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Despite effective countermeasures, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists worldwide because of its ability to diversify and evade human immunity. This evasion stems from amino acid substitutions, particularly in the receptor binding domain (RBD) of the spike protein that confers resistance to vaccine-induced antibodies and antibody therapeutics. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different RBD sites into multispecific antibodies. Here, we describe multispecific antibodies, including a trivalent trispecific antibody that potently neutralized diverse SARS-CoV-2 variants and prevented virus escape more effectively than single antibodies or mixtures of the parental antibodies. Despite being generated before the appearance of Omicron, this trispecific antibody neutralized all major Omicron variants through BA.4/BA.5 at nanomolar concentrations. Negative stain electron microscopy suggested that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated binding across more than one spike protein. Moreover, a tetravalent trispecific antibody containing the same variable regions as the trivalent trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2, and BA.5 challenge, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. These results demonstrated that multispecific antibodies have the potential to provide broad SARS-CoV-2 coverage, decrease the likelihood of escape, simplify treatment, and provide a strategy for antibody therapies that could help eliminate pandemic spread for this and other pathogens.

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; η 2 = 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., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: Research reported in this manuscript was supported by funding from the University of North Carolina's Department of Orthopaedics Laurence E. Dahners Research Grant, the National Institute of Arthritis and Musculoskeletal and Skin Disease of the National Institutes of Health (1R03 AR066840-01A1 and P30 AR072580), the North Carolina Translational and Clinical Sciences (TraCS) Institute, and the National Athletic Trainers’ Association Research and Education Foundation (14NewINV001). R.A.C. has received consulting fees from Arthrex and support for education from SouthTech Orthopedics. G.K. has received compensation for services other than consulting from Arthrex. J.S. has received support for education from SouthTech Orthopedics. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

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 (F 9,206  = 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., (© 2024. The Author(s), under exclusive licence to International League of Associations for Rheumatology (ILAR).)

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., (Copyright © 2023 by the American College of Sports Medicine.)

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/m 2 , 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 (∆R 2  = 0.29, p = 0.02) but not medial (∆R 2  < 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 (∆R 2 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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

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., (© 2022. The Author(s).)

Despite effective countermeasures, SARS-CoV-2 persists worldwide due to its ability to diversify and evade human immunity 1 . This evasion stems from amino-acid substitutions, particularly in the receptor-binding domain of the spike, that confer resistance to vaccines and antibodies 2-16 . To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different receptor binding domain (RBD) sites 17,18 into multispecific antibodies. Here, we describe multispecific antibodies, including a trispecific that prevented virus escape >3000-fold more potently than the most effective clinical antibody or mixtures of the parental antibodies. Despite being generated before the evolution of Omicron, this trispecific antibody potently neutralized all previous variants of concern and major Omicron variants, including the most recent BA.4/BA.5 strains at nanomolar concentrations. Negative stain electron microscopy revealed that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated inter-spike binding. An optimized trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2 and BA.5, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. Such multispecific antibodies decrease the likelihood of SARS-CoV-2 escape, simplify treatment, and maximize coverage, providing a strategy for universal antibody therapies that could help eliminate pandemic spread for this and other pathogens.

The processes leading to the emergence of new species are poorly understood in marine plankton, where weak physical barriers and homogeneous environmental conditions limit spatial and ecological segregation. Here, we combine molecular and ecological information from a long-term time series and propose Pseudo-nitzschia allochrona , a new cryptic planktonic diatom, as a possible case of speciation by temporal segregation. The new species differs in several genetic markers (18S, 28S and ITS rDNA fragments and rbc L) from its closest relatives, which are morphologically very similar or identical, and is reproductively isolated from its sibling species P. arenysensis . Data from a long-term plankton time series show P. allochrona invariably occurring in summer-autumn in the Gulf of Naples, where its closely related species P. arenysensis , P. delicatissima , and P. dolorosa are instead found in winter-spring. Temperature and nutrients are the main factors associated with the occurrence of P. allochrona , which could have evolved in sympatry by switching its phenology and occupying a new ecological niche. This case of possible speciation by time shows the relevance of combining ecological time series with molecular information to shed light on the eco-evolutionary dynamics of marine microorganisms., Competing Interests: We declare no conflict of interest., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

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., (© by the National Athletic Trainers' Association, Inc.)

