Delayed cartilage oligomeric matrix protein response to loading is associated with femoral cartilage composition post-ACLR.

PURPOSE: To determine associations between immediate and delayed response of serum cartilage oligomeric matrix protein (sCOMP) to loading (i.e., 3000 walking steps) and femoral cartilage interlimb T1ρ relaxation times in individual's post-anterior cruciate ligament reconstruction (ACLR). METHODS: This cross-sectional study included 20 individuals 6-12 months following primary ACLR (65% female, 20.5 ± 4.0 years old, 24.9 ± 3.0 kg/m2, 7.3 ± 1.5 months post-ACLR). Serum samples were collected prior to, immediately following, and 3.5 h following walking 3000 steps on a treadmill at habitual walking speed. sCOMP concentrations were processed using enzyme-linked immunosorbent assays. Immediate and delayed absolute sCOMP responses to loading were evaluated immediately and 3.5 h post-walking, respectively. Participants underwent bilateral magnetic resonance imaging with T1ρ sequences to calculate resting femoral cartilage interlimb T1ρ relaxation time ratios between limbs (i.e., ACLR/Uninjured limb). Linear regression models were fitted to determine associations between sCOMP response to loading and femoral cartilage T1ρ outcomes controlling for pre-loading sCOMP concentrations. RESULTS: Greater increases in delayed sCOMP response to loading were associated with greater lateral (∆R2 = 0.29, p = 0.02) but not medial (∆R2 < 0.01, p = 0.99) femoral cartilage interlimb T1ρ ratios. Associations between immediate sCOMP response to loading with femoral cartilage interlimb T1ρ ratios were weak and non-significant (∆R2 range = 0.02-0.09, p range = 0.21-0.58). CONCLUSION: Greater delayed sCOMP response to loading, a biomarker of cartilage breakdown, is associated with worse lateral femoral cartilage composition in the ACLR limb compared to the uninjured limb. Delayed sCOMP response to loading may be a more indicative metabolic indicator linked to deleterious changes in composition than immediate sCOMP response.

Impaired Annulus Fibrosus Development and Vertebral Fusion Cause Severe Scoliosis in Mice with Deficiency of c-Jun NH2-Terminal Kinases 1 and 2.

Mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK), play an important role in the development and function of a large variety of tissues. The skeletal phenotype of JNK1 and JNK2 double-knockout (dKO) mice (JNK1fl/flCol2-Cre/JNK2-/-) and control genotypes were analyzed at different embryonic and postnatal stages. JNK1/2 dKO mice displayed a severe scoliotic phenotype beginning during development that was grossly apparent around weaning age. Alcian blue staining at embryonic day 17.5 showed abnormal fusion of the posterior spinal elements. In adult mice, fusion of vertebral bodies and of spinous and transverse processes was noted by micro-computed tomography, Alcian blue/Alizarin red staining, and histology. The long bones developed normally, and histologic sections of growth plate and articular cartilage revealed no significant abnormalities. Histologic sections of the vertebral column at embryonic days 15.5 and 17.5 revealed an abnormal organization of the annulus fibrosus in the dKOs, with chondrocyte-like cells and fusion of dorsal processes. Spinal sections in 10-week-old dKO mice showed replacement of intervertebral disk structures (annulus fibrosus and nucleus pulposus) by cartilage and bone tissues, with cells staining for markers of hypertrophic chondrocytes, including collagen X and runt-related transcription factor 2. These findings demonstrate a requirement for both JNK1 and JNK2 in the normal development of the axial skeleton. Loss of JNK signaling results in abnormal endochondral bone formation and subsequent severe scoliosis.

Synovial joints: from development to homeostasis.

Synovial joint morphogenesis occurs through the condensation of mesenchymal cells into a non-cartilaginous region known as the interzone and the specification of progenitor cells that commit to the articular fate. Although several signaling molecules are expressed by the interzone, the mechanism is poorly understood. For treatments of cartilage injuries, it is critical to discover the presence of joint progenitor cells in adult tissues and their expression gene pattern. Potential stem cell niches have been found in different joint regions, such as the surface zone of articular cartilage, synovium, and groove of Ranvier. Inherited joint malformations as well as joint-degenerating conditions are often associated with other skeletal defects and may be seen as the failure of morphogenic factors to establish the correct microenvironment in cartilage and bone. Therefore, exploring how joints form can help us understand how cartilage and bone are damaged and develop drugs to reactivate this developing mechanism.

