Osteoarthritis and Cartilage

Background: Knee osteoarthritis (OA) is characterized by progressive joint degradation and altered gait biomechanics. Inter-limb load asymmetry during daily living tasks may accelerate joint deterioration, particularly when combined with physical fatigue. This study investigates the relationship between daily physical fatigue and inter-limb load asymmetry patterns during functional activities in individuals with unilateral knee OA. Objective: To evaluate the impact of fatigue on inter-limb load asymmetry and joint loading rates during daily living tasks in patients with unilateral knee osteoarthritis, and to identify biomechanical and clinical factors associated with load distribution patterns. Methods: One hundred eighteen participants with unilateral knee OA (mean age 62.6 (SD 7.4) years, BMI 29.9 (SD 4.2) kg/m2) completed biomechanical assessments during non-fatigued and fatigued conditions. Ground reaction force measurements were obtained during functional tasks including stair climbing, sit-to-stand transitions, and level walking. Load asymmetry was quantified as the inter-limb difference in peak vertical ground reaction forces. Statistical analysis was conducted using Python (v3.9), including paired t-tests, analysis of variance, and linear regression modeling. Results: Participants demonstrated significantly increased load asymmetry under fatigue conditions (mean difference 8.7% (SD 12.3) %, p < 0.001). Fatigue-induced asymmetry was task-dependent, with stair negotiation showing the greatest increase (11.2% (SD 14.1) %). Peak loading rates on the affected knee were inversely correlated with time-to-peak load during fatigued conditions (r = -0.68, p < 0.001). Disease severity (WOMAC score) and age significantly predicted asymmetry changes (R2 = 0.52, p < 0.001). Conclusions: Physical fatigue exacerbates inter-limb load asymmetry in unilateral knee OA, potentially accelerating joint degeneration. Task-specific interventions targeting fatigue management and load redistribution may improve long-term outcomes. Future longitudinal studies should examine the temporal relationship between acute biomechanical changes and progressive structural joint damage. Keywords: Knee osteoarthritis, load asymmetry, fatigue, biomechanics, ground reaction forces, functional tasks.

Even though osteoarthritis is considered a disease of the whole joint, integrated analysis of articular calcified cartilage (ACC) behaviour with adjacent tissues remains a persistent limitation. Herein, we performed synchrotron X-ray computed tomography in intact loaded joints and coupled anatomical analysis with digital volume correlation to examine whether microarchitectural ACC features serve as predictive mechano-biomarkers of osteoarthritis. We reveal that male osteoarthritis-prone STR/Ort mice have thicker yet more porous ACC that contains larger but less spherical chondrocyte lacunae than their healthy parental-control CBA mice. This was linked to asymmetrical distribution of tensile and compressive strains generated under physiological loads in the ACC and underlying subchondral bone in tibial epiphyses of STR/Ort mice. Together these data suggest that murine ACC microarchitecture has a mechano-predictive role in age-related osteoarthritis.

Objective: To determine how fibronectin fragment (Fn-f), a matrikine that induces an osteoarthritis (OA)-like phenotype, reshapes chondrocyte states and transcriptional programs at single-cell resolution. Design: Primary human ankle chondrocytes from two cadaveric donors without OA were treated with PBS or purified recombinant Fn-f for 18 hours and profiled by single-cell RNA sequencing. Transcriptional states were characterized by marker genes and functional enrichment, compared with classifications used for OA chondrocytes, and evaluated for Fn-f-dependent changes in abundance and transcriptional programs. Results: Five transcriptionally distinct populations were identified with features that matched previous classifications of effector, inflammatory, pre-hypertrophic/fibrochondrocyte, reparative, and homeostatic chondrocytes. Fn-f markedly redistributed cells among these states, enriching inflammatory chondrocytes while reducing effector and reparative populations. Within transcriptional states, Fn-f differentially altered matrix/cartilage, inflammatory, and stress/immediate-early programs. These responses did not uniformly correspond to assigned subtype identities, demonstrating that transcriptional programs can be dynamically deployed across chondrocyte states. Conclusions: Controlled perturbations of healthy chondrocytes complement observational OA atlases by resolving stimulus-dependent changes in chondrocyte state abundance and transcriptional programs. Discordance between subtype annotations and dynamic functional programs further highlights the need for harmonized definitions of chondrocytes across phenotypic states.

