase p85 was determined by immunohistochemistry. Effect of PFE and inhibitors of MMP-13, mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB was studied in IL-1 ß-stimulated rabbit articular chondrocytes. RESULTS: Safranin-O-staining and chondrocyte cluster formation was significantly reduced in the anterior cruciate ligament transaction plus PFE fed groups. Expression of MMP-3, MMP-9, and MMP-13 mRNA was higher in the cartilage of rabbits given water alone but was significantly lower in the animals fed PFE. PFE-fed rabbits had lower IL-6, MMP-13, and PGE2 levels in the synovial fluid and plasma, respectively, and showed higher expression of aggrecan and COL2A1 mRNA. Significantly higher numbers of chondrocytes were positive for markers of apoptosis in the joints of rabbits with OA given water only compared with those in the PFE-fed groups. PFE pretreatment significantly reduced IL-1 ß induced IL-6 and MMPs expression in rabbit articular chondrocytes. These effects were also mimicked using MMP-13, MAPK, and NF-κB inhibitors in IL-1 ß-stimulated rabbit chondrocytes. In an in vitro activity assay, PFE blocked the activity of MMP-13. Like MAPK and NF-κB inhibitors, PFE was also effective in inhibiting IL-1 ß-induced PGE2 production in rabbit chondrocytes. PFE also reversed the inhibitory effect of IL-1ß on COL2A1 mRNA and protein expression in IL-1 ß-stimulated rabbit chondrocytes. CONCLUSION: The present data highlight the chondroprotective effects of PFE oral consumption in a model of posttraumatic OA and suggest that PFE-derived compounds may have potential value in the management of OA.

Panax notoginseng saponins promote wound repair of anterior cruciate ligament through phosphorylation of PI3K, AKT and ERK.

Panax notoginseng saponins (PNS) are components derived from Chinese herb panax notoginseng and play important roles in the cure of wounds. However, how PNS plays this function is still unclear. In this study, we used MTT assay, wound healing assay, western blot, quantitative real time PCR and enzyme-linked immunosorbent assay to detect the effects of PNS on the proliferation, migration and expression of collagen and fibronectin of anterior cruciate ligament (ACL) fibroblasts as well as the underlying mechanism. We found that PNS promoted the proliferation and migration of ACL fibroblasts and increased the expression levels of collagen and fibronectin. Further mechanism study indicates that PNS might play its function through the phosphorylation of PI3K, AKT and ERK. This study provides a possible mechanism for the function of PNS and lays foundation for further study on the function of panax notoginseng.

Effects of low-level laser therapy on joint pain, synovitis, anabolic, and catabolic factors in a progressive osteoarthritis rabbit model.

The aim of this study was to investigate the effect of low-level laser therapy (LLLT) on short-term and long-term joint pain, synovitis, anabolic, and catabolic factors in the cartilage of a rabbit model with progressive osteoarthritis (OA) induced by anterior cruciate ligament transection (ACLT). A total of 160 New Zealand white rabbits were randomly assigned into two groups (ACLT group and LLLT group). All rabbits received ACLT surgery, and 2-, 4-, 6-, and 8-week treatment after the surgery, with 20 rabbits being tested biweekly over every study period. The LLLT group received LLLT with a helium-neon (He-Ne) laser (830 nm) of 1.5 J/cm(2) three times per week, and the ACLT group received placebo LLLT with the equipment switched off. Long-term and short-term pain was tested via weight-bearing asymmetry; synovitis was assessed histologically; and knee joint cartilage was evaluated by gross morphology, histology, and gene expression analysis of anabolic and catabolic factors. The histological assessment of pain and synovitis showed that at least 6-week intermittent irradiation of LLLT could relief knee pain and control synovium inflammation. Gross morphologic inspection and histological evaluation showed that 6 weeks of LLLT could decrease cartilage damage of medical femoral condyle and 8 weeks of LLLT could decrease cartilage damage of medical and lateral femoral condyles and medical tibial plateau. Gene expression analysis revealed two results: At least 6 weeks of LLLT could decrease production of catabolic factors, for example, interleukin 1ß (IL-1ß), inducible nitric oxide synthase (iNOS), and MMP-3, and slow down the loss of anabolic factors, mainly TIMP-1. Eight weeks of LLLT treatment could slow down the loss of collagen II, aggrecan, and anabolic factors, mainly transforming growth factor beta (TGF-ß). The study suggests that LLLT plays a protective role against cartilage degradation and synovitis in rabbits with progressive OA by virtue of the regulation of catabolic and anabolic factors in the cartilage.

