Destabilization Of The Medial Meniscus Surgery Research Articles

Low-level cyclic tibial compression attenuates early osteoarthritis progression after joint injury in mice

Mechanical loading and joint health have a unique relationship in osteoarthritis (OA) onset and progression. Although high load levels adversely affect cartilage health, exercise that involves low to moderate load levels can alleviate OA symptoms. We sought to isolate the beneficial effects of mechanical loading using controlled invivo cyclic tibial compression. We hypothesized that low-level cyclic compression would attenuate post-traumatic OA symptoms induced by destabilization of the medial meniscus (DMM). 10-week-old C57Bl/6J male mice underwent DMM surgery (n=51). After a 5-day post-operative recovery period, we applied daily cyclic tibial compression to the operated limbs at low (1.0N or 2.0N) or moderate (4.5N) magnitudes for 2 or 6 weeks. At the completion of loading, we compared cartilage and peri-articular bone features of mice that underwent DMM and loading to mice that only underwent DMM. Compared to DMM alone, low-level cyclic compression for 6 weeks attenuated DMM-induced cartilage degradation (OARSI score, P=0.008, 95% confidence interval (CI): 0.093 to 0.949). Low-level loading attenuated DMM-induced osteophyte formation after 2 weeks (osteophyte size, P=0.033, 95% CI: 3.27-114.45μm), and moderate loading attenuated subchondral bone sclerosis after 6 weeks (tissue mineral density (TMD), P=0.011, 95% CI: 6.32-70.60mg HA/ccm) compared to limbs that only underwent DMM. Finally, loading had subtle beneficial effects on cartilage cellularity and aggrecanase activity after DMM. Low-level cyclic compression is beneficial to joint health after an injury. Therefore, the progression of early OA may be attenuated by applying well controlled, low-level loading shortly following joint trauma.

Disruption of glucocorticoid signalling in osteoblasts attenuates age-related surgically induced osteoarthritis

Aging is a major risk factor for osteoarthritis (OA). Skeletal expression and activity of the glucocorticoid-activating enzyme 11β-hydroxysteroid-dehydrogenase type 1 increases progressively with age in humans and rodents. Here we investigated the role of endogenous osteocytic and osteoblastic glucocorticoid (GC) signalling in the development of osteoarthritic bone and cartilage damage in mice. We utilized transgenic (tg) mice in which glucocorticoid signalling is disrupted in osteoblasts and osteocytes via overexpression of the glucocorticoid-inactivating enzyme, 11β-hydroxysteroid-dehydrogenase type 2. Osteoarthritis was induced in 10- and 22-week-old male transgenic mice (tg-OA, n=6/group) and their wildtype littermates (WT-OA, n=7-8/group) by surgical destabilization ofthemedial meniscus (DMM). Sham-operated mice served as controls (WT- & tg-Sham, n=3-5 and 6-8/group at 10- and 22-weeks of age, respectively). Sixteen weeks after DMM surgery, mice developed features of cartilage degradation, subchondral bone sclerosis and osteophyte formation. These changes did not differ between WT and tg mice when OA was induced at 10-weeks of age. However, when OA was induced at 22-weeks of age, cartilage erosion was significantly attenuated in tg-OA mice compared to WT-OA littermates. Similarly, subchondral bone volume (-5.2%, 95% confidence intervals (CI) -9.1 to-1.2%, P=0.014) and osteophyte size (-4.0mm2, 95% CI -7.5 to-0.5mm2, P=0.029) were significantly reduced in tg-OA compared to WT-OA mice. Glucocorticoid signalling in cells of the osteoblast lineage promotes the development of surgically-induced osteoarthritis in older, but not younger, male mice. These data implicate osteoblasts and osteocytes in the progression of DMM-OA, via a glucocorticoid-dependent and age-related pathway.

Rapamycin Inhibits Nf-ΚB Activation by Autophagy to Reduce Catabolism in Human Chondrocytes

Background and Aim: Osteoarthritis is a disease that is accompanied by inflammation and catabolic disorders in the cartilage. Rapamycin is a good autophagy activator and has an inhibitory effect on inflammation, and autophagy can remove waste generated by catabolic disorders and delay the destruction of cartilage by inflammation. This study was designed to evaluate the effect of rapamycin upon the catabolism in human chondrocytes and unravel underlying mechanism. Methods and Results: C57 mice and SW1353 cells were cultured and osteoblastic arthritis was formed by destabilization of the medial meniscus surgery; 10 ng/ml of IL-1β was added to the cells to produce inflammatory chondrocytes, and a non-cytotoxic concentration of 20 nM Rapamycin was used as the self-activator. Autophagy levels were verified by quantitative analysis of autophagy markers of LC3 and ATG5, and it was verified that rapamycin can activate autophagy. Autophagy inhibition was induced by ATG5 siRNA by comparing the presence of rapamycin treatment in inflammatory chondrocytes. The expression of the degrading gene and chemokine was evaluated by qPCR. Rapamycin down-regulated the expression of MMP-3 and -9, ADAMTS5, CCL-1, -2, and -5 induced by inflammation. Quantitative analysis of IκBα and P-P65 was used to analyze the interaction between the NF-κB pathway and autophagy in inflammation. Activation of the NF-κB pathway by inflammatory stimulation, P-P65 nuclear translocation, and degradation of IκBα protein were attenuated, respectively. Autophagy inhibited the activation of NF-κB signaling pathway in inflammatory chondrocytes. Conclusions: Rapamycin can inhibit the overexpression of inflammatory catabolic genes by activating autophagy, and can suppress the NF-κB signaling pathway in chondrocytes to break the positive feedback loop with inflammatory factors and reduce the rate and level of inflammation progression.