Skeletal muscle dysfunction is a major contributor to disability and incomplete functional recovery in patients with end-stage knee osteoarthritis (KOA), yet the spatial components of disease-associated molecular remodeling remain poorly understood. Here, we applied spatial transcriptomics to paired skeletal muscle biopsies obtained from the surgical (Sx) and contralateral (Ct) limbs of individuals undergoing total knee arthroplasty (TKA) to define the cellular architecture of periarticular muscle and determine how muscle inflammation susceptibility (MuIS) shapes local transcriptional programs. Integrated analysis of 27,087 spots obtained from 22 muscle histological cross-sections (11 Sx-Ct pairs) revealed seven spatially resolved transcriptional domains corresponding to slow and fast myofiber states, an extracellular matrix/fibroadipogenic-enriched domain, a pericyte/smooth muscle domain, and a satellite cell/myogenic-enriched domain. Despite advanced unilateral disease, the major annotated cellular compartments were similarly represented between Sx and Ct limbs. KOA-associated remodeling was reflected primarily by within-cluster transcriptional changes, with the most informative differences observed in fibroadipogenic, pericyte/smooth muscle, and satellite/myogenic domains. Within Sx, MuIS stratification identified a coordinated transcriptional program characterized by denervation- and regeneration-associated genes and altered contractile and metabolic features. Neighborhood analysis localized denervation-associated signals primarily to fast-myofiber-rich regions, while local adjacency patterns among the examined myofiber, fibroadipogenic, and pericyte/smooth muscle domains were broadly preserved. Our findings provide the first spatial transcriptomic analysis of periarticular skeletal muscle in end-stage KOA and identify MuIS as a distinct local transcriptional phenotype in diseased muscle.

Cartilage thickness on MRI is a structural biomarker of osteoarthritis (OA), yet anatomical variation and aging can mask early disease, including changes occurring 12-36 months before incident OA. We propose longitudinal local centile maps that use normative growth charts as prior information and integrate a knee's earlier scans to identify cartilage that is thinner or thicker than expected. We built the reference charts from 5,045 scans of 957 persistently radiographically normal knees in the Osteoarthritis Initiative. Under participant-separated nested cross-validation, longitudinal centile profiles achieved higher 12- and 24-month AUCs for predicting incident radiographic OA than regional PCA, radiomics, cross-sectional centile features, and raw-map deep features across most classifiers. These conventional descriptors added little when combined with centile profiles, and cross-sectional centiles added little beyond longitudinal centiles. Deep centile features achieved the best AUCs (0.954 [95% CI 0.931-0.974] at 12 months and 0.969 [0.949-0.985] at 24 months; balanced accuracy 0.917 and 0.931) and outperformed raw-map deep features in all 12 classifiers after correction for multiple comparisons. No representation consistently improved 36-month prediction. Longitudinal centiles transform cartilage thickness maps into reference-based deviation maps: deep longitudinal centile features provide the highest predictive accuracy, while regional longitudinal centile profiles provide interpretable prognosis with the second-highest accuracy.