Expression levels of insulin receptor substrate-1 modulate the osteoblastic differentiation of mesenchymal stem cells and osteosarcoma cells.

The insulin-like growth factor-1 system, including its critical mediator insulin receptor substrate-1 (IRS-1), is involved in regulating osteosarcoma (OS) cell proliferation or differentiation. The aim of this study is to define the role of IRS-1 in OS cells by assessing the contribution of IRS-1 in the differentiation of human and murine OS cell lines and mouse mesenchymal stem cells (MSCs) and found that the basal level of IRS-1 is important for the initiation of differentiation. Both down-regulation and over-expression of IRS-1 inhibited osteoblastic differentiation. In vivo studies showed that OS cells over-expressing IRS-1 have increased metastatic potential and tumor growth. The proteasome inhibitor MG-132 led to an increase in IRS-1 protein level that inhibited osteoblastic differentiation, suggesting a role for proteasomal regulation in maintaining the appropriate expression level of IRS-1. Thus, precise regulation of IRS-1 expression level is critical for determining the differentiating capacity of MSCs and OS cells, and that derangement of IRS-1 levels can be a critical step in OS transformation.

Physical Activity Associates with T1rho MRI of Femoral Cartilage After Anterior Cruciate Ligament Reconstruction.

PURPOSE: Less physical activity has been associated with systemic biomarkers of cartilage breakdown after anterior cruciate ligament reconstruction (ACLR). However, previous research lacks analysis of deleterious cartilage compositional changes and objective physical activity after ACLR. The purpose of this study was to determine the association between physical activity quantified via accelerometer-based measures of daily steps and time in moderate-to-vigorous physical activity (MVPA), and T1rho magnetic resonance imaging (MRI) of the femoral articular cartilage, a marker of proteoglycan density in individuals with ACLR. METHODS: Daily steps and MVPA were assessed over 7 d using an accelerometer worn on the hip in 26 individuals between 6 and 12 months after primary unilateral ACLR. Resting T1rho MRI was collected bilaterally, and T1rho MRI interlimb ratios (ILR: ACLR limb/contralateral limb) were calculated for lateral and medial femoral condyle regions of interest. We conducted univariate linear regression analyses to determine associations between T1rho MRI ILRs and daily steps and MVPA with and without controlling for sex. RESULTS: Greater T1rho MRI ILR of the central lateral femoral condyle, indicative of less proteoglycan density in the ACLR limb, was associated with greater time in MVPA ( R2 = 0.178, P = 0.032). Sex-adjusted models showed significant interaction terms between daily steps and sex in the anterior ( P = 0.025), central ( P = 0.002), and posterior ( P = 0.002) medial femoral condyle. CONCLUSIONS: Lesser physical activity may be a risk factor for maintaining cartilage health after ACLR; additionally, the relationship between physical activity and cartilage health may be different between males and females.

Association of Serum Biochemical Biomarker Profiles of Joint Tissue Inflammation and Cartilage Metabolism With Posttraumatic Osteoarthritis-Related Symptoms at 12 Months After ACLR.