Murine chondrocytes within intact medial femoral condyles have dysregulated calcium responses to mechanical loading after dmm surgery

Purpose: Intracellular calcium mobilization is known to be a downstream signaling response to different types of mechanical loading in a variety of cells, including chondrocytes. Osteoarthritis involves aberrant loading, however, the calcium responses of chondrocytes to the mechanical loading changes induced by osteoarthritis have not been examined. Therefore, the purpose of this study was to generate novel methods to begin to address this question using the destabilization of the medial meniscus (DMM) mouse model of osteoarthritis. By better understanding how chondrocyte response to loading is altered in osteoarthritis, novel targets for therapies may be uncovered. Methods: We created a novel line of mice by crossing Collagen II cre mice with GCaMP6s loxp mice. GCaMP6s is an ultra-sensitive fluorescent calcium reporter that can be genetically expressed in specific cell types using cre-loxp recombination (in this case, chondrocytes). In these Collagen II-GCaMP6s mice, when intracellular calcium increases in chondrocytes, an increase in green fluorescence can be measured. DMM surgery in these mice causes joint damage similar to wild-type mice. To apply mechanical loading to mouse femoral condyle cartilage while performing calcium imaging, we built a system using a Zeiss Axio Observer inverted fluorescent microscope and an Aurora Scientific dual-mode lever system (300C) in order to apply compressive strain with 1 μm resolution (Fig 1). This setup enables us to compress murine medial femoral cartilage against a glass coverslip while imaging from below. We verified that our applied displacement resulted in accurate movement of our sample by an Eddy sensor (Micro-Epsilon ES04) with a resolution of 0.04 μm. DMM (n=4) or sham (n=3) surgery was performed in the right knee of 10-week old male Collagen II-GCaMP6 mice. Sixteen weeks after surgery, each femur was freshly dissected and prepared for calcium imaging. Calcium Imaging and Data Analysis: A defined sodium-calcium buffer, commonly used for calcium imaging, was used for all imaging and was slowly perfused through the chamber during each experiment. Images were taken at a frequency of 1 Hz, with an exposure time of 20 ms, using a GFP filter set. For each femur, baseline images were acquired without loading for 5 min, loading using a square waveform with amplitude 7 μm and frequency 0.05 Hz was applied for 5 min, 5 min of imaging was acquired post loading with the femur out of contact, and 8 min of imaging was performed while a 50% hypo-osmotic buffer was perfused past the femur. Images were processed using custom macros in Fiji to correct for minor movement as well as to calculate (F-Fo)/Fo, where Fo = the first 2-20 frames imaged. Responses in individual chondrocytes during the different imaging phases were compared between sham and DMM using unpaired, two-tailed t-tests (GraphPad Prism 6), where p<0.05 was considered statistically significant. Results: Seven microns of compression (∼18% strain; our sham mouse medial condyle cartilage was on average 39 μm thick) in medial femoral condyles resulted in greater numbers of chondrocytes generating intracellular calcium responses in sham mice compared to DMM mice 16 weeks after surgery (3.8-fold higher; p=0.03) (Fig 2). However, on average, DMM chondrocyte calcium responses were greater in amplitude and duration than sham chondrocyte responses (2.5-fold higher; p=0.03) (Fig 2). The average peak forces observed during compression were similar for sham (13.4±2.6 mN) and DMM mice (16.3±1.6 mN) (p = 0.35). Exposure to hypo-osmotic salt solution has been used previously as a positive control for inducing intracellular calcium responses in chondrocytes as it causes cells to stretch. Here, we found that 50% hypo-osmotic solution induced similar numbers of chondrocyte responses in both sham (54±10) and DMM (42±11; p=0.46) mice 16 weeks after surgery, and similar amplitude and duration of responses (sham: 6.9±0.2 ΔF/Fo x seconds; DMM: 9.7±2.6 ΔF/Fo x seconds; p=0.36). Conclusions: Here, we have generated a novel strain of mice and a novel mechanical stimulation and imaging system in order to image real-time responses of chondrocytes to mechanical loading within intact mouse femoral condyles after DMM surgery. Our results suggest that after DMM surgery, femoral condyle chondrocytes are still able to generate intracellular calcium responses to changes in osmolarity, but they have become dysregulated in response to mild levels of cartilage compression. Future work will seek to understand which calcium signaling pathways may be mediating these changes and how these changes may contribute to other pathological cartilage changes in osteoarthritis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Nociceptive neuroplasticity of the murine knee joint precedes severe structural joint damage in a surgical model of OA

