Osteoclasts (OCs) are multinucleated cells that play a pivotal role in skeletal development and bone remodeling. Abnormal activation of OCs contributes to the development of bone-related diseases, such as osteoporosis, bone metastasis and osteoarthritis. Restoring the normal function of OCs is crucial for bone homeostasis. Recently, RNA therapeutics emerged as a new field of research for osteoarticular diseases. The aim of this study is to use non-coding RNAs (ncRNAs) to molecularly engineer OCs and modulate their function. Specifically, we investigated the role of the microRNAs (namely miR-16) and long ncRNAs (namely DLEU1) in OCs differentiation and fusion. DLEU1/DLEU2 region, located at chromosome 13q14, also encodes miR-15 and miR-16. Our results show that levels of these ncRNA transcripts are differently expressed at distinct stages of the OCs differentiation. Specifically, silencing of DLEU1 by small interfering RNAs (siDLEU1) and overexpression of miR-16 by synthetic miRNA mimics (miR-16-mimics) led to a significant reduction in the number of OCs formed per field (OC/field), both at day 5 and 9 of the differentiation stage. Importantly, time-lapse analysis, used to track OCs behavior, revealed a significant decrease in fusion events after transfection with siDLEU1 or miR-16-mimics and an alteration in the fusion mode and partners. Next, we investigated the migration profile of these OCs, and the results show that only miR-16-mimics-OCs, but not siDLEU-OCs, have a lower percentage of immobile cells and an increase in cells with mobile regime, compared with controls. No differences in cell shape were found. Moreover, mass-spectrometry quantitative proteomic analysis revealed independent effects of siDLEU1 and miR-16-mimics at the protein levels. Importantly, DLEU1 and miR-16 act by distinct processes and pathways. Collectively, our findings support the ncRNAs DLEU1 and miR-16 as therapeutic targets to modulate early stages of OCs differentiation and, consequently, to impair OC fusion, advancing ncRNA-therapeutics for bone-related diseases. Acknowledgements: Authors would like to thank to AO CMF / AO Foundation (AOCMFS-21-23A). SRM and MIA are supported by FCT (SFRH/BD/147229/2019 and BiotechHealth Program; CEECINST/00091/2018/CP1500/CT0011, respectively)

Introduction. Cartilage homeoprotein 1 (CART-1) is a homeoprotein which has been suggested to play a role in chondrocyte differentiation and in skeletal development. It is expressed mainly in prechondrocytic mesenchymal condensations. Patients with mutations in the CART-1 gene display several craniofacial abnormalities, suggesting that CART-1 has a functional role in craniofacial skeletal development. However, its target genes and position in the established chondrogenic pathways is poorly documented. Given the fact that CART-1 is expressed predominantly in the chondrocyte lineage and its role in skeletal development, we hypothesized that CART-1 regulates expression of several pivotal genes involved in chondrogenic differentiation. Methods. The coding sequence of human CART-1 was custom synthesized with optimized codon usage and cloned into a p3XFLAG-CMV-7.1 expression vector. FLAG-CART-1 was transiently overexpressed in SW1353 cells by polyethyleneimine-mediated transfection (1,000 ng of plasmid/well in 12-well plates). FLAG-Empty vector was used as a negative control. FLAG-CART-1 overexpression was confirmed by means of anti-FLAG immunoblotting. To investigate a potential connection between CART-1 and established key chondrogenic pathways, TGFβ3 (10 ng/mL) was added to SW1353 cells in CART-1 overexpression cultures or their appropriate controls. Cells were harvested 48 hours after transfection and mRNA expression of several genes involved in chondrogenic differentiation was determined by qRT-PCR. Data represent three separate experiments performed in technical triplicate. Results. Overexpression of CART-1 was confirmed on protein level. CART-1 significantly upregulated the expression of hypertrophic markers MMP13 and COLX, while the expression of RUNX2, ALP and COL1 was significantly downregulated. The expression of COL2A1 and SOX9 was not altered in the presence of CART1. TGFβ3 significantly decreased MMP13 expression in SW1353 cultures, but induced the expression of COLX, RUNX2 and COL1. This TGFβ3 dependent behaviour was reversed when CART-1 was overexpressed in these cultures. Conclusion. Our results implicate a functional role for the homeodomain protein CART-1 in controlling the expression of several markers involved in chondrocyte differentiation and show important interactions with other signaling pathways involved in chondrogenic differentiation. Current efforts focus on further elucidating the connection between CART-1 and other chondrogenic pathways

