Journal of Orthopaedic Research

Rotator cuff tendinopathy is highly prevalent in aging populations, yet the mechanisms leading to age-dependent tendon degeneration are not well understood. In addition to tensile loading, tendons are subjected to compressive forces at certain anatomical sites (e.g., Achilles, rotator cuff), where altered adaptive responses may contribute to degenerative remodeling. The objective of this study was to investigate age-related differences in tendon responses to dynamic compressive loading using an ex vivo model. Murine flexor tendon explants from young and aged animals were cultured in a biaxial bioreactor and subjected to different levels of dynamic compressive loading. We then observed changes in metabolic activity, matrix composition, matrix biosynthesis, matrix structure, and gene expression. Young tendons exposed to moderate levels of compression maintained homeostasis, whereas high compression induced a robust adaptive response characterized by increased glycosaminoglycan accumulation, elevated collagen content, and upregulation of remodeling-associated genes including collagen I, decorin, and MMP-9, as well as inflammatory and apoptotic markers. In contrast, aged tendons demonstrated a qualitatively different response, with transcriptional downregulation of key remodeling markers alongside elevated secretion of matrix-degrading enzymes and pro-inflammatory cytokines, indicative of a maladaptive mechanobiological response even at low compressive levels. These findings reveal that impaired mechanosensitivity and a lower threshold for injury may predispose chronically loaded tissues to degenerative pathology associated with excessive compressive loading.

ObjectiveRecently, alternatives to animal testing, such as new approach methodologies, are being developed in the orthopedic research field; animal models still provide valuable insights into the pathogenesis of knee osteoarthritis (OA). However, commonly used models develop OA much more rapidly and severely than those observed in human patients. We aimed to develop a novel murine model that closely mimics the slow progression of human OA with posterior Cruciate ligament (PCL) rupture. Design12-week-old C57BL/6 mice were induced to PCL-rupture (PCL-R) by manually applying an external tibial posterior translation force. We analyzed joint kinematics, histological observations, and bone structure to confirm the absence of concurrent injury on day 0. Then, joint stability and the pathophysiological progression of knee OA were analyzed at 8, 16, and 34 weeks post-PCL-R. The destabilized medial meniscus (DMM) model was also analyzed to compare the OA progression. ResultsNon-invasive PCL-R intervention induced the complete rupture in the central region of PCL without concurrent injury. The PCL-R group showed larger posterior tibial deviation than the INTACT (P=0.008). Regarding the range of motion in the PCL-R group, there was no limitation in range of motion on day 0, but extension limitations occurred at weeks 16 and 34 weeks. Histologically, articular cartilage degeneration in PCL-R was milder than DMM. In the subchondral bone, micro-CT reconstruction images indicated that, compared with the INTACT group, the DMM group observed progressive subchondral bone formation from 16 weeks post-surgery. In contrast, the PCLR group maintained the subchondral bone structure even at 34 weeks. ConclusionsPCL-R model induced mild abnormal mechanical stress depending on posterior instability, and cartilage degeneration occurred more slowly in this model than in DMM models.

ObjectiveAnterior cruciate ligament (ACL) tears increase the risk for developing posttraumatic osteoarthritis (PTOA). Females have greater risk for both. However, studies defining sex-specific protein responses in human cartilage after ACL injury are lacking. We hypothesize that articular cartilages response to an injurious environment differs depending on sex. DesignWe compared the proteomic profiles of normal cartilage with injured cartilage harvested from the intercondylar area during ACL surgery. Sex-specific injury effects were estimated through contrasts between Injured Male and Normal Male and between Injured Female and Normal Female. Pathway enrichment analysis was done using gene ontology (GO) and compared against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Extracellular matrix (ECM) proteins were further analyzed using the Matrisome AnalyzeR. ResultsFrom the 2,188 proteins identified, males and females shared 1,121 upregulated and 23 downregulated proteins in injured compared to normal cartilage. Analysis of ECM proteins and enriched pathways revealed mostly similar male and female responses to an injurious environment, with evidence of early cartilage remodeling in both sexes. Nevertheless, more than 240 proteins were affected specifically by sex, and significant sex differences were found in inflammation, ECM-related, and metabolic pathways. Males were enriched mostly in "ECM-receptor interaction", while females were enriched in "Citrate cycle (TCA cycle)", "Fatty acid degradation", and "Fatty acid metabolism" pathways. ConclusionArticular cartilage shows signs of remodeling soon after ACL injury, even when only exposed to an injurious environment rather than being physically impacted. Sex differences were observed in inflammation, metabolic pathways, and ECM synthesis.