Phytoplankton play a pivotal role in global biogeochemical and trophic processes and provide essential ecosystem services. However, there is still no broad consensus on how and to what extent their community composition responds to environmental variability. Here, high-frequency oceanographic and biological data collected over more than 25 years in a coastal Mediterranean site are used to shed light on the temporal patterns of phytoplankton species and assemblages in their environmental context. Because of the proximity to the coast and due to large-scale variations, environmental conditions showed variability on the short and long-term scales. Nonetheless, an impressive regularity characterised the annual occurrence of phytoplankton species and their assemblages, which translated into their remarkable stability over decades. Photoperiod was the dominant factor related to community turnover and replacement, which points at a possible endogenous regulation of biological processes associated with species-specific phenological patterns, in analogy with terrestrial plants. These results highlight the considerable stability and resistance of phytoplankton communities in response to different environmental pressures, which contrast the view of these organisms as passively undergoing changes that occur at different temporal scales in their habitat, and show how, under certain conditions, biological processes may prevail over environmental forcing., (© 2022. The Author(s).)

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/m 2 , 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 β-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 β-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., (© 2021, American College of Rheumatology.)

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 C cr2 , 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., Competing Interests: Authors do not have conflicts of interest to disclose., (© 2021 The Authors.)

Introduction: Conflicting results regarding the impact of left common pulmonary vein (LCPV) on clinical outcome of atrial fibrillation (AF) ablation with cryoballoon technology have been reported., Methods: We systematically searched PubMed and Cochrane library for articles that compared the arrhythmia recurrence rate after cryoballoon ablation between patients with normal pattern PVs and patients with LCPV. Studies of first ablation for persistent and paroxysmal AF using the 28 mm Arctic Front Advance, Medtronic cryoballoon (CB-A) reporting clinical success rates at a mean follow-up of ≥12 months were included. Data were analyzed by applying a random effects model., Results: A total of 5 studies with a total of 1178 patients met our predefined inclusion criteria. After a mean follow-up of 18.4 months, the overall success rate of CB-A ablation among patients with persistent and paroxysmal AF was 57%; in the LCPV group the success rate was 46% and in the normal anatomical pattern group it was 61%. No significant heterogeneity was noted among the studies (I 2  = 35.8%; Q (df = 3) = 6.23 p-value = 0.18). Arrhythmia recurrence after CB-A ablation was not statistically significant between the two groups (LogOR 0.24; 95% CI [-0.16-0.63]; p-value = 0.23). No significant difference in PNI was observed between the two groups (p-value = 0.693)., Conclusion: The presence of LCPV does not affect the long-term outcome of paroxysmal and persistent atrial fibrillation ablation with 28 mm CB-A compared to normal left PVs pattern., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Indian Heart Rhythm Society. Production and hosting by Elsevier B.V. All rights reserved.)

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-β 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-β 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., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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 (JNK1 fl/fl Col2-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., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

The cellular and humoral responses that orchestrate fracture healing are still elusive. Here we report that bone morphogenic protein 2 (BMP2)-dependent fracture healing occurs through a tight control of chemokine C-X-C motif-ligand-12 (CXCL12) cellular, spatial, and temporal expression. We found that the fracture repair process elicited an early site-specific response of CXCL12(+)-BMP2(+) endosteal cells and osteocytes that was not present in unfractured bones and gradually decreased as healing progressed. Absence of a full complement of BMP2 in mesenchyme osteoprogenitors (BMP2(cKO/+)) prevented healing and led to a dysregulated temporal and cellular upregulation of CXCL12 expression associated with a deranged angiogenic response. Healing was rescued when BMP2(cKO/+) mice were systemically treated with AMD3100, an antagonist of CXCR4 and agonist for CXCR7 both receptors for CXCL12. We further found that mesenchymal stromal cells (MSCs), capable of delivering BMP2 at the endosteal site, restored fracture healing when transplanted into BMP2(cKO/+) mice by rectifying the CXCL12 expression pattern. Our in vitro studies showed that in isolated endosteal cells, BMP2, while inducing osteoblastic differentiation, stimulated expression of pericyte markers that was coupled with a decrease in CXCL12. Furthermore, in isolated BMP2(cKO/cKO) endosteal cells, high expression levels of CXCL12 inhibited osteoblastic differentiation that was restored by AMD3100 treatment or coculture with BMP2-expressing MSCs that led to an upregulation of pericyte markers while decreasing platelet endothelial cell adhesion molecule (PECAM). Taken together, our studies show that following fracture, a CXCL12(+)-BMP2(+) perivascular cell population is recruited along the endosteum, then a timely increase of BMP2 leads to downregulation of CXCL12 that is essential to determine the fate of the CXCL12(+)-BMP2(+) to osteogenesis while departing their supportive role to angiogenesis. Our findings have far-reaching implications for understanding mechanisms regulating the selective recruitment of distinct cells into the repairing niches and the development of novel pharmacological (by targeting BMP2/CXCL12) and cellular (MSCs, endosteal cells) interventions to promote fracture healing., (© 2015 American Society for Bone and Mineral Research.)

