Autoimmunity to stromal-derived autoantigens in rheumatoid ectopic germinal centers exacerbates arthritis and affects clinical response.

Ectopic lymphoid structures (ELSs) in the rheumatoid synovial joints sustain autoreactivity against locally expressed autoantigens. We recently identified recombinant monoclonal antibodies (RA-rmAbs) derived from single, locally differentiated rheumatoid arthritis (RA) synovial B cells, which specifically recognize fibroblast-like synoviocytes (FLSs). Here, we aimed to identify the specificity of FLS-derived autoantigens fueling local autoimmunity and the functional role of anti-FLS antibodies in promoting chronic inflammation. A subset of anti-FLS RA-rmAbs reacting with a 60 kDa band from FLS extracts demonstrated specificity for HSP60 and partial cross-reactivity to other stromal autoantigens (i.e., calreticulin/vimentin) but not to citrullinated fibrinogen. Anti-FLS RA-rmAbs, but not anti-neutrophil extracellular traps rmAbs, exhibited pathogenic properties in a mouse model of collagen-induced arthritis. In patients, anti-HSP60 antibodies were preferentially detected in RA versus osteoarthritis (OA) synovial fluid. Synovial HSPD1 and CALR gene expression analyzed using bulk RNA-Seq and GeoMx-DSP closely correlated with the lympho-myeloid RA pathotype, and HSP60 protein expression was predominantly observed around ELS. Moreover, we observed a significant reduction in synovial HSP60 gene expression followed B cell depletion with rituximab that was strongly associated with the treatment response. Overall, we report that synovial stromal-derived autoantigens are targeted by pathogenic autoantibodies and are associated with specific RA pathotypes, with potential value for patient stratification and as predictors of the response to B cell-depleting therapies.

Stable Sulforaphane Targets the Early Stages of Osteoclast Formation to Engender a Lasting Functional Blockade of Osteoclastogenesis.

Sulforaphane, the native but unstable form of SFX-01, is an antioxidant that activates the NRF2 and inhibits the NF-KB pathways to achieve its actions. Resolving the mechanism(s) by which SFX-01 serves to control the various osteoclastogenic stages may expose pathways that could be explored for therapeutic use. Here we seek to identify the stage of osteoclastogenesis targeted by SFX-01 and explore whether, like SFN, it exerts its actions via the NRF2 and NF-KB pathways. Osteoclasts generated from the bone marrow (BM) of mice were cultured with SFX-01 at different timepoints to examine each phase of osteoclastogenesis separately. This showed that SFX-01 exerted actions throughout the process of osteoclastogenesis, but had its largest effects in the early osteoclast precursor differentiation stage. Thus, treatment with SFX-01 for the duration of culture, for the initial 3 days differentiation or for as little as the first 24 h was sufficient for effective inhibition. This aligned with data suggesting that SFX-01 reduced DC-STAMP levels, osteoclast nuclear number and modified cytoskeletal architecture. Pharmacological regulation of the NRF2 pathways, via selective inhibitors/activators, supported the anti-osteoclastogenic roles of an SFX-01-mediated by NRF2 activation, as well as the need for tight NF-KB pathway regulation in osteoclast formation/function.

Ablation of collagen XII disturbs joint extracellular matrix organization and causes patellar subluxation.

Collagen XII, belonging to the fibril-associated collagens, is a homotrimeric secreted extracellular matrix (ECM) protein encoded by the COL12A1 gene. Mutations in the human COL12A1 gene cause an Ehlers-Danlos/myopathy overlap syndrome leading to skeletal abnormalities and muscle weakness. Here, we studied the role of collagen XII in joint pathophysiology by analyzing collagen XII deficient mice and human patients. We found that collagen XII is widely expressed across multiple connective tissue of the developing joint. Lack of collagen XII in mice destabilizes tendons and the femoral trochlear groove to induce patellar subluxation in the patellofemoral joint. These changes are associated with an ECM damage response in tendon and secondary quadriceps muscle degeneration. Moreover, patellar subluxation was also identified as a clinical feature of human patients with collagen XII deficiency. The results provide an explanation for joint hyperlaxity in mice and human patients with collagen XII deficiency.