Purpose: Surgical destabilization of the medial meniscus (DMM) in the mouse knee results in slowly progressive osteoarthritis (OA), characterized by mild-to-moderate joint damage by week 8 and severe damage by week 16 after surgery. Progressive joint damage is associated with pain-related behaviors. Using NaV1.8-tdTomato reporter mice, which express a bright red fluorescent tdTomato reporter in >90% of C-nociceptors (pain-sensing neurons), we recently reported that DMM surgery led to changes in the anatomical distribution of nociceptors in the medial compartment of the knee. Specifically, in association with severe structural joint damage 16 weeks after DMM, the medial compartment of OA knees showed increased NaV1.8+ innervation in the medial synovium and meniscus, and NaV1.8+ nociceptors in subchondral bone channels. Here, we analyzed the nociceptive innervation of the knee in the early stages of experimental OA, 4 and 8 weeks after DMM. Methods: DMM or sham surgery was performed in the right knee of 10-week old male C57BL/6 NaV1.8-tdTomato mice. Four and eight weeks after surgery, n=3 mice/group mice were perfused transcardially with paraformaldehyde, and the right knees collected, post fixed and decalcified. Twenty-μm thick coronal frozen sections were collected at mid-joint level, and imaged using a confocal microscope. NaV1.8-tdTomato signal of the medial synovium was quantified using ImageJ. Channels observed in the subchondral bone of the medial femoral condyles and tibial plateaux that contained NaV1.8+ nerves were quantified as follows: two mid-joint sections (80 μm apart) per knee were used to count NaV1.8+ channels; the counts were averaged for each knee. Results: As previously reported, 4 and 8 weeks after DMM surgery, cartilage damage in the femur and tibia was very mild and limited to slight loss of proteoglycans. In addition, DMM mice had maximal subchondral bone sclerosis in the medial compartment by week 4. Despite the limited cartilage damage at this stage, analysis of the NaV1.8+ signal showed striking changes in the nociceptive innervation after DMM surgery, compared to controls. Firstly, we detected an increase in NaV1.8 innervation of the medial synovium, as early as 4 weeks after DMM, and no further increase weeks 8-16 (Fig. 1). Secondly, NaV1.8+ nociceptors were observed in the subchondral bone pointing toward calcified cartilage, and this as early as week 4. Conclusions: These findings suggest that the NaV1.8 nociceptive innervation of the mouse knee changes very rapidly after DMM surgery, and this before the onset of significant joint damage. While the biological significance of this observation needs to be established, it suggests that neuroplasticity of sensory nerves is an early feature of OA joint damage, which may contribute to joint disease.

B-cell-specific mammalian target of rapamycin complex 1 activation results in severe osteoarthritis in mice

This study aims to investigate the effect of enriched plasma cells on the production of inflammatory cytokines and development of osteoarthritis (OA) in mice with B-cell-specific conditional deletion of the tuberous sclerosis 1 gene (TSC1). OA was induced by destabilization of the medial meniscus (DMM) in mice with TSC1 disruption in B cells (CD19-TSC1) and in littermate control mice (CON). The effects of DMM and incidence of OA were evaluated histologically, mRNA levels of inflammatory cytokines were detected by polymerase chain reaction, and serum cytokine levels were detected by enzyme-linked immunosorbent assay. Deletion of TSC1 caused constitutive activation of mechanistic target of rapamycin complex 1 mTORC1 in B cells. CON mice subjected to DMM exhibited a severe OA phenotype with increased inflammatory cytokines in B cells, serum, and the synovial membrane. Importantly, inflammatory cytokine production was also increased in B cells from the spleen of CD19-TSC1 conditional KO mice, but the OA phenotype was significantly elevated in conditional KO mice after DMM surgery compared with CON mice, as indicated by more severe articular cartilage destruction, increased protein expression of matrix metalloproteinase-13 and mRNA of type X collagen in the articular cartilage, decreased mRNA expression of type II collagen in the articular cartilage, and increased inflammatory cytokines in serum and the synovial membrane. The results demonstrate that inflammatory cytokine synthesis by B cells was enriched in CD19-TSC1 conditional KO mice, and this enhanced synthesis of inflammatory cytokines accelerated the incidence of OA.

Bioluminescence and second harmonic generation imaging reveal dynamic changes in the inflammatory and collagen landscape in early osteoarthritis

Osteoarthritis (OA) is a leading cause of chronic disability whose mechanism of pathogenesis is largely elusive. Local inflammation is thought to play a key role in OA progression, especially in injury-associated OA. While multiple inflammatory cytokines are detected, the timing and extent of overall inflammatory activities in early OA and the manner by which joint inflammation correlates with cartilage structural damage are still unclear. We induced OA via destabilization of the medial meniscus (DMM) in NFκB luciferase reporter mice, whose bioluminescent signal reflects the activity of NFκB, a central mediator of inflammation. Bioluminescence imaging data showed that DMM and sham control joints had a similar surge of inflammation at 1-week post-surgery, but the DMM joint exhibited a delay in resolution of inflammation in subsequent weeks. A similar trend was observed with synovitis, which we found to be mainly driven by synovial cell density and inflammatory infiltration rather than synovial lining thickness. Interestingly, an association between synovitis and collagen structural damage was observed in early OA. Using Second Harmonic Generation (SHG) imaging, we analyzed collagen fiber organization in articular cartilage. Zonal differences in collagen fiber thickness and organization were observed as soon as OA initiated after DMM surgery, and persisted over time. Even at 1-week post-surgery, the DMM joint showed a decrease in collagen fiber thickness in the deep zone and an increase in collagen fiber disorganization in the superficial zone. Since we were able detect and quantify collagen structural changes very early in OA development by SHG imaging, we concluded that SHG imaging is a highly sensitive tool to evaluate pathological changes in OA. In summary, this study uncovered a dynamic profile of inflammation and joint cartilage damage during OA initiation and development, providing novel insights into OA pathology.