Comparison of light-emitting diode wavelength on activity and migration of rabbit ACL cells.

The purpose of this study was to evaluate the biological response and gene expression of New Zealand White Rabbit anterior cruciate ligament (ACL) fibroblasts for different wave lengths of light-emitting diode (LED) irradiation. In other words, this study was undertaken to evaluate the effects of different wavelengths of LED irradiation on cell growth, expression of extracellular matrix and growth factors, migration, and expression of actin and integrin. Proliferation assay showed that red (630 nm, 9.5 J/cm(2)) and green LED (530 nm, 9.8 J/cm(2)) irradiated cells were more increased than control group but there was no difference between the control group and the blue LED (460 nm, 27 J/cm(2)) irradiated group. Moreover, the expression of insulin-like growth factor, transforming growth factor-beta (TGF-ß1), and collagen I were significantly increased in the red and green LED-irradiated group, but the expression of collagen was decreased in the blue LED-irradiated group. The results of staining showed that collagen and TGF-ß1 were weaker in the control group and blue LED-irradiated cells, but stronger in the red and green LED-irradiated cells. Also, in the red and green LED-irradiated group, the expression of actin and integrin was not changed compared to the control group, but the expression of actin and integrin was decreased in the blue irradiated group. This study revealed that irradiation with a wavelength of 460 nm (blue LED) is cytotoxic to ACL cells, but irradiation with nontoxic fluencies of 530 (green LED) and 630 nm (red LED) wavelengths induced cell growth in cultured ACL cells.

Extra-virgin olive oil diet and mild physical activity prevent cartilage degeneration in an osteoarthritis model: an in vivo and in vitro study on lubricin expression.

Mediterranean diet includes a relatively high fat consumption mostly from monounsaturated fatty acids mainly provided by olive oil, the principal source of culinary and dressing fat. The beneficial effects of olive oil have been widely studied and could be due to its phytochemicals, which have been shown to possess anti-inflammatory properties. Lubricin is a chondroprotective glycoprotein and it serves as a critical boundary lubricant between opposing cartilage surfaces. A joint injury causes an initial flare of cytokines, which decreases lubricin expression and predisposes to cartilage degeneration such as osteoarthritis. The aim of this study was to evaluate the role of extra-virgin olive oil diet and physical activity on inflammation and expression of lubricin in articular cartilage of rats after injury. In this study we used histomorphometric, histological, immunocytochemical, immunohistochemical, western blot and biochemical analysis for lubricin and interleukin-1 evaluations in the cartilage and in the synovial fluid. We report the beneficial effect of physical activity (treadmill training) and extra-virgin olive oil supplementation, on the articular cartilage. The effects of anterior cruciate ligament transection decrease drastically the expression of lubricin and increase the expression of interleukin-1 in rats, while after physical activity and extra-virgin olive oil supplemented diet, the values return to a normal level compared to the control group. With our results we can confirm the importance of the physical activity in conjunction with extra-virgin olive oil diet in medical therapy to prevent osteoarthritis disease in order to preserve the articular cartilage and then the entire joint.

Treatment with SiMiaoFang, an anti-arthritis chinese herbal formula, inhibits cartilage matrix degradation in osteoarthritis rat model.

A Chinese herbal preparation, SiMiaoFang (SMF), has been used clinically for treating arthralgia by virtue of its anti-inflammatory and pain-relieving activities. However, no evidence base links SMF to anti-osteoarthritis (OA), particularly its link to inhibiting cartilage matrix degradation. In this study, we undertook a characterization of anti-OA activity of SMF using an in vivo rat model induced by anterior cruciate ligament transection and medial meniscus resection (ACLT+MMx) together with in vitro studies with chondrocytes for further molecular characterization. ACLT+MMx rats were treated with SMF at doses of 0.63, 1.25, and 2.5 grams/kg per day for 6 weeks. SMF treatments significantly inhibited cartilage matrix degradation, as indicated by increasing proteoglycan and collagen content, particularly type II collagen expression in articular cartilage, decreasing CTX-II (collagen type II degradation marker), and increasing CPII (collagen type II synthesis marker) in circulation. Moreover, SMF suppressed synovial inflammation and inhibited release of interleukin-1ß (IL-1ß) and tumor necrosis factor-α in serum. The levels of serum prostaglandin E2 and nitric oxide productions were decreased via suppression of the production of cyclooxygenase-2 and inducible nitric oxide synthase, respectively. Importantly, SMF interfered with OA-augmented expression of matrix metalloproteinases (MMPs) -3 and -13 and aggrecanases (ADAMTS) -4 and -5, which are considered to be key enzymes in cartilage matrix degradation, and simultaneously augmented OA-reduced tissue inhibitors of metalloproteinases (TIMPs) -1 and -3 expression in the joints. The largest changes in these parameters were found at the highest dose. Meanwhile, SMF significantly decreased MMP-3 and -13 and increased TIMP-1 and -3 at mRNA and protein levels in IL-1ß-induced chondrocytes. These findings provide the first evidence that SMF effectively treats OA by inhibiting cartilage matrix degradation.

