Rat Knee Cartilage Research Articles

MicroRNA-27b targets CBFB to inhibit differentiation of human bone marrow mesenchymal stem cells into hypertrophic chondrocytes

BackgroundHuman bone marrow-derived mesenchymal stem cells (hBMSCs) have chondrocyte differentiation potential and are considered to be a cell source for cell-transplantation-mediated repair of cartilage defects, including those associated with osteoarthritis (OA). However, chondrocyte hypertrophic differentiation is a major obstacle for the application of hBMSCs in articular cartilage defect treatment. We have previously shown that microRNA-27b (miR-27b) inhibits hypertrophy of chondrocytes from rat knee cartilage. In this study, we investigated the role of miR-27b in chondrocyte hypertrophic differentiation of hBMSCs.MethodsChondrogenic marker and microRNA expression in hBMSC chondrogenic pellets were evaluated using RT-qPCR and immunohistochemistry. The hBMSCs were transfected with miR-27b before inducing differentiation. Gene and protein expression levels were analyzed using RT-qPCR and western blot. Coimmunoprecipitation was used to confirm interaction between CBFB and RUNX2. Luciferase reporter assays were used to demonstrate that CBFB is a miR-27b target. Chondrogenic differentiation was evaluated in hBMSCs treated with shRNA targeting CBFB. Chondrogenic hBMSC pellets overexpressing miR-27b were implanted into cartilage lesions in model rats; therapeutic effects were assessed based on histology and immunohistochemistry.ResultsThe hBMSCs showed typical MSC differentiation potentials. During chondrogenic differentiation, collagen 2 and 10 (COL2 and COL10), SOX9, and RUNX2 expression was upregulated. Expression of miR-140, miR-143, and miR-181a increased over time, whereas miR-27b and miR-221 were downregulated. Cartilage derived from hBMSC and overexpressing miR-27b exhibited higher expression of COL2 and SOX9, but lower expression of COL10, RUNX2, and CBFB than did the control cartilage. CBFB and RUNX2 formed a complex, and CBFB was identified as a novel miR-27b target. CBFB knockdown by shRNA during hBMSC chondrogenic differentiation led to significantly increased COL2 and SOX9 expression and decreased COL10 expression. Finally, miR-27b-overexpressing hBMSC chondrogenic pellets had better hyaline cartilage morphology and reduced expression of hypertrophic markers and tend to increase repair efficacy in vivo.ConclusionMiR-27b plays an important role in preventing hypertrophic chondrogenesis of hBMSCs by targeting CBFB and is essential for maintaining a hyaline cartilage state. This study provides new insights into the mechanism of hBMSC chondrocyte differentiation and will aid in the development of strategies for treating cartilage injury based on hBMSC transplantation.

Effects of in vivo cyclic compressive loading on the distribution of local Col2 and superficial lubricin in rat knee cartilage.

This study aimed to examine the effects of an episode of in vivo cyclic loading on rat knee articular cartilage (AC) under medium-term observation, while also investigating relevant factors associated with the progression of post-traumatic osteoarthritis (PTOA). Twelve-week-old Wistar rats underwent one episode comprising 60 cycles of 20 N or 50 N dynamic compression on the right knee joint. Spatiotemporal changes in the AC after loading were evaluated using histology and immunohistochemistry at 3 days and 1, 2, 4, and 8 weeks after loading (n = 6 for each condition). Chondrocyte vitality was assessed at 1, 3, 6, and 12 hours after loading (n = 2 for each condition). A localized AC lesion on the lateral femoral condyle was confirmed in all subjects. The surface and intermediate cartilage in the affected area degenerated after loading, but the calcified cartilage remained intact. Expression of type II collagen in the lesion cartilage was upregulated after loading, whereas the superficial lubricin layer was eroded in response to cyclic compression. However, the distribution of superficial lubricin gradually recovered to the normal level 4 weeks after loading-induced injury. We confirmed that 60 repetitions of cyclic loading exceeding 20 N could result in cartilage damage in the rat knee. Endogenous repairs in well-structured joints work well to rebuild protective layers on the lesion cartilage surface, which may be the latent factor delaying the progression of PTOA.