Elf3 Contributes to Cartilage Degradation in vivo in a Surgical Model of Post-Traumatic Osteoarthritis

The E-74 like factor 3 (ELF3) is a transcription factor induced by inflammatory factors in various cell types, including chondrocytes. ELF3 levels are elevated in human cartilage from patients with osteoarthritis (OA), and ELF3 contributes to the IL-1β-induced expression of genes encoding Mmp13, Nos2, and Ptgs2/Cox2 in chondrocytes in vitro. Here, we investigated the contribution of ELF3 to cartilage degradation in vivo, using a mouse model of OA. To this end, we generated mouse strains with cartilage-specific Elf3 knockout (Col2Cre:Elf3f/f) and Comp-driven Tet-off-inducible Elf3 overexpression (TRE-Elf3:Comp-tTA). To evaluate the contribution of ELF3 to OA, we induced OA in 12-week-old Col2Cre:Elf3f/f and 6-month-old TRE-Elf3:Comp-tTA male mice using the destabilization of the medial meniscus (DMM) model. The chondrocyte-specific deletion of Elf3 led to decreased levels of IL-1β- and DMM-induced Mmp13 and Nos2 mRNA in vitro and in vivo, respectively. Histological grading showed attenuation of cartilage loss in Elf3 knockout mice compared to wild type (WT) littermates at 8 and 12 weeks following DMM surgery that correlated with reduced collagenase activity. Accordingly, Elf3 overexpression led to increased cartilage degradation post-surgery compared to WT counterparts. Our results provide evidence that ELF3 is a central contributing factor for cartilage degradation in post-traumatic OA in vivo.

Use of Contralateral Knee as Control in the Destabilization of Medial Meniscus Osteoarthritis Rat Model

Destabilization of the medial meniscus (DMM) is a common surgical procedure used to induce osteoarthritis (OA) in a rodent model. However, there is a lack of evidence on using the contralateral limb as a control. Contralateral rat knees have been used as the control in noninvasive OA pain models, but not DMM models; and the contralateral limb of guinea pigs has been used to study the development of OA after DMM surgery. In a previous study, we examined the effects of DMM and obesity on the development and progression of OA in a rat model. In that study, 42 female Lewis rats were randomly assigned to 4 groups: Group 1 received a sham surgery (control) and was on a regular diet, Group 2 received a DMM surgery and was on a regular diet, Group 3 received a sham surgery and was on a high‐fat diet, and Group 4 received a DMM surgery and was on a high‐fat diet. The rats were on their respective diets for 4 weeks, underwent surgery, and were sacrificed 4 weeks after surgery. The knee joints of surgical and contralateral limbs were harvested. The Osteoarthritis Research Society International (OARSI) scoring system was used to score the damage to the articular cartilage of the knee. In the surgical limbs, DMM resulted in significantly greater OARSI scores than the sham surgery in the lateral and medial compartments of the knee in both diet groups. After that study, the contralateral knee tissues were stored in 10% formalin. For the current study, we used those stored tissues to determine whether the contralateral limbs could serve as a control in future studies that involve DMM in a rat model. Knees were decalcified, embedded in paraffin, and sectioned coronally at 10 μm. Slides were stained using Safranin O and fast green for counterstain. Four articular surfaces of the knee (medial and lateral femoral condyles and medial and lateral tibial plateaus) were histologically evaluated for degenerative changes by a blinded scorer. A generalized linear mixed model was used to analyze differences between the rat knee groups. Comparison of sham and DMM rat knees across both diet groups showed no significant difference in OARSI scores (P=.61). When comparing within each of the diet groups, there was no significant difference between the DMM and sham scores in the high‐fat diet groups (P&gt;.99) or regular diet groups (P=.49). After comparing the knee surface scores for all groups, the medial tibial plateau had significantly lower scores than the other 3 surfaces (P&lt;.001). Based on results of the current study, we believe the contralateral limb can be used as a control in short‐term (4 weeks or less) OA studies using DMM in a rat model. This will save time and resources by achieving an adequate sample size with fewer animals. Our results also showed that the medial tibial plateau was significantly less susceptible to the development of spontaneous OA compared to the other 3 articular surfaces studied.Support or Funding InformationSupport for this project was received from Dr. Peter Kondrashov, Department of Anatomy, A.T. Still University, Kirksville College of Osteopathic Medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Male and female gait compensations in a mouse model of medial meniscus destabilization