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.

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.

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., (© 2013 Wiley Periodicals, Inc.)

TGF-β type II receptor (Tgfbr2) signaling plays an essential role in joint-element development. The Tgfbr2(PRX-1KO) mouse, in which the Tgfbr2 is conditionally inactivated in developing limbs, lacks interphalangeal joints and tendons. In this study, we used the Tgfbr2-β-Gal-GFP-BAC mouse as a LacZ/green fluorescent protein (GFP)-based read-out to determine: the spatial and temporally regulated expression pattern of Tgfbr2-expressing cells within joint elements; their expression profile; and their slow-cycling labeling with bromodeoxyuridine (BrdU). Tgfbr2-β-Gal activity was first detected at embryonic day (E) 13.5 within the interphalangeal joint interzone. By E16.5, and throughout adulthood, Tgfbr2-expressing cells clustered in a contiguous niche that comprises the groove of Ranvier and the synovio-entheseal complex including part of the perichondrium, the synovium, the articular cartilage superficial layer, and the tendon's entheses. Tgfbr2-expressing cells were found in the synovio-entheseal complex niche with similar temporal pattern in the knee, where they were also detected in meniscal surface, ligaments, and the synovial lining of the infrapatellar fat pad. Tgfbr2-β-Gal-positive cells were positive for phospho-Smad2, signifying that the Tgfbr2 reporter was accurate. Developmental-stage studies showed that Tgfbr2 expression was in synchrony with expression of joint-morphogenic genes such as Noggin, GDF5, Notch1, and Jagged1. Prenatal and postnatal BrdU-incorporation studies showed that within this synovio-entheseal-articular-cartilage niche most of the Tgfbr2-expressing cells labeled as slow-proliferating cells, namely, stem/progenitor cells. Tgfbr2-positive cells, isolated from embryonic limb mesenchyme, expressed joint progenitor markers in a time- and TGF-β-dependent manner. Our studies provide evidence that joint Tgfbr2-expressing cells have anatomical, ontogenic, slow-cycling trait and in-vivo and ex-vivo expression profiles of progenitor joint cells.

Careful filtering is necessary for observations of quantum phenomena in superconducting circuits at low temperatures. Measurements of coherence between quantum states require extensive filtering to protect against noise coupled from room temperature electronics. We demonstrate distributed transmission line filters which cut off exponentially at GHz frequencies and can be anchored at the base temperature of a dilution refrigerator. The compact design makes them suitable to filter many different bias lines in the same setup, necessary for the control and measurement of superconducting qubits.