BACKGROUND: Anterior cruciate ligament injury and anterior cruciate ligament reconstruction (ACLR) are risk factors for symptomatic posttraumatic osteoarthritis (PTOA). After ACLR, individuals demonstrate altered joint tissue metabolism indicative of increased inflammation and cartilage breakdown. Serum biomarker changes have been associated with tibiofemoral cartilage composition indicative of worse knee joint health but not with PTOA-related symptoms. PURPOSE/HYPOTHESIS: The purpose of this study was to determine associations between changes in serum biomarker profiles from the preoperative sample collection to 6 months after ACLR and clinically relevant knee PTOA symptoms at 12 months after ACLR. It was hypothesized that increases in biomarkers of inflammation, cartilage metabolism, and cartilage degradation would be associated with clinically relevant PTOA symptoms after ACLR. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: Individuals undergoing primary ACLR were included (N = 30). Serum samples collected preoperatively and 6 months after ACLR were processed to measure markers indicative of changes in inflammation (ie, monocyte chemoattract protein 1 [MCP-1]) and cartilage breakdown (ie, cartilage oligomeric matrix protein [COMP], matrix metalloproteinase 3, ratio of type II collagen breakdown to type II collagen synthesis). Knee injury and Osteoarthritis Outcome Score surveys were completed at 12 months after ACLR and used to identify participants with and without clinically relevant PTOA-related symptoms. K-means cluster analyses were used to determine serum biomarker profiles. One-way analyses of variance and logistic regressions were used to assess differences in Knee injury and Osteoarthritis Outcome Score subscale scores and clinically relevant PTOA-related symptoms between biomarker profiles. RESULTS: Two profiles were identified and characterized based on decreases (profile 1: 67% female; age, 21.4 ± 5.1 years; body mass index, 24.4 ± 2.4) and increases (profile 2: 33% female; age, 21.3 ± 3.2 years; body mass index, 23.4 ± 2.6) in sMCP-1 and sCOMP preoperatively to 6 months after ACLR. Participants with profile 2 did not demonstrate differences in knee pain, symptoms, activities of daily living, sports function, or quality of life at 12 months after ACLR compared to those with profile 1 (P = .56-.81; η2 = 0.002-0.012). No statistically significant associations were noted between biomarker profiles and clinically relevant PTOA-related symptoms (odds ratio, 1.30; 95% CI, 0.23-6.33). CONCLUSION: Serum biomarker changes in MCP-1 and sCOMP within the first 6 months after ACLR were not associated with clinically relevant PTOA-related symptoms.

Modifying loading during gait leads to biochemical changes in serum cartilage oligomeric matrix protein concentrations in a subgroup of individuals with anterior cruciate ligament reconstruction.

PURPOSE: Strong observational evidence has linked changes in limb loading during walking following anterior cruciate ligament reconstruction (ACLR) to posttraumatic osteoarthritis (PTOA). It remains unknown if manipulating peak loading influences joint tissue biochemistry. Thus, the purpose of this study is to determine whether manipulating peak vertical ground reaction force (vGRF) during gait influences changes in serum cartilage oligomeric matrix protein (sCOMP) concentrations in ACLR participants. METHODS: Forty ACLR individuals participated in this randomized crossover study (48% female, age = 21.0 ± 4.4 years, BMI = 24.6 ± 3.1). Participants attended four sessions, wherein they completed one of four biofeedback conditions (habitual loading (no biofeedback), high loading (5% increase in vGRF), low loading (5% decrease in vGRF), and symmetrical loading (between-limb symmetry in vGRF)) while walking on a treadmill for 3000 steps. Serum was collected before (baseline), immediately (acute post), 1 h (1 h post), and 3.5 h (3.5 h post) following each condition. A comprehensive general linear mixed model was constructed to address the differences in sCOMP across all conditions and timepoints in all participants and a subgroup of sCOMP Increasers. RESULTS: No sCOMP differences were found across the entire cohort. In the sCOMP Increasers, a significant time × condition interaction was found (F9,206 = 2.6, p = 0.009). sCOMP was lower during high loading than low loading (p = 0.009) acutely (acute post). At 3.5 h post, sCOMP was higher during habitual loading than symmetrical loading (p = 0.001). CONCLUSION: These data suggest that manipulating lower limb loading in ACLR patients who habitually exhibit an acute increase in sCOMP following walking results in improved biochemical changes linked to cartilage health. Key Points • This study assesses the mechanistic link between lower limb load modification and joint tissue biochemistry at acute and delayed timepoints. • Real-time biofeedback provides a paradigm to experimentally assess the mechanistic link between loading and serum biomarkers. • Manipulating peak loading during gait resulted in a metabolic effect of lower sCOMP concentrations in a subgroup of ACLR individuals. • Peak loading modifications may provide an intervention strategy to mitigate the development of PTOA following ACLR.

Stachydrine ameliorates high-glucose induced endothelial cell senescence and SIRT1 downregulation.