Purpose: Destabilization of the medial meniscus (DMM) has become a common method for modeling the development of post-traumatic knee osteoarthritis in mice. Moreover, following a DMM surgery, male and female mice develop histologic signs of cartilage damage at different rates. Less is known about the development of disability in this model, as characterizations of the mouse gait pattern have been limited and primarily focused on spatiotemporal gait patterns. Recently, our group developed a highly sensitive gait analysis platform capable of detecting rodent spatiotemporal gait patterns in conjunction with ground reaction forces. Therefore, the aim of this study is to evaluate quadrupedal gait compensations and joint degeneration in male and female mice after DMM. Methods: For 20 C57/Bl6 mice, a medial para-patellar ligament incision was made on the right hind limb, and blunt dissection revealed the medial meniscotibial ligament. Sham animals (5 male, 5 female) were then closed. For DMM animals (5 male, 5 female), the meniscotibial ligament was transected and animals were closed. Ground reaction force data and spatiotemporal gait data were collected at 2, 4, 6, 9, and 12 weeks post-surgery. Animals were placed in a gait arena and allowed to freely explore. Ultra-high speed video (500fps) recorded sagittal and ventral views as the animal crossed the arena and walked over force panels. A linear regression was used to describe the expected gait for male or female sham animals at a given velocity and body weight. Residuals were calculated from this control line to determine the expected gait pattern at a given velocity and body weight. Groups were compared using a 2-way ANOVA with interaction (α = 0.05), while gait symmetries and imbalances were compared to expected values using Bonferroni-corrected student's t-tests. After 12 weeks, animals were euthanized, and knees were processed for histological grading. Results: Male DMM mice used higher hind limb duty factors in both limbs compared to sham animals (Fig 1A, left P = 0.001, right P = 0.037). Male DMM mice also used shorter stride lengths (P = 0.007). Both fore and hind limb peak vertical forces were lower in male DMM mice compared to sham mice (Fig 1C & 1E, fore left P = 0.001, fore right P = 0.029, hind left P = 0.010). Female DMM mice walked with lower hind limb duty factors compared to sham animals (Fig 1B, left P = 0.045, right P = 0.001), were temporally asymmetric in the hind limbs at week 12 (P = 0.005), and significantly loaded the right hind limb more than sham animals (Fig 1F, P = 0.039). Female DMM mice also took longer to load the right hind limb (P = 0.004). Histology confirmed no cartilage loss or osteophyte formation in sham animals. Male and female DMM animals presented similar cartilage matrix loss at the surface and at 50% lesion depths at 12 weeks. Additionally, total cartilage degeneration width was similar between genders (male = 69.81 ± 18.5%, female = 69.9 ± 28.9%). Conclusions: Male and female mice selected different gait compensations following meniscus destabilization. Male mice developed higher hind limb duty factors, indicating more time spent in stance on both limbs. Additionally, male DMM mice had shorter stride lengths and normal gait symmetries. Male DMM mice also had lower peak vertical forces on both hind limbs. Combined, these data indicate a bilateral shuffle-step compensation. Shuffle-stepping may protect the limb by reducing time where either leg must support load without contralateral support. Female DMM mice used lower hind limb duty factors in the right hind limb (injured). Additionally, female mice placed more load on the right hind limb, but also took longer to load the injured limb. Combined, these data may indicate a steppage gait compensation. It is not clear why male shuffle-step and female mice use a steppage-like compensation. These gender differences warrant further exploration, but may be important to how male and female animals respond to orthopaedic injuries. Finally, our histological data demonstrate the development of OA lesions in both male and female mice at 12 weeks. While prior reports have detailed more severe OA lesions in male mice relative to female mice, these histological investigations largely focused on post-operative timepoints of 8 weeks or less. Thus, it is plausible that the severity of OA in female mice had approached that of male mice by 12 weeks. Regardless, the two sexes developed different gait compensations in response to the injury, further highlighting sex differences in the progression of OA in mice.

Inhibition of Wnt/β-catenin signaling ameliorates osteoarthritis in a murine model of experimental osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease involving both cartilage and synovium. The canonical Wnt/β-catenin pathway, which is activated in OA, is emerging as an important regulator of tissue repair and fibrosis. This study seeks to examine Wnt pathway effects on synovial fibroblasts and articular chondrocytes as well as the therapeutic effects of Wnt inhibition on OA disease severity. Mice underwent destabilization of the medial meniscus surgery and were treated by intra-articular injection with XAV-939, a small-molecule inhibitor of Wnt/β-catenin signaling. Wnt/β-catenin signaling was highly activated in murine synovial fibroblasts as well as in OA-derived human synovial fibroblasts. XAV-939 ameliorated OA severity associated with reduced cartilage degeneration and synovitis in vivo. Wnt inhibition using mechanistically distinct small-molecule inhibitors, XAV-939 and C113, attenuated the proliferation and type I collagen synthesis in synovial fibroblasts in vitro but did not affect human OA-derived chondrocyte proliferation. However, Wnt modulation increased COL2A1 and PRG4 transcripts, which are downregulated in chondrocytes in OA. In conclusion, therapeutic Wnt inhibition reduced disease severity in a model of traumatic OA via promoting anticatabolic effects on chondrocytes and antifibrotic effects on synovial fibroblasts and may be a promising class of drugs for the treatment of OA.

Visualization of Peripheral Neuron Sensitization in a Surgical Mouse Model of Osteoarthritis by In Vivo Calcium Imaging.

