Altered mechanical loading is a widely suggested, but poorly understood potential cause of cartilage degeneration in osteoarthritis. In rodents, osteoarthritis is induced following destabilization of the medial meniscus (DMM). This study estimates knee kinematics and contact forces in rats with DMM to gain better insight into the specific mechanisms underlying disease development in this widely-used model. Unilateral knee surgery was performed in adult male Sprague-Dawley rats (n=5 with DMM, n=5 with sham surgery). Radio-opaque beads were implanted on their femur and tibia. 8 weeks following knee surgery, rat gait was recorded using the 3D²YMOX setup (Sanctorum et al. 2019, simultaneous acquisition of biplanar XRay videos and ground reaction forces). 10 trials (1 per rat) were calibrated and processed in XMALab (Knörlein et al. 2016). Hindlimb bony landmarks were labeled on the XRay videos using transfer learning (Deeplabcut, Mathis et al. 2019; Laurence-Chasen et al. 2020). A generic OpenSim musculoskeletal model of the rat hindlimb (Johnson et al. 2008) was adapted to include a 3-degree-of-freedom knee. Inverse kinematics, inverse dynamics, static optimization of muscle forces, and joint reaction analysis were performed. In rats with DMM, knee adduction was lower compared to sham surgery. Ground reaction forces were less variable with DMM, resulting in less variability in joint external moments. The mediolateral ground reaction force was lower, resulting in lower hip adduction moment, thus less force was produced by the rectus femoris. Rats with DMM tended to break rather than propel, resulting in lower hip flexion moment, thus less force was produced by the semimembranosus. These results are consistent with lower knee contact forces in the anteroposterior and axial directions. These preliminary data indicate no overloading of the knee joint in rats with DMM, compared with sham surgery. We are currently expanding our workflow to finite element analysis, to examine mechanical cues in the cartilage of these rats (Fig1G)

The effect of high-fat diet and testosterone replacement therapy upon bone remodelling was investigated in orchiectomised male APOE-/- mice. Mice were split in to three groups: sham surgery + placebo treatment (control, n=9), orchiectomy plus placebo treatment (n=8) and orchiectomy plus testosterone treatment (n=10). Treatments were administered via intramuscular injection once a fortnight for 17 weeks before sacrifice at 25 weeks of age. Tibiae were scanned ex-vivo using µCT followed by post-analysis histology and immunohistochemistry. Previously presented µCT data demonstrated orchiectomised, placebo treated mice exhibited significantly reduced trabecular bone volume, number, thickness and BMD compared to control mice despite no significant differences in body weight. Trabecular parameters were rescued back to control levels in orchiectomised mice treated with testosterone. No significant differences were observed in the cortical bone. Assessment of TRAP stained FFPE sections revealed no significant differences in osteoclast or osteoblast number along the endocortical surface. IHC assessment of osteoprotegerin (OPG) expression in osteoblasts is to be quantified alongside markers of osteoclastogenesis including RANK and RANKL. Results support morphological analysis of cortical bone where no change in cortical bone volume or density between groups is in line with no significant change in osteoblast or osteoclast number and percentage across all three groups. Future work will include further IHC assessment of bone remodelling and adiposity, as well as utilisation of mechanical testing to establish the effects of observed morphological differences in bone upon mechanical properties. Additionally, the effects of hormone treatments upon murine-derived bone cells will be investigated to provide mechanistic insights

Little is known on how sensory nerves and osteoclasts affect degenerative processes in subchondral bone in osteoarthritis (OA). Substance P (SP) effects on bone are ambivalent but physiological levels are critical for proper bone quality whereas α-calcitonin gene-related peptide (αCGRP) has anabolic effects. Here, we aimed to analyse the influence of an altered sensory neuropeptide microenvironment on subchondral bone in murine OA. Transection of the medial meniscotibial ligament (DMM) of the right hind leg induced joint instability leading to development of OA. Subchondral bone of tibiae from wildtype (WT), alendronate-treated WT (ALN, osteoclast inhibition), αCGRP- and SP- (Tachykinin (Tac)1) knockout mice was analysed by micro-computed tomography 4 and 12 weeks after DMM or sham surgery. Bone resorption marker CTX-I was measured in serum. We observed osteophytosis in all DMM groups and ALN sham mice 4 weeks after surgery but also in sham groups 12 weeks after surgery. In subchondral bone, bone volume density (BV/TV) increased from 4 to 12 weeks after surgery in DMM WT and Tac1-/− mice. DMM WT mice additionally had increased trabecular numbers (Tb.N.) and decreased trabecular space (Tb.Sp.) over time. Sham mice also showed time-dependent alterations in subchondral bone. In sham WT and αCGRP-/− mice specific bone surface (BS/BV) decreased and trabecular thickness (Tb.Th.) increased from 4 to 12 weeks after surgery while subchondral BV/TV of αCGRP-/− mice increased. Comparison of subchondral bone parameters at each time point showed elevated BV/TV in ALN DMM compared to WT DMM mice 4 weeks after surgery. In addition, both ALN sham and DMM mice showed a reduced BS/BV compared to WT. 4 weeks after sham surgery Tb.Th. was highest in ALN mice. In DMM WT mice Tb.Sp. was higher compared to ALN and αCGRP-/−. 12 weeks after surgery (late OA stage), BS/BV of ALN sham mice was significantly reduced in relation to ALN DMM, WT and Tac1-/− sham, while Tb.Th. increased compared to WT. DMM significantly decreased Tb.N. and increased Tb.Sp. in Tac1-/− compared to sham 12 weeks after surgery. CTX-I concentrations were significantly higher in ALN compared to Tac1-/− mice 4 weeks after sham surgery. 12 weeks after sham surgery CTX-I concentrations of WT mice were increased compared to αCGRP-/− and Tac1-/− mice. Over time, DMM induced stronger changes in subchondral bone of WT mice compared to knockout strains. WT and αCGRP-/− sham mice also show alterations in bone parameters over time indicating age-related effects on bone structure. SP deficiency enhanced DMM-induced structural bone alterations in late stage OA emphasizing the importance of SP under pathophysiological conditions. Osteoclast inhibition with alendronate proved to be preservative for time-dependent changes of subchondral bone observed in both, DMM and sham mice. Interestingly, ALN treatment did not reduce bone turnover marker CTX-I, and additionally promoted early osteophyte formation in sham mice