An important concern in the study of fracture healing is the ability to assess mechanical integrity in response to candidate therapeutics in small-animal systems. In recent reports, it has been proposed that microCT image-derived densitometric parameters could be used as a surrogate for mechanical property assessment. Recently, we have proposed an inverse methodology that iteratively reconstructs the modulus of elasticity of the lumped soft callus/hard callus region by integrating both intrinsic mechanical property (from biomechanical testing) and geometrical information (from microCT) within an inverse finite element analysis (FEA) to define a callus quality measure. In this paper, data from a therapeutic system involving mesenchymal stem cells is analyzed within the context of comparing traditional microCT densitometric and mechanical property metrics. In addition, a novel multi-parameter regression microCT parameter is analyzed as well as our inverse FEA metric. The results demonstrate that the inverse FEA approach was the only metric to successfully detect both longitudinal and therapeutic responses. While the most promising microCT-based metrics were adequate at early healing states, they failed to track late-stage mechanical integrity. In addition, our analysis added insight to the role of MSCs by demonstrating accelerated healing and was the only metric to demonstrate therapeutic benefits at late-stage healing. In conclusion, the work presented here indicates that microCT densitometric parameters are an incomplete surrogate for mechanical integrity. Additionally, our inverse FEA approach is shown to be very sensitive and may provide a first-step towards normalizing the often challenging process of assessing mechanical integrity of healing fractures., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Dissipation encodes the interaction of a quantum system with the environment and regulates the activation regimes of a Brownian particle. We have engineered grain boundary biepitaxial YBaCuO junctions to drive a direct transition from a quantum activated running state to a phase diffusion regime. The crossover to the quantum regime is tuned by the magnetic field and dissipation is described by a fully consistent set of junction parameters. To unravel phase dynamics in moderately damped systems is of general interest for advances in the comprehension of retrapping phenomena and in view of quantum hybrid technology.

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.

Despite its clinical significance, the mechanisms of joint morphogenesis are elusive. By combining laser-capture microdissection for RNA sampling with microarrays, we show that the setting in which joint-forming interzone cells develop is distinct from adjacent growth plate chondrocytes and is characterized by downregulation of chemokines, such as monocyte-chemoattractant protein-5 (MCP-5). Using in vivo, ex vivo, and in vitro approaches, we show that low levels of interzone-MCP-5 are essential for joint formation and contribute to proper growth plate organization. Mice lacking the TGF-β-type-II-receptor (TβRII) in their limbs (Tgfbr2(Prx1KO)), which lack joint development and fail chondrocyte hypertrophy, show upregulation of interzone-MCP-5. In vivo and ex vivo blockade of the sole MCP-5 receptor, CCR2, led to the rescue of joint formation and growth plate maturation in Tgfbr2(Prx1KO) but an acceleration of growth plate mineralization in control mice. Our study characterized the TβRII/MCP-5 axis as an essential crossroad for joint development and endochondral growth., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 AlphaMed Press.)

Importance of the Field: Mesenchymal stem cells have the ability to differentiate into osteoblasts, chondrocytes and adipocytes. Along with differentiation, MSCs can modulate inflammation, home to damaged tissues and secrete bioactive molecules. These properties can be enhanced through genetic-modification that would combine the best of both cell and gene therapy fields to treat monogenic and multigenic diseases., Areas Covered in This Review: Findings demonstrating the immunomodulation, homing and paracrine activities of MSCs followed by a summary of the current research utilizing MSCs as a vector for gene therapy, focusing on skeletal disorders, but also cardiovascular disease, ischemic damage and cancer., What the Reader Will Gain: MSCs are a possible therapy for many diseases, especially those related to the musculoskeletal system, as a standalone treatment, or in combination with factors that enhance the abilities of these cells to migrate, survive or promote healing through anti-inflammatory and immunomodulatory effects, differentiation, angiogenesis or delivery of cytolytic or anabolic agents., Take Home Message: Genetically-modified MSCs are a promising area of research that would be improved by focusing on the biology of MSCs that could lead to identification of the natural and engrafting MSC-niche and a consensus on how to isolate and expand MSCs for therapeutic purposes.