Objectives. Observational studies report large, conflicting associations between common drugs and hip or knee replacement. We tested them in the serious adverse event (SAE) tables of randomized trials on ClinicalTrials.gov. Methods. We included randomized drug trials with at least 500 participants. A language model classified arms from summaries without outcome counts (masked by instruction only). We pooled osteoarthritis-related SAEs by drug class as Mantel-Haenszel risk ratios (RRs). Because the plan followed a preliminary analysis of 72 trials, we report estimates rather than tests, checked by positive and negative controls and calibration. Results. The 881 placebo- or usual-care-controlled trials included 1.75 million participants and 9,408 osteoarthritis-related SAEs. The tables reproduced CANTOS's own canakinumab effect (RR 0.57, 95% CI 0.39 to 0.82), the anti-nerve growth factor excess (2.31, 1.82 to 2.92) and two of three modest effects specified before extraction: gallbladder disease with glucagon-like peptide-1 receptor agonists (GLP-1RAs; 1.36, 1.22 to 1.51) and ketoacidosis with sodium-glucose cotransporter-2 (SGLT2) inhibitors (1.96, 1.48 to 2.60). In the same trials, osteoarthritis-related SAEs did not differ detectably with GLP-1RAs (0.97, 0.86 to 1.10; calibrated 0.87, 0.54 to 1.41) or SGLT2 inhibitors (0.96, 0.82 to 1.12; calibrated 0.86, 0.53 to 1.40). Other proposed drugs gave imprecise estimates. Post hoc, 8 of 21 published observational estimates differed from randomized ones, in both directions (3 to 6 after calibration). Conclusions. Over about two years, GLP-1RAs and SGLT2 inhibitors had no detectable effect on osteoarthritis-related SAEs in trials that detected their known effects. Slow or modest structural effects remain possible.

Introduction Autologous and allogeneic chondrocyte cell sheet transplantation has shown safety and potential efficacy for cartilage defects associated with knee osteoarthritis. Although polydactyly-derived chondrocyte (PDC) cell sheets have been used clinically for allogeneic transplantation, a scalable and reliable alternative cell source is needed. This study aimed to establish a proof-of-concept protocol for generating chondrocyte cell sheets from clinical-grade human induced pluripotent stem cells (iPS cells). Methods Cartilaginous tissue was generated from human iPS cells, and isolated chondrocytes were used to fabricate iPS cell-derived chondrocyte sheets. In Phase 1, sheets were produced in high-serum medium under normoxic, hypoxic, or hypoxic/high-seeding-density conditions and compared with PDC sheets using a three-dimensional in vitro model and xenogeneic orthotopic transplantation in an immunodeficient rat model. In Phase 2, sheets were produced in low-serum medium under hypoxic/high-seeding-density conditions and evaluated for cartilage-related gene expression, secreted factors, cell surface marker expression, and in vivo cartilage repair. Results In Phase 1, hypoxic/high-seeding-density culture enhanced in vitro chondrogenic properties. However, after transplantation, iPS cell-derived chondrocyte sheets formed fibrotic repair tissue, whereas PDC sheets generated hyaline-like cartilage. In Phase 2, low-serum culture increased expression of cartilage-related genes, including collagen type II alpha 1 (COL2A1) and SRY-box transcription factor 9 (SOX9), enhanced secretion of transforming growth factor beta 1 and melanoma inhibitory activity, and upregulated CD9, CD26, CD56, CD201, CD227, and GD2. Low-serum iPS cell-derived chondrocyte sheets regenerated hyaline cartilage at 4 weeks, and the repair tissue was maintained at 12 weeks without bone formation. Conclusions A robust fabrication protocol enabled human iPS cell-derived chondrocyte sheets to regenerate hyaline-like cartilage in vivo. These findings support their potential as a scalable cell source for clinical cartilage repair.

A persistent challenge in cartilage tissue engineering is identifying cell sources capable of regenerating cartilage with native-like properties. Variability in chondrogenic potential among human- and animal-derived cells complicates graft development and disease modeling. Here we benchmarked ten chondrogenic cell sources under standardized culture conditions using hyaluronan-alginate biomaterials and implementing a functionality index combining histological and mechanical metrics referenced to native tissue. Mesenchymal stem cells and fetal chondroprogenitors demonstrated superior regenerative capacity, achieving 103% and 95% of native cartilage functionality, respectively. In contrast, adult articular chondrocytes from healthy and osteoarthritic donors produced fibrocartilaginous tissue with functionality scores below 50%. These findings provide a quantitative framework to guide cell source selection and optimize cartilage graft standardization for clinical applications and physiologically relevant disease models, addressing regulatory demands for product characterization.