INTRODUCTION. Endochondral ossification in the growth plate is directly responsible for skeletal growth and its de novo bone-generating activity. Growth plates are vulnerable to disturbances that may lead to abnormal skeletal development. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used analgesics but have been reported to impair endochondral ossification-driven fracture healing. Despite the general awareness that NSAIDs affect endochondral ossification, the consequences of NSAIDs on skeletal development are unknown. We hypothesise that the NSAID celecoxib leads to impaired growth plate development and consequently impairs skeletal development. METHODS. Healthy skeletally immature (5 weeks old) C57BL/6 mice were treated for ten weeks with celecoxib (daily oral administration 10 mg/kg) or placebo (water) (institutional approval 2013–094) (n=12 per group). At 15 weeks postnatally, total growth plate thickness, the thickness of specific growth plate zones, (immuno)histological analysis of extracellular matrix composition in the growth plate, cell number and cell size, longitudinal bone growth and bone micro-architecture by micro-CT were analysed. Inhibition of COX-2 activity was confirmed by determining PGE2 levels in plasma using an ELISA. RESULTS. No significant difference in total growth plate thickness or thickness of the resting zone, proliferative or hypertrophic zone was found between groups. Staining of growth plate extracellular matrix components revealed, however, a significantly higher proteoglycan content and less collagen type II staining in the proliferative zone. In the hypertrophic zone of the growth plates of celecoxib treated mice collagen type X was hardly detectable as compared to placebo mice. In addition, a significantly decreased cell number was observed in the hypertrophic zone of the growth plate and cells were significantly smaller in the celecoxib group. Micro-CT analysis of the subchondral bone region directly beneath the growth plate showed significantly higher bone density, bone volume density and trabecular thickness following celecoxib treatment. Despite the detected differences in extracellular matrix composition of the growth plate, no difference was found in the length of the tibia in celecoxib treated mice. DISCUSSION. In summary, there are no measurable differences found in murine skeletal formation as a result of treatment with celecoxib in this study. However, there are notable phenotypic features found in the maturation of the growth plate (hypertrophic zone and subchondral bone) as a result from the celecoxib treatment, of which the potential consequences we do not yet understand. SIGNIFICANCE. When follow-up actions from the use of celecoxib on the growing individual are found this may warrant re-evaluation for the use of celecoxib in these individuals

TGF-beta signaling has a well established role not only in adult organ homeostasis but also in skeletal development. Follistatin-like 3 (FSTL3), related to follistatin, is an inhibitor of TGF-beta ligands, with an established role in glucose and fat metabolism. However it has not previously been studied in skeletal development. Using a FSTL3 knock-out (KO) mouse model we have studied both embryonic skeletal development and adult bone phenotypes. Staining for skeletal and cartilage markers during development shows acceleration of skeletal tissue differentiation, with an eventual normalization at E18.5 (which is just prior to birth). Acceleration of bone mineralization occurs during both endochondral and intramembranous ossification. Use of micro-CT imaging highlighted the development of a scoliosis in the KO animals, along with abnormal shape of cranium and cranial sutures. Further investigation of the cranial phenotype in adult KO mice reveals craniosynastosis, with atypical fusion of the frontal suture. These mice have a change in overall cranial shape with shortening of the anterior head and a compensatory expansion of the posterior cranial bones, in a similar fashion to brachyencephaly. Our study therefore highlights a significant role of FSTL3 in skeletal tissue development and mineralization, as well as the development of clinically significant skeletal developmental disorders such as scoliosis, craniosynastosis and brachyencephaly