Transcriptomics for Clinical and Experimental Biology Research: Hang on a Seq.

Sequencing the human genome empowers translational medicine, facilitating transcriptome-wide molecular diagnosis, pathway biology, and drug repositioning. Initially, microarrays are used to study the bulk transcriptome; but now short-read RNA sequencing (RNA-seq) predominates. Positioned as a superior technology, that makes the discovery of novel transcripts routine, most RNA-seq analyses are in fact modeled on the known transcriptome. Limitations of the RNA-seq methodology have emerged, while the design of, and the analysis strategies applied to, arrays have matured. An equitable comparison between these technologies is provided, highlighting advantages that modern arrays hold over RNA-seq. Array protocols more accurately quantify constitutively expressed protein coding genes across tissue replicates, and are more reliable for studying lower expressed genes. Arrays reveal long noncoding RNAs (lncRNA) are neither sparsely nor lower expressed than protein coding genes. Heterogeneous coverage of constitutively expressed genes observed with RNA-seq, undermines the validity and reproducibility of pathway analyses. The factors driving these observations, many of which are relevant to long-read or single-cell sequencing are discussed. As proposed herein, a reappreciation of bulk transcriptomic methods is required, including wider use of the modern high-density array data-to urgently revise existing anatomical RNA reference atlases and assist with more accurate study of lncRNAs.

A non-coding insertional mutation of Grhl2 causes gene over-expression and multiple structural anomalies including cleft palate, spina bifida and encephalocele.

Orofacial clefts, including cleft lip and palate (CL/P) and neural tube defects (NTDs) are among the most common congenital anomalies, but knowledge of the genetic basis of these conditions remains incomplete. The extent to which genetic risk factors are shared between CL/P, NTDs and related anomalies is also unclear. While identification of causative genes has largely focused on coding and loss of function mutations, it is hypothesized that regulatory mutations account for a portion of the unidentified heritability. We found that excess expression of Grainyhead-like 2 (Grhl2) causes not only spinal NTDs in Axial defects (Axd) mice but also multiple additional defects affecting the cranial region. These include orofacial clefts comprising midline cleft lip and palate and abnormalities of the craniofacial bones and frontal and/or basal encephalocele, in which brain tissue herniates through the cranium or into the nasal cavity. To investigate the causative mutation in the Grhl2Axd strain, whole genome sequencing identified an approximately 4 kb LTR retrotransposon insertion that disrupts the non-coding regulatory region, lying approximately 300 base pairs upstream of the 5' UTR. This insertion also lies within a predicted long non-coding RNA, oriented on the reverse strand, which like Grhl2 is over-expressed in Axd (Grhl2Axd) homozygous mutant embryos. Initial analysis of the GRHL2 upstream region in individuals with NTDs or cleft palate revealed rare or novel variants in a small number of cases. We hypothesize that mutations affecting the regulation of GRHL2 may contribute to craniofacial anomalies and NTDs in humans.

Bone marrow lesions: plugging the holes in our knowledge using animal models.

Bone marrow lesions (BMLs), which are early signs of osteoarthritis (OA) that are associated with the presence, onset and severity of pain, represent an emerging imaging biomarker and clinical target. Little is known, however, regarding their early spatial and temporal development, structural relationships or aetiopathogenesis, because of the sparsity of human early OA imaging and paucity of relevant tissue samples. The use of animal models is a logical approach to fill the gaps in our knowledge, and it can be informed by appraising models in which BMLs and closely related subchondral cysts have already been reported, including in spontaneous OA and pain models. The utility of these models in OA research, their relevance to clinical BMLs and practical considerations for their optimal deployment can also inform medical and veterinary clinicians and researchers alike.

3D profiling of mouse epiphyses across ages reveals new potential imaging biomarkers of early spontaneous osteoarthritis.