Hyperglycaemia, a characteristic feature of diabetes mellitus, induces endothelial dysfunction and vascular complications by accelerating endothelial cell (EC) senescence and limiting the proliferative potential of these cells. Here we aimed to investigate the effect of stachydrine, a proline betaine present in considerable quantities in juices from fruits of the Citrus genus, on EC under high-glucose stimulation, and its underlying mechanism. The senescence model of EC was set up by treating cells with high-glucose (30 mM) for different times. Dose-dependent (0.001-1 mM) evaluation of cell viability revealed that stachydrine does not affect cell proliferation with a similar trend up to 72 h. Noticeable, stachydrine (0.1 mM) significantly attenuated the high-glucose induced EC growth arrest and senescence. Indeed, co-treatment with high-glucose and stachydrine for 48 h kept the percentage of EC in the G0 /G1 cell cycle phase near to control values and significantly reduced cell senescence. Western blot analysis and confocal-laser scanning microscopy revealed that stachydrine also blocked the high-glucose induced upregulation of p16(INK4A) and downregulation of SIRT1 expression and enzyme activity. Taken together, results here presented are the first evidence that stachydrine, a naturally occurring compound abundant in citrus fruit juices, inhibits the deleterious effect of high-glucose on EC and acts through the modulation of SIRT1 pathway. These results may open new prospective in the identification of stachydrine as an important component of healthier eating patterns in prevention of cardiovascular diseases.

Sustained inhibition of CC-chemokine receptor-2 via intraarticular deposition of polymeric microplates in post-traumatic osteoarthritis.

Posttraumatic osteoarthritis (PTOA) is mostly treated via corticosteroid administration, and total joint arthroplasty continues to be the sole effective intervention in severe conditions. To assess the therapeutic potential of CCR2 targeting in PTOA, we used biodegradable microplates (µPLs) to achieve a slow and sustained intraarticular release of the CCR2 inhibitor RS504393 into injured knees and followed joint damage during disease progression. RS504393-loaded µPLs (RS-µPLs) were fabricated via a template-replica molding technique. A mixture of poly(lactic-co-glycolic acid) (PLGA) and RS504393 was deposited into 20 × 10 µm (length × height) wells in a polyvinyl alcohol (PVA) square-patterned template. After physicochemical and toxicological characterizations, the RS504393 release profile from µPL was assessed in PBS buffer. C57BL/6 J male mice were subjected to destabilization of the medial meniscus (DMM)/sham surgery, and RS-µPLs (1 mg/kg) were administered intraarticularly 1 week postsurgery. Administrations were repeated at 4 and 7 weeks post-DMM. Drug free-µPLs (DF-µPLs) and saline injections were performed as controls. Mice were euthanized at 4 and 10 weeks post-DMM, corresponding to the early and severe PTOA stages, respectively. Knees were evaluated for cartilage structure score (ACS, H&E), matrix loss (safranin O score), osteophyte formation and maturation from cartilage to bone (cartilage quantification), and subchondral plate thickness. The RS-µPL architecture ensured the sustained release of CCR2 inhibitors over several weeks, with ~ 20% of RS504393 still available at 21 days. This prolonged release improved cartilage structure and reduced bone damage and synovial hyperplasia at both PTOA stages. Extracellular matrix loss was also attenuated, although with less efficacy. The results indicate that local sustained delivery is needed to optimize CCR2-targeted therapies.

CC-Chemokine Receptor-2 Expression in Osteoblasts Contributes to Cartilage and Bone Damage during Post-Traumatic Osteoarthritis.

In osteoarthritis (OA), bone changes are radiological hallmarks and are considered important for disease progression. The C-C chemokine receptor-2 (CCR2) has been shown to play an important role in bone physiology. In this study, we investigated whether Ccr2 osteoblast-specific inactivation at different times during post-traumatic OA (PTOA) progression improves joint structures, bone parameters, and pain. We used a tamoxifen-inducible Ccr2 inactivation in Collagen1α-expressing cells to obtain osteoblasts lacking Ccr2 (CCR2-Col1αKO). We stimulated PTOA changes in CCR2-Col1αKO and CCR2+/+ mice using the destabilization of the meniscus model (DMM), inducing recombination before or after DMM (early- vs. late-inactivation). Joint damage was evaluated at two, four, eight, and twelve weeks post-DMM using multiple scores: articular-cartilage structure (ACS), Safranin-O, histomorphometry, osteophyte size/maturity, subchondral bone thickness and synovial hyperplasia. Spontaneous and evoked pain were assessed for up to 20 weeks. We found that early osteoblast-Ccr2 inactivation delayed articular cartilage damage and matrix degeneration compared to CCR2+/+, as well as DMM-induced bone thickness. Osteophyte formation and maturation were only minimally affected. Late Collagen1α-Ccr2 deletion led to less evident improvements. Osteoblast-Ccr2 deletion also improved static measures of pain, while evoked pain did not change. Our study demonstrates that Ccr2 expression in osteoblasts contributes to PTOA disease progression and pain by affecting both cartilage and bone tissues.