Effect of light-emitting diode (LED) therapy on the development of osteoarthritis (OA) in a rabbit model.

OBJECTIVE: The objective of this study was to evaluate whether light-emitting diodes (LEDs) could be effective in a noninvasive, therapeutic device for the treatment of osteoarthritic (OA) knee joints. DESIGN: Five weeks following the anterior cruciate ligament transection (ACLT) of mature New Zealand White rabbits, the animal knees were exposed to LED stimulation at intervals of 10 min/day, 5 days/week for 5 weeks in the experimental group (n=7). The device used high intensity red and infrared (IR) LEDs with a total amount of energy delivered to the skin of 2.4 J/cm(2). Animals were sacrificed at 9 weeks postoperatively. Femoral surface gross morphology was evaluated with a modified Outerbridge classification and mRNA expression of catabolic and anabolic markers from femoral condyle cartilage and synovial tissue was assessed using RT-PCR. A control group was harvested 9 weeks following untreated ACLT. RESULTS: Gross morphometry of the control group showed four Grade II, two Grade III and one Grade IV (average 2.6) condyles macroscopically. The experimental group showed two Grade I and five Grade II (average 1.7) (Table 1). mRNA expression of aggrecan in the cartilage showed no difference between the groups, however type II collagen expression increased in the experimental group compared with control. TNF-α expression was significantly decreased in the experimental group compared to control. CONCLUSIONS: There was general preservation of the articular surface and decreased levels of inflammation in the osteoarthritic joints with the application of LED therapy. This may provide potential application as a noninvasive treatment.

Characterization of the structure-function relationship at the ligament-to-bone interface.

Soft tissues such as ligaments and tendons integrate with bone through a fibrocartilaginous interface divided into noncalcified and calcified regions. This junction between distinct tissue types is frequently injured and not reestablished after surgical repair. Its regeneration is also limited by a lack of understanding of the structure-function relationship inherent at this complex interface. Therefore, focusing on the insertion site between the anterior cruciate ligament (ACL) and bone, the objectives of this study are: (i) to determine interface compressive mechanical properties, (ii) to characterize interface mineral presence and distribution, and (iii) to evaluate insertion site-dependent changes in mechanical properties and matrix mineral content. Interface mechanical properties were determined by coupling microcompression with optimized digital image correlation analysis, whereas mineral presence and distribution were characterized by energy dispersive x-ray analysis and backscattered scanning electron microscopy. Both region- and insertion-dependent changes in mechanical properties were found, with the calcified interface region exhibiting significantly greater compressive mechanical properties than the noncalcified region. Mineral presence was only detectable within the calcified interface and bone regions, and its distribution corresponds to region-dependent mechanical inhomogeneity. Additionally, the compressive mechanical properties of the tibial insertion were greater than those of the femoral. The interface structure-function relationship elucidated in this study provides critical insight for interface regeneration and the formation of complex tissue systems.

COL-3 inhibition of collagen fragmentation in ruptured cranial cruciate ligament explants from dogs with stifle arthritis.

Inhibition of collagen fragment generation in canine cranial cruciate ligament (CCL) explant cultures by the matrix metalloprotease inhibitor (6-demethyl)-6-deoxy-4-dedimethylamino tetracycline (COL-3) was studied. Cranial cruciate ligament specimens were collected from dogs with inflammatory stifle arthritis/CCL rupture and dogs with normal stifles. Explant cultures from each CCL specimen included one COL-3 treated explant and a baseline control; explants from 12 ruptured CCLs were prepared in triplicate and a protease inhibitor cocktail positive control was used. Explant supernatants were analyzed for generation of collagen fragments after two days. Treatment of ruptured CCL explants with 10(-4)M COL-3 decreased generation of collagen fragments. The extent of this inhibition was increased in explants treated with a protease inhibitor cocktail. Generation of collagen fragments was increased in ruptured CCLs, when compared with intact CCLs. It is concluded that generation of collagen fragments was increased in pathological ruptured CCL explants. This degradation could be significantly inhibited in vitro by 10(-4)M COL-3.