Worldwide research groups and funding bodies have highlighted the need for imaging biomarkers to predict osteoarthritis (OA) progression and treatment effectiveness. Changes in trabecular architecture, which can be detected with non-destructive high-resolution CT imaging, may reveal OA progression before apparent articular surface damage. Here, we analysed the tibial epiphyses of STR/Ort (OA-prone) and CBA (healthy, parental control) mice at different ages to characterise the effects of mouse age and strain on multiple bony parameters. We isolated epiphyseal components using a semi-automated method, and measured the total epiphyseal volume; cortical bone, trabecular bone and marrow space volumes; mean trabecular and cortical bone thicknesses; trabecular volume relative to cortical volume; trabecular volume relative to epiphyseal interior (trabecular BV/TV); and the trabecular degree of anisotropy. Using two-way ANOVA (significance level ≤0.05), we confirmed that all of these parameters change significantly with age, and that the two strains were significantly different in cortical and trabecular bone volumes, and trabecular degree of anisotropy. STR/Ort mice had higher cortical and trabecular volumes and a lower degree of anisotropy. As the two mouse strains reflect markedly divergent OA predispositions, these parameters have potential as bioimaging markers to monitor OA susceptibility and progression. Additionally, significant age/strain interaction effects were identified for total epiphyseal volume, marrow space volume and trabecular BV/TV. These interactions confirm that the two mouse strains have different epiphyseal growth patterns throughout life, some of which emerge prior to OA onset. Our findings not only propose valuable imaging biomarkers of OA, but also provide insight into ageing 3D epiphyseal architecture bone profiles and skeletal biology underlying the onset and development of age-related OA in STR/Ort mice.

Sexing Bones: Improving Transparency of Sex Reporting to Address Bias Within Preclinical Studies.

Despite knowledge that sexually dimorphic mechanisms regulate bone homeostasis, sex often remains unreported and unconsidered in preclinical experimental design. Failure to report sex could lead to inappropriate generalizations of research findings and less effective translation into clinical practice. Preclinical sex bias (preferential selection of one sex) is present across other fields, including neuroscience and immunology, but remains uninvestigated in skeletal research. For context, we first summarized key literature describing sexually dimorphic bone phenotypes in mice. We then investigated sex reporting practices in skeletal research, specifically how customary it is for murine sex to be included in journal article titles or abstracts and then determined whether any bias in sex reporting exists. Because sex hormones are important regulators of bone health (gonadectomy procedures, ie, ovariectomy [OVX] and orchidectomy [ORX], are common yet typically not reported with sex), we incorporated reporting of OVX and ORX terms, representing female and male mice, respectively, into our investigations around sex bias. Between 1999 and 2020, inclusion of sex in titles or abstracts was low in murine skeletal studies (2.6%-4.06%). Reporting of OVX and ORX terms was low (1.44%-2.64%) and reporting of OVX and ORX with sex uncommon (0.4%-0.3%). When studies were combined to include both sexes and OVX (representing female) and ORX terms (representing male), a bias toward reporting of female mice was evident. However, when the terms OVX and ORX were removed, a bias toward the use of male mice was identified. Thus, studies focusing on sex hormones are biased toward female reporting with all other studies biased in reporting of male mice. We now call upon journal editors to introduce consistent guidance for transparent and accessible reporting of murine sex in skeletal research to better monitor preclinical sex bias, to diversify development of treatments for bone health, and to enable global skeletal health equity. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Increased PHOSPHO1 and alkaline phosphatase expression during the anabolic bone response to intermittent parathyroid hormone delivery.