Clinical translation of mesenchymal stromal cell (MSC) therapies derived from primary tissue sources is limited by manufacturing inconsistencies driven by donor-to-donor variability, which reduces predictability of clinical response. Induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) offer a promising donor-independent, clonal alternative strategy that is also more amenable to engineering, but whether engineered iMSCs reproduce the potency attributes of primary MSCs remains unclear. Here, we benchmarked a doxycycline-inducible, hTERT-immortalized iMSC line engineered to overexpress TGFB1 (TGFB1-iMSCs), a key regulator of macrophage polarization, against multiple adipose tissue-derived MSC (MSC(AT)) donors across predefined immunomodulatory and angiogenic potency attributes. TGFB1-iMSCs were smaller and more circular with comparable or higher proliferative rates than primary MSC(AT) donors. Interestingly, they had a distinct angiogenic signature (EDIL3, EDN1, PDGFA), and nine differentially expressed microRNAs were predicted to target secretory trafficking and growth factor receptor signalling. Functionally, however, TGFB1-iMSCs secreted less VEGF and showed intermediate HUVEC tube formation, but matched or exceeded all primary MSC(AT) donors in a monocyte-macrophage transwell immunomodulatory assay. Notably, in a murine destabilization of the medial meniscus (DMM) model of post-traumatic osteoarthritis, a single intra-articular injection of TGFB1-iMSCs, but not MSC(AT), reduced total synovial macrophage numbers and lowered the MHCII/CD206 ratio at eight weeks, although it did not reduce cartilage degeneration, synovitis, or fibrosis. Together, these findings show that TGFB1-engineered iMSCs have a macrophage-directed potency profile distinct from that of primary MSC(AT). Immortalized iMSCs therefore provide a reproducible platform for mechanistically dissecting engineered MSC potency and its effects on the immune environment.

Purpose: To determine whether a cobalt-chrome genetic reactivity (CoGR) score - originally developed to identify patients at greater risk of developing adverse reaction to metal-on-metal hips - is associated with poorer patient-reported outcomes after knee arthroplasty (KA). Methods: In a prospective multicentre study, patients at a minimum two years post all cobalt-chrome KA were invited to research clinics. Participants provided samples for blood metal ion measurements and DNA sequencing and completed Forgotten Joint Scores (FJS). Component alignment was assessed on AP and lateral plain radiographs. The association between CoGR and FJS was assessed using regression adjusted for time since surgery, age, gender, BMI and tested for modification by blood cobalt. In a case-control analysis, CoGR scores of revised patients with histologically confirmed adverse reactions to metal debris (and specifically aseptic lymphocyte-dominated vasculitis-associated lesions (ALVAL)) were compared with controls (revised patients with no signs of ARMD and unrevised patients with FJS of 75 or more) using Firth logistic regression. Results: 376 patients were included: 312 unrevised patients and 64 patients who underwent revision. Each one-unit increase in CoGR score was associated with a 9.8 point lower FJS (95% confidence interval (CI) 4.2 to 15.4 points; p < 0.001). This increased to a 17.5 (9.4 to 25.5) point reduction per CoGR score unit in patients with blood cobalt above the median level. Comparing 28 ARMD cases (12 with ALVAL) with 177 controls, each one-unit CoGR increase was associated with odds ratios of 1.90 (CI 1.34 to 2.75) for ARMD and 3.05 (CI 1.89 to 5.34) for ALVAL. Conclusions: A higher CoGR score was associated with worse patient-reported outcome and higher odds of histologically confirmed adverse reaction to metal debris at revision. Adverse immune responses may be a more significant factor in unexplained pain after KA than is generally recognised.

