Early patellofemoral cartilage and bone pathology in a rat model of noninvasive anterior cruciate ligament rupture.

OBJECTIVE: Anterior cruciate ligament rupture (ACLR) is a risk factor for the development of post-traumatic osteoarthritis (PTOA). While PTOA in the tibiofemoral joint compartment is well-characterized, very little is known about pathology in the patellofemoral compartment after ACL injury. Here, we evaluated the extent to which ACLR induces early patellofemoral joint damage in a rat model. METHODS: Adult female Lewis rats were randomized to noninvasive ACLR or Sham. Two weeks post-injury, contrast-enhanced micro-computed tomography (µCT) of femoral and patellar cartilage was performed using 20% v/v ioxaglate. Morphometric parameters of femoral trochlear and patellar cartilage, subchondral bone, and trabecular bone were derived from µCT. Sagittal Safranin-O/Fast-Green-stained histologic sections were graded using the OARSI score in a blinded fashion. RESULTS: Cartilage and bone remodelling consistent with an early PTOA phenotype were observed in both femoral trochleas and patellae. ACLR caused osteophyte formation of the patella and pathology in the superficial zone of articular cartilage, including surface fibrillation, fissures, increased cellularity, and abnormal chondrocyte clustering. There were significant increases in thickness of patellar and trochlear cartilage. Loss of subchondral bone thickness, bone volume fraction, and tissue mineral density, as well as changes to patellar and trochlear trabecular microarchitecture, were indicative of catabolic bone remodelling. Several injury-induced changes, including increased cartilage thickness and trabecular spacing and decreased trabecular number were more severe in the patella compared to the trochlea. CONCLUSION: The patellofemoral joint develops mild but evident pathology in the early period following ACL rupture, extending the utility of this model to the study of patellofemoral PTOA.

Traumatic joint injury induces acute catabolic bone turnover concurrent with articular cartilage damage in a rat model of posttraumatic osteoarthritis.

Assess acute alterations in bone turnover, microstructure, and histomorphometry following noninvasive anterior cruciate ligament rupture (ACLR). Twelve female Lewis rats were randomized to receive noninvasive ACLR or Sham loading (n = 6/group). In vivo µCT was performed at 3, 7, 10, and 14 days postinjury to quantify compartment-dependent subchondral (SCB) and epiphyseal trabecular bone remodeling. Near-infrared (NIR) molecular imaging was used to measure in vivo bone anabolism (800 CW BoneTag) and catabolism (Cat K 680 FAST). Metaphyseal bone remodeling and articular cartilage morphology was quantified using ex vivo µCT and contrast-enhanced µCT, respectively. Calcein-based dynamic histomorphometry was used to quantify bone formation. OARSI scoring was used to assess joint degeneration, and osteoclast number was quantified on TRAP stained-sections. ACLR induced acute catabolic bone remodeling in subchondral, epiphyseal, and metaphyseal compartments. Thinning of medial femoral condyle (MFC) SCB was observed as early as 7 days postinjury, while lateral femoral condyles (LFCs) exhibited SCB gains. Trabecular thinning was observed in MFC epiphyseal bone, with minimal changes to LFC. NIR imaging demonstrated immediate and sustained reduction of bone anabolism (~15%-20%), and a ~32% increase in bone catabolism at 14 days, compared to contralateral limbs. These findings were corroborated by reduced bone formation rate and increased osteoclast numbers, observed histologically. ACLR-injured femora had significantly elevated OARSI score, cartilage thickness, and cartilage surface deviation. ACL rupture induces immediate and sustained reduction of bone anabolism and overactivation of bone catabolism, with mild-to-moderate articular cartilage damage at 14 days postinjury.

Pharmacological tools to mobilise mesenchymal stromal cells into the blood promote bone formation after surgery.

Therapeutic approaches requiring the intravenous injection of autologous or allogeneic mesenchymal stromal cells (MSCs) are currently being evaluated for treatment of a range of diseases, including orthopaedic injuries. An alternative approach would be to mobilise endogenous MSCs into the blood, thereby reducing costs and obviating regulatory and technical hurdles associated with development of cell therapies. However, pharmacological tools for MSC mobilisation are currently lacking. Here we show that ß3 adrenergic agonists (ß3AR) in combination with a CXCR4 antagonist, AMD3100/Plerixafor, can mobilise MSCs into the blood in mice and rats. Mechanistically we show that reversal of the CXCL12 gradient across the bone marrow endothelium and local generation of endocannabinoids may both play a role in this process. Using a spine fusion model we provide evidence that this pharmacological strategy for MSC mobilisation enhances bone formation.

