Carmen Huesa

Micro-computed tomography (µCT) is widely used to assess trabecular bone microarchitecture, with trabecular separation (Tb.Sp) among the core parameters recommended for reporting. Tb.Sp is typically expressed as a single volume-weighted mean derived from maximal sphere fitting, although the underlying distribution of local separation values is rarely examined. Here, we show that Tb.Sp distributions in metaphyseal trabecular bone are frequently non-Gaussian and bimodal or multimodal. Using µCT datasets from three established models of osteoporosis, spinal cord injury (SCI), ovariectomy (OVX), and ageing, we demonstrate that this behaviour is most evident in metaphyseal trabecular bone and is less apparent in epiphyseal trabecular bone or trabecular thickness distributions. We further show that multimodal metaphyseal Tb.Sp distributions correspond to two spatially distinct contributions within the marrow space: lower-diameter local separation within the residual trabecular network, and higher-diameter regions associated with larger contiguous marrow cavities. Based on this observation, we introduce a simple extension to standard morphometric analysis in which Tb.Sp is decomposed into local trabecular separation (Tb.Sp _L ) and marrow cavity separation (Tb.Sp _M ). Tb.Sp decomposition revealed model-specific patterns of trabecular deterioration. SCI was characterised predominantly by increased Tb.Sp _M , consistent with expansion of larger marrow cavities, whereas OVX showed a more subtle or distributed alteration. Ageing showed changes in both Tb.Sp _L and Tb.Sp _M , with the higher-diameter component becoming most prominent in older animals. Together, these findings demonstrate that mean Tb.Sp can mask structurally distinct forms of metaphyseal marrow-space organisation and support reporting distributional descriptors, and where appropriate Tb.Sp _L and Tb.Sp _M , alongside conventional Tb.Sp.

Enhancing osteogenesis in mesenchymal stromal (stem) cells is essential for advancing cellular therapies that target and alleviate skeletal pathologies. By employing a bespoke bioreactor capable of delivering nano-amplitude vibration (30 nm, 1kHz) to human adipose-derived mesenchymal stromal cells, we observed osteo-specific differentiation. We related this to the mechanotransductive mechanism by showing that inhibition of intracellular tension results in the loss of cytoskeletal organisation and myosin activation driven by nanovibration. Further, we dissect the mechanism of osteogenesis using a panel of Wnt agonists and antagonists and highlight the role of non-canonical Wnt. Then, using Bcl3 ^-/- cells and by stimulating with BCL3 peptide, we show that non-canonical Wnt, osteogenesis-related inflammation and osteogenesis itself are all regulated by BCL3. This is important as nanoscale direct cell-stimulation is gaining interest, and there is an emerging consensus that such signals can be osteogenic. While prior research has only broadly hinted at how nanovibrational signals convert to an osteogenic phenotype, this new work pinpoints critical mechanistic insights, thereby advancing our understanding of this promising avenue in musculoskeletal cell therapy.

Protease-activated receptor 2 (PAR2) plays a pivotal role in the early stages of surgery-induced murine osteoarthritis OA. It remains to be determined however, whether PAR2 contributes to later stages of disease pathology and which cellular compartments drive pathological changes. Thus, we characterised OA pathology in global, chondrocyte- or osteoblast-specific PAR2 knockout mice up to 12 months after OA induction. While wild-type mice display a gradual increase in cartilage damage/loss, PAR2 knockout mice had significantly reduced cartilage pathology. Notably, removing PAR2 specifically in osteoblasts, but not in chondrocytes, substantially improved cartilage health. Interrogation of the osteoblast compartment revealed that PAR2 has a divergent role during osteoblast development and maturation compared to its function in already differentiated cells. This suggests that PAR2 expression in the bone compartment promotes joint deterioration in later stages of OA, highlighting the important role of bone in OA and the therapeutic potential of targeting PAR2.