To correct lower limb length discrepancies, orthopedic surgeons often perform limb lengthening procedures. These procedures involve directly lengthening a primary bone (e.g., femur), which also stretches the muscles and tendons spanning the elongation site. Limb lengthening procedures improve lower-limb length symmetry, often at the expense of chronic joint-level decrements. For example, knee joint range of motion and strength does not always return to pre-surgery values following femur lengthening. These joint-level decrements governed by altered muscle force-length characteristics, but it is unestablished how human muscles remodel during surgical limb lengthening. Here, we characterized bilateral human vastus lateralis fascicle force-length profiles following femur lengthening. At 2.2 {+/-} 1.7 years (Avg {+/-} SE) post-initial surgery, the lengthened limb's vastus lateralis fascicles were longer, effectively stiffer, and weaker than the contralateral limb. The lengthened limb's vastus lateralis fascicles were 38 mm longer (Avg) than that of the contralateral limb, even though the lengthened limb's femur was only 31 mm longer (Avg). The onset of vastus lateralis fascicle passive force production began at 22% shorter (Avg) operating lengths in lengthened versus contralateral limb muscles. Additionally, vastus lateralis peak active force production was 34% less (Avg) in the lengthened versus contralateral limbs, and in-series quadriceps tendon stiffness was not stiffer in lengthened limbs. These data suggest that active muscle tissues lengthen more than passive muscle tissues following limb lengthening, effectively creating stiffer muscles. Years after limb lengthening, patient muscles exhibit numerous differences in their force-length properties versus that of natural growth, likely contributing to impaired joint-function.

Chronic pain is frequently accompanied by anxiety and other affective disturbances, but the molecular and cellular changes linking peripheral injury to persistent alterations in brain function remain incompletely defined. The hippocampal extracellular matrix (ECM) regulates synaptic plasticity, cellular signaling, and neuroimmune interactions and may therefore contribute to pain-related central plasticity. We used a tibial fracture/cast immobilization model to examine temporal changes in hippocampal ECM, glial morphology, and affective behavior following peripheral injury. Behavioral testing was performed at 3, 7, and 20 weeks after injury, while high-dimensional morphometric analysis of microglia and astrocytes was performed at 1, 3, 7, and 20 weeks. Hippocampal ECM associated molecular signals were mapped at the chronic stage using decellularization coupled with MALDI-TOF mass spectrometry imaging. Injured mice developed mechanical hypersensitivity and reduced exploration in two anxiety related paradigms during the early and chronic stages, with these behavioral phenotypes no longer detected at 20 weeks. At 7 weeks, injury was associated with changes in hippocampal ECM-associated peptide signals, including reduced signals corresponding to four ECM-associated proteins. Microglia and astrocytes exhibited distinct, time-dependent changes in density and multidimensional morphology, with some alterations persisting after behavioral measures returned toward control levels. In vitro, BV2 microglia exhibited rigidity-dependent changes in morphology, phagocytic behavior, and calcium signaling. Together, these findings define a temporally evolving hippocampal ECM and glial phenotype following peripheral injury and identify matrix biomechanics as a candidate regulator of microglial function.