Systemically delivered insulin-like growth factor-I enhances mesenchymal stem cell-dependent fracture healing.

In this study, we examined the effectiveness of systemic subcutaneous delivery of recombinant Insulin-like growth factor (IGF)-I concurrently with primary cultured bone marrow-derived mesenchymal stem cell (MSC) transplant on fracture repair. We found that the fracture callus volume increased in mice with a stabilized tibia fracture that received IGF-I+MSC when compared with that in either untreated or MSC alone treated mice. In evaluating the callus tissue components, we found that the soft and new bone tissue volumes were significantly increased in IGF-I+MSC recipients. Histological and in-situ hybridization analyses confirmed a characteristic increase of newly forming bone in IGF-I+MSC recipients and that healing progressed mostly through endochondral ossification. The increase in soft and new bone tissue volumes correlated with increased force and toughness as determined by biomechanical testing. In conclusion, MSC transplant concurrent with systemic delivery of IGF-I improves fracture repair suggesting that IGF-I+MSC could be a novel therapeutic approach in patients who have inadequate fracture repair.

Mesenchymal stem cells expressing insulin-like growth factor-I (MSCIGF) promote fracture healing and restore new bone formation in Irs1 knockout mice: analyses of MSCIGF autocrine and paracrine regenerative effects.

Failures of fracture repair (nonunions) occur in 10% of all fractures. The use of mesenchymal stem cells (MSC) in tissue regeneration appears to be rationale, safe, and feasible. The contributions of MSC to the reparative process can occur through autocrine and paracrine effects. The primary objective of this study is to find a novel mean, by transplanting primary cultures of bone marrow-derived MSCs expressing insulin-like growth factor-I (MSC(IGF)), to promote these seed-and-soil actions of MSC to fully implement their regenerative abilities in fracture repair and nonunions. MSC(IGF) or traceable MSC(IGF)-Lac-Z were transplanted into wild-type or insulin-receptor-substrate knockout (Irs1(-/-)) mice with a stabilized tibia fracture. Healing was assessed using biomechanical testing, microcomputed tomography (µCT), and histological analyses. We found that systemically transplanted MSC(IGF) through autocrine and paracrine actions improved the fracture mechanical strength and increased new bone content while accelerating mineralization. We determined that IGF-I adapted the response of transplanted MSC(IGF) to promote their differentiation into osteoblasts. In vitro and in vivo studies showed that IGF-I-induced osteoglastogenesis in MSCs was dependent of an intact IRS1-PI3K signaling. Furthermore, using Irs1(-/-) mice as a nonunion fracture model through altered IGF signaling, we demonstrated that the autocrine effect of IGF-I on MSC restored the fracture new bone formation and promoted the occurrence of a well-organized callus that bridged the gap. A callus that was basically absent in Irs1(-/-) left untransplanted or transplanted with MSCs. We provided evidence of effects and mechanisms for transplanted MSC(IGF) in fracture repair and potentially to treat nonunions.

TGF-beta signaling is essential for joint morphogenesis.