Joint tissues release extracellular vesicles (EVs) that potentially sustain joint homeostasis and contribute to osteoarthritis (OA) pathogenesis. EVs are putative novel therapeutics for OA, and transport biologically active molecules (including small non-coding RNAs (SNCRNAs)) between cells. This study identified altering SNCRNA cargo in EVs in OA which may act as early diagnostic markers and treatment targets. OA was surgically induced in four skeletally mature Standardbred horses using an osteochondral fragment model in the left middle carpal joint. The right joint underwent sham surgery. Synovial fluid (SF) and plasma were obtained weekly throughout the 70-day study. EVs were isolated using size exclusion chromatography and characterised using nanoparticle tracking (Nanosight), and exosome fluorescence detection and tetraspanin phenotyping (Exoview). RNA was extracted from EVs derived from SF (sham and OA joints) and plasma collected at days 10, 35, 42, 49, 56, 63, and subjected to small RNA sequencing on a NovaSeq SP100 flow cell (Illumina). Nanosight-derived EV characteristics of size and concentration were not significantly different following disease induction. The diameter of the temporal population of plasma and SF-derived exosomes changed significantly for CD9 and CD81 following OA induction with significant temporal, and disease-related changes in CD63 and CD81 protein expressin in plasma and SF. In SF and plasma-derived EVs snoRNAs, snRNAs, tRNAs, lncRNA, y-RNA, piRNAs and scRNA were found. Following pairwise analysis of all-time points we identified 27 miRs DE in plasma and 45 DE miRs in SF. Seven were DE in plasma and SF; miR-451, miR-25, miR-215, miR-92a, miR-let-7c, miR-486-5p, miR-23a. In plasma and SF 35 and 21 snoRNAs were DE with four DE in plasma and SF; U3, snord15, snord46, snord58. This work has identified alterations to OA EV sncRNAs in plasma and SF providing a greater understanding of the role of EVs in early OA

Introduction and Objective. Global prevalence of obesity has risen almost three-fold between 1975 and 2016. Alongside the more well-known health implications of obesity such as cardiovascular disease, cancer and type II diabetes, is the effect of male obesity on testosterone depletion and hypogonadism. Hypogonadism is a well-known contributor to the acceleration of bone loss during aging, and obesity is the single biggest risk factor for testosterone deficiency in men. Understanding the micro and macro structural changes to bone in response to testosterone depletion in combination with a high fat ‘Western’ diet, will advance our understanding of the relationship between obesity and bone metabolism. This study investigated the impact of surgically induced testosterone depletion and subsequent testosterone treatment upon bone remodelling in mice fed a high fat diet. Materials and Methods. Male ApoE. −/−. mice were split into 3 groups at 7 weeks of age and fed a high fat diet: Sham surgery with placebo treatment, orchiectomy surgery with placebo treatment, and orchiectomy surgery with testosterone treatment. Surgeries were performed at 8 weeks of age, followed by fortnightly testosterone treatment via injection. Mice were sacrificed at 25 weeks of age. Tibiae were collected and scanned ex-vivo at 4.3μm on a SkyScan 1272 Micro-CT scanner (Bruker). Left tibiae were used for assessment of trabecular and cortical Volumes of Interest (VOIs) 0.2mm and 1.0mm respectively from the growth-plate bridge break. Tibiae were subsequently paraffin embedded and sectioned at 4μm prior to immunohistochemical evaluation of alkaline phosphatase. Results. Trabecular bone volume and mineral density were significantly reduced in orchiectomised mice compared to sham-operated controls; and these parameters were normalised to control levels in orchiectomised mice treated with testosterone. In contrast, Trabecular thickness was significantly higher in testosterone depleted animals. Cortical bone parameters and body weights did not significantly differ between groups. Levels of alkaline phosphatase did not differ significantly in cortical or trabecular osteoblasts between groups. Conclusions. Findings suggest that testosterone deficiency significantly reduces trabecular bone parameters, and testosterone therapy may be a useful intervention for the loss of bone mass in testosterone deficient males. These results indicate that testosterone therapy may be useful for the treatment of trabecular bone frailty in testosterone deficient males. Observed changes in trabecular bone do not appear to be due to decreased mineralisation caused by osteoblast alkaline phosphatase. Ongoing work includes histology analysis to elucidate the mechanisms underpinning the changes seen in the bones of testosterone deficient animals