ObjectivesTo identify synovial transcriptional clusters in human knee osteoarthritis (OA) and determine how these relate to synovial histologic features, cell-type-associated gene expression, and cartilage degeneration severity. MethodsBulk RNA sequencing (RNA-seq) of synovial tissue from n = 135 patients with knee OA was analyzed using consensus clustering. Clusters were compared by clinical and histologic features, including cartilage degeneration severity (OARSI score). Single-cell RNA-seq (n = 18) and spatial transcriptomics were used to relate cartilage degeneration-associated gene expression patterns to synovial cell populations. ResultsFour synovial transcriptional clusters that differed in synovial histologic features and cartilage degeneration severity were identified. Greater cartilage degeneration was associated with enrichment of lining fibroblast- and inflammatory myeloid-associated gene expression, whereas lesser cartilage degeneration was associated with enrichment of sublining fibroblast, endothelial, mural cell, and adipocyte-associated gene expression. ConclusionsHuman knee OA synovium segregates into transcriptional clusters associated with cartilage degeneration severity. Synovial transcriptional heterogeneity corresponds to cell-type-associated gene expression. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSO_LIOsteoarthritis synovium exhibits marked histologic and molecular heterogeneity. C_LIO_LISynovial inflammation detected by MRI correlates with cartilage degeneration and predicts progressive cartilage loss in knee OA. C_LIO_LIPrior transcriptomic studies have identified molecular subsets of OA synovium, but their relationship to cartilage degeneration severity remains unclear. C_LI What this study addsO_LIOA synovium segregates into four transcriptional clusters: Sublining (C1), Lymphomyeloid (C2), Myeloid (C3), and Major trauma (C4). C_LIO_LIGreater cartilage degeneration is associated with enrichment of inflammatory myeloid and lining fibroblast gene expression, whereas lesser degeneration is associated with enrichment of adipocyte, sublining fibroblast, endothelial, and mural cell-associated gene expression. C_LI How this study might affect research, practice or policyO_LIProvides a framework for a clinically relevant biological stratification of OA patients based on synovial molecular features. C_LIO_LIInforms future efforts to link synovial biology with OA prognosis, cartilage degeneration, treatment allocation, and development of targeted therapeutic strategies. C_LI

The surface layer of articular cartilage provides for low-friction joint movement and protects the tissue from mechanical wear. The superficial zone chondrocytes (SZCs) of the surface layer produce proteoglycan-4 (PRG4), which is a lubricant that is necessary to reduce friction. Articular cartilage has limited capacity for self-repair and cell-based therapies, such as autologous chondrocyte implantation (ACI), is used to stimulate repair. However, in ACI, cells are expanded on tissue culture polystyrene where SZC poorly attach, proliferate slowly and dedifferentiate. Consequently, expanded SZC produce fibrocartilage tissue with insufficient PRG4. We previously demonstrated that culturing SZC on chondrocyte-derived decellularized extracellular matrix (CM) enhances SZC attachment and preserves phenotype. Since fibronectin (FN) was identified as the most abundant matrix protein within CM, here we tested the hypothesis that FN-coated culture surfaces would partially reproduce the beneficial effects of CM. We found that, similar to CM, SZC on FN-coated polystyrene increased SZC attachment and proliferation. However, unlike CM, SZCs expanded on FN-coated polystyrene remained more dedifferentiated as indicated by spread cells, elevated fibroblastic and contractile mRNA levels, and increased formation of SMA positive stress fibers. Consistent with the dedifferentiated phenotype, SZC on FN-coated polystyrene displayed extensive stress fibers, and higher nuclear myocardin-related-transcription-factor-a (MRTF-A). In contrast, CM reduced stress fiber formation and diminished nuclear MRTF-A in SZC. CM provides matrix cues beyond FN that suppress dedifferentiation and preserve the SZC phenotype. Identifying the matrix cues necessary to improve SZC expansion could lead to the generation of a superior surface in ACI repair tissue.

