V. Pitelka

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.

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.

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.

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.

Measurement of kidney stone formation in the rat model using micro-computed tomography

Kidney stones were induced in 5 rats by treating them with 1% ethylene glycol and 1% ammonium chloride through free drinking water for six weeks. The animals were anesthetized and imaged in vivo before the treatment at week 0, to obtain baseline data, then at weeks 2 and 6 to monitor the kidney stone formation. Micro-CT imaging was performed with x-ray tube voltage of 90 kV and a current of 40 mA. At week 2, kidney stone formation was observed. A micro-computed tomography methodology of estimating the volume and hydroxyapatite-equivalent mineral content of the kidney stone is presented. It determines the threshold CT number (390 HU) that separates the kidney stone from the tissue. The mean volume of the stones in the 10 kidneys significantly increased from 3.81±0.72 mm3 at week 2 to 23.96±9.12 mm3 at week 6 (p<0.05, r2=0.34). Measurement precision error was about 4%. This method allows analysis of the kidney stone formation to be carried out in vivo, with fewer experimental animals compared with other ex vivo methods, in which animals are sacrificed. It is precise, accurate, non-destructive, and could be used in pre-clinical research to study the formation of kidney stones in live small animals.

Global gene expression analyses in early experimental osteoarthritis reveal novel players in articular cartilage degenerations

Introduction: Articular cartilage degeneration is a hallmark of osteoarthritis (OA). We sought to identify dysregulated genes in degenerating cartilage and hypothesized that altered growth factor expression causes cartilage degradation in OA. Methods: Genome-wide microarray analysis of RNA harvested directly from sham (control) and degenerating articular cartilage was performed using our previously characterized pre-clinical rat model of knee OA1. Known OA genes were validated using RNA samples (real-time PCR) and histological sections (immunofluorescence) from independent animals. Functional studies in chondrocytes and articular cartilage explants investigated the effects of one identified factor (transforming growth factor alpha (TGF-α)) on cartilage degeneration. The effects of TGF-α on primary chondrocyte morphology, proliferation, gene expression, and SOX9 transcription factor expression were assessed. Results: Dysregulated gene expression profiles in degenerating cartilage included known OA genes2. Microarray expression profiles were consistently validated at RNA and protein levels by alternative methods. Several genes previously unstudied in OA cartilage were upregulated, including growth factors (e.g. TGF-α and kit ligand), cell surface receptors (e.g. endothelin type A receptor), and proteases (e.g. cathepsin S). Functional studies demonstrated that TGF-α alters chondrocyte morphology through re-organization of the actin cytoskeleton. TGF-α also stimulated primary chondrocyte proliferation and chondrocyte cluster formation in cartilage explants. Chondrocyte expression of anabolic genes and total collagen protein levels were reduced by TGF-α, while expression of catabolic factors increased. Finally, TGF-α reduced both the expression of total SOX9 and levels of phosphorylated (active) SOX9. Conclusions: Our microarray study identified numerous factors previously unstudied in OA cartilage, including increased levels of TGF-α. Functional studies determined that TGF-α promotes cartilage degeneration through chondrocyte proliferation and catabolic factor expression. Further, TGF-α inhibits chondrocyte anabolism, likely through a mechanism involving suppression of SOX9. References: 1C.T.G. Appleton et al. (2007). Arthritis Res Ther 9(1):R13. 2C.T.G. Appleton et al. (2007), Arthritis Rheum In Press.