Current treatment of orthopedic ligament injury relies principally on non-regenerative materials and surgical repair rather than strategies that promote intrinsic tissue regeneration. This study evaluated whether biologic augmentation with XWRAP, a human amniotic membrane allograft, combined with a cryopreserved amniotic-fluid-derived biologic containing micronized human amnion membrane, promotes regeneration of ligament architecture in a rabbit medial collateral ligament (MCL) gap-defect model. Standardized 4-mm mid-substance MCL gap defects were created in New Zealand White rabbits (n=3) and either treated with the amniotic membrane/amniotic-fluid biologic combination or left untreated, with intact contralateral or adjacent MCL serving as native controls; ligaments were evaluated by transmission electron microscopy at 2 and 6 weeks post-procedure, with collagen fibril diameter quantified from calibrated high-magnification micrographs. Treated defects developed significantly larger mean collagen fibril diameters than untreated defects at both time points (75 vs. 50 nm at 2 weeks and 88 vs. 62 nm at 6 weeks; p<0.0001 for each comparison), reaching approximately 90% of native, uninjured ligament fibril diameter (98 nm) by 6 weeks. Treated defects also developed a broader, bimodal fibril-diameter distribution resembling native ligament architecture, whereas untreated defects remained characterized by a narrow, unimodal population of small-diameter fibrils typical of scar-associated repair. These findings indicate that biologic augmentation with XWRAP combined with an amniotic-fluid-derived biologic promotes a regenerative, rather than purely reparative, healing response following ligament injury, progressively restoring collagen fibril architecture toward that of native tissue in this preclinical model.

In the murine osteochondral unit, morphology of chondrocyte lacunae (CCL) within calcified articular cartilage and osteocyte lacunae (OCL) within the underlying subchondral bone reflects local biomechanical conditions that are altered in disease, making reliable discrimination of both lacunar populations important for studying osteochondral physiology and pathology. Histology is generally considered the gold standard for identifying CCL and OCL. However, it is inherently limited to two-dimensional assessment and a relatively small tissue section, restricting comprehensive characterisation of three-dimensional lacunar structure. Synchrotron-radiation micro-computed tomography (SR-microCT) enables volumetric quantification of lacunae; where classification is typically based on the position of each lacuna relative to the cement line separating calcified cartilage from bone, requiring manual delineation of this convoluted interface. Such approaches are labour-intensive, operator-dependent, and rely on access to specialised synchrotron infrastructure that is not widely available. To address these limitations, we present a fully automated, annotation-free pipeline for classifying OCL and CCL in the mouse osteochondral unit based on desktop micro-computed tomography (microCT), independent of cement line identification. The proposed unsupervised workflow requires neither tissue separation nor labelled training data, enabling objective and scalable discrimination of lacunar populations directly from volumetric image data. Seven morphometric and positional features per lacuna were extracted from SR-microCT and desktop microCT datasets and analysed using Uniform Manifold Approximation and Projection (UMAP). Validation of the pipeline outcome on SR-microCT against manual classification across specimens yielded very-high agreement, with Gwet's AC1, the Matthews correlation coefficient, and balanced accuracy each approaching 0.99, in both healthy and post-traumatic osteoarthritis specimens. Such a workflow can be readily adapted for the investigation of osteochondral interface in various other species.

Leisure time physical activity (LTPA) in mid-life may have beneficial effects on risk factors for hip fracture in later life. Conversely, physical activity may increase fracture risk through risk of injury, and the optimal amount of physical activity for reducing hip fracture risk remains unclear. We explored the associations between volume and intensity of LTPA in mid-life, and subsequent risk of hip fracture ascertained from linked health data. This prospective observational study used middle aged adults aged 40- 65 years from UK Biobank. LTPA volume (LTPAv) was derived from self-reported walking, moderate and vigorous activity at baseline (2006- 2010). Cox proportional hazard and Fine-Gray sub-distribution models were used to estimate the association between activity and hip fracture, which was sex-stratified. From 324,284 participants with complete data 53% were female and mean age at baseline was 54.5 (SD 7.3). Across 16 years of follow-up there were 2,674 hip fractures. In terms of LTPA volume, the lowest risk of hip fracture occurred in quintile 3 for women (HR 0.78, 95% CI 0.67-0.91), and men (HR 0.74, 95% CI 0.60- 0.90), relating to approximately one hour/day of leisure time physical activity. When activity intensities were examined separately, the lowest estimated hazards were observed for vigorous activity, at quintile 4 in women (HR 0.65, 95% CI 0.55 - 0.76) and quintile 3 in men (HR 0.64, 95% CI 0.52- 0.79), corresponding to approximately 60- 90 minutes of vigorous activity per week. In conclusion, moderately high volumes of mid-life physical activity, particularly vigorous activity, were related to lower risk of subsequent hip fracture, but these relationships appear to attenuate at higher volumes.