Despite its clinical significance, joint morphogenesis is still an obscure process. In this study, we determine the role of transforming growth factor beta (TGF-beta) signaling in mice lacking the TGF-beta type II receptor gene (Tgfbr2) in their limbs (Tgfbr2(PRX-1KO)). In Tgfbr2(PRX-1KO) mice, the loss of TGF-beta responsiveness resulted in the absence of interphalangeal joints. The Tgfbr2(Prx1KO) joint phenotype is similar to that in patients with symphalangism (SYM1-OMIM185800). By generating a Tgfbr2-green fluorescent protein-beta-GEO-bacterial artificial chromosome beta-galactosidase reporter transgenic mouse and by in situ hybridization and immunofluorescence, we determined that Tgfbr2 is highly and specifically expressed in developing joints. We demonstrated that in Tgfbr2(PRX-1KO) mice, the failure of joint interzone development resulted from an aberrant persistence of differentiated chondrocytes and failure of Jagged-1 expression. We found that TGF-beta receptor II signaling regulates Noggin, Wnt9a, and growth and differentiation factor-5 joint morphogenic gene expressions. In Tgfbr2(PRX-1KO) growth plates adjacent to interphalangeal joints, Indian hedgehog expression is increased, whereas Collagen 10 expression decreased. We propose a model for joint development in which TGF-beta signaling represents a means of entry to initiate the process.

Association of Quality of Life With Moderate-to-Vigorous Physical Activity After Anterior Cruciate Ligament Reconstruction.

CONTEXT: Better knee function is linked to psychological readiness to return to sport after anterior cruciate ligament reconstruction (ACLR). Individuals with ACLR participate in less physical activity than matched uninjured control individuals, yet the association between knee function and physical activity post-ACLR remains unclear. OBJECTIVE: To determine the associations between (1) patient-reported knee function measured using the Knee Injury and Osteoarthritis Outcome Score Knee-Related Quality of Life (KOOS-QOL), daily steps, and minutes spent in moderate-to-vigorous physical activity (MVPA) of individuals with ACLR and (2) KOOS-QOL and daily steps and MVPA in individuals with ACLR who presented with (ie, symptomatic) or without (ie, asymptomatic) clinically meaningful knee-related symptoms. DESIGN: Cross-sectional study. SETTING: Laboratory, free-living conditions. PATIENTS OR OTHER PARTICIPANTS: A total of 66 individuals with primary unilateral ACLR (36 women, 30 men; age = 22 ± 4 years, height = 1.71 ± 0.1 m, mass = 71.3 ± 12.6 kg, body mass index = 24.2 ± 2.9, time post-ACLR = 28 ± 33 months). MAIN OUTCOME MEASURE(S): We collected KOOS data and retrospectively stratified participants into those with (symptomatic group, n = 30) or without (asymptomatic group, n = 36) clinically meaningful knee-related symptoms based on previously defined KOOS cutoffs. We assessed daily steps and MVPA using accelerometers that participants wore on the right hip for 7 days. We conducted linear regressions to determine associations between KOOS-QOL and daily steps and MVPA. RESULTS: In the entire sample, no associations existed between KOOS-QOL and daily steps (ΔR2 = 0.01, P = .50) or MVPA (ΔR2 = 0.01, P = .36). In the symptomatic group, a greater KOOS-QOL was associated with more time in MVPA (ΔR2 = 0.12, P = .05). In the asymptomatic group, no associations were identified between the KOOS-QOL and daily steps and MVPA. CONCLUSIONS: Individuals with symptoms post-ACLR who spent more time in MVPA reported higher QOL.

Association of Increased Serum Lipopolysaccharide, But Not Microbial Dysbiosis, With Obesity-Related Osteoarthritis.

OBJECTIVE: To test the hypothesis that an altered gut microbiota (dysbiosis) plays a role in obesity-associated osteoarthritis (OA). METHODS: Stool and blood samples were collected from 92 participants with a body mass index (BMI) ≥30 kg/m2 , recruited from the Johnston County Osteoarthritis Project. OA patients (n = 50) had hand and knee OA (Kellgren/Lawrence [K/L] grade ≥2 or arthroplasty). Controls (n = 42) had no hand OA and a K/L grade of 0-1 for the knees. Compositional analysis of stool samples was carried out by 16S ribosomal RNA amplicon sequencing. Alpha- and beta-diversity and differences in taxa relative abundances were determined. Blood samples were used for multiplex cytokine analysis and measures of lipopolysaccharide (LPS) and LPS binding protein. Germ-free mice were gavaged with patient- or control-pooled fecal samples and fed a 40% fat, high-sucrose diet for 40 weeks. Knee OA was evaluated histologically. RESULTS: On average, OA patients were slightly older than the controls, consisted of more women, and had a higher mean BMI, higher mean Western Ontario and McMaster Universities Osteoarthritis Index pain score, and higher mean K/L grade. There were no significant differences in α- or ß-diversity or genus level composition between patients and controls. Patients had higher plasma levels of osteopontin (P = 0.01) and serum LPS (P < 0.0001) compared to controls. Mice transplanted with patient or control microbiota exhibited a significant difference in α-diversity (P = 0.02) and ß-diversity, but no differences in OA severity were observed. CONCLUSION: The lack of differences in the gut microbiota, but increased serum LPS levels, suggest the possibility that increased intestinal permeability allowing for greater absorption of LPS, rather than a dysbiotic microbiota, may contribute to the development of OA associated with obesity.