The administration of intermittent parathyroid hormone (iPTH) is anabolic to the skeleton. Recent studies with cultured osteoblasts have revealed that the expression of PHOSPHO1, a bone-specific phosphatase essential for the initiation of mineralisation, is regulated by PTH. Therefore, this study sought to determine whether the bone anabolic response to iPTH involves modulation of expression of Phospho1 and of other enzymes critical for bone matrix mineralisation. To mimic iPTH treatment, primary murine osteoblasts were challenged with 50 nM PTH for 6 h in every 48 h period for 8 days (4 cycles), 14 days (7 cycles) and 20 days (10 cycles) in total. The expression of both Phospho1 and Smpd3 was almost completely inhibited after 4 cycles, whereas 10 cycles were required to stimulate a similar response in Alpl expression. To explore the in vivo role of PHOSPHO1 in PTH-mediated osteogenesis, the effects of 14- and 28-day iPTH (80 µg/kg/day) administration was assessed in male wild-type (WT) and Phospho1-/- mice. The expression of Phospho1, Alpl, Smpd3, Enpp1, Runx2 and Trps1 expression was enhanced in the femora of WT mice following iPTH administration but remained unchanged in the femora of Phospho1-/- mice. After 28 days of iPTH administration, the anabolic response in the femora of WT was greater than that noted in Phospho1-/- mice. Specifically, cortical and trabecular bone volume/total volume, as well as cortical thickness, were increased in femora of iPTH-treated WT but not in iPTH-treated Phospho1-/- mice. Trabecular bone osteoblast number was also increased in iPTH-treated WT mice but not in iPTH-treated Phospho1-/-  mice. The increased levels of Phospho1, Alpl, Enpp1 and Smpd3 in WT mice in response to iPTH administration is consistent with their contribution to the potent anabolic properties of iPTH in bone. Furthermore, as the anabolic response to iPTH was attenuated in mice deficient in PHOSPHO1, this suggests that the osteoanabolic effects of iPTH are at least partly mediated via bone mineralisation processes.

A new method for segmentation and analysis of bone callus in rodent fracture models using micro-CT.

Fracture burden has created a need to better understand bone repair processes under different pathophysiological states. Evaluation of structural and material properties of the mineralized callus, which is integral to restoring biomechanical stability is, therefore, vital. Microcomputed tomography (micro-CT) can facilitate noninvasive imaging of fracture repair, however, current methods for callus segmentation are only semiautomated, restricted to defined regions, time/labor intensive, and prone to user variation. Herein, we share a new automatic method for segmenting callus in micro-CT tomograms that will allow for objective, quantitative analysis of the bone fracture microarchitecture. Fractured and nonfractured mouse femurs were scanned and processed by both manual and automated segmentation of fracture callus from cortical bone after which microarchitectural parameters were analyzed. All segmentation and analysis steps were performed using CTAn (Bruker) with automatic segmentation performed using the software's image-processing plugins. Results showed automatic segmentation reliably and consistently segmented callus from cortical bone, demonstrating good agreement with manual methods with low bias: tissue volume (TV): -0.320 mm3 , bone volume (BV): 0.0358 mm3 , and bone volume/tissue volume (BV/TV): -3.52%, and was faster and eliminated user-bias and variation. Method scalability and translatability across rodent models were verified in scans of fractured rat femora showing good agreement with manual methods with low bias: TV: -3.654 mm3 , BV: 0.830 mm3 , and BV/TV: 7.81%. Together, these data validate a new automated method for segmentation of callus and cortical bone in micro-CT tomograms that we share as a fast, reliable, and less user-dependent tool for application to study bone callus in fracture, and potentially elsewhere.

Biofabrication of the osteochondral unit and its applications: Current and future directions for 3D bioprinting.

Multiple prevalent diseases, such as osteoarthritis (OA), for which there is no cure or full understanding, affect the osteochondral unit; a complex interface tissue whose architecture, mechanical nature and physiological characteristics are still yet to be successfully reproduced in vitro. Although there have been multiple tissue engineering-based approaches to recapitulate the three dimensional (3D) structural complexity of the osteochondral unit, there are various aspects that still need to be improved. This review presents the different pre-requisites necessary to develop a human osteochondral unit construct and focuses on 3D bioprinting as a promising manufacturing technique. Examples of 3D bioprinted osteochondral tissues are reviewed, focusing on the most used bioinks, chosen cell types and growth factors. Further information regarding the applications of these 3D bioprinted tissues in the fields of disease modelling, drug testing and implantation is presented. Finally, special attention is given to the limitations that currently hold back these 3D bioprinted tissues from being used as models to investigate diseases such as OA. Information regarding improvements needed in bioink development, bioreactor use, vascularisation and inclusion of additional tissues to further complete an OA disease model, are presented. Overall, this review gives an overview of the evolution in 3D bioprinting of the osteochondral unit and its applications, as well as further illustrating limitations and improvements that could be performed explicitly for disease modelling.