Traumatic brain injury (TBI) is a leading cause of long-term neurological disability, affecting 50-60 million people annually. Cascading secondary injury mechanisms, including oxidative stress and neuroinflammation, impair motor and cognitive function, while current therapies often manage symptoms rather than restore lost neurological function. This study investigated an ultrasound-activated piezoelectric hydrogel scaffold composed of barium titanate nanoparticles (BTNPs), sodium alginate (SA), and polyethylene oxide (PEO). The hypothesis was that TBI flies receiving the scaffold and ultrasound would display a higher climbing assay pass rate than TBI flies receiving no treatment or BTNPs alone. TBI was induced in Drosophila melanogaster with a high-impact trauma (HIT) apparatus. Treatments were applied by opening and resealing the fly head cuticle, followed by ultrasound stimulation. Motor recovery was quantified with a climbing assay across four groups (N=10 flies per group), which measured the percentage of flies that could cross a 5 cm mark within 30 seconds. Assay validity was supported by a statistically significant difference between flies without TBI and those with TBI using Mann-Whitney U testing (p=0.0035). Although TBI treatment groups did not differ significantly (p>0.05), median pass rates increased from injury control (35%) to BTNPs (45%) to scaffold (50%). However, high inter-trial variation, a 45.2% procedural mortality rate, and small sample size limited statistical power. These results suggest that piezoelectric electrospun hydrogel scaffolds may be a pathway for safer and more biocompatible restoration after TBI, but provide insufficient evidence to conclude that the scaffold significantly improved motor recovery.

The pace of fracture repair declines with age, increasing the risk of delayed union and nonunion. Delayed union and nonunion are associated with significant morbidity and even mortality with few effective non-surgical interventions. Here, we identify cathelicidin antimicrobial peptide (CAMP) as an age-dependent regulator of bone repair. Single-cell RNA sequencing and immunohistochemistry demonstrated higher CAMP expression during fracture healing in young compared with aged mice. Global or myeloid lineage-depletion of Camp delayed fracture repair in young mice, whereas Camp depletion in aged mice increased callus bone formation. Heterochronic bone marrow transplantation using the conditional genetic knockout further implicated hematopoietic-lineage derived cells in these effects and demonstrated that myeloid-specific Camp expression is important in the rejuvenation of the pace of fracture healing. CAMP promotes osteogenic differentiation through P2X7 receptor (P2X7R)-dependent signaling, influencing the downstream regulation of NLRP3 expression. NLRP3 expression is also correlated with osteogenesis following Camp modulation in both young and old mice, supporting CAMP-P2X7R-NLRP3 signaling as a regulator of osteogenesis. Together, these findings identify CAMP as a hematopoietic-derived regulator of bone repair whose function is age-dependent, promoting fracture healing in young animals while constraining it in aged animals.

Objective Single-cell RNA sequencing has transformed our understanding of cellular heterogeneity within osteoarthritic (OA) joint tissues, yet transcript abundance provides only an indirect measure of cellular phenotype and function. Recent advances in mass spectrometry now enable proteins to be measured directly at single-cell resolution. Here, we applied single-cell proteomics (SCP) to primary human OA cartilage and synovium to determine whether cellular heterogeneity within the diseased joint can be resolved directly at the proteome level. Design Cartilage and synovium were obtained from patients undergoing total knee arthroplasty for OA. Low-input proteomic profiling was first performed to establish proteome depth from limited cell numbers and validate biologically relevant clustering. SCP was then applied to individual cells, followed by unsupervised clustering, differential protein abundance analysis and iterative biological annotation. Results Low-input proteomics identified 7,800 proteins from 1,000 cells while retaining tissue- and cell-associated biological differences. Across the SCP dataset, ~2,300 unique proteins were detected, resolving chondrocyte, synovial fibroblast and immune-cell populations that closely paralleled those previously defined by single-cell transcriptomics. Higher-resolution analysis distinguished lining and sublining fibroblasts, discrete immune populations and multiple protein-defined chondrocyte states. Conclusions SCP enables direct characterisation of cellular heterogeneity within the human OA joint at the level of the proteome. By independently recovering established cellular populations while revealing protein-defined chondrocyte states, our findings establish SCP as a powerful complement to single-cell transcriptomics and open a new route to investigating functional cellular states and disease mechanisms that cannot be inferred from RNA abundance alone.