Objective: IĸB protein B cell lymphoma 3‐encoded protein (BCL3) is a regulator of the NF‐κB family of transcription factors. NF‐κB signaling fundamentally influences the fate of bone‐forming osteoblasts and bone‐resorbing osteoclasts, but the role of BCL3 in bone biology has not been investigated. The objective of this study was to evaluate BCL3 in skeletal development, maintenance, and osteoarthritic pathology.Methods: To assess the contribution of BCL3 to skeletal homeostasis, neonatal mice (n = 6–14) lacking BCL3 (Bcl3^−/−) and wild‐type (WT) controls were characterized for bone phenotype and density. To reveal the contribution to bone phenotype by the osteoblast compartment in Bcl3^−/− mice, transcriptomic analysis of early osteogenic differentiation and cellular function (n = 3–7) were assessed. Osteoclast differentiation and function in Bcl3^−/− mice (n = 3–5) was assessed. Adult 20‐week Bcl3^−/− and WT mice bone phenotype, strength, and turnover were assessed. A destabilization of the medial meniscus model of osteoarthritic osteophytogenesis was used to understand adult bone formation in Bcl3^−/− mice (n = 11–13).Results: Evaluation of Bcl3^−/− mice revealed congenitally increased bone density, long bone dwarfism, increased bone biomechanical strength, and altered bone turnover. Molecular and cellular characterization of mesenchymal precursors showed that Bcl3^−/− cells displayed an accelerated osteogenic transcriptional profile that led to enhanced differentiation into osteoblasts with increased functional activity, which could be reversed with a mimetic peptide. In a model of osteoarthritis‐induced osteophytogenesis, Bcl3^−/− mice exhibited decreased pathological osteophyte formation (P < 0.05).Conclusion: Cumulatively, these findings demonstrate that BCL3 controls developmental mineralization to enable appropriate bone formation, whereas in a pathological setting, it contributes to skeletal pathology. image

Osteoarthritis is the most prevalent systemic musculoskeletal disorder characterised by articular cartilage degeneration and subchondral bone (SCB) sclerosis. Here we sought to examine the contribution of accelerated growth to osteoarthritis development using a murine model of excessive longitudinal growth. Suppressor of cytokine signalling 2 (SOCS2) is a negative regulator of growth hormone (GH) signalling, thus mice deficient in SOCS2 ( Socs2 ^-/- ) display accelerated bone growth. We examined vulnerability of Socs2 ^-/- mice to osteoarthritis following surgical induction of disease (destabilisation of the medial meniscus (DMM)), and with ageing, by histology and micro-CT. We observed significant increase in number (WT DMM: 532±56; WT sham: 495±45; KO DMM: 169±49; KO sham: 187±56; PSocs2 ^-/- in comparison to wild-type (WT). Histological examination of WT and Socs2 ^-/- knees revealed articular cartilage damage with DMM in comparison to sham (WT DMM: 3.4±0.4; WT sham: 0.3±0.05 (PSocs2 ^-/- mice with either DMM, or with ageing. Micro-CT analysis revealed significant decreases in SCB thickness, epiphyseal trabecular number and thickness in the medial compartment of Socs2 ^-/- , in comparison to WT (P<0.001). DMM had no effect on the SCB thickness in comparison to sham in either genotype. Together these data suggest that enhanced GH signalling through SOCS2 deletion accelerates growth plate fusion, however this has no effect on osteoarthritis vulnerability in this model. Summary statement: Deletion of SOCS2 results in accelerated growth plate fusion, however this has no effect on osteoarthritis vulnerability.

Periodontitis is the inflammation-associated bone loss disease of the alveolar bone that surrounds teeth. Classically, the emphasis on the etiology of periodontitis has been on the products of periodontal pathogens that lead to an inflammatory response of the soft tissues of the periodontium, eventually leading to activation of osteoclasts that degrade the alveolar bone. Until recently, the response of osteocytes that populate the alveolar bone, and that are known for their regulatory role in bone anabolism and catabolism, has not been addressed.

This chapter describes the surgical procedure for destabilization of medial meniscus in mice. Details on subsequent microCT and histological analysis are also provided, as well as details on osteoarthritis evaluation.

Protease-activated receptor-2 (PAR2) is one member of a small family of transmembrane, G-protein-coupled receptors. These receptors are activated via cleavage of their N terminus by serine proteases (e.g., tryptase), unveiling an N terminus tethered ligand which binds to the second extracellular loop of the receptor. Increasing evidence has emerged identifying key pathophysiological roles for PAR2 in both rheumatoid arthritis (RA) and osteoarthritis (OA). Importantly, this includes both pro-inflammatory and destructive roles. For example, in murine models of RA, the associated synovitis, cartilage degradation, and subsequent bone erosion are all significantly reduced in the absence of PAR2. Similarly, in experimental models of OA, PAR2 disruption confers protection against cartilage degradation, subchondral bone osteosclerosis, and osteophyte formation. This review focuses on the role of PAR2 in rheumatic disease and its potential as an important therapeutic target for treating pain and joint degradation.