CCL2 induces articular chondrocyte MMP expression through ERK and p38 signaling pathways.

Objective: In previous studies, we determined an association between increased serum and articular cartilage levels of CCL2 with osteoarthritis (OA) progression, cartilage damage and increased MMP13 in cartilage. Here we analyzed CCL2 downstream signaling mediators that lead to gene expression of cartilage catabolic markers, in healthy and OA human articular chondrocytes. Design: Human articular chondrocytes obtained from healthy or OA subjects were treated with or without recombinant human CCL2; cell lysates or mRNA were collected for immunoblotting or qRT-PCR. For pathway analysis, chondrocytes were pre-incubated with an inhibitor of CCR2 (the unique CCL2 receptor), ERK inhibitor or p38 inhibitor prior to CCL2 treatment. Results: CCL2 treatment of both healthy and OA chondrocytes activated ERK and p38 via CCR2. In healthy chondrocytes, short (6h) and prolonged (24-72h) CCL2 treatments led to Ccr2, Mmp-1, Mmp-3, Mmp-13 and Timp1 upregulation. In OA chondrocytes, CCL2 induced expression of Ccr2, Mmp-1 and Mmp-3, but not Mmp1 and Timp1, and only following longer treatments (72h). In both healthy and OA chondrocytes, the CCL2-mediated upregulation of Ccr2 and cartilage catabolic markers was mediated by ERK and p38 signaling. Conclusions: The triggering of the CCL2/CCR2 axis in articular chondrocytes activates specific MAPK pathways leading to gene expression of cartilage degrading enzymes. However, some differences in the response to CCL2 stimulation are detected in healthy vs OA chondrocytes with respect to the number of activated genes and to the time of exposure to CCL2, suggesting that CCL2 action in articular cartilage may be dependent on OA stage and severity.

Regenerative effects of transplanted mesenchymal stem cells in fracture healing.

Mesenchymal stem cells (MSC) have a therapeutic potential in patients with fractures to reduce the time of healing and treat nonunions. The use of MSC to treat fractures is attractive for several reasons. First, MSCs would be implementing conventional reparative process that seems to be defective or protracted. Secondly, the effects of MSCs treatment would be needed only for relatively brief duration of reparation. However, an integrated approach to define the multiple regenerative contributions of MSC to the fracture repair process is necessary before clinical trials are initiated. In this study, using a stabilized tibia fracture mouse model, we determined the dynamic migration of transplanted MSC to the fracture site, their contributions to the repair process initiation, and their role in modulating the injury-related inflammatory responses. Using MSC expressing luciferase, we determined by bioluminescence imaging that the MSC migration at the fracture site is time- and dose-dependent and, it is exclusively CXCR4-dependent. MSC improved the fracture healing affecting the callus biomechanical properties and such improvement correlated with an increase in cartilage and bone content, and changes in callus morphology as determined by micro-computed tomography and histological studies. Transplanting CMV-Cre-R26R-Lac Z-MSC, we found that MSCs engrafted within the callus endosteal niche. Using MSCs from BMP-2-Lac Z mice genetically modified using a bacterial artificial chromosome system to be beta-gal reporters for bone morphogenic protein 2 (BMP-2) expression, we found that MSCs contributed to the callus initiation by expressing BMP-2. The knowledge of the multiple MSC regenerative abilities in fracture healing will allow design of novel MSC-based therapies to treat fractures.

Subcellular localization of IRS-1 in IGF-I-mediated chondrogenic proliferation, differentiation and hypertrophy of bone marrow mesenchymal stem cells.