Mechanical stability critically influences bone fracture healing, yet how the early mechanical environment directs the transition from inflammation to regeneration remains unclear. Using a murine femoral osteotomy model, we compared rigid, semirigid, and dynamically adjusted semirigid-to-rigid fixation. Semirigid fixation delayed healing on day 21, whereas a strategy of early compliance followed by increased stiffness after 7 days restored bridging and improved bone regeneration beyond constant rigid fixation, pointing to a critical early mechanosensitive window after fracture. Single-cell RNA sequencing across the first week, comparing semirigid with rigid fixation, showed that fixation stiffness altered intercellular signaling within one day of injury. Signaling among myeloid populations was more broadly increased under semirigid fixation, with monocytes the single exception, and signaling associated with resolution of inflammation was reduced. From day 5, periosteal and skeletal progenitor populations increasingly directed cartilage-associated matrix and growth factor signaling toward macrophages and chondrocytes, accompanied by increased Sox9 regulon activity and increased COL10+ hypertrophic cartilage matrix by day 7. Reduced fixation stiffness therefore promotes a chondrogenic trajectory of repair, but increasing stiffness is required to redirect this response toward bone formation. Piezo1 was expressed across the responding populations and shifted between compartments as healing progressed. PIEZO1 activation with Yoda1 under rigid fixation enhanced bone formation but provided no additional benefit under semirigid or dynamically adjusted fixation, whereas inhibition of mechanosensitive signaling with GsMTx4 impaired repair across all conditions. Together, these findings establish the early mechanical environment as a determinant of fracture-healing trajectory, shaping the transition from inflammation to regeneration, and show that successful repair depends not on maximal stability but on when stiffness is applied. Increasing either early mechanical stimulation or cellular mechanosensitivity enhanced regeneration, supporting mechanosensitive signaling as a key component of the early healing response.

Seroma is a common complication following abdominoplasty and other procedures involving extensive soft tissue dissection. Although animal seroma models have been reported, the stromal pathology underlying post-abdominoplasty seroma, particularly the role of oxidative stress, remains poorly characterized. Here, we developed a rat abdominal seroma model by surgically separating the subcutaneous tissue from the underlying muscle fascia and investigated the temporal evolution of the associated stromal response. Seroma developed in 9 of 20 operated rats, corresponding to a formation rate of 45.0%. Longitudinal ultrasound demonstrated that fluid accumulation peaked around postoperative day (POD) 7 and largely resolved by POD30. Despite macroscopic fluid resolution, substantial stromal remodeling persisted. Compared with normal abdominal tissue, seroma-associated tissues at POD14 and POD35 exhibited increased collagen deposition, macrophage accumulation, oxidative injury, and vascular remodeling. Additionally, at POD35, macrophage accumulation and 4-hydroxynonenal staining were greater than at POD14, whereas collagen deposition and vascular density remained elevated with no significant differences between postoperative time points. To our knowledge, this study presents the first rat model specifically designed to reproduce post-abdominoplasty seroma and the first temporal characterization of oxidative stress within seroma-associated tissue. These findings reveal that seroma fluid resolution does not coincide with stromal recovery and identify persistent oxidative stress, inflammation, fibrosis, and vascular remodeling as important features of seroma pathology.