Nondestructive, indirect assessment of the biomechanical properties of the rat intervertebral disc using contrast-enhanced µCT.

Mechanical characterization of the intervertebral disc involves labor-intensive and destructive experimental methodology. Contrast-enhanced micro-computed tomography is a nondestructive imaging modality for high-resolution visualization and glycosaminoglycan quantification of cartilaginous tissues. The purpose of this study was to determine whether anionic and cationic contrast-enhanced micro-computed tomography of the intervertebral disc can be used to indirectly assess disc mechanical properties in an ex vivo model of disc degeneration. L3/L4 motion segments were dissected from female Lewis rats. To deplete glycosaminoglycan, samples were treated with 0 U/ml (Control) or 5 U/ml papain. Contrast-enhanced micro-computed tomography was performed following incubation in 40% Hexabrix (anionic) or 30 mg I/ml CA4+ (cationic) for 24 h (n = 10/contrast agent/digestion group). Motion segments underwent cyclic mechanical testing to determine compressive and tensile modulus, stiffness, and hysteresis. Glycosaminoglycan content was determined using the dimethylmethylene blue assay. Correlations between glycosaminoglycan content, contrast-enhanced micro-computed tomography attenuation, and mechanical properties were assessed via the Pearson correlation. The predictive accuracy of attenuation on compressive properties was assessed via repeated random sub-sampling cross validation. Papain digestion produced significant decreases in glycosaminoglycan content and corresponding differences in attenuation and mechanical properties. Attenuation correlated significantly to glycosaminoglycan content and to all compressive mechanical properties using both Hexabrix and CA4+ . Predictive linear regression models demonstrated a predictive accuracy of attenuation on compressive modulus and stiffness of 79.8-86.0%. Contrast-enhanced micro-computed tomography was highly predictive of compressive mechanical properties in an ex vivo simulation of disc degeneration and may represent an effective modality for indirectly assessing disc compressive properties. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2030-2038, 2018.

Contrast-enhanced µCT of the intervertebral disc: A comparison of anionic and cationic contrast agents for biochemical and morphological characterization.

The objective of this study was to quantify and compare the contrast-enhancing properties of the anionic contrast agent ioxaglate/Hexabrix, and cationic contrast agent CA4+ for biochemical and morphological characterization of the intervertebral disc (IVD) via µCT. Optimal contrast agent concentrations were determined by incubating rat lumbar IVDs in dilutions of Hexabrix-320 (20%, 30%, 40%, and 50%) and CA4+ (10, 20, 30, and 40 mg I/ml). µCT imaging was performed at 70 kVp, 114 µA, and 250 ms integration time, 12 µm voxel size. The kinetics of contrast enhancement were quantified with cumulative incubations for 0.5, 1, 2, 12, 16, 20, and 24 h using both agents. Agreement in morphological quantification was assessed via serial scans of the same IVDs. Correlation of attenuation to glycosaminoglycan (GAG) content was determined by enzymatic digestion of IVDs, subsequent µCT imaging, and GAG quantification via dimethylmethylene blue assay. Forty percent Hexabrix and 30 mg I/ml CA4+ were chosen as optimal concentrations. Hexabrix enabled greater delineation of the IVD from surrounding tissues, and CA4+ had the lowest uptake in surrounding soft tissue. Twenty-four hour incubation was sufficient for >99% equilibration of both agents. A high level of agreement was observed in the quantification of IVD volume (ICC = 0.951, r = 0.997) and height (ICC = 0.947, r = 0.991). Both agents exhibited strong linear correlations between µCT attenuation and GAG content (Hexabrix: r = -0.940; CA4+ : r = 0.887). Both agents enable biochemical and morphological quantification of the IVD via contrast-enhanced µCT and are effective tools for preclinical characterization. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1067-1075, 2017.