Although osteoporosis reduces overall bone mass causing bone fragility, our recent studies have shown that bone tissue composition is altered at the microscopic level, which is undetectable by conventional diagnostic techniques (DEXA) but may contribute to bone fracture. However, the time sequence of changes in bone microarchitecture, mechanical environment and mineral distribution are not yet fully understood. This study quantified the longitudinal effects of estrogen deficiency on the trabecular microarchitecture and mineral distribution in the tibia of Female Wistar rats (6 months) that underwent ovariectomy (OVX, n=10) or sham surgery (SHAM, n=10). Weekly micro-CT scans of the proximal tibia were conducted at 15µm resolution for the first month of estrogen deficiency. Morphometric analysis was conducted to characterise the trabecular bone microarchitecture. The bone mineral composition was characterised with analysis of bone mineral density distributions (BMDD). There was significantly reduced trabecular bone volume fraction at 2 weeks in OVX rats compared to controls (p<0.01). There was no difference in mineral distribution between the OVX and control animals. This study provides the first evidence in uncovering the temporal nature of changes in bone microarchitecture and mineral distribution, showing that structure changes before composition. In-vivo µCT analysis for later time points (week 8, 14 and 34) is ongoing to comprehensively examine these longitudinal compositional changes. Moreover, we are conducting ex-vivo mechanical analysis (nanoindentation), and together these will uncover the time-sequence and respective contribution of changes in bone mass and composition to the integrity of the bone tissue at these stages of estrogen deficiency

The superior analgesic effects of minodronate compared with other bisphosphonates has been previously reported. However, to our knowledge, there are no studies analyzing the analgesic effects of bisphosphonates on chronic pain. The purpose of the present study was to evaluate the analgesic effects of minodronate (MIN), alendronate (ALN), and pregabalin (PRG) on chronic pain caused by chronic constriction injury (CCI) of the sciatic nerve. Four-week-old female Wister rats underwent ovariectomy. At 8 weeks old, the left sciatic nerve was ligated to induce the chronic pain model (CCI side), and sham surgery was performed on the right posterior limb as a CCI control (control side). The rats were divided into the following four groups: 1) MIN group, administered with minodronate (0.15 mg/kg/week) (n = 10); 2) ALN group, administered with alendronate (0.15 mg/kg/week) (n = 10); 3) PRG group, administered with pregabalin (10 mg/kg) (n = 9); and 4) Control group, administered with vehicle (n = 10). Treatments were administered subcutaneously every week for 2 weeks immediately after CCI. To quantify the sensitivity to a tactile stimulus, paw withdrawal in response to a tactile stimulus was measured using von Frey filaments at 0, 1, and 2 weeks after CCI. Von Frey filaments were applied to the plantar surface of the hindpaws for 3 s, and this was repeated three times. Paw withdrawal in response to the stimulus was evaluated by scoring as follows: 0, no response; 1, a slow and/ or slight response to the stimulus; 2, a quick withdrawal response; 3, an intense withdrawal response away from the stimulus. The mean value of the score was adopted as the pain score. After evaluating the response, bilateral femurs were harvested for bone mineral density (BMD) measurements. The pain score of the CCI side was significantly higher than that of the sham side in all groups (p < 0.05) at each time point. The pain score for the MIN group, but not the ALN group, of the CCI side was significantly lower (p = 0.05) at 0 and 1 week after CCI. Total femoral BMD of the CCI side was significantly lower in the PRG and Control groups than those of the MIN and ALN groups (p < 0.05). No significant difference was identified for BMD between the MIN and ALN groups. Minodronate showed a significant analgesic effect on chronic pain and suppressed osteoporotic changes caused by CCI

Objectives

The role of mechanical stress and transforming growth factor beta 1 (TGF-β1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known.

Objectives

Sustained intra-articular delivery of pharmacological agents is an attractive modality but requires use of a safe carrier that would not induce cartilage damage or fibrosis. Collagen scaffolds are widely available and could be used intra-articularly, but no investigation has looked at the safety of collagen scaffolds within synovial joints. The aim of this study was to determine the safety of collagen scaffold implantation in a validated in vivo animal model of knee arthrofibrosis.