Patellar osteochondral allograft (OCA) transplantation is widely used to treat large full-thickness cartilage defects, yet long-term failure and reoperation rates remain high. Although surface congruity and osseous integration are emphasized clinically, cartilage thickness and mechanical compatibility between donor and recipient are not considered. Our previous work suggests that cartilage thickness mismatch can amplify local deformation at the graft boundary, potentially compromising graft longevity. This study investigates how combined mismatches in cartilage thickness and mechanical properties influence the local strain environment at the patellar OCA interface. Simplified two-dimensional axisymmetric finite element models of patellar OCA repair were developed in ABAQUS. Donor-to-recipient cartilage thickness ratios ranging from 0.33 to 3.25 were evaluated together with donor-recipient Youngs modulus mismatches (2.5-7.0 MPa). Cartilage was modeled using homogeneous linear elastic and functionally graded material formulations to account for depth-dependent stiffness. A compressive pressure of 1.0 MPa was applied to represent patellofemoral joint loading, and peak compressive and shear strains were quantified at the graft boundary. Cartilage thickness mismatch produced localized high-strain regions (HSR) of compressive and shear strain at the donor-recipient interface that were absent in thickness-matched constructs. Strain amplification increased with both thickness and mechanical property mismatch. Compressive strain exhibited directional asymmetry, with donor-side-thicker configurations producing greater amplification than recipient-side-thicker configurations. Incorporating depth-dependent cartilage stiffness reduced peak strain magnitudes but did not eliminate mismatch-driven strain amplification. These findings demonstrate that cartilage thickness and mechanical disparity can create HSR at the patellar OCA graft boundary that may predispose grafts to impaired integration and long-term failure.

Fracture nonunion remains a major cause of morbidity, yet patient-specific factors associated with impaired healing remain incompletely characterized. Anemia has been associated with adverse orthopaedic outcomes, but its relationship with fracture nonunion is poorly understood. We examined whether pre-fracture anemia, anemia burden, and clinically relevant anemia subtypes were associated with nonunion following tibial or femoral fractures. Using commercial and Medicare fee-for-service claims from 2016 through 2023, we identified adults aged 19 years or older with a tibial or femoral fracture, continuous enrollment during the preceding year and for at least six months after fracture, and no baseline cancer. Pre-fracture anemia was evaluated as any anemia, the number of distinct anemia diagnoses, and nutritional, hemolytic, aplastic, and other anemia subgroups. Nonunion occurring six to eighteen months after fracture was assessed using incidence rates and multivariable-adjusted hazard models. Among 326,673 adults, 149,704 had pre-fracture anemia and 176,969 did not. The crude incidence of nonunion was 42% higher among individuals with anemia than among those without anemia (incidence rate ratio, 1.42; 95% confidence interval, 1.32 to 1.53) and increased with greater anemia burden. After adjustment for demographic and clinical characteristics, including prior fractures at other anatomical sites, pre-fracture anemia remained associated with nonunion following tibial and femoral fractures, with hazard ratios of 1.83 (95% confidence interval, 1.54 to 2.18) and 1.38 (95% confidence interval, 1.26 to 1.50), respectively. Associations were also observed for nutritional and other anemias, whereas estimates for hemolytic and aplastic anemias were limited by few nonunion events. Within the femur, the association was strongest for distal fractures. These findings demonstrate that pre-fracture anemia is independently associated with nonunion. The increase in risk with greater anemia burden and findings across evaluable subgroups suggest that pre-fracture anemia may help identify patients at increased risk of impaired fracture healing.

BackgroundThe pivot shift (PS) test is the most specific clinical examination for anterolateral rotational instability in ACL-deficient knees, yet grading remains subjective, as evidenced by poor inter-observer reliability, particularly for Grade 2. Since low-grade (Grade 1) versus high-grade (Grades 2/3) PS is the threshold for recommending lateral extra-articular augmentation, performing the test in awake clinic patients limits grading reproducibility and introduces variability in surgical decision-making. Existing methods to quantify the pivot shift usually require examiner-performed testing under general anaesthesia. No prior approach has ascertained PS grading from a separate patient-performed functional movement. PurposeTo evaluate the feasibility of a machine learning (ML) classifier, trained on kinematic ultrasound bone-tracking signals acquired during a patients sit-stand-sit (SSS) knee movement, to predict their PS grade, and to clinically validate its ability to differentiate low versus high-grade PS. MethodsUltrasound bone-tracking kinematic data were collected during SSS manoeuvres in 23 ACL-injured patients using the GATOR device, and ground truth PS grades (0-3) were assigned under general anaesthesia by fellowship-trained orthopaedic sports surgeons. From the data collected, Leave-one-out cross-validation (LOOCV) was used to train the ML classifier. Clinical SSS data from 6 ACL-deficient patients was used for independent held-out validation of their low-grade (Grade 1) versus high-grade (Grade 2/3) PS. Multiple deep learning architectures (XceptionTime, InceptionTime, FCN, ResNet, ResCNN) and training strategies (including mixup augmentation and supervised contrastive learning) were tested. Performance was measured by one-versus-rest (OVR) AUC under LOOCV and by AUC (low vs high grade PS) from the held-out patient sessions. ResultsThe ML classifier achieved a maximum OVR AUC of 0.928 {+/-} 0.084 under LOOCV. Classifier performance increased with pivot-shift severity: Grade 3 was identified most reliably (AUC ~0.81; sensitivity 0.70-0.80), whereas Grade 2 remained the most challenging boundary (sensitivity 0.20-0.75 across configurations). For the clinically relevant binary classification of low-versus high-grade pivot shift, the classifier generalised well to a completely unseen patient cohort (AUC 0.889; accuracy 0.860; sensitivity 0.850; minimum-class sensitivity 0.767). ConclusionThe study demonstrates that kinematic ultrasound bone-tracking during sit-stand-sit contains transferable information about rotational instability severity in ACL-deficient patients, and represents the first reported approach to predict pivot shift grade from a patient-performed functional movement. The strong cross-validation performance confirms that the signals contain meaningful PS grade-discriminative information, but larger datasets targeting 50-100 sessions per grade will be required to achieve patient-level generalisation and advance this novel rotational instability assessment tool toward full clinical adoption. Level of EvidenceLevel IV, diagnostic feasibility study.