High bone mass in mice can be linked to lower osteoclast formation, resorptive capacity, and restricted in vitro sensitivity to inhibition by stable sulforaphane.

Mouse strains can have divergent basal bone mass, yet this phenotype is seldom reflected in the design of studies seeking to identify new modulators of bone resorption by osteoclasts. Sulforaphane exerts inhibitory effects on in vitro osteoclastogenesis in cells from C57BL/6 mice. Here, we explore whether a divergent basal bone mass in different mouse strains is linked both to in vitro osteoclastogenic potential and to SFX-01 sensitivity. Accordingly, osteoclasts isolated from the bone marrow (BM) of C57BL/6, STR/Ort and CBA mice with low, high, and intermediate bone mass, respectively, were cultured under conditions to promote osteoclast differentiation and resorption; they were also treated with chemically stabilised sulforaphane (SFX-01) and respective sensitivity to inhibition evaluated by counting osteoclast number/resorption activity on dentine discs. We observed that osteoclastogenesis exhibited different macrophage colony-stimulating factor/receptor activator of nuclear factor kappa-Β ligand sensitivity in these mouse strains, with cells from C57BL/6 and CBA generating higher osteoclast numbers than STR/Ort; the latter formed only half as many mature osteoclasts. We found that 100 nM SFX-01 exerted a potent and significant reduction in osteoclast number and resorptive activity in cells derived from C57BL/6 mice. In contrast, 10-fold higher SFX-01 concentrations were required for similar inhibition in CBA-derived cells and, strikingly, a further 2.5-fold greater concentration was required for significant restriction of osteoclast formation/function in STR/Ort. These data are consistent with the notion that the BM osteoclast precursor population contributes to the relative differences in mouse bone mass and that mice with higher bone mass exhibit lower in vitro osteoclastogenic potential as well as reduced sensitivity to inhibition by SFX-01.

Structural clues to articular calcified cartilage function: A descriptive review of this crucial interface tissue.

Articular calcified cartilage (ACC) has been dismissed, by some, as a remnant of endochondral ossification without functional relevance to joint articulation or weight-bearing. Recent research indicates that morphologic and metabolic ACC features may be important, reflecting knee joint osteoarthritis (OA) predisposition. ACC is less investigated than neighbouring joint tissues, with its component chondrocytes and mineralised matrix often being either ignored or integrated into analyses of hyaline articular cartilage and subchondral bone tissue respectively. Anatomical variation in ACC is recognised between species, individuals and age groups, but the selective pressures underlying this variation are unknown. Consequently, optimal ACC biomechanical features are also unknown as are any potential locomotory roles. This review collates descriptions of ACC anatomy and biology in health and disease, with a view to revealing its structure/function relationship and highlighting potential future research avenues. Mouse models of healthy and OA joint ageing have shown disparities in ACC load-induced deformations at the knee joint. This raises the hypothesis that ACC response to locomotor forces over time may influence, or even underlie, the bony and hyaline cartilage symptoms characteristic of OA. To effectively investigate the ACC, greater resolution of joint imaging and merging of hierarchical scale data will be required. An appreciation of OA as a 'whole joint disease' is expanding, as is the possibility that the ACC may be a key player in healthy ageing and in the transition to OA joint pathology.

Increased PHOSPHO1 expression mediates cortical bone mineral density in renal osteodystrophy.

Patients with advanced chronic kidney disease (CKD) often present with skeletal abnormalities, a condition known as renal osteodystrophy (ROD). While tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1 are critical for bone mineralization, their role in the etiology of ROD is unclear. To address this, ROD was induced in both WT and Phospho1 knockout (P1KO) mice through dietary adenine supplementation. The mice presented with hyperphosphatemia, hyperparathyroidism, and elevated levels of FGF23 and bone turnover markers. In particular, we noted that in CKD mice, bone mineral density (BMD) was increased in cortical bone (P < 0.05) but decreased in trabecular bone (P < 0.05). These changes were accompanied by decreased TNAP (P < 0.01) and increased PHOSPHO1 (P < 0.001) expression in WT CKD bones. In P1KO CKD mice, the cortical BMD phenotype was rescued, suggesting that the increased cortical BMD of CKD mice was driven by increased PHOSPHO1 expression. Other structural parameters were also improved in P1KO CKD mice. We further investigated the driver of the mineralization defects, by studying the effects of FGF23, PTH, and phosphate administration on PHOSPHO1 and TNAP expression by primary murine osteoblasts. We found both PHOSPHO1 and TNAP expressions to be downregulated in response to phosphate and PTH. The in vitro data suggest that the TNAP reduction in CKD-MBD is driven by the hyperphosphatemia and/or hyperparathyroidism noted in these mice, while the higher PHOSPHO1 expression may be a compensatory mechanism. Increased PHOSPHO1 expression in ROD may contribute to the disordered skeletal mineralization characteristic of this progressive disorder.