Cytokines profile in knee cartilage of rats during monoiodoacetate-induced osteoarthritis and administration of probiotic

Cytokines profile in knee cartilage of rats during monoiodoacetate-induced osteoarthritis and administration of probiotic

A2A adenosine receptor stimulation regenerates cartilage in osteoarthritis animal model

Purpose: Many studies have been shown that obesity along with joint injury is one of the most common risk factor in the development of osteoarthritis (OA). In a previous study we described that intra-articular injections of liposomal preparations of adenosine completely prevent progression and reverse cartilage loss in post-traumatic OA. TGF-β signaling plays dual and opposing roles in cartilage health and chondrocyte life depending on the signals activated downstream. Activation of downstream signaling pathways for TGF-β leading to localization of phospho-SMAD2/3 associated with maintenance of cartilage. In contrast nuclear localization of phospho-SMAD1/5/8 results in chondrocyte hypertrophy. Here we report that intraarticular injections of liposomal adenosine and A2AR agonist reverses OA in a post-traumatic OA model in rat and in an obesity related mice model. We moreover explore the role of TGF-β signaling in this phenomenon. Methods: Obesity-induced OA model: C57Bl6 mice (5-6 for each group, 12 weeks old) were fed a 60% fat diet (HFF mice) for 3months, after which received intraarticular injections (10 μl) of empty liposomes (Lipo) or liposomes containing the A2AR agonist CGS21680 (Lipo-CGS) or Adenosine (Lipo-Ado) into the knee every 10 days for 4 injections. Post-traumatic OA (PTOA) was induced in Sprague Dawley rats following non-surgical rupture of anterior cruciate ligament (ACL). Four weeks later rats were injected in the knee with 100ul of saline, Lipo or Lipo-CGS every 10 days (6 injections). RNA was isolated from chondrocytes in knee cartilage of rats treated as described above (3 from each group X 3 replicates) and subjected to RNAseq analysis. TC28a2 human chondrocyte cell line was used for in vitro experiments. Results: Lipo-CGS and Lipo-Ado reversed OA in the obesity and post traumatic OA model. Mouse knees had an OARSI score of 5.17±1.84 before treatment. Treatment with LIPO-Ado and lipo-CGS decreased OA severity (OARSI score 1.33±0.81 and 1.83±0.98, respectively, p<0.001 vs pre-treatment; figure 1). In the PTOA model the OARSI score significantly decreases after Lipo-CGS and Lipo-Ado treatment compare to the saline group (OARSI: 1.28±0.39; 1.42±0.69; 2.97±0.0.75 respectively; p<0.05). Analysis of the transcriptome suggests that the treatment of Lipo-CGS promotes the up-regulation of genes involved in proliferation process and downregulations of genes responsible of apoptosis, cartilage catabolism and chondrocyte hypertrophy (Figure 2). TGF-β expression was increased in deep layers of cartilage in the Lipo-CGS-treated rats and there was notable nuclear localization of phospho-SMAD2/3 in these chondrocytes. In contrast, phospho-SMAD1/5/8 was expressed in the nuclei of chondrocytes in the saline and LIPO-treated rats but not in the LIPO-CGS treated rats. Identical changes were observed in the knees of obese mice. To determine whether the effect of A2AR stimulation on TGF-β signaling was direct or indirect we studied the effect of CGS21680 on nuclear phospho-SMAD expression in TC28a2 cells and found that CGS21680 increased nuclear phospho-SMAD2/3 and reduced nuclear phospho-SMAD1/5/8, as detected by immunofluorescence. Conclusions: Administration of an A2AR agonist to established OA knees reverses OA in rats and mice and shifts TGF-β signaling from ALK1/SMAD1/5/8 to ALK5/SMAD2/3 in OA chondrocytes after activation of A2AR in 2 OA animal models.

Synovium extra cellular matrices seeded with transduced mesenchymal stem cells stimulate chondrocyte maturation in vitro and cartilage healing in clinically-induced rat-knee lesions in vivo.