We performed a new set of experiments to confirm findings reported in our previous publication regarding the role of Sulf-1 and Sulf-2 in regulating BMP-7 and FGF-2 signaling in human articular chondrocytes. Using primary chondrocytes from five independent human donors, we examined the effects of Sulf knockdown on Smad1/5 and Erk1/2 phosphorylation. Sulf-1 and Sulf-2 knockdown consistently reduced BMP-7-induced Smad1/5 phosphorylation and enhanced FGF-2-induced Erk1/2 phosphorylation. Although the magnitude of Erk1/2 activation was somewhat lower than originally reported, the direction and statistical significance of the effects were preserved. These results confirm the original conclusions and support the role of Sulfs as dual regulators of BMP-7 and FGF-2 signaling pathways in human chondrocytes.

1.Cancer-induced bone pain (CIBP) occurs in a majority of patients when primary or metastatic cancer develops within the bone. This pain has a significant impact on quality of life, yet there are limited effective treatment options available. Nerve sprouting is a complex mechanism that has been implicated in CIBP. Netrin-1 is a neuronal guidance molecule that is produced by numerous cell types, including cancer cells. In this study we aimed to determine whether netrin-1 inhibition (with NP137 - a humanized IGg1 monoclonal antibody) could ameliorate nerve sprouting, and nociception by extension, in three models of CIBP - osteosarcoma, metastatic breast cancer, and metastatic prostate cancer. Sustained administration of NP137 failed to produce an anti-nociceptive effect in these models, but a delayed onset was observed in the osteosarcoma model. NP137 did not produce a disease-modifying effect, as micro-computed tomography did not reveal reduced bone destruction in the NP137-treated groups. Additionally, there was no nerve fibre density reduction in any of the groups at the late-stage of the disease, suggesting that nerve sprouting occurs in early- to mid-stage CIBP development. Investigation of NP137 exposure indicated that serum levels of NP137 were comparable between the sham and cancer groups. Our study indicates that netrin-1 may play a role in early-stage CIBP development, but inhibition of this mechanism does not produce robust anti-nociception.

ObjectiveThis study investigates joint injury-induced angiogenesis and the effects of genetic deficiency of thrombospondin-2 (TSP2), an anti-angiogenic factor, in joint homeostasis and post-traumatic osteoarthritis (PTOA). MethodWe utilized a murine non-invasive anterior cruciate ligament rupture (ACLR) model of PTOA and mined published synovial transcriptomics datasets to investigate injury-induced synovial angiogenesis. Spatial transcriptomics and flow cytometry of TSP2-GFP reporter mice were used to assess injury-induced thrombospondin-2 and its cellular origins in synovium. Global TSP2 knockout mice (TSP2-KO) were used to assess the effect of TSP2 deficiency on early and late stages of PTOA development via molecular imaging of inflammation and angiogenesis, histopathology, micro-computed tomography, Raman spectroscopy, and synovium bulk RNA-sequencing. ResultsIntra-articular angiogenesis peaked at 7d post-ACLR and declined but remained elevated above baseline at 28d post-ACLR. We identified synovial crosstalk between endothelial cells and sublining fibroblasts as a key driver of angiogenesis and source of thrombospondin-2 signaling, with TSP2 primarily upregulated in sublining fibroblasts. TSP2-KO mice exhibited increased peri-articular inflammation at 7d post-ACLR and inferior bone quality. Histopathology revealed greater PTOA severity but paradoxically lower synovitis in TSP2-KOs. Additionally, aberrant structural remodeling of the entire knee joint was observed in uninjured and ACLR TSP2-KO limbs. The uninjured TSP2-KO synovial transcriptome demonstrated elevated immune, fibrotic, and angiogenic activation; however, TSP2-KO and WT synovial transcriptomes partially converged upon injury. ConclusionTSP2 is essential for joint homeostasis and trauma response. Global TSP2 deficiency causes premature OA and worsened PTOA, suggesting that therapeutic targeting with TSP2 mimetic could be used to prevent OA.