Murine embryonic limb cultures have invaluable roles in studying skeletogenesis. Substance delivery is an underdeveloped area in developmental biology that has primarily relied on Affi-Gel-Blue-agarose-beads. However, the lack of information about the efficiency of agarose-bead loading and release and difficulties for a single-bead implantation represent significant limitations. We optimized the use of glycol chitosan-5beta-cholanic acid conjugates (HGC) as a novel protein delivery system in mouse embryonic limbs. To this purpose, we loaded HGC either with recombinant Noggin, or bovine serum albumin (BSA). The size, morphology and stability of the protein-loaded-HGC were determined by transmission electron microscopy and dynamic-light-scattering. HGC-BSA and HGC-Noggin loading efficiencies were 80-90%. Time-course study revealed that Noggin and BSA were 80-90% released after 48 h. We developed several techniques to implant protein-loaded-HGC into murine embryonic joints from embryonic age E13.5 to E15.5, including a micro-injection system dispensing nanoliters. HGC did not interfere with skeletogenesis. Using CBR-3BA staining, we detected HGC nanoparticles within implanted tissues. Furthermore, a sustained release of BSA and Noggin was demonstrated in HGC-BSA and HGC-Noggin injected regions. HGC-released Noggin was biologically active in blocking the BMP signaling in in vitro mesenchyme limb micromasses as well as in ex-vivo limb cultures. Results reveal that HGC is a valuable protein-delivery system in developmental biology., (Copyright 2010 Elsevier B.V. All rights reserved.)

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.

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.

Background: Mesenchymal stem cells (MSC) are multipotent cells with the ability to differentiate into mesenchyme-derived cells including osteoblasts and chondrocytes., Objective: To provide an overview and expert opinion on the in vivo ability of MSC to home into tissues, their regenerative properties and potential applications for cell-based therapies to treat bone and cartilage disorders., Methods: Data sources including the PubMed database, abstract booklets and conference proceedings were searched for publications pertinent to MSC and their properties with emphasis on the in vivo studies and clinical use in cartilage and bone regeneration and repair. The search included the most current information possible., Conclusion: MSC can migrate to injured tissues and some of their reparative properties are mediated by paracrine mechanisms including their immunomodulatory actions. MSC possess a critical potential in regenerative medicine for the treatment of skeletal diseases, such as osteoarthritis or fracture healing failure, where treatments are partially effective or palliative.

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.

Unlabelled: A novel role for IGF-I in MSC chondrogenesis was determined. IGF-I effects were evaluated in the presence or absence of TGF-beta signaling by conditionally inactivating the TGF-beta type II receptor. We found that IGF-I had potent chondroinductive actions on MSCs. IGF-I effects were independent from and additive to TGF-beta., Introduction: Mesenchymal stem cells (MSCs) can be isolated from adult bone marrow (BM), expanded, and differentiated into several cell types, including chondrocytes. The role of IGF-I in the chondrogenic potential of MSCs is poorly understood. TGF-beta induces MSC chondrogenic differentiation, although its actions are not well defined. The aim of our study was to define the biological role of IGF-I on proliferation, chondrogenic condensation, apoptosis, and differentiation of MSCs into chondrocytes, alone or in combination with TGF-beta and in the presence or absence of TGF-beta signaling., Materials and Methods: Mononuclear adherent stem cells were isolated from mouse BM. Chondrogenic differentiation was induced by culturing high-density MSC pellets in serum- and insulin-free defined medium up to 7 days, with or without IGF-I and/or TGF-beta. We measured thymidine incorporation and stained 2-day-old pellets with TUNEL, cleaved caspase-3, peanut-agglutinin, and N-cadherin. Seven-day-old pellets were measured in size, stained for proteoglycan synthesis, and analyzed for the expression of collagen II and Sox-9 by quantitative real time PCR. We obtained MSCs from mice in which green fluorescent protein (GFP) was under the Collagen2 promoter and determined GFP expression by confocal microscopy. We conditionally inactivated the TGF-beta type II receptor (TbetaRII) in MSCs using a cre-lox system, generating TbetaRII knockout MSCs (RIIKO-MSCs)., Results and Conclusions: IGF-I modulated MSC chondrogenesis by stimulating proliferation, regulating cell apoptosis, and inducing expression of chondrocyte markers. IGF-I chondroinductive actions were equally potent to TGF-beta1, and the two growth factors had additive effects. Using RIIKO-MSCs, we showed that IGF-I chondrogenic actions are independent from the TGF-beta signaling. We found that the extracellular signal-related kinase 1/2 mitogen-activated protein kinase (Erk1/2 MAPK) pathway mediated the TGF-beta1 mitogenic response and in part the IGF-I proliferative action. Our data, by showing the role of IGF-I and TGF-beta1 in the critical steps of MSC chondrogenesis, provide critical information to optimize the therapeutic use of MSCs in cartilage disorders.