Spatial links between subchondral bone architectural features and cartilage degeneration in osteoarthritic joints.

Early diagnosis of osteoarthritis (OA), before the onset of irreversible changes is crucial for understanding the disease process and identifying potential disease-modifying treatments from the earliest stage. OA is a whole joint disease and affects both cartilage and the underlying subchondral bone. However, spatial relationships between cartilage lesion severity (CLS) and microstructural changes in subchondral plate and trabecular bone remain elusive. Herein, we collected femoral heads from hip arthroplasty for primary osteoarthritis (n = 7) and femoral neck fracture (n = 6; non-OA controls) cases. Samples were regionally assessed for cartilage lesions by visual inspection using Outerbridge classification and entire femoral heads were micro-CT scanned. Scans of each femoral head were divided into 4 quadrants followed by morphometric analysis of subchondral plate and trabecular bone in each quadrant. Principal component analysis (PCA), a data reduction method, was employed to assess differences between OA and non-OA samples, and spatial relationship between CLS and subchondral bone changes. Mapping of the trabecular bone microstructure in OA patients with low CLS revealed trabecular organisation resembling non-OA patients, whereas clear differences were identifiable in subchondral plate architecture. The OA-related changes in subchondral plate architecture were summarised in the first principle component (PC1) which correlated with CLS in all quadrants, whilst by comparison such associations in trabecular bone were most prominent in the higher weight-bearing regions of the femoral head. Greater articular cartilage deterioration in OA was regionally-linked with lower BV/TV, TMD and thickness, and greater BS/BV and porosity in the subchondral plate; and with thinner, less separated trabeculae with greater TMD and BS/BV in the trabecular bone. Our findings suggest that impairment of subchondral bone microstructure in early stage of OA is more readily discernible in the cortical plate and that morphological characterisation of the femoral head bone microstructure may allow for earlier OA diagnosis and monitoring of progression.

Moving Beyond the Limits of Detection: The Past, the Present, and the Future of Diagnostic Imaging in Canine Osteoarthritis.

Osteoarthritis (OA) is the most common orthopedic condition in dogs, characterized as the chronic, painful end-point of a synovial joint with limited therapeutic options other than palliative pain control or surgical salvage. Since the 1970s, radiography has been the standard-of-care for the imaging diagnosis of OA, despite its known limitations. As newer technologies have been developed, the limits of detection have lowered, allowing for the identification of earlier stages of OA. Identification of OA at a stage where it is potentially reversible still remains elusive, however, yet there is hope that newer technologies may be able to close this gap. In this article, we review the changes in the imaging of canine OA over the past 50 years and give a speculative view on future innovations which may provide for earlier identification, with the ultimate goal of repositioning the limit of detection to cross the threshold of this potentially reversible disease.

The tendon interfascicular basement membrane provides a vascular niche for CD146+ cell subpopulations.