Context: Although knee extensor and flexor strength deficits are well-documented after anterior cruciate ligament reconstruction, limited data exist characterizing how strength recovery evolves over time. Understanding the temporal patterns of recovery, and how they differ by autograft type, is critical for optimizing rehabilitation and return-to-sport decision-making. Objective: To characterize temporal trends in knee extensor and flexor strength recovery during the first year post-ACLR and evaluate differences between patellar tendon and hamstring tendon autografts. Design: Case series. Setting: Sports physical therapy clinics within a large health system. Participants: Five hundred three patients (17.8 {+/-} 3.0 y) who underwent primary reconstruction with either patellar tendon or hamstring tendon autografts and completed a combined 730 return-to-sport tests within 12 months postoperatively. Main Outcome Measures: Normalized peak isokinetic concentric knee extension and flexion torques for involved and uninvolved limbs, and normalized symmetry indices for knee extension and flexion strength. Results: Knee extension strength on both limbs and extension strength symmetry improved over time. Patients with hamstring autografts demonstrated superior involved leg knee extension strength and better extension strength symmetry compared with those receiving patellar tendon autografts, although uninvolved leg strength was similar between autografts. Knee flexion strength on both limbs and flexion strength symmetry also improved over time. Patellar tendon autograft patients exhibited greater strength symmetry, despite no between autografts for flexion strength for the involved or uninvolved limb. Conclusions: Autograft significantly influences muscle strength recovery following anterior cruciate ligament reconstruction. Hamstring tendon autografts are associated with superior recovery of knee extension strength and strength symmetry compared to patellar tendon autografts. These findings underscore the need for graft-specific rehabilitation strategies and earlier identification of patients at risk for delayed recovery.

PurposeEstrogen deficiency drives bone loss through interacting endocrine, oxidative, inflammatory and bone-remodeling disturbances. Ergothioneine (EGT) is a diet-derived thiol/thione antioxidant whose effects on the estrogen-deficient skeleton are unknown. We evaluated whether EGT, alone or combined with vitamin K2, vitamin D3 and magnesium L-threonate, attenuates the skeletal and systemic consequences of ovariectomy (OVX) in mice. MethodsForty-eight female C57BL/6J mice underwent sham surgery or OVX and received daily oral gavage for 12 weeks of vehicle, alendronate (1.53 mg/kg), EGT (30 mg/kg/day), EGT with vitamin K2 (40 {micro}g/kg/day) and vitamin D3 (500 IU/kg/day), or EGT with vitamin K2, magnesium L-threonate (350 mg/kg/day) and vitamin D3 (n = 5-6 analysed per group). Outcomes included the uterine index, tibial micro-computed tomography, distal-femoral histology, and serum bone turnover markers (CTX-I, PINP, osteocalcin), sex hormones, TNF-, IL-6, SOD and MDA. OVX lowered the uterine index and induced tibial trabecular deterioration, with increased CTX-I, decreased PINP and osteocalcin, elevated TNF- and IL-6, reduced SOD and increased MDA (all P < 0.01 vs sham). Alendronate restored tibial micro-CT bone-volume fraction (BV/TV) and trabecular number (P < 0.01 vs OVX). The EGT-based regimens did not significantly restore tibial micro-CT BV/TV, trabecular thickness or trabecular number (all P > 0.05 vs OVX), but significantly increased trabecular area on distal-femoral histology (OVX 7.6% vs 14.2-15.0% across regimens; P < 0.05 vs OVX) and lowered CTX-I, TNF-, IL-6 and MDA while raising SOD and partially restoring PINP and osteocalcin (P < 0.05-0.01 vs OVX). Because the histological and micro-CT endpoints were assessed at different skeletal sites, structural interpretation is cautious. Apparent increases in serum estradiol were assay-dependent and are regarded as exploratory. Ergothioneine-based nutritional regimens improved the systemic oxidative, inflammatory and bone-turnover environment of estrogen-deficient bone loss and preserved distal-femoral trabecular area on histology, although tibial three-dimensional microarchitecture by micro-CT was not restored. Because the histological and micro-CT endpoints were assessed at different skeletal sites, the structural interpretation is necessarily cautious. These findings support further evaluation of EGT as a dietary adjunct, with mechanistic and dose-optimization studies warranted.