To develop a method for analyzing sensory neuron responses to mechanical stimuli in vivo, and to evaluate whether these neuronal responses change after destabilization of the medial meniscus (DMM). DMM or sham surgery was performed in 10-week-old male C57BL/6 wild-type or Pirt-GCaMP3+/- mice. All experiments were performed 8 weeks after surgery. Knee and hind paw hyperalgesia were assessed in wild-type mice. The retrograde label DiI was injected into the ipsilateral knee to quantify the number of knee-innervating neurons in the L4 dorsal root ganglion (DRG) in wild-type mice. In vivo calcium imaging was performed on the ipsilateral L4 DRG of Pirt-GCaMP3+/- mice as mechanical stimuli (paw pinch, knee pinch, or knee twist) were applied to the ipsilateral hind limb. Eight weeks after surgery, mice subjected to DMM had more hyperalgesia in the knee and hind paw compared to mice subjected to sham surgery. Intraarticular injection of DiI labeled similar numbers of neurons in the L4 DRG of mice subjected to sham surgery and mice subjected to DMM. Increased numbers of sensory neurons responded to all 3 mechanical stimuli in mice subjected to DMM, as assessed by in vivo calcium imaging. The majority of responses in mice subjected to sham surgery and mice subjected to DMM were in small to medium-sized neurons, consistent with the size of nociceptors. The magnitude of responses was similar between mice subjected to sham surgery and mice subjected to DMM. Our findings indicate that increased numbers of small to medium-sized DRG neurons respond to mechanical stimuli 8 weeks after DMM surgery, suggesting that nociceptors have become sensitized by lowering the response threshold.

Yougui Pills Attenuate Cartilage Degeneration via Activation of TGF-β/Smad Signaling in Chondrocyte of Osteoarthritic Mouse Model

Yougui pills (YGPs) have been used for centuries in the treatment of Chinese patients with Kidney-Yang Deficiency Syndrome. Despite the fact that the efficiency of YGPs on treating osteoarthritis has been verified in clinic, the underlying mechanisms are not totally understood. The present study observes the therapeutic role of YGPs and mechanisms underlying its chondroprotective action in osteoarthritic cartilage. To evaluate the chondroprotective effects of YGPs, we examined the impact of orally administered YGPs in a model of destabilization of the medial meniscus (DMM). Male C57BL/6J mice were provided a daily treatment of YGPs and a DMM surgery was performed on the right knee. At 12 weeks post-surgery, the joints were harvested for tissue analyses, including histomorphometry, OARSI scoring, micro-CT and immunohistochemistry for COL-2, MMP-13 and pSMAD-2. We also performed the relative experiments mentioned above in mice with Tgfbr2 conditional knockout (TGF-βRIICol2ER mice) in articular cartilage. To evaluate the safety of YGPs, hematology was determined in each group. Amelioration of cartilage degradation was observed in the YGPs group, with increases in cartilage area and thickness, proteoglycan matrix, and decreases in OARSI score at 12 weeks post surgery. In addition, reduced BV/TV and Tb. Th, and elevated Tb. Sp were observed in DMM-induced mice followed by YGPs treatment. Moreover, the preservation of cartilage correlated with reduced MMP-13, and elevated COL-2 and pSMAD-2 protein expressional levels were also revealed in DMM-induced mice treated with YGPs. Similarly, TGF-βRIICol2ER mice exhibited significant OA-like phenotype. However, no significant difference in cartilage structure was observed in TGF-βRIICol2ER mice after YGPs treatment. Interestingly, no obvious adverse effects were observed in mice from each group based on the hematologic analyses. These findings suggested that YGPs could inhibit cartilage degradation through enhancing TGF-β/Smad signaling activation, and be considered a good option for the treatment of osteoarthritis.

Chemokine receptor-7 (CCR7) deficiency leads to delayed development of joint damage and functional deficits in a murine model of osteoarthritis.

Elevated chemokine receptor Ccr7 is observed in knee osteoarthritis (OA) and associated with severity of symptoms. In this study, we confirmed that CCR7 protein expression is elevated in synovial tissue from OA patients by immunohistochemical staining. We then investigated whether Ccr7 deficiency impacted structural and functional joint degeneration utilizing a murine model of OA. OA-like disease was induced in male C57BL/6 and Ccr7-deficient (Ccr7-/- ) mice by destabilization of the medial meniscus (DMM). Functional deficits were measured by computer integrated monitoring of spontaneous activity every 4 weeks after DMM surgery up 16 weeks. Joint degeneration was evaluated at 6 and 19 weeks post-surgery by histopathology, and subchondral bone changes analyzed by microCT. Results showed reduction in locomotor activities in DMM-operated C57BL/6 mice by 8 weeks, while activity decreases in Ccr7-/- mice were delayed until 16 weeks. Histopathologic evaluation showed minimal protection from early cartilage degeneration (p = 0.06) and osteophytosis (p = 0.04) in Ccr7-/- mice 6 weeks post-DMM compared to C57BL/6 controls, but not at 19 weeks. However, subchondral bone mineral density (p = 0.03) and histologic sclerosis (p = 0.02) increased in response to surgery in C57BL/6 mice at 6 weeks, while Ccr7-/- mice were protected from these changes. Our results are the first to demonstrate a role for Ccr7 in early development of functional deficits and subchondral bone changes in the DMM model. Understanding the mechanism of Ccr7 receptor signaling in the initiation of joint pathology and disability will inform the development of innovative therapies to slow symptomatic OA development after injury. Published 2017. This article is a U.S. Government work and is in the public domain in the USA. J Orthop Res 36:864-875, 2018.

Anemonin attenuates osteoarthritis progression through inhibiting the activation of IL-1β/NF-κB pathway.

