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Objective: Osteoarthritis (OA) is characterized by articular cartilage erosion, pathological subchondral bone changes, and signs of synovial inflammation and pain. We previously identified p[63-82], a bone morphogenetic protein 7 (BMP7)-derived bioactive peptide that attenuates structural cartilage degeneration in the rat medial meniscal tear-model for posttraumatic OA. This study aimed to evaluate the cartilage erosion-attenuating activity of p[63-82] in a different preclinical model for OA (anterior cruciate ligament transection-partial medial meniscectomy [anterior cruciate ligament transection (ACLT)-pMMx]). The disease-modifying action of the p[63-82] was followed-up in this model for 5 and 10 weeks., Design: Skeletally mature male Lewis rats underwent ACLT-pMMx surgery. Rats received weekly intra-articular injections with either saline or 500 ng p[63-82]. Five and 10 weeks postsurgery, rats were sacrificed, and subchondral bone characteristics were determined using microcomputed tomography (µCT). Histopathological evaluation of cartilage degradation and Osteoarthritis Research Society International (OARSI)-scoring was performed following Safranin-O/Fast Green staining. Pain-related behavior was measured by incapacitance testing and footprint analysis., Results: Histopathological evaluation at 5 and 10 weeks postsurgery showed reduced cartilage degeneration and a significantly reduced OARSI score, whereas no significant changes in subchondral bone characteristics were found in the p[63-82]-treated rats compared to the saline-treated rats. ACLT-pMMx-induced imbalance of static weightbearing capacity in the p[63-82] group was significantly improved compared to the saline-treated rats at weeks 5 postsurgery. Footprint analysis scores in the p[63-82]-treated rats demonstrated improvement at week 10 postsurgery., Conclusions: Weekly intra-articular injections of p[63-82] in the rat ACLT-pMMx posttraumatic OA model resulted in reduced degenerative cartilage changes and induced functional improvement in static weightbearing capacity during follow-up., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: MC and TW are listed as the inventor on filed patents: WO2017178251 and WO2017178253. TW, PE, and LR have shares in Chondropeptix BV.

Objective: Transforming growth factor α (TGFα) is increased in osteoarthritic (OA) cartilage in rats and humans and modifies chondrocyte phenotype. CCL2 is increased in OA cartilage and stimulates proteoglycan loss. This study was undertaken to test whether TGFα and CCL2 cooperate to promote cartilage degradation and whether inhibiting either reduces disease progression in a rat model of posttraumatic OA., Methods: Microarray analysis was used to profile expression of messenger RNA (mRNA) for Tgfa, Ccl2, and related genes in a rat model of posttraumatic OA. Rat primary chondrocytes and articular cartilage explants were treated with TGFα in the presence or absence of MEK-1/2, p38, phosphatidylinositol 3-kinase, Rho-associated protein kinase, or CCR2 inhibitors and immunostained for markers of cartilage degradation. The rat model was used to administer pharmacologic inhibitors of TGFα (AG1478) and CCL2 (RS504393) signaling for up to 10 weeks and assess histopathology and serum biomarkers of cartilage synthesis (C-propeptide of type II collagen [CPII]) and breakdown (C2C)., Results: Tgfa and Ccl2 mRNA were simultaneously up-regulated in articular cartilage in the rat model of posttraumatic OA. TGFα induced expression of CCL2, Mmp3, and Tnf in primary chondrocytes. Cleavage of type II collagen and aggrecan (by matrix metalloproteinases and ADAMTS-4/5, respectively) induced by TGFα was blocked by pharmacologic inhibition of CCL2 in cartilage explants. In vivo pharmacologic inhibition of TGFα or CCL2 signaling reduced Osteoarthritis Research Society International cartilage histopathology scores and increased serum CPII levels, but only TGFα inhibition reduced C2C levels intreated versus untreated rat OA cartilage., Conclusion: TGFα signaling stimulates cartilage degradation via a CCL2-dependent mechanism, but pharmacologic inhibition of the TGFα-CCL2 axis reduces experimental posttraumatic OA progression in vivo., (© 2015, American College of Rheumatology.)