Osteoarthritis (OA) is a progressive disease associated with cartilage injury and its inherently limited repair capability. Synovium-based cellular constructs (sConstructs) are proposed as possible treatments. Equine sConstructs were produced from decellularized synovium-based extracellular matrix scaffolds (sECM) seeded with synovium-derived mesenchymal stem cells (sMSC), and engineered to express green fluorescent protein (GFP), or bone morphogenetic protein-2 (BMP-2). Survival, distribution, and chondrogenic potential of the sConstructs in vitro and in vivo were assessed. sConstructs in co-culture with chondrocytes increased chondrocyte proliferation, viability, and Col II production, greatest in BMP-2-sConstructs. Chondrocyte presence increased the production of hyaluronic acid (HA), proteoglycan (PG), and BMP-2 by the sConstructs in a positive feedback loop. sECM alone, or GFP- or BMP-2-sConstructs were implanted in synovium adjacent to clinically created full-thickness rat-knee cartilage lesions. At 5 weeks, the lesion area and implants were resected. Gross anatomy, adjacent articulate cartilage growth and subchondral bone repair were scored; and peripheral, central and cartilage lesion measurements taken. For all scores and measurements, sConstruct implants were significantly greater than controls, greatest with the BMP-2-sConstructs. Immunohistochemistry demonstrated migration of endogenous cells into the sECM, with greater cellularity in the constructs with intense positive GFP staining confirming engraftment of implanted sMSC and continued gene expression. In summary, exposing cartilage to sConstructs was chondrogenic in vitro and in vivo, and resulted in substantially increased growth in vivo. This effect was mediated, in part, by soluble ECM and cell factors and upregulation of anabolic growth proteins, such as BMP-2. This work is “proof of concept” that sConstructs surgically implanted adjacent to cartilage damage can significantly improve cartilage and subchondral bone repair, and potentially prevent the progression of OA.

A novel, self-assembled artificial cartilage-hydroxyapatite conjugate for combined articular cartilage and subchondral bone repair: histopathological analysis of cartilage tissue engineering in rat knee joints.

PurposeWe previously created a self-assembled cartilage-like complex in vitro from only three cartilage components, hyaluronic acid (HA), aggrecan (AG) and type II collagen, without other materials such as cross-linking agents. Based on this self-organized AG/HA/collagen complex, we have created three novel types of biphasic cartilage and bone-like scaffolds combined with hydroxyapatite (HAP) for osteochondral tissue engineering. These scaffolds have been developed from self-assembled cartilage component molecules and HAP at the nanometer scale by manipulating the intermolecular relations.Patients and methodsThe surface structure of each self-organized biphasic cartilage and bone-like scaffold was evaluated by scanning electron microscopy, whereas the viscoelasticity was also analyzed in vitro. Three types of artificial cartilage–HAP conjugates were implanted into an osteochondral defect in rat knee joints, and bone and cartilage tissues of the implanted site were examined 4 and 8 weeks after implantation. The tissues were examined histopathologically to evaluate the effects of the implantation on the articular cartilage and subchondral bone tissues.ResultsOur in vitro and in vivo data reveal that the self-organized biphasic cartilage and bone-like scaffold conjugated with HAP are superior to the scaffold with no HAP in both cartilage regeneration and subchondral bone regeneration.ConclusionOur present study indicates that the self-organized biphasic cartilage and bone-like scaffold, which is conjugated with an HAP layer, may have potential not only to repair articular cartilage defects but also to ameliorate the degeneration of subchondral bone in the diseases with osteochondral defect.