Abstract Background: Management of high sagittal split fractures of the mandibular condyle remains a formidable surgical chal-lenge due to limited visualization, technical difficulties in direct in-situ fixation, and the high risk of secondary avascular necrosis or temporomandibular joint (TMJ) ankylosis. Objectives: To evaluate the clinical outcomes and technical efficacy of the "Motamed Technique, "a standardized protocol involving extracorporeal rigid internal fixation followed by anatomical re-implantation for complex high condylar split fractures. Methods: A retrospective evaluation was conducted on a clinical series of 11 consecutive patients (9 males, 2 females) presenting with severe, displaced high sagittal split condylar fractures secondary to high velocity trauma. In all cases, the fragmented condylar segments were completely retrieved, stabilized ex vivo on a back table using a titanium X-shaped 3D mini-plate system (1.5 mm), and meticulously reimplanted into the glenoid fossa. Total cold ischemia time was strictly maintained between 10 to 20 minutes. The postoperative longitudinal follow-up period ranged from 6 to 11 months (mean duration: 8.6 months). Comprehensive post-operative tracking included clinical parameter checking, 3D Computed Tomography (3D-CT), and high-resolution dynamic Magnetic Resonance Imaging (MRI) to analyze ony union, vertical ramus height restoration, and articular disc kinetics. Results: All 11 patients achieved predictable and stable functional outcomes. At the definitive milestones, the mean maximum mouth opening (MMO) was 37.3 mm (range, 33-45 mm), demonstrating excellent vertical clearance and stable lateral/protrusive excursions. Pre-traumatic stable centric occlusion was perfectly restored and maintained in 100% of cases (n=11), with zero incidence of postoperative open bite or crossbite. Facial nerve motor function was entirely pre-served across the cohort (100% House-Brackmann Grade I at final follow-up). Longitudinal 3D-CT scans confirmed complete osseous union and anatomical alignment in all cases by the 4th postoperative month, with no radiographic evidence of condylar head resorption or hardware failure. Follow-up MRI findings demonstrated the preservation of TMJ dynamics, functional articular disc movement (with stable reduction in 3 cases), and a total absence of avascular necrosis or intra-articular effusion. No cases of TMJ ankylosis were reported. Conclusion: The Motamed Technique provides a reliable, reproducible, and biologically sound approach for managing intricate high condylar split fractures. By utilizing systematic extracorporeal mini-fixation, this protocol effectively overcomes intraoperative spatial limitations while ensuring excellent long-term anatomical stability, stable occlusion, and functional joint mobility without compromising facial nerve integrity.

ObjectiveAdults with knee osteoarthritis often experience movement-evoked pain (MEP), and that pain has the potential to alter gait mechanics and influence disease progression. However, the associations between MEP and gait biomechanics have only been assessed in typical lab settings. Gait mechanics differ in the lab compared to in the real-world, thus it is unknown whether these associations between pain and gait translate to real-world settings. Therefore, this study aimed to measure concurrent changes in MEP and gait mechanics across three days of typical real-world activity. DesignSeventeen participants with self-reported physician-diagnosed symptomatic knee osteoarthritis wore inertial measurement units on their more symptomatic limbs thigh and shank, as well as on both feet for three days of typical activity. Participants were sent 5 automated text messages a day and were instructed to complete a short 3-5 minute walk and self-report their MEP via a Numeric Rating Scale (0-10) during each of the walks. A random coefficients model was used to determine how gait speed, stride length, and knee and ankle range of motion was related to changes in pain intensity. ResultsThe average MEP experienced during the instructed walks was 1.4 {+/-} 1.3 with individual participant average pain intensities ranging from 0 to 4.8. Greater MEP was associated with a 2.7{degrees} decrease in knee range of motion per unit increase in pain (95% CI [-4.8 -0.5], p = 0.02). Seven of the seventeen participants never reported a pain level of 0. Speed, stride length, and ankle range of motion did not differ by pain intensity. ConclusionsIncreases in MEP were associated with decreases in knee range of motion. A 2.7{degrees} decrease in knee range of motion in response to a 1-unit change in pain is meaningful as 5{degrees} is generally considered the threshold for a meaningful difference in joint angles. With a change in pain intensity of 2 being common with daily activity, individuals may be experiencing meaningful changes in knee joint angles regularly. With gait mechanics being associated with disease progression, these daily acute fluctuations in pain may be influencing disease progression rates.