Introduction: Subchondral bone cysts (SBC) have been identified in patients with knee osteoarthritis (OA) as a cause of greater pain, loss of cartilage and increased chance of joint replacement surgery. Few studies monitor SBC longitudinally, and clinical research using three-dimensional imaging techniques, such as magnetic resonance imaging (MRI), is limited to retrospective analyses as SBC are identified within an OA patient cohort. The purpose of this study was to use dual-modality, preclinical imaging to monitor the initiation and progression of SBC occurring within an established rodent model of knee OA., Methods: Eight rodents underwent anterior cruciate ligament transection and partial medial meniscectomy (ACLX) of the right knee. In vivo 9.4 T MRI and micro-computed tomography (micro-CT) scans were performed consecutively prior to ACLX and 4, 8, and 12 weeks post-ACLX. Resultant images were co-registered using anatomical landmarks, which allowed for precise tracking of SBC size and composition throughout the study. The diameter of the SBC was measured, and the volumetric bone mineral density (vBMD) was calculated within the bone adjacent to SBC. At 12 weeks, the ACLX and contralateral knees were processed for histological analysis, immunohistochemistry, and Osteoarthritis Research Society International (OARSI) pathological scoring., Results: At 4 weeks post-ACLX, 75% of the rodent knees had at least 1 cyst that formed in the medial tibial plateau; by 12 weeks all ACLX knees contained SBC. Imaging data revealed that the SBC originate in the presence of a subchondral bone plate breach, with evolving composition over time. The diameter of the SBC increased significantly over time (P = 0.0033) and the vBMD significantly decreased at 8 weeks post-ACLX (P = 0.033). Histological analysis demonstrated positive staining for bone resorption and formation surrounding the SBC, which were consistently located beneath the joint surface with the greatest cartilage damage. Trabecular bone adjacent the SBC lacked viable osteocytes and, combined with bone marrow changes, indicated osteonecrosis., Conclusions: This study provides insight into the mechanisms leading to SBC formation in knee OA. The expansion of these lesions is due to stress-induced bone resorption from the incurred mechanical instability. Therefore, we suggest these lesions can be more accurately described as a form of OA-induced osteonecrosis, rather than 'subchondral cysts'.

Purpose: Preclinical research protocols often require insertion of needles to specific targets within small animal brains. To target biologically relevant locations in rodent brains more effectively, a robotic device has been developed that is capable of positioning a needle along oblique trajectories through a single burr hole in the skull under volumetric microcomputed tomography (micro-CT) guidance., Methods: An x-ray compatible stereotactic frame secures the head throughout the procedure using a bite bar, nose clamp, and ear bars. CT-to-robot registration enables structures identified in the image to be mapped to physical coordinates in the brain. Registration is accomplished by injecting a barium sulfate contrast agent as the robot withdraws the needle from predefined points in a phantom. Registration accuracy is affected by the robot-positioning error and is assessed by measuring the surface registration error for the fiducial and target needle tracks (FRE and TRE). This system was demonstrated in situ by injecting 200 microm tungsten beads into rat brains along oblique trajectories through a single burr hole on the top of the skull under micro-CT image guidance. Postintervention micro-CT images of each skull were registered with preintervention high-field magnetic resonance images of the brain to infer the anatomical locations of the beads., Results: Registration using four fiducial needle tracks and one target track produced a FRE and a TRE of 96 and 210 microm, respectively. Evaluation with tissue-mimicking gelatin phantoms showed that locations could be targeted with a mean error of 154 +/- 113 microm., Conclusions: The integration of a robotic needle-positioning device with volumetric micro-CT image guidance should increase the accuracy and reduce the invasiveness of stereotactic needle interventions in small animals.

The rodent calvarial defect model is commonly used to investigate bone regeneration and wound healing. This study presents a micro-computed tomography (micro-CT) methodology for measuring the bone mineral content (BMC) in a rat calvarial defect and validates it by estimating its precision error. Two defect models were implemented. A single 6 mm diameter defect was created in 20 rats, which were imaged in vivo for longitudinal experiments. Three 5 mm diameter defects were created in three additional rats, which were repeatedly imaged ex vivo to determine precision. Four control rats and four rats treated with bone morphogenetic protein were imaged at 3, 6, 9 and 12 weeks post-surgery. Scan parameters were 80 kVp, 0.45 mA and 180 mAs. Images were reconstructed with an isotropic resolution of 45 microm. At 6 weeks, the BMC in control animals (4.37 +/- 0.66 mg) was significantly lower (p < 0.05) than that in treated rats (11.29 +/- 1.01 mg). Linear regression between the BMC and bone fractional area, from 20 rats, showed a strong correlation (r(2) = 0.70, p < 0.0001), indicating that the BMC can be used, in place of previous destructive analysis techniques, to characterize bone growth. The high precision (2.5%) of the micro-CT methodology indicates its utility in detecting small BMC changes in animals.