Bone marrow derived mesenchymal stem cells (BM-MSC) can differentiate into chondrocytes. Understanding the mechanisms and growth factors that control the MSC stemness is critical to fully implement their therapeutic use in cartilage diseases. The activated type 1 insulin-like growth factor receptor (IGF-IR), interacting with the insulin receptor substrate-1 (IRS-1), can induce cancer cell proliferation and transformation. In cancer or transformed cells, IRS-1 has been shown to localize in the cytoplasm where it activates the canonical Akt pathway, as well as in the nucleus where it binds to nuclear proteins. We have previously demonstrated that IGF-I has distinct time-dependent effect on primary BM-MSC chondrogenic pellets: initially (2-day culture), IGF-I induces proliferation; subsequently, IGF-I promotes chondrocytic differentiation (7-day culture). In the present study, by using MSC from the BM of IRS-1(- / - ) mice we show that IRS-1 mediates almost 50% of the IGF-I mitogenic response and the MAPK-MEK/ERK signalling accounts for the other 50%. After stimulation with IGF-I, we found that in 2-day old human and mouse derived BM-MSC pellets, IRS-1 (total and phosphorylated) is nuclearly localized and that proliferation prevails over differentiation. The IGF-I mitogenic effect is Akt-independent. In 7-day MSC pellets, IGF-I stimulates the chondrogenic differentiation of MSC into chondrocytes, pre-hypertrophic and hypertrophic chondrocytes and IRS-1 accumulates in the cytoplasm. IGF-I-dependent differentiation is exclusively Akt-dependent. Our data indicate that in the physiologically relevant model of primary cultured MSC, IGF-I induces a temporally regulated nuclear or cytoplasmic localization of IRS-1 that correlate with the transition from proliferation to chondrogenic differentiation.

TGF-ß type 2 receptor-mediated modulation of the IL-36 family can be therapeutically targeted in osteoarthritis.

Mechanisms that govern the shift from joint homeostasis to osteoarthritis (OA) remain unknown. Here, we identify a pathway used for joint development and homeostasis, and its role in OA. Using a combination of transgenic, pharmacological, and surgical conditions in mouse and human tissues, we found that TGF-ß signaling promotes joint homeostasis through regulation of the IL-36 family. We identified IL-36 receptor antagonist (IL-36 in mice and IL-36RN in humans) as a potential disease-modifying OA drug. Specifically, OA development was associated with IL-36α up-regulation and IL-36Ra down-regulation in mice with tissue-specific postnatally induced ablation of Tgfbr2, mice treated with a TGF-ß signaling inhibitor, mice with posttraumatic OA, and aging mice with naturally occurring OA. In human cartilage, OA severity was associated with decreased TGFBR2 and IL-36RN, whereas IL-36α increased. Functionally, intra-articular treatment with IL-36Ra attenuated OA development in mice, and IL-36RN reduced MMP13 in human OA chondrocytes. These findings highlight the relevance of TGFBR2-IL-36 interplay in joint homeostasis and IL-36RN as a potential therapeutic agent for OA.

A mouse model for chronic pain-induced increase in ethanol consumption.

Chronic pain conditions are often comorbid with alcohol abuse. "Self-medication" with alcohol introduces a host of problems associated with the abuse of alcohol which over time has the potential of exacerbating the painful condition. Despite the prevalence of chronic pain being associated with alcohol abuse, rodent models which mimic the comorbid conditions are lacking. In this study, we model osteoarthritis (OA) in C57BL/6J mice by surgically destabilizing the medial meniscus (DMM). Sham-operated mice served as controls. Thirteen weeks after surgery, DMM but not sham-operated mice exhibited pronounced incapacitance of the surgically manipulated hind limb compared with the nonsurgically manipulated hind limb. At this time, the mice were exposed to the 2-bottle ethanol choice, beginning with 2.5% with a gradual increasing to 20%. Compared with sham controls, DMM mice consumed more EtOH and preferred EtOH over water at the 20% EtOH concentration. Histological analysis verified that the DMM mice exhibited significant damage to the articular cartilage and osteophyte growth compared with sham controls and these measures of the severity of OA correlated with the amount of ethanol intake. Thus, the combination of the DMM model of OA with the enhanced two-bottle ethanol choice is a potential preclinical approach in mice by which the basis of the comorbid association of alcohol abuse and chronic pain conditions can be explored.