Mesenchymal stem cells (MSCs) have been long studied for their role in skeletal development. MSCs are unique in adult physiology in that they exhibit pluripotency and differentiate into cells that can evolve into various skeletal tissue as a result have been extensively employed as a viable alternative to terminally differentiated cells in engineering of cartilage and bone tissue ex vivo and in vivo. In spite of decades of effort in this direction, our understanding of what drives MSC fate choices is rather narrow in that it places heavy emphasis on a role for morphogens and cytokines (TGF-beta super family, FGF-2). In recent years it has become evident that MSCs also play an important role in wound healing, immunomodulation (immune suppression) and in tumour progression. However, what becomes of an MSC when it arrives at or exits an environment is less understood. We hypothesize that activation of differentiation programs in MSCs have an autocrine and paracrine component involving interplay between MSC-MSC (cell-cell contact) and MSC-(environment), and in this signalling paradigm the biophysical aspects of their microenvironment play a dominant role. We have tested this premise in several aspects of MSC behaviour (proliferation, migration, differentiation, chondrogenesis) and have gathered compelling evidence for biophysics and mechanobiology in MSC fate decisions. This talk will present some of our latest findings in this broad arena

Dynamic loading is necessary for the preservation of native cartilage, but mechanical disuse is one major risk factor for osteoarthritis (OA). As post-transcriptional regulators, microRNAs (miRs) represent promising molecules to quickly adjust the cellular transcriptome in a stimulus-dependent manner. Several miR clusters were related to skeletal development, joint homeostasis and OA pathophysiology but whether miRs are associated with mechanosensitivity and regulated by mechanotransduction is so far unknown. We aimed to investigate the influence of mechanical loading on miR expression and to identify mechanosensitive miR clusters characteristic for non-beneficial loading regimes which may serve as future tools for improved diagnosis or intervention during OA development. Loading regimes leading to an anabolic or catabolic chondrocyte response were established based on an increase or decrease of proteoglycan synthesis after loading of human engineered cartilage. miR microarray profiling at termination of loading revealed only small changes of miR expression (7 significantly upregulated miRs) by an anabolic loading protocol while catabolic stimulation produced a significant regulation of 80 miRs with a clear separation of control and compressed samples by hierarchical clustering. Overall regulation of 8/14 miR was confirmed by qRT-PCR with mean amplitudes of up to 2.5-fold for catabolic loading. Cross-testing revealed that 2 miRs were upregulated by both loading conditions and 6 were specifically elevated by the catabolic loading regime. Conclusively, this study defines the first mechanosensitive miR cluster associated with non-beneficial compressive cyclic loading of human engineered cartilage which can now be tested for its diagnostic potential in healthy versus OA-affected human cartilage