BackgroundPain with movement is common in adults with knee osteoarthritis (OA), but the effect of movement-evoked pain on gait is not well understood. This relationship is vital to understand as gait mechanics are associated with OA initiation and progression. Our current understanding of acute changes in pain and gait stems from extended bouts of walking, however these bouts likely dont represent real-world behavior. Therefore, understanding how gait changes with shorter, more intense bouts of activity may provide valuable insight into the pain experience. MethodsAdults with (n=19) and without (n=19) knee OA wore inertial measurement units (IMUs) while completing bouts of walking before and after two bouts of stair navigation (two flights). We tested whether pain and gait (speed, stride length, and lower extremity joint ranges of motion (ROM)) changed differently between adults with and without knee OA in response to multiple bouts of stair activity. FindingsThere were no significant interactions between group and stair bouts for any variable. When stratifying the OA group by those who did and did not experience pain, those who experienced a change in pain also had a greater change in early stance knee ROM in response to bouts of stairs. InterpretationThe observed changes suggest that knee kinematics may be more sensitive to acute changes in pain than gait speed or stride length. These differences were detectable using IMUs and therefore our results support the use of IMUs to measure concurrent pain and gait mechanics in less controlled and real-world settings.

Osteoarthritis (OA) drug development remains constrained by preclinical models that fail to recapitulate the multicellular interactions that regulate human joint inflammation and extracellular matrix degeneration in response to investigational drugs. Tanezumab, a humanized anti-nerve growth factor monoclonal antibody developed for non-opioid pain relief, advanced to late-stage clinical trials but was discontinued due to unresolved joint-localized safety concerns, including rapidly progressive OA. This study evaluated whether a human microfluidic joint-on-a-chip co-culture system could detect early biomarker responses to tanezumab exposure that were not apparent in conventional chondrocyte monoculture. Tanezumab was first tested in human chondrocyte monoculture under untreated and disease-like (i.e., IL-1{beta}-treated) conditions. Across a 20-analyte panel of inflammatory and matrix-remodeling biomarkers, statistically significant monoculture responses to tanezumab were limited to decreased IL-1{beta} from 335 to 132 pg/mL ([~]0.39-fold) and increased IL-8 from 575 to 675 pg/mL ([~]1.17-fold). Major OA-associated matrix-remodeling markers, including MMP-1, MMP-3, and MMP-13, remained largely unchanged, indicating that monoculture conditions are insufficiently sensitive to detect clinically predictive drug-related molecular changes. Tanezumab was then evaluated in co-cultures containing chondrocytes, osteoblasts, fibroblast-like cells, and macrophages under low-inflammation (i.e., M0 macrophage-based) and high-inflammation (i.e., M1 macrophage-based) conditions. In the M0-based co-culture, tanezumab increased MMP-1 from [~]4.20 x 104 to [~]6.20 x 104 pg/mL ([~]1.48-fold), MMP-3 from [~]8.00 x 104 to [~]1.20 x 105 pg/mL ([~]1.50-fold), and MCP-1 from 2.85 x 103 to 4.31 x 103 pg/mL ([~]1.51-fold). In contrast, the M1-based co-culture showed decreases in MMP-13 from [~]1.66 x 104 to [~]1.17 x 104 pg/mL ([~]0.70-fold) and IFN-{gamma} from [~]1.95 x 104 to [~]1.56 x 104 pg/mL ([~]0.80-fold), changes that may appear beneficial despite the drugs known clinical risks. Collectively, these findings show that low-inflammation multicellular co-culture revealed coordinated matrix remodeling and inflammatory responses to NGF blockade that were missed in monoculture and were partly obscured in highly stimulated disease-like conditions. This platform may provide a useful, human-relevant approach for safety signal assessment and early evaluation of OA therapeutics within a defined context of use focused on joint-specific, tissue-level drug-response testing.