Increased Substance P Immunoreactivity in Ipsilateral Knee Cartilage of Rats Exposed to Lumbar Spine Injury

The present study aimed to investigate whether experimentally induced lumbar facet-joint OA lead to degenerative changes and enhanced SP expression within the ipsilateral neurosegmentally linked tibiofemoral cartilage. Adult male Sprague-Dawley rats were assigned to left side L5-L6 facet mechanical compression injury (surgery) (n = 6), L5-L6 facet exposure with no compression (sham) (n = 5), or naïve (no surgery) (n = 4) groups. The morphology of the tibiofemoral articular cartilage was assessed using a modified Mankin scoring system. Immunohistochemistry was used to examine the density of chondrocytes stained positive for SP (cells/cm2) in the ipsilateral tibiofemoral cartilage at 28 days postintervention. Tibiofemoral cartilage in the surgery group showed consistent loss of superficial zone chondrocytes, mild roughening of the articular surface and occasional chondrocyte clusters as well as a greater density of SP mainly in the superficial cartilage zone compared with sham and naïve groups, although they also had a basic SP-expression. Our results support the hypothesis that neurogenic mechanisms may mediate the spread of SP to neurosegmentally linked heterologous joints affecting the distal cartilage homeostasis. These findings contribute additional insight into the potential role of neurogenic inflammation with implications in the pathophysiology of chronic inflammatory joint disease and OA.

Expression of dishevelled-2 in cartilage of rheumatoid arthritis and its effect on cartilage destruction

Objective: To study the expression of dishevelled-2 (DVL2) in rheumatoid arthritis cartilage and its effect on cartilage destruction. Methods: Cartilage DVL2 expression in rat models of rheumatoid arthritis (RA), osteoarthritis(OA) and collagen-induced arthritis(CIA) were tested by Western blotting. DVL2 overexpressed lentivirus was transfected into the knee of CIA rats. Primary chondrocytes were extracted from RA patients by knee arthroplasty and transfected with DVL2 overexpressed lentivirus. Gene expression of related inflammation related cytokines was detected by real-time polymerase chain reaction (PCR) . Results: Compared with knee articular cartilage in OA patients and normal rats, DVL2 protein was highly expressed in knee cartilage of RA patients and CIA rats (P values 0.041 and 0.032, respectively). DVL2 did not significantly affect the destruction of knee cartilage in CIA rats (P=0.885). DVL2 overexpression in chondrocytes enhanced gene expression of cyclo-oxygenase-2 (COX-2), inducible nitric oxide synthase (NOS), matrix metalloproteinase (MMP) 2, MMP-3, and MMP-9, which could be more pronounced when tumor necrosis factor alpha was added. Conclusions: DVL2 is highly expressed in RA articular cartilage and promotes the expression of inflammatory cytokines and MMP gene in chondrocytes by activating Wnt/β-catenin pathway, which involves in the destruction of articular cartilage in RA.

The effects of long-term low selenium diet on the expression of CHST-3, CHST-12 and UST in knee cartilage of growing rats

ObjectivesTo investigate the effect of low selenium diet on rat´s knee cartilage and expression of chondroitin sulfate (CS) sulfated enzymes in articular and epiphyseal-plate cartilage of rats’ femur and tibia. MethodsTwenty-four SD rats were randomly divided into two groups with six female and six male in each group: control group (selenium 0.18 mg/kg), and low selenium group (selenium 0.02 mg/kg). After 109 days, the rats were sacrificed. The ultrastructural changes in chondrocytes of rat knee cartilage were observed by transmission electron microscopy (TEM). The morphology and pathology changes of knee cartilage were examined by hematoxylin-eosin (HE) and toluidine blue (TB) staining. The localization and expression of enzymes involved in CS sulfation, including chondroitin 6-O-sulfotransferase 1 (CHST-3), chondroitin 4-O-sulfotransferase 2 (CHST-12) and uronyl 2-O-sulfotransferase (UST) were examined by immunohistochemical staining and semi-quantitative analysis. ResultsIn low selenium group, ultrastructural changes of chondrocytes were observed in articular cartilage of femur (AF), articular cartilage of tibia (AT), epiphyseal-plate cartilage of femur (EF) and epiphyseal-plate cartilage of tibia (ET); however, no significant changes in chondrocytes number were observed in the above AF, AT, EF or ET. Moreover, reduced thickness of cartilage layer in AF, EF and ET was detected along with reduced staining areas of sulfated glycosaminoglycan in EF and ET in low selenium group. In addition, positive staining rate of CHST-3 was lower in AF, AT and EF, while positive staining rates of CHST-12 and UST were lower in AF, AT, EF and ET in low selenium group when compared with control group. ConclusionsLow selenium undermines the ultrastructure of chondrocytes, inhibits the normal development of cartilage and the expression of CS sulfated enzymes.