The osteoarthritis (OA) progression is now considered to be related to inflammation. Anemonin (ANE) is a small natural molecule extracted from various kinds of Chinese traditional herbs and has been shown to inhibiting inflammation response. In this study, we examined whether ANE could attenuate the progression of OA via suppression of IL‐1β/NF‐κB pathway activation. Destabilization of the medial meniscus (DMM) was performed in 10‐week‐old male C57BL/6J mice. ANE was then intra‐articularly injected into joint capsule for 8 and 12 weeks. Human articular chondrocytes and cartilage explants challenged with interleukin‐1β (IL‐1β) were treated with ANE. We found that ANE delayed articular cartilage degeneration in vitro and in vivo. In particular, proteoglycan loss and chondrocyte hypertrophy were significantly decreased in ANE ‐treated mice compared with vehicle‐treated mice. ANE decreased the expressions of matrix metalloproteinase‐13 (MMP13), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), collagen X (Col X) while increasing Aggrecan level in murine with DMM surgery. ANE treatment also attenuated proteoglycan loss in human cartilage explants treated with IL‐1β ex vivo. ANE is a potent protective molecule for OA; it delays OA progression by suppressing ECM loss and chondrocyte hypertrophy partially by suppressing IL‐1β/NF‐κB pathway activation.

Chemogenetic Inhibition of Pain Neurons in a Mouse Model of Osteoarthritis.

To determine the ability of drugs that activate inhibitory G protein-coupled receptors (GPCRs) expressed in peripheral voltage-gated sodium channel 1.8 (NaV 1.8)-positive sensory neurons to control osteoarthritis (OA)-associated pain. We used designer receptors exclusively activated by a designer drug (DREADD) technology, which employs engineered GPCRs to activate or inhibit neurons upon binding the synthetic ligand clozapine N-oxide (CNO). NaV 1.8-Pdi C57BL/6 mice were generated to express the inhibitory DREADD receptor Pdi in NaV 1.8-expressing sensory neurons. Destabilization of the medial meniscus (DMM) surgery was performed in 10-week-old male mice. Four, 8, 12, or 16 weeks after surgery, knee hyperalgesia or hind paw mechanical allodynia was tested. Subsequently, CNO or vehicle was administered, and the effect on pain-related behaviors was measured by a blinded observer. Morphine was used as a control. Immunohistochemistry and electrophysiology confirmed functional expression of the inhibitory DREADD receptor Pdi by NaV 1.8-positive sensory neurons. Acute inhibition of NaV 1.8-expressing neurons in mice treated with CNO reduced knee hyperalgesia 4 weeks after DMM surgery and reduced mechanical allodynia 8 weeks after DMM surgery. Inhibition had no effect on pain-related behaviors 12 and 16 weeks after DMM surgery. Morphine, a drug that activates GPCRs in the peripheral and central nervous systems, was still effective in the later stage of experimental OA. Chemogenetic inhibition of NaV 1.8-expressing neurons blocks knee hyperalgesia and mechanical allodynia in early experimental OA, but is no longer efficacious in the later stages. These data indicate that activation of inhibitory GPCRs located solely outside the central nervous system may be ineffective in treating chronic OA pain.

Deletion of Runx2 in Articular Chondrocytes Decelerates the Progression of DMM-Induced Osteoarthritis in Adult Mice

Runx2 may play an important role in development of osteoarthritis (OA). However, the specific role of Runx2 in articular chondrocyte function and in OA development in adult mice has not been fully defined. In this study, we performed the destabilization of the medial meniscus (DMM) surgery at 12-week-old mice to induce OA in adult Runx2Agc1CreER mice, in which Runx2 was specifically deleted in Aggrecan-expressing chondrocytes by administering tamoxifen at 8-weeks of age. Knee joint samples were collected 8- and 12-weeks post-surgery and analyzed through histology, histomorphometry and micro-computed tomography (μCT). Our results showed that severe OA-like defects were observed after DMM surgery in Cre-negative control mice, including articular cartilage degradation and subchondral sclerosis, while the defects were significantly ameliorated in Runx2Agc1CreER KO mice. Immunohistochemical (IHC) results showed significantly reduced expression of MMP13 in Runx2Agc1CreER KO mice compared to that in Cre-negative control mice. Results of quantitative reverse-transcription PCR (qRT-PCR) demonstrated that expression of the genes encoding for matrix degradation enzymes was significantly decreased in Runx2Agc1CreER KO mice. Thus, our findings suggest that inhibition of Runx2 in chondrocytes could at least partially rescue DMM-induced OA-like defects in adult mice.

Osteoarthritis induced by destabilization of the medial meniscus (DMM) is reduced in germ-free mice

