The BMP7-Derived Peptide p[63-82] Reduces Cartilage Degeneration in the Rat ACLT-pMMx Model for Posttraumatic Osteoarthritis.

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.

Integration and evaluation of a needle-positioning robot with volumetric microcomputed tomography image guidance for small animal stereotactic interventions.

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.

In vivo micro-CT analysis of bone remodeling in a rat calvarial defect model.

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.

Reduction in disease progression by inhibition of transforming growth factor α-CCL2 signaling in experimental posttraumatic osteoarthritis.

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.

An in vivo investigation of the initiation and progression of subchondral cysts in a rodent model of secondary osteoarthritis.

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'.

Forced mobilization accelerates pathogenesis: characterization of a preclinical surgical model of osteoarthritis.

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.