Procaine and saline have similar effects on articular cartilage and synovium in rat knee

BackgroundIntra-articular local anaesthetics are widely used for providing postoperative analgesia and decreasing the need for opioids. Procaine has proven positive effects in carpal tunnel syndrome and chondromalacia patella. However, the effect of procaine on articular cartilage has not yet been studied. The aim of this study was to evaluate the effects of intra-articular procaine injection on the articular cartilage and the synovium.MethodsTwenty adult Sprague-Dawley rats were enrolled in the study. After providing anaesthesia and aseptic conditions, 0.25 ml of 10% procaine was injected to the right knee joint, and 0.25 ml of normal saline (as control group) was injected to the left knee joint. Knee joint samples were obtained from four rats in each group after appropriate euthanasia on days 1, 2, 7, 14 and 21. The histological sections of the articular and periarticular regions and the synovium were evaluated by two histologists, and inflammatory changes were graded according to a five-point scale in a blinded manner. The apoptosis of chondrocytes was determined by the caspase-3 indirect immunoperoxidase method.ResultsThere were no significant differences in inflammation between procaine and saline groups at any of the time intervals. Slight inflammatory infiltration due to injection was seen in both groups on the 1st day. Haemorrhage was observed in both groups at days 1 and 2, and the difference between groups was not found to be significant. No significant difference was detected in the percentage of apoptotic chondrocytes between groups at any of the time intervals.ConclusionsInjection of procaine seems safe to use intra-articularly based on this in vivo study on rat knee cartilage. However, further studies investigating both the analgesic and histopathological effects of procaine on damaged articular cartilage and synovium models are needed.

Link Protein N-Terminal Peptide as a Potential Stimulating Factor for Stem Cell-Based Cartilage Regeneration.

Background Link protein N-terminal peptide (LPP) in extracellular matrix (ECM) of cartilage could induce synthesis of proteoglycans and collagen type II in cartilaginous cells. Cartilage stem/progenitor cells (CSPCs), the endogenous stem cells in cartilage, are important in cartilage degeneration and regeneration. We hypothesized that LPP could be a stimulator for stem cell-based cartilage regeneration by affecting biological behaviors of CSPC. Methods CSPCs were isolated from rat knee cartilage. We evaluated the promoting effect of LPP on proliferation, migration, and chondrogenic differentiation of CSPCs. The chondrogenic differentiation-related genes and proteins were quantitated. Three-dimensional culture of CSPC was conducted in the presence of TGF-β3 or LPP, and the harvested pellets were analyzed to assess the function of LPP on cartilage regeneration. Results LPP stimulated the proliferation of CSPC and accelerated the site-directional migration. Higher expression of SOX9, collagen II, and aggrecan were demonstrated in CSPCs treated with LPP. The pellets treated with LPP showed more distinct characteristics of chondroid differentiation than those with TGF-β3. Conclusion LPP showed application prospect in cartilage regeneration medicine by stimulating proliferation, migration, and chondrogenic differentiation of cartilage stem/progenitor cells.

Guidelines for an optimized indentation protocol for measurement of cartilage stiffness: The effects of spatial variation and indentation parameters