Purpose: Recent studies have suggested a role for the (gut) microbiota in a number of inflammatory conditions, including rheumatoid arthritis, but little is known in osteoarthritis. For this project, we used the DMM model of OA and investigated the difference in OA development between mice maintained in a germ-free environment (GF) and mice housed in standard specific pathogen free (SPF) conditions. We hypothesized that if the gut microbiota produces factors that promote systemic inflammation, then GF mice would develop less severe OA than SPF mice. Methods: DMM surgery was performed on the right leg in C57BL/6 male mice using 23 SPF mice and 20 GF mice housed in a gnotobiotic facility. A sham group was not included since GF mice are in limited supply and we wanted to maximize the numbers of mice in the DMM groups. We have previously shown that OA does not develop after sham surgery in wild-type mice and so the contralateral knee (left) was used as an unoperated control. We analyzed 2 age groups -13.5 weeks AVG age at DMM (17 SPF and 15 GF) and 43 weeks AVG age at DMM (6 SPF and 5 GF). Mice were sacrificed 8 weeks following surgery and both knee joints were processed for histology. Serial sections were cut throughout the joint and the mid- coronal region was identified by the shape of the menisci. Two slides (mid- coronal and another slide ∼ 50 um posteriorly) were stained with Safranin O (Saf O) and scored for cartilage lesions using a modified articular cartilage structure (ACS) score in all four quadrants of the knee. The MAX score was the highest OA severity score of the four quadrant scores and the SUM score was the SUM of the four quadrant scores for the 2 slides. The mid-coronal region slide was also scored for proteoglycan loss (Saf O staining score- using a modified scoring system that follows the ACS score), osteophyte size (medial femur, medial tibia and MAX medial side) and synovial hyperplasia (MAX joint). The data were analyzed using one- way ANOVA and Tukey's post- hoc test; a multiplicity adjusted p value <0.05 was considered significant. Results: Several of the OA severity measurements showed a level of protection in the DMM knees of the GF mice compared to SPF mice. The MAX_ ACS score was decreased in the GF mice by 28% (AVG SPF = 8.652; AVG GF = 6.250; p = 0.036), the MAX_ Saf O score showed no difference while the SUM_Saf O score was decreased by 31% in GF mice (20.87 SPF; 14.35 GF; p = 0.0089). When analyzed in all the animals, the MAX osteophyte size score showed no significant change. When the scores were separated by age, the level of significance increased in the younger mice: the GF showed a reduction in the MAX_ACS score by 45% (9.294 SPF; 5.133 GF; p = 0.0003 - Fig.1), the SUM_ Saf O score by 44% (22.59 SPF; 12.67 GF; p = 0.0004), the MAX_Saf O score by 30% (10.18 SPF; 7.133 GF; p = 0.0380 ) and the MAX osteophyte size score by 36% (2.412 SPF; 1.533 GF; p = 0.0119 ), while the older GF mice showed no difference when compared to the age- matched SPF mice. While the synovial hyperplasia score was higher in the DMM knee compare to the control knee, no difference was seen between the SPF and GF mice, even if analyzed separately based on age. There were no differences in any of the measures in the unoperated control joints. Conclusions: These results suggest a role for the microbiome in OA development in an injury-induced OA model in mice. The finding that the older group of GF mice, compared to age-matched SPF mice, showed less protection from OA than the younger group might be explained by additional pathologies in the GF mice that appear with age (such as bone loss) that could override the effect of the microbiome on the rest of the joint. These results support further investigation into the role of the microbiome in OA, including studies in humans and other mouse models that are ongoing.

Chondroprotective activity of N-acetyl phenylalanine glucosamine derivative on knee joint structure and inflammation in a murine model of osteoarthritis

Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by joint structure changes and inflammation, both mediated by the IκB kinase (IKK) signalosome complex. The ability of N-acetyl phenylalanine derivative (NAPA) to increase cartilage matrix components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity, has been observed invitro. The present study aims to further clarify the effect of NAPA in counteracting OA progression, in an invivo mouse model after destabilization of the medial meniscus (DMM). 26 mice were divided into three groups: (1) DMM surgery without treatment; (2) DMM surgery treated after 2weeks with one intra-articular injection of NAPA (2.5mM) and (3) no DMM surgery. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and microhardness of subchondral bone (SB) tests. The injection of NAPA significantly improved cartilage thickness (CT) and reduced Chambers and Mankin modified scores and fibrillation index (FI), with weaker MMP13, ADAMTS5, MMP10 and IKKα staining. The microhardness measurements did not shown statistically significant differences between the different groups. NAPA markedly improved the physical structure of articular cartilage while reducing catabolic enzymes, extracellular matrix (ECM) remodeling and IKKα expression, showing to be able to exert a chondroprotective activity invivo.

Fibroblast Growth Factor Receptor 3 Inhibits Osteoarthritis Progression in the Knee Joints of Adult Mice.

Fibroblast growth factor (FGF) signaling is involved in articular cartilage homeostasis. This study was undertaken to investigate the role and mechanisms of FGF receptor 3 (FGFR-3) in the pathogenesis of osteoarthritis (OA) caused by surgery and aging in mice. FGFR-3 was conditionally deleted or activated in articular chondrocytes in adult mice subjected to surgical destabilization of the medial meniscus (DMM). A mouse model of human achondroplasia was also used to assess the role of FGFR-3 in age-associated spontaneous OA. Knee joint cartilage was histologically evaluated and scored using the Osteoarthritis Research Society International system. The expression of genes associated with articular cartilage maintenance was quantitatively evaluated in hip cartilage explants. The effect of inhibiting Indian hedgehog (IHH) signaling in Fgfr3-deficient explants was analyzed. Conditional Fgfr3 deletion in mice aggravated DMM-induced cartilage degeneration. Matrix metalloproteinase 13 and type X collagen levels were up-regulated, while type II collagen levels were down-regulated, in the articular cartilage of these mice. Conversely, FGFR-3 activation attenuated cartilage degeneration induced by DMM surgery and age. IHH signaling and runt-related transcription factor 2 levels in mouse articular chondrocytes were up-regulated in the absence of Fgfr3, while inhibition of IHH signaling suppressed the increases in the expression of Runx2, Mmp13, and other factors in Fgfr3-deficient mouse cartilage explants. Our findings indicate that FGFR-3 delays OA progression in mouse knee joints at least in part via down-regulation of IHH signaling in articular chondrocytes.