Introduction: The interfascicular matrix (IFM; also known as the endotenon) is critical to the mechanical adaptations and response to load in energy-storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT). We hypothesized that the IFM is a tendon progenitor cell niche housing an exclusive cell subpopulation. Methods: Immunolabelling of equine superficial digital flexor tendon was used to identify the interfascicular matrix niche, localising expression patterns of CD31 (endothelial cells), Desmin (smooth muscle cells and pericytes), CD146 (interfascicular matrix cells) and LAMA4 (interfascicular matrix basement membrane marker). Magnetic-activated cell sorting was employed to isolate and compare in vitro properties of CD146+ and CD146- subpopulations. Results: Labelling for CD146 using standard histological and 3D imaging of large intact 3D segments revealed an exclusive interfascicular cell subpopulation that resides in proximity to a basal lamina which forms extensive, interconnected vascular networks. Isolated CD146+ cells exhibited limited mineralisation (osteogenesis) and lipid production (adipogenesis). Discussion: This study demonstrates that the interfascicular matrix is a unique tendon cell niche, containing a vascular-rich network of basement membrane, CD31+ endothelial cells, Desmin+ mural cells, and CD146+ cell populations that are likely essential to tendon structure and/or function. Contrary to our hypothesis, interfascicular CD146+ subpopulations did not exhibit stem cell-like phenotypes. Instead, our results indicate CD146 as a pan-vascular marker within the tendon interfascicular matrix. Together with previous work demonstrating that endogenous tendon CD146+ cells migrate to sites of injury, our data suggest that their mobilisation to promote intrinsic repair involves changes in their relationships with local interfascicular matrix vascular and basement membrane constituents.

Restraint upon Embryonic Metatarsal Ex Vivo Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR Pathway.

Mechanical cues play a vital role in limb skeletal development, yet their influence and underpinning mechanisms in the regulation of endochondral ossification (EO) processes are incompletely defined. Furthermore, interactions between endochondral growth and mechanics and the mTOR/NF-ĸB pathways are yet to be explored. An appreciation of how mechanical cues regulate EO would also clearly be beneficial in the context of fracture healing and bone diseases, where these processes are recapitulated. The study herein addresses the hypothesis that the mTOR/NF-ĸB pathways interact with mechanics to control endochondral growth. To test this, murine embryonic metatarsals were incubated ex vivo in a hydrogel, allowing for the effects of quasi-static loading on longitudinal growth to be assessed. The results showed significant restriction of metatarsal growth under quasi-static loading during a 14-day period and concentration-dependent sensitivity to hydrogel-related restriction. This study also showed that hydrogel-treated metatarsals retain their viability and do not present with increased apoptosis. Metatarsals exhibited reversal of the growth-restriction when co-incubated with mTOR compounds, whilst it was found that these compounds showed no effects under basal culture conditions. Transcriptional changes linked to endochondral growth were assessed and downregulation of Col2 and Acan was observed in hydrogel-treated metatarsi at day 7. Furthermore, cell cycle analyses confirmed the presence of chondrocytes exhibiting S-G2/M arrest. These data indicate that quasi-static load provokes chondrocyte cell cycle arrest, which is partly overcome by mTOR, with a less marked interaction for NF-ĸB regulators.

CD146 Delineates an Interfascicular Cell Sub-Population in Tendon That Is Recruited during Injury through Its Ligand Laminin-α4.

The interfascicular matrix (IFM) binds tendon fascicles and contains a population of morphologically distinct cells. However, the role of IFM-localised cell populations in tendon repair remains to be determined. The basement membrane protein laminin-α4 also localises to the IFM. Laminin-α4 is a ligand for several cell surface receptors, including CD146, a marker of pericyte and progenitor cells. We used a needle injury model in the rat Achilles tendon to test the hypothesis that the IFM is a niche for CD146+ cells that are mobilised in response to tendon damage. We also aimed to establish how expression patterns of circulating non-coding RNAs alter with tendon injury and identify potential RNA-based markers of tendon disease. The results demonstrate the formation of a focal lesion at the injury site, which increased in size and cellularity for up to 21 days post injury. In healthy tendon, CD146+ cells localised to the IFM, compared with injury, where CD146+ cells migrated towards the lesion at days 4 and 7, and populated the lesion 21 days post injury. This was accompanied by increased laminin-α4, suggesting that laminin-α4 facilitates CD146+ cell recruitment at injury sites. We also identified a panel of circulating microRNAs that are dysregulated with tendon injury. We propose that the IFM cell niche mediates the intrinsic response to injury, whereby an injury stimulus induces CD146+ cell migration. Further work is required to fully characterise CD146+ subpopulations within the IFM and establish their precise roles during tendon healing.