Mechanical properties of articular cartilage that are vital to its function are often determined by indentation tests, which can be performed at different scales. Cartilage tissue exhibits various types of structural, geometrical, and spatial variations that pose strict demands on indentation protocols. This study aims to define a reproducible micro-indentation protocol for measuring the effective (average) stiffness of the cartilage surface in a region around 1mm2. We elucidated how different parameters such as indenter size, indenter depth, and the location of the indentation influence the effective elastic modulus measured in micrometer scale on rat knee cartilage. When an indentation was performed (50μm radial probe, ≈10μm indentation depth) at exactly the same location, the variability was less than 10%, even with a recovery period of 30s. However, there was a high spatial variation and a small change of around 60μm in location could change the modulus values up to as much as 10-20 fold. The effective elastic modulus of cartilage surface layer cannot therefore be reproducibly determined from a few indentations on a cartilage sample, and requires at least 144 (12×12) indentations for a soft spherical probe with a 50μm radius. With higher depths, the spatial variation is slightly lower, allowing slightly lower number of indentations (≈80 measurements or a 9×9 frame) to provide a representative elastic modulus. Using this protocol, we determined an elastic modulus of 2.6±1.9N/mm2 at the medial side versus a higher modulus of 4.2±2.6N/mm2 at the lateral side of the tibia of 12 weeks old Wistar rats. Optimized indentation protocols similar to the one presented here are required for revealing such variations in the mechanical properties of cartilage with anatomical location.

Achyranthes bidentata polysaccharides activate the Wnt/β-catenin signaling pathway to promote chondrocyte proliferation.

Achyranthes bidentata polysaccharides (ABPS) are the active components of Radix Achyranthis Bidentatae (AB), which has been extensively used in Traditional Chinese medicine (TCM) in the treatment of osteoarthritis (OA). Our previous study provided evidence that ABPS regulated the G1/S transition to promote chondrocyte proliferation. However, the precise mechanisms involved remain to be elucidated. In the present study, we aimed to investigate the effects of ABPS on the Wnt/β‑catenin signaling pathway in chondrocytes. Chondrocytes, obtained from the knee cartilage of Sprague-Dawley rats, were identified by type II collagen immunohistochemistry. ABPS upregulated the expression of Wnt-4, Frizzled-2, β-catenin and cyclin D1, and downregulated the expression of glycogen synthase kinase 3β (GSK-3β), as shown by reverse transcription PCR (RT-PCR) and western blot analysis. Using immunofluorescence, we also found that ABPS induced β-catenin nuclear translocation. Importantly, the expression of β-catenin and cyclin D1 was partly inhibited by Dickkopf-1 (DKK-1), an inhibitor of the Wnt/β-catenin signaling pathway. In addition, we found that ABPS increased the expression of type II collagen in chondrocytes. These results suggest that ABPS promote chondrocyte proliferation by activating the Wnt/β-catenin signaling pathway.

Sustained release of PTH(1–34) from PLGA microspheres suppresses osteoarthritis progression in rats

We previously reported that PTH(1–34) inhibits the terminal differentiation of articular chondrocytes and, in turn, suppresses the progression of osteoarthritis (OA). However, this treatment requires an injection of PTH(1–34) once every 3days over the treatment period. In this study, we studied the effect of sustained administration of PTH(1–34) in a papain-induced OA rat model. We developed an effective controlled-release system for prolonging the treatment duration of an intra-articular injection for OA treatment in rats. The effects of released PTH(1–34) from PLGA(65:35)-encapsulated PTH(1–34) microspheres (PTH/PLGA) on papain-induced OA in rat knees were studied. Microsphere morphology was observed in vitro by scanning electron microscopy, and microsphere size was determined with a particle size analyzer. The PTH(1–34) encapsulation efficiency and release profile, as well as the toxicity of PTH/PLGA, were examined. The bioactivity of released PTH(1–34) was tested by examining cAMP levels in MC3T3E1 cells. In vivo, we evaluated the changes of localized GAG, Col II, and Col X in the articular cartilage of rat knees. Our results demonstrated that the surface of the PLGA microspheres was smooth, and the size of the microspheres was in the range of 51–127μm. PTH/PLGA microspheres sustainably released PTH(1–34) for 19days with a concentration range of 0.01–100nM that covered the expected concentration of 10nM at 37°C. The cAMP levels of MC3T3E1 cells were elevated in the response to released PTH(1–34) from PTH/PLGA microspheres, indicating that the released PTH(1–34) is bioactive. Most importantly, intra-articular treatment with either PTH(1–34) (0.1–100nM) 3days/injection or PTH/PLGA microspheres (15days/injection) for 5weeks revealed the similar effect on suppressing papain-induced OA changes (decreasing GAG and Col II and increasing Col X) in rat knee cartilage. The effect of PTH/PLGA microspheres on suppressing OA progression was similar to that of a once-every-three-day injection of PTH(1–34), indicating that both the sustained and intermittent action of PTH(1–34) effectively suppress OA progression. The developed PLGA microspheres with sustained release and long-term effect may be potent carriers for PTH(1–34) used to treat early OA.