Summary Statement. Bone stress fracture triggers a rapid increase in blood flow in association with mast cell production of inducible nitric oxide synthase (iNOS). NOS inhibition blocks the increase in blood flow and reduces woven bone formation needed for stress fracture healing. Introduction. Vascular-bone interactions are critical in skeletal development and fracture healing. We recently showed that angiogenesis is required for stress fracture healing. However, the changes in vascularity that occur in the first 72 hours after stress fracture can not be explained by angiogenesis. Here, we evaulated early changes in blood flow and vasodilation after either damaging (stress fracture) or non-damaging mechanical loading in rats. Methods. The right forelimbs of adult rats were subjected to cyclic axial compression in vivo. We used two established protocols: damaging loading that creates a stress fracture and leads to woven bone formation (WBF loading), or non-damaging loading that stimulates lamellar bone formation (LBF loading). PET imaging was used to evaluate blood flow and fluoride kinetics based on uptake of . 15. O water and . 18. F fluoride radioisotopes, respectively, at the site of bone formation. To quantify vasodilation, the area of the anterior interosseous artery was measured. Inducible nitric oxide synthase (iNOS) expression was evaluated by immunostaining. Finally, NO production was impaired by administration of L-NAME (N. ω. -nitro-L-arginine methyl ester), a NOS inhibitor. Results. PET Imaging: Damaging WBF loading induced early and persistent increases in blood flow. Blood flow rate was increased ∼30% at 4 hours through 14 days in WBF loaded limbs. Fluoride uptake peaked 7 days after WBF loading, then declined from 7 to 14 days, consistent with the dynamics of woven bone formation described previously. Non-damaging LBF loading did not affect blood flow or fluoride kinetics. Histology: WBF loaded limbs had significantly increased arterial area (+50%) compared to non-loaded limbs at days 1 and 3, with return to normal by day 7. LBF loading did not affect arterial area. Since mast cells are a possible effector of vasodilation, mast cell infiltration and iNOS expression were quantified following loading. iNOS+ mast cells in WBF-loaded limbs were significantly increased on days 1 and 3, with return to normal by day 7. LBF loading was not associated with increases in iNOS+ mast cells. NOS Inhibition: L-NAME blocked the expression of iNOS in mast cells following WBF loading. Additionally, L-NAME treatment abolished the increase in blood flow rate at days 1 and 3, and diminished fluoride uptake at day 3. Finally, L-NAME treatment decreased woven bone formation, with significant decreases in woven bone volume (−27%) and BMD (−26%), compared to vehicle controls. Discussion/Conclusion. Damaging loading produces a stress fracture and leads to woven bone formation (WBF). Prior to bone formation, there is a rapid increase in blood flow rate in association with vasodilation and infiltration of iNOS+ mast cells in the expanded periosteum. Inhibition of NOS blocks the increase in blood flow rate, and ultimately impairs woven bone formation. In contrast, non-damaging (LBF) loading does not affect blood flow rate, vasodilation, or iNOS expression in mast cells. Thus, the vascular response after stress fracture involves an early increase in blood flow by vasodilation, followed by angiogenesis to maintain increased blood flow. Disruption of either response affects subsequent bone formation during stress fracture healing

Bone morphogenetic protein (BMP) has a crucial role in osteochondrogenesis of bone formation as well as in the repair of fractures. The interaction between hedgehog protein and BMPs is inferred from recent molecular studies. Hedgehog genes encode secreted proteins which mediate patterning and growth during skeletal development. We have shown that Indian hedgehog gene (Ihh) is expressed in cartilage anlage and later in mature and hypertrophic chondrocytes. This finding suggests that Ihh may regulate the development of chondrocytes. Our results in this study have shown that Ihh transcripts were expressed in hypertrophic chondrocytes in mice at three days but not at three weeks, although a similar expression pattern of α1 (X) collagen could be observed in both types of cartilage. To investigate the possibility that there are direct and age-dependent functions of Ihh in chondrocytes, cultured chondrocytes were treated with the amino-terminal fragment of Sonic hedgehog protein (Shh-N) which can functionally substitute for Ihh protein. Shh-N did not affect the proliferation and differentiation of chondrocytes from three-week-old mice but had a significant effect on three-day-old mice. It enhanced proliferation up to 128% of the control culture in a dose-dependent manner. Although there was no effect in Shh-N-treated cultures, Shh-N enhanced the stimulatory effect of parathyroid hormone (PTH) on the synthesis of proteoglycans. Because the effects of Shh-N on chondrocyte differentiation in this culture system differed from those of bone morphogenetic protein-2 (BMP2) and PTH, in terms of proteoglycan synthesis and ALPase activity, it is unlikely that BMP2 or PTH/PTH-related protein mediates the direct effects of Ihh in chondrocytes. Our study shows that Ihh can function in chondrocytes in a direct and age-dependent fashion

Objectives

Adult mice lacking the transcription factor NFAT1 exhibit osteoarthritis (OA). The precise molecular mechanism for NFAT1 deficiency-induced osteoarthritic cartilage degradation remains to be clarified. This study aimed to investigate if NFAT1 protects articular cartilage (AC) against OA by directly regulating the transcription of specific catabolic and anabolic genes in articular chondrocytes.

Aim

Osteoarthritis (OA) is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control the expression of genes and are likely to regulate the OA transcriptome. We performed integrative genomic analyses to define methylation-gene expression relationships in osteoarthritic cartilage.

