Functional Characterization of Ovine Dorsal Root Ganglion Neurons Reveal Peripheral Sensitization after Osteochondral Defect.

Knee joint trauma can cause an osteochondral defect (OD), a risk factor for osteoarthritis (OA) and cause of debilitating pain in patients. Rodent OD models are less translatable because of their smaller joint size and open growth plate. This study proposes sheep as a translationally relevant model to understand the neuronal basis of OD pain. A unilateral 6-mm deep OD was induced in adult female sheep. Two to six weeks after operation, lumbar dorsal root ganglia (DRG) neurons were collected from the contralateral (Ctrl) and OD side of operated sheep. Functional assessment of neuronal excitability and activity of the pain-related ion channels transient receptor potential vanilloid receptor 1 (TRPV1) and P2X3 was conducted using electrophysiology and Ca2+ imaging. Immunohistochemistry was used to verify expression of pain-related proteins. We observed that an increased proportion of OD DRG neurons (sheep, N = 3; Ctrl neurons, n = 15, OD neurons, n = 16) showed spontaneous electrical excitability (Ctrl: 20.33 ± 4.5%; OD: 50 ± 10%; p = 0.009, unpaired t test) and an increased proportion fired a greater number of spikes above baseline in response to application of a TRPV1 agonist (capsaicin) application (Ctrl: 40%; OD: 75%; p = 0.04, χ2 test). Capsaicin also produced Ca2+ influx in an increased proportion of isolated OD DRG neurons (Ctrl: 25%; OD: 44%; p = 0.001, χ2 test). Neither protein expression, nor functionality of the P2X3 ion channel were altered in OD neurons. Overall, we provide evidence of increased excitability of DRG neurons (an important neural correlate of pain) and TRPV1 function in an OD sheep model. Our data show that functional assessment of sheep DRG neurons can provide important insights into the neural basis of OD pain and thus potentially prevent its progression into arthritic pain.

Computed Tomographic Assessment of Block Recession Trochleoplasty and Partial Parasagittal Patellectomy in Cats.

OBJECTIVE: The aim of this study was to assess computed tomography (CT) images of cadaveric feline stifles and record the relationship between the patella and femoral trochlea in normal stifles; then to investigate the effect of block recession trochleoplasty (BRT) with and without partial parasagittal patellectomy (PPP) on patellofemoral contact, depth of patellar recession and size of trochlea and patella. MATERIALS AND METHODS: The sample population included six cat cadavers (12 stifles). Preoperative CT scans with stifles in three positions: extended (155-165°), neutral (85-95°) and flexed (35-45°). Lateral stifle arthrotomy was followed by BRT. CT scans were taken as above. PPP was performed so the width of the patella was 1 mm narrower than the BRT. CT scans were repeated and CT measurements were taken. RESULTS: The area of contact between the patella and femoral trochlea was significantly reduced after BRT and then significantly increased after PPP. The depth of patellar recession was unchanged after BRT, but then was significantly higher after PPP. The patella was wider than the trochlea preoperatively and post-BRT and narrower post-PPP. Reduction in bone volume following PPP (mean: 20%) was 50% the reduction in patella width (mean: 40%). CONCLUSION: Block recession trochleoplasty did not improve patellar recession and decreased patellofemoral contact area. Following BRT, the patella rides the trochlear ridges and loses contact with the trochlear sulcus. Block recession trochleoplasty alone should not be performed in cats. Instead, BRT should be combined with PPP.

Ex vivo MRI cell tracking of autologous mesenchymal stromal cells in an ovine osteochondral defect model.

BACKGROUND: Osteochondral injuries represent a significant clinical problem requiring novel cell-based therapies to restore function of the damaged joint with the use of mesenchymal stromal cells (MSCs) leading research efforts. Pre-clinical studies are fundamental in translating such therapies; however, technologies to minimally invasively assess in vivo cell fate are currently limited. We investigate the potential of a MRI- (magnetic resonance imaging) and superparamagnetic iron oxide nanoparticle (SPION)-based technique to monitor cellular bio-distribution in an ovine osteochondral model of acute and chronic injuries. METHODS: MSCs were isolated, expanded and labelled with Nanomag, a 250-nm SPION, and using a novel cell-penetrating technique, glycosaminoglycan-binding enhanced transduction (GET). MRI visibility thresholds, cellular toxicity and differentiation potential post-labelling were assessed in vitro. A single osteochondral defect was created in the medial femoral condyle in the left knee joint of each sheep with the contralateral joint serving as the control. Cells, either GET-Nanomag labelled or unlabelled, were delivered 1 week or 4.5 weeks later. Sheep were sacrificed 7 days post implantation and immediately MR imaged using a 0.2-T MRI scanner and validated on a 3-T MRI scanner prior to histological evaluation. RESULTS: MRI data demonstrated a significant increase in MRI contrast as a result of GET-Nanomag labelling whilst cell viability, proliferation and differentiation capabilities were not affected. MRI results revealed evidence of implanted cells within the synovial joint of the injured leg of the chronic model only with no signs of cell localisation to the defect site in either model. This was validated histologically determining the location of implanted cells in the synovium. Evidence of engulfment of Nanomag-labelled cells by leukocytes is observed in the injured legs of the chronic model only. Finally, serum c-reactive protein (CRP) levels were measured by ELISA with no obvious increase in CRP levels observed as a result of P21-8R:Nanomag delivery. CONCLUSION: This study has the potential to be a powerful translational tool with great implications in the clinical translation of stem cell-based therapies. Further, we have demonstrated the ability to obtain information linked to key biological events occurring post implantation, essential in designing therapies and selecting pre-clinical models.

"Patient reported outcomes" following experimental surgery-using telemetry to assess movement in experimental ovine models.

Many potential treatments for orthopedic disease fail at the animal to human translational hurdle. One reason for this failure is that the majority of pre-clinical outcome measurements emphasize structural changes, such as gross morphology and histology, and do not address pain or its alleviation, which is a key component of treatment success in man. With increasing emphasis on "patient reported outcome measurements (PROM)" in clinical practice, in this study we have used two different telemetric methods (geolocation and Fitbark activity trackers, Kansas City, MO) to measure movement behavior, i.e., an indirect PROM, in an ovine osteoarthritis induction and an osteochondral defect model performed in adult female Welsh Mountain sheep. This study demonstrates that both systems can be used to track movement and activity of experimental sheep before and after surgery and that the Geolocator system recorded a decrease in distance moved and activity at the end of the experimental period in both models. The Fitbark activity tracker also recorded significant alterations in movement behavior at the end of these studies and this method of recording showed a correlation between Fitbark data and radiography, macroscopic and histological scoring (well recognized outcome measurements), particularly in animals with large (10 mm) defects, i.e., more severe pathology. These results suggest that telemetry is able to track movement behavior in experimental sheep and that the methodology should be considered for inclusion in outcome measures in preclinical orthopedic research. © 2017 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 36:1498-1507, 2018.