Sutures are the primary sites of cranial bone growth, allowing the skull to accommodate the growing brain. External mechanical stimulus has been shown to delay suture fusion and induce tissue remodelling. Recent studies suggest that in vivo cyclic bone loading increased proliferation of mesenchymal stem cells (MSC) in the coronal suture. The overall aim of this study was to understand how many loading sessions (exposure-response) and how long after loading (time-course) did MSC proliferation increase in the coronal suture. In the exposure-response analysis, mice underwent 1, 3, or 5 loading sessions between Postnatal day 7 (P7) and P11, and in the time-course analysis, treated mice underwent 10 loading sessions between P7 and P21. Loading sessions were 10 minutes at a frequency of 1 Hz and a force of 10 g (0.1 N). The loading tip was positioned on the posterior aspect of the left frontal bone, dorsal to the coronal suture. The EdU marker shows a statistically significant increase in proliferation after one loading session and a decrease after three loading sessions. The PCNA marker shows a statistically significant increase after three and five loading sessions. The exposure-response analysis showed that when the results of both markers are combined, levels of proliferation cannot be interpreted until at least five loading sessions have been completed, after which a clear increase in proliferation was observed. In the time-course analysis, proliferation was highest immediately after the final treatment session and 24 hours after the final loading session the effects of mechanical bone loading gradually returned to baseline.

Mechanical cues are key regulators of bone formation, yet their potential as therapeutic stimuli remains incompletely explored in vivo. Nanovibrational stimulation, which delivers low-amplitude, high-frequency mechanical input, has been shown to promote osteogenic differentiation in vitro, and rodent studies have similarly demonstrated osteogenic effects. However, its impact on other cellular systems at the whole-organism level remains poorly understood. Here, we demonstrate that nanovibrational stimulation enhances myogenic differentiation in vitro before investigating its effects on skeletal development and tissue specificity in zebrafish. Larval zebrafish exposed to nanovibrational stimulation exhibited increased osteoblast numbers and enhanced bone formation relative to controls. In adult zebrafish, nanovibration increased the osteoblast response to a fracture-like injury, indicating enhanced osteogenic activity during repair. To assess tissue specificity, we examined additional cell types and systems relevant to skeletal regeneration, including cartilage, muscle, vasculature and innate immune cells. Although nanovibrational stimulation promoted myogenic differentiation of C2C12 cells in vitro, its effects on muscle and other non-skeletal tissues in zebrafish larvae were comparatively limited. These findings suggest that nanovibrational stimulation exerts a preferential effect on osteogenic processes in vivo. These findings demonstrate that nanovibrational stimulation preferentially enhances osteoblast-mediated bone formation and skeletal injury responses in zebrafish, without causing significant perturbations in other tissues. Our results establish zebrafish as a tractable in vivo model for investigating vibration-induced mechanobiological processes, providing a more physiologically relevant representation of tissue-level mechanotransduction than conventional two-dimensional culture systems. Furthermore, these findings highlight the potential of nanovibrational stimulation as a non-invasive strategy to promote bone regeneration and fracture repair.

Schwann cells (SCs) provide support for nerves throughout the body. Despite importance for nerve function and repair, the morphology and distribution of SCs in bone remains largely undefined. In this study we used a "Schwann Cell Mapper" mouse (Mpz-Cre+/-;TdT+/+;Ngfr-eGFP+/+) and a "p75 Lineage Tracer" mouse (Ngfr-CreERT2+/-;ZsGreen1+/+) to study SC localization and morphology within the adult mouse calvaria, limb, and vertebrae. We found that all nerves in bone were covered by mature myelinating or non-myelinating SCs, labeled by MPZ and p75-NGFR, respectively. Mature SCs populated the periosteum and entered the bone marrow through transcortical canals. Non-myelinating SCs outnumbered myelinating SCs in bone, with a ratio of [~]2:1 by length density. Non-myelinating SCs in periosteum had more branching and increased size relative to myelinating SCs. In addition, we identified two candidate populations of MPZ lineage+ and p75-NGFR+ immature SCs (iSCs) that were distributed throughout the calvarial periosteum. Similar to mature SCs, p75-NGFR+ candidate iSCs were more prevalent at a ratio of [~]3:1. Overall, neural crest-derived calvarial bone had evidence of increased SC maturity relative to mesoderm-derived sites, identifying niche-level differences in SC maturation. Lastly, lineage tracing revealed that mesenchymal lineages in bone were largely negative for both MPZ and p75-NGFR (<0.1% labeling). These findings provide a framework of SC organization in bone and highlight previously unrecognized diversity across skeletal compartments. By defining distribution and morphology, this work lays the foundation for future studies investigating how SCs contribute to bone biology, including roles in repair, pain, and homeostasis.