Objectives

This study aimed to investigate the functional effects of microRNA (miR)-214-5p on osteoblastic cells, which might provide a potential role of miR-214-5p in bone fracture healing.

Objectives

Excessive acetabular coverage is the most common cause of pincer-type femoroacetabular impingement. To date, an association between acetabular over-coverage and genetic variations has not been studied. In this study we investigated the association between single nucleotide polymorphisms (SNPs) of paralogous Homeobox (HOX)9 genes and acetabular coverage in Japanese individuals to identify a possible genetic variation associated with acetabular over-coverage.

Post-natal vasculogenesis, the process by which vascular committed bone marrow stem cells or endothelial precursor cells migrate, differentiate and incorporate into the nacent endothelium and thereby contribute to physiological and pathological neurovascularisation, has stimulated much interest. Its contribution to neovascularisation of tumours, wound healing and revascularisation associated with ischaemia of skeletal and cardiac muscles is well established. We evaluated the responses of endothelial precursor cells in bone marrow to musculoskeletal trauma in mice.

Bone marrow from six C57 Black 6 mice subjected to a standardised, closed fracture of the femur, was analysed for the combined expression of cell-surface markers stem cell antigen 1 (sca-1⁺) and stem cell factor receptor, CD117 (c-kit⁺) in order to identify the endothelial precursor cell population. Immunomagnetically-enriched sca-1⁺ mononuclear cell (MNC^sca-1+) populations were then cultured and examined for functional vascular endothelial differentiation. Bone marrow MNC^{sca-1+,c-kit+} counts increased almost twofold within 48 hours of the event, compared with baseline levels, before decreasing by 72 hours.

Sca-1⁺ mononuclear cell populations in culture from samples of bone marrow at 48 hours bound together Ulex Europus-1, and incorporated fluorescent 1,1′-dioctadecyl- 3,3,3,’3′-tetramethylindocarbocyanine perchlorate-labelled acetylated low-density lipoprotein intracellularily, both characteristics of mature endothelium.

Our findings suggest that a systemic provascular response of bone marrow is initiated by musculoskeletal trauma. Its therapeutic manipulation may have implications for the potential enhancement of neovascularisation and the healing of fractures.

We attempted to repair full-thickness defects in the articular cartilage of the trochlear groove of the femur in 30 rabbit knee joints using allogenic cultured chondrocytes embedded in a collagen gel. The repaired tissues were examined at 2, 4, 8, 12 and 24 weeks after operation using histological and histochemical methods. The articular defect filling index measurement was derived from safranin-O stained sections. Apoptotic cellular fractions were derived from analysis of apoptosis in situ using TUNEL staining, and was confirmed using caspase-3 staining along with quantification of the total cellularity. The mean articular defect filling index decreased with time. After 24 weeks it was 0.7 (sd 0.10), which was significantly lower than the measurements obtained earlier (p < 0.01). The highest mean percentage of apoptotic cells were observed at 12 weeks, although the total cellularity decreased with time. Because apoptotic cell death may play a role in delamination after chondrocyte transplantation, anti-apoptotic gene therapy may protect transplanted chondrocytes from apoptosis.

We have investigated in vitro the release kinetics and bioactivity of fibroblast growth factor-2 (FGF-2) released from a carrier of fibrin sealant. In order to evaluate the effects of the FGF-2 delivery mechanism on the repair of articular cartilage, full-thickness cylindrical defects, 5 mm in diameter and 4 mm in depth, which were too large to undergo spontaneous repair, were created in the femoral trochlea of rabbit knees. These defects were then filled with the sealant.

Approximately 50% of the FGF-2 was released from the sealant within 24 hours while its original bioactivity was maintained. The implantation of the fibrin sealant incorporating FGF-2 successfully induced healing of the surface with hyaline cartilage and concomitant repair of the subchondral bone at eight weeks after the creation of the defect.

Our findings suggest that this delivery method for FGF-2 may be useful for promoting regenerative repair of full-thickness defects of articular cartilage in humans.