A Dedicated Two-Channel Phased-Array Receiver Coil for High-Resolution MRI of the Rat Knee Cartilage at 7 T

In the field of small animal studies, the array coil imaging has become increasingly important. In this paper, a dedicated two-channel array coil operating at 300 MHz (7 T) for high-resolution MRI (HR-MRI) of the rat knee cartilage is presented. The average gain in signal-to-noise ratio (SNR) compared to a 15-mm multipurpose surface coil was 2.2. This SNR gain was used to improve the spatial resolution of 3-D acquisitions by decreasing the voxel size from 59 x 59 x 156 microm(3) to 51 x 51 x 94 microm(3) without time penalty. Also, a set of two array coils was used to perform a simultaneous acquisition of both knee joints of a rat, maintaining the same scanning time without SNR or spatial resolution degradation compared to the single knee joint acquisition. This two-channel array coil is a key element to perform HR-MRI and extract cartilage morphological parameters such as thickness and volume.

The response to oestrogen deprivation of the cartilage collagen degradation marker, CTX-II, is unique compared with other markers of collagen turnover

IntroductionThe urinary level of the type II collagen degradation marker CTX-II is increased in postmenopausal women and in ovariectomised rats, suggesting that oestrogen deprivation induces cartilage breakdown. Here we investigate whether this response to oestrogen is also true for other type II collagen turnover markers known to be affected in osteoarthritis, and whether it relates to its presence in specific areas of cartilage tissue.MethodsThe type II collagen degradation markers CTX-II and Helix-II were measured in the body fluids of premenopausal and postmenopausal women and in those of ovariectomised rats receiving oestrogen or not. Levels of PIIANP, a marker of type II collagen synthesis, were also measured in rats. Rat knee cartilage was analysed for immunoreactivity of CTX-II and PIIANP and for type II collagen expression.ResultsAs expected, urinary levels of CTX-II are significantly increased in postmenopausal women and also in oestrogen-deprived rats, although only transiently. However, in neither case were these elevations paralleled by a significant increase of Helix-II levels and PIIANP levels did not change at any time. CTX-II immunoreactivity and collagen expression were detected in different cartilage areas. The upper zone is the area where CTX-II immunoreactivity and collagen expression best reflected the differences in urinary levels of CTX-II measured in response to oestrogen. However, correlations between urinary levels of CTX-II and tissue immunostainings in individual rats were not statistically significant.ConclusionsWe found only a small effect of oestrogen deprivation on cartilage. It was detected by CTX-II, but not by other type II collagen turnover markers typically affected in osteoarthritis.

F-spondin, a neuroregulatory protein, is up-regulated in osteoarthritis and regulates cartilage metabolism via TGF-beta activation.

In osteoarthritis (OA) articular chondrocytes undergo phenotypic changes culminating in the progressive loss of cartilage from the joint surface. The molecular mechanisms underlying these changes are poorly understood. Here we report enhanced (approximately 7-fold) expression of F-spondin, a neuronal extracellular matrix glycoprotein, in human OA cartilage (P<0.005). OA-specific up-regulation of F-spondin was also demonstrated in rat knee cartilage following surgical menisectomy. F-spondin treatment of OA cartilage explants caused a 2-fold increase in levels of the active form of TGF-beta1 (P<0.01) and a 10-fold induction of PGE2 (P<0.005) in culture supernatants. PGE2 induction was found to be dependent on TGF-beta and the thrombospondin domain of the F-spondin molecule. F-spondin addition to cartilage explant cultures also caused a 4-fold increase in collagen degradation (P<0.05) and a modest reduction in proteoglycan synthesis (approximately 20%; P<0.05), which were both TGF-beta and PGE2 dependent. F-spondin treatment also led to increased secretion and activation of MMP-13 (P<0.05). Together these studies identify F-spondin as a novel protein in OA cartilage, where it may act in situ at lesional areas to activate latent TGF-beta and induce cartilage degradation via pathways that involve production of PGE2.