Preclinical osteoarthritis (OA) models are often employed in studies investigating disease-modifying OA drugs (DMOADs). In this study we present a comprehensive, longitudinal evaluation of OA pathogenesis in a rat model of OA, including histologic and biochemical analyses of articular cartilage degradation and assessment of subchondral bone sclerosis. Male Sprague-Dawley rats underwent joint destabilization surgery by anterior cruciate ligament transection and partial medial meniscectomy. The contralateral joint was evaluated as a secondary treatment, and sham surgery was performed in a separate group of animals (controls). Furthermore, the effects of walking on a rotating cylinder (to force mobilization of the joint) on OA pathogenesis were assessed. Destabilization-induced OA was investigated at several time points up to 20 weeks after surgery using Osteoarthritis Research Society International histopathology scores, in vivo micro-computed tomography (CT) volumetric bone mineral density analysis, and biochemical analysis of type II collagen breakdown using the CTX II biomarker. Expression of hypertrophic chondrocyte markers was also assessed in articular cartilage. Cartilage degradation, subchondral changes, and subchondral bone loss were observed as early as 2 weeks after surgery, with considerable correlation to that seen in human OA. We found excellent correlation between histologic changes and micro-CT analysis of underlying bone, which reflected properties of human OA, and identified additional molecular changes that enhance our understanding of OA pathogenesis. Interestingly, forced mobilization exercise accelerated OA progression. Minor OA activity was also observed in the contralateral joint, including proteoglycan loss. Finally, we observed increased chondrocyte hypertrophy during pathogenesis. We conclude that forced mobilization accelerates OA damage in the destabilized joint. This surgical model of OA with forced mobilization is suitable for longitudinal preclinical studies, and it is well adapted for investigation of both early and late stages of OA. The time course of OA progression can be modulated through the use of forced mobilization.

1. Microvascular endothelial cells (MVECs) form a barrier between circulating metabolites, such as adenosine, and the surrounding tissue. We hypothesize that MVECs have a high capacity for the accumulation of nucleosides, such that inhibition of the endothelial nucleoside transporters (NT) would profoundly affect the actions of adenosine in the microvasculature. 2. We assessed the binding of [(3)H]nitrobenzylmercaptopurine riboside (NBMPR), a specific probe for the inhibitor-sensitive subtype of equilibrative NT (es), and the uptake of [(3)H]formycin B (FB), by MVECs isolated from rat skeletal muscle. The cellular expression of equilibrative (ENT1, ENT2, ENT3) and concentrative (CNT1, CNT2, CNT3) NT subtypes was also determined using both qualitative and quantitative polymerase chain reaction techniques. 3. In the absence of Na(+), MVECs accumulated [(3)H]FB with a V(max) of 21+/-1 pmol microl(-1) s(-1). This uptake was mediated equally by es (K(m) 260+/-70 microm) and ei (equilibrative inhibitor-insensitive; K(m) 130+/-20 microm) NTs. 4. A minor component of Na(+)-dependent cif (concentrative inhibitor-insensitive FB transporter)/CNT2-mediated [(3)H]FB uptake (V(i) 0.008+/-0.005 pmol microl(-1) s(-1) at 10 microm) was also observed at room temperature upon inhibition of ENTs with dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]pyrimidine)/NBMPR. 5. MVECs had 122,000 high-affinity (K(d) 0.10 nm) [(3)H]NBMPR binding sites (representing es transporters) per cell. A lower-affinity [(3)H]NBMPR binding component (K(d) 4.8 nm) was also observed that may be related to intracellular es-like proteins. 6. Rat skeletal muscle MVECs express es/ENT1, ei/ENT2, and cif/CNT2 transporters with characteristics typical of rat tissues. This primary cell culture model will enable future studies on factors influencing NT subtype expression, and the consequent effect on adenosine bioactivity, in the microvasculature.