Cartilage formation is driven by mesenchymal chondroprogenitor cells (MCCs) that proliferate and differentiate into chondrocytes. The molecular mechanisms by which growth factors regulate MCC fate are not well defined. Insulin-like growth factor binding protein-3 (IGFBP-3) has intrinsic bioactivity that is independent of IGF binding. We previously reported that IGFBP-3 has IGF-independent antiproliferative and apoptotic effects in MCCs, and requires STAT-1 activation to mediate its apoptotic effect. Transforming growth factor-beta (TGF-beta) is a key chondroinductive growth factor. The objective of the study is to define the interactions between IGFBP-3 and TGF-beta in MCC growth and their intracellular signaling pathways. We used the RCJ3*1C5*18 mesenchymal chondrogenic cells that without biochemical or oncogenic transformation progress in culture from MCCs to differentiated chondrocytes. Cell proliferation was assessed in MCCs treated with IGFBP-3 or transfected with IGFBP-3, in the presence or absence of TGF-beta. To demonstrate that IGFBP-3 effects were IGF-independent an IGFBP-3 analog that lacks IGF binding was used (GGG-IGFBP-3). To determine the functional roles of the TGF-beta-mediated signaling and the STAT-1 pathway, cells were either stably transfected with a dominant negative TGF-beta type II receptor (MCC-DNTbetaRII) or treated with a STAT-1 morpholino antisense oligonucleotide. We found that in MCCs, TGF-beta antagonized the antiproliferative effect of IGFBP-3. IGFBP-3 increased the cyclin-dependent kinase inhibitor p21 expression and this effect was abolished by TGF-beta. Furthermore, TGF-beta inhibited STAT-1 phosphorylation induced by IGFBP-3. Similarly to TGF-beta, STAT-1 antisense oligonucleotide inhibited the IGFBP-3 antiproliferative action. Although TGF-beta in MCC-DNTbetaRII lacked Smad-mediated signaling, it persistently antagonized the IGFBP-3 antiproliferative action. However, TGF-beta even in MCC-DNTbetaRII cells induced ERK1/2 phosphorylation, and treatment with MEK inhibitor, UO126, inhibited the antagonistic effects of TGF-beta on IGFBP-3. Furthermore, UO126 blocked the TGF-beta inhibition of STAT-1 phosphorylation induced by IGFBP-3. Collectively, these results demonstrate cross-talk between the IGFBP-3-dependent STAT-1 signaling and the TGF-beta-dependent ERK pathway that regulates MCC proliferation.

Background: SEG-1, a Barrett's-derived esophageal adenocarcinoma cell line, is not responsive to transforming growth factor beta (TGF-beta) growth effects. We hypothesize that SEG-1 cells lack the tumor-suppressor gene Runt domain transcription factor 3 (RUNX3) and that its reinstatement can restore the antiproliferative and apoptotic effects of TGF-beta., Methods: RUNX3 expression was assessed by immunoblotting. SEG-1 cells were transfected with RUNX3 and treated with TGF-beta. The effects of RUNX3 transfection on cell proliferation and apoptosis were determined. Smad-mediated TGF-beta transcriptional activity was evaluated with the use of dual-luciferase assay., Results: SEG-1 cells are not responsive to TGF-beta. SEG-1 cells lack RUNX3 protein expression, while RUNX3 is highly expressed in normal human gastric and esophageal epithelium. Although the Smad-2 signaling is activated by TGF-beta, SEG-1 cells lack Smad-mediated TGF-beta transcriptional activity. In cells transfected with RUNX3, TGF-beta acquired an antiproliferative effect and induced apoptosis (P = .001). RUNX3 transfection, in the absence of TGF-beta, had no effect on proliferation and apoptosis of SEG-1 cells. RUNX3 expression dramatically increases SMAD-mediated TGF-beta-induced transcriptional activity when compared with controls (P = .0001)., Conclusions: RUNX3 is not expressed in SEG-1 cells, while it is present in normal esophageal mucosa. RUNX3 is essential for the antiproliferative and apoptotic effects of TGF-beta in SEG-1 cells and for the Smad-mediated transcriptional activity of TGF-beta., (Copyright 2004 Elsevier Inc.)