In vivo rat knee cartilage volume measurement using quantitative high resolution MRI (7 T): Feasibility and reproducibility

to assess reliability and reproducibility of quantitative MRI (7 T) in assessing rat femoro-tibial cartilage volume. 5 healthy rat knees were scanned in vivo using a 7 T experimental imager. Sagittal high resolution 3D Gradient Echo with fat suppression sequences were performed with a dedicated home-made 2-elements array coil. 3D MRI sets were used to perform manual segmentation of the 3 cartilage compartments (femoral groove, medial and lateral tibial plateaus) by using a tactile screen. To evaluate inter- and intra-observer reproducibilities, the segmentation procedure was done blindly by two trained observers. One observer repeated the operation twice, with a period of 10 months between both readings. the mean duration to manually segment all the slices covering the cartilaginous joint was 4 hours. On the one hand, the inter-observer root mean square of coefficients of variation was 9.1%, 6.2%, 9.6% for the femoral, medial and lateral tibial compartments respectively. On the other hand, the intra-observer reproducibility was 2.1%, 3.2%, 2.5% for these cartilage compartments cited above. the image quality obtained at 7 Teslas with our dedicated coil allowed segmentation of the cartilage compartments with good reproducibility. This study demonstrated that MRI is a useful technology to provide a non-invasive and reliable assessment of rat knee cartilage volume.

Immunohistochemical localization of calcitonin gene-related peptide and substance P in the rat knee cartilage at birth.

Substance P (SP) and calcitonin gene-related peptide (CGRP) have been found in the perichondrium and within the cartilage canals. It is still unknown whether they exert a direct effect on chondrocytes during joint development. We processed 28 knees of newborn Wistar rats in 7 different fashions to perform histology and immunohistochemistry studies. Positive immunoreactivity against CGRP and SP was found in the inner aspect of the perichondrium in a close contact with chondrocytes. The presence of CGRP and SP indicates the presence of nerves fibers, and precedes the development of cartilage canals. Nerve fibers may play a role in the development of synovial joints before and during the presence of cartilage canals. The presence of CGRP and SP in the cartilage at birth may be involved in the early postnatal maturation of synovial joints. It remains to be determined whether autonomic innervation is later involved in age-related degenerative joint disease.

High resolution MRI relaxation measurements of water in the articular cartilage of the meniscectomized rat knee at 4.7 T.

Measurements by magnetic resonance imaging (MRI) of the spin-spin (T2), spin-lattice (T1) and spin-density (M0) parameters of water protons, optimized by using the Cramér-Rao Lower Bound (CRLB) theory, were made to quantify the effect of surgically induced osteoarthritis on rat knee cartilage at 4.7 T. Partial meniscectomy was performed on the right medial condyle of four Sprague Dawley rats, leaving the left medial condyle as a control. The animals were euthanized 3 weeks after the operation; the entire limbs were removed and T2 and T1 relaxation measurements and M0 measurements of the protons of water were obtained using conventional Carr-Purcell-Meiboom-Gill (CPMG) and saturation recovery methods. M0 was normalized with respect to a water phantom, to obtain the relative spin-density M0%. Weight-bearing cartilage areas on the meniscectomized medial condyles exhibited a significant increase of T2 relaxation time (p < 0.001) and of M0% (p < 0.01) with respect to the control; T1 relaxation times did not show any statistically significant changes. CRLB-based sampling optimization offered an insight to improved measurement precision and a reduction of scanning time against conventional sampling methods methods. Quantitative MRI assessment of the meniscectomized rat knee shows that cartilage exhibits changes in T2 and M0 values 3 weeks after operation.