We hypothesized that macrophage activation results in nitric oxide (NO) production and that this NO acts directly on Leydig cells (LC) to alter androgen synthesis. Both peritoneal macrophages and a murine macrophage cell line (RAW 264.7) were activated in vitro by sequential exposure to interferon-gamma (50 U/ml) and then bacterial lipopolysaccharide (LPS; 100 ng/ml) for 24 h each. At various times after initiation of activation, selected wells were harvested for identification of messenger RNA for inducible NO synthase by RT-PCR. Amplicons of the predicted 651-bp product were isolated, cloned, and sequenced to validate the PCR procedure. Such amplicons first appeared between 2-4 h after exposure to LPS, and staining increased in intensity for the rest of the study. Nitrite accumulation followed a similar time course. Similarly treated wells were washed after 24-h activation and cocultured with purified LC for a final 24-h incubation in the absence of interferon-gamma and LPS. Basal and LH-stimulated production of androgen was estimated by RIA. In some experiments the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester or the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO) was added during activation and coculture. Coculture of LC with quiescent macrophages altered neither basal nor LH-stimulated androgen production. Coculture with either type of activated macrophage did not alter basal, but significantly reduced (by 50%) LH-stimulated, androgen production. N(omega)-Nitro-L-arginine methyl ester and C-PTIO blocked the inhibitory effect. The NO donor S-nitroso-N-acetyl penicillamine at concentrations greater than 10(-5) M significantly inhibited LH-stimulated androgen production by purified LC (P < 0.01). The inhibitory effect of S-nitroso-N-acetyl penicillamine was evident when exposure exceeded 4 h. Intermediates of steroidogenesis were added to elucidate the site of NO inhibition. The enzymatic inhibition occurred at least in part at 17alpha-hydroxylase/C(17/20) lyase (P450c17). Enzyme inhibition was reversed by C-PTIO. Northern blot analysis indicated that accumulation of messenger RNA for P450c17 was not significantly altered. Therefore, activation of macrophages results in decreased androgen production by cocultured LC. The inhibition is mediated in part by macrophage-derived NO acting directly on the LC via inhibition of at least one of the P450 steroidogenic enzymes.

The site(s) of the negative feedback action of estrogen on gonadotropin secretion were studied in the ovariectomized rhesus monkey by observing the serum LH and FSH responses to intravenous GnRH injections at various times after implantation of Silastic capsules filled with estrogen. Circulating estrogen concentrations produced by the capsules were within the normal midcycle range for this species. Four h after estrogen implantation, no LH or FSH response was seen to the GnRH injection, indicating a suppressive effect of the steroid directly on the pituitary. Twelve and 22 h after estrogen implantation, however, the LH and FSH responses were equal to or larger than control responses. Since preinjection LH and FSH levels were below control values at these times and the pituitary responded to exogenous GnRH, it appears that endogenous GnRH secretion was affected, indicating an inhibitory action of estrogen on the central nervous system. Thus these experiments suggest 2 separate negative feedback actions of estrogen: a transient one directly on the pituitary and a longer lasting effect on the central nervous system.

Experiments were performed to study the responsiveness of the pituitary to gonadotropin-releasing hormone (GnRH) during the dynamic changes in gonadotropin secretion associated with the estrogen-induced luteinizing hormone (LH) surge in the ovariectomized (OVX) rhesus monkey. Silastic capsules filled with estradiol-17-beta were implanted subcutaneously in ovariectomized rhesus monkeys, resulting in an initial lowering of circulating LH and follicle-stimulating hormone (FSH) concentrations followed by an LH-FSH surge. GnRH was injected intravenously just before estrogen implantation, during the negative feedback response and during the rising, the peak, and the declining phases of the LH surge. The LH and FSH responses during the negative feedback phase were as large as those before estrogen treatment (control responses). During the rising phase of the LH surge, the acute response to GnRH injection did not differ significantly from the control response, but the responses 60 and 120 min after injection were somewhat increased. During the declining phase of the LH surge, the pituitary was not responsive to exogenous GnRH, although LH probably continued to be secreted at this time since the LH surge decreased more slowly than predicted by the normal rate of disappearance of LH in the monkey. We conclude that an increased duration of response to GnRH may be an important part of the mechanism by which estrogen induces the LH surge, but we do not see evidence of increased sensitivity of the pituitary to GnRH as an acute releasing factor at that time.