PAR2 deletion in the osteoblast lineage affords long-term cartilage protection in experimental osteoarthritis

doi:10.21203/rs.3.rs-5078785/v1

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PAR2 deletion in the osteoblast lineage affords long-term cartilage protection in experimental osteoarthritis

https://doi.org/10.21203/rs.3.rs-5078785/v1

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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.

Health sciences/Rheumatology/Musculoskeletal system/Bone

Health sciences/Rheumatology/Musculoskeletal system/Cartilage

Osteoarthritis (OA) stands as one of the most widespread musculoskeletal diseases globally, constituting a significant public health challenge. Its prevalence is on the rise across numerous countries, particularly among the elderly and women. Notably, the percentage of individuals aged 60 and above affected by OA increased from 9.2% in 1990 to approximately 13.5% in 2019 ¹. Incidence and prevalence of OA are expected to rise due to age and obesity trends. OA is mainly characterised by degradation of articular cartilage, development of osteosclerosis, osteophyte formation and localized inflammation². These structural changes can be associated with life-impacting symptoms such as pain and reduced joint mobility. Whether it is primary (naturally occurring) or secondary OA (exacerbated by a specific trigger), the joint experiences a gradual repair process, leading to structural changes while remaining symptom-free³. However, this repair process often fails, leading to OA pain and joint failure for which there are currently no disease-modifying drugs. Current treatments for symptomatic OA consist of pain targeted therapeutics, exercise, weight loss and, as a last resort, surgical intervention³.

A range of pre-clinical models are used to study OA pathophysiology and assess possible treatments^4–7. Destabilisation of the medial meniscus (DMM)⁸ is a surgical model of post-traumatic OA (PTOA; a form of secondary OA) that consistently displays the structural changes seen clinically. Notably, the majority of studies have focussed on the characterisation of disease pathology in the early phase of disease; 4 to 12 weeks after surgery. The model displays many of the features of OA, such as damage and loss of articular cartilage, subchondral bone osteosclerosis, osteophyte formation, low level inflammation and, at the later stages, pain^8,9.

Protease activated receptor 2 (PAR2) is a transmembrane G-coupled receptor, activated by serine protease cleavage of the PAR2 N-terminus, allowing the “tethered ligand” to bind to the second extracellular loop ^10,11. Utilising the DMM model, it has been demonstrated that deletion or inhibition of PAR2 protects against development of OA in the early stages of disease (e.g. 4 and 8 weeks after surgery)^12–15 Moreover, it has also been demonstrated that loss of PAR2 significantly reduces pain-associated behaviour in DMM mice^13; which is partly attributed to the known and important role of PAR2 in neurogenic inflammation ¹⁶ and nociception ¹⁷. However, there are many PAR2-related unanswered questions remaining including the contribution of chondrocytes and/or celsl within bone to the physical and neurological pathological features of OA¹³. The hypothesis in the present study was that modulation of PAR2 offers long-term protection against OA, either delaying or preventing the development of chronic/symptomatic disease. Accordingly, this study investigated the role of PAR2 in late disease, by characterising the post-surgical longitudinal (6–12 months) pathological changes in DMM in global PAR2^−/− mice compared to wild-type littermates. To further elucidate the underlying PAR2-pathophysiological mechanisms in late-stage OA, pathology and physical manifestations of pain were investigated in mice with targeted deletion of PAR2 specifically in chondrocytes or osteoblasts. Combined these data highlighted the dominant dual role of skeletal PAR2 in driving OA-related pathogenesis and pain.

Absence of PAR2 delays evolution of symptomatic/late-stage OA in mice

Mean total joint cartilage damage score was assessed temporally in wild-type PAR2^+/+ mice following DMM (Fig. 1A), where we observed an increase in joint cartilage damage from 6 months post-surgery, up to a complete loss of cartilage at 12 months (Figs. 1B and C, Supplemental Fig. 1). The joint cartilage showed linear increased degeneration on the medial side of the joint (Supplemental Fig. 1). PAR2^−/− mice showed overall a lower joint damage (Figs. 1B and C). Pain-associated behaviour was measured via dynamic weight bearing (DWB) assessment. This quantified, amongst other parameters, the amount of time the mice spent with the osteoarthritic hindlimb raised in comparison to the unoperated hindlimb (Fig. 1D) and the differential load between both rear legs (Supplemental Fig. 2). Measurements across the time points revealed that wild-type littermates showed pain symptoms from 11 months post-induction, whilst PAR2^−/− mice did not (Fig. 1D). Interestingly, no correlation was found between cartilage damage and the degree of pain in wild-type mice (Fig. 1E).

Upregulation of bone remodelling in murine late-stage OA

To further investigate the impact of long-term OA on joint structure, we evaluated the impact on bone parameters. Both wild-type and PAR2^−/− mice displayed changes to bone structure around the joint following induction of OA (Fig. 2). The most noticeable change was the emergence of gross calcifications around the knee, starting with an enlargement of the displaced medial meniscus at 6 months (Fig. 2A). This calcification expanded around the joint, ultimately leading to ligament calcification. Quantification of the calcification volume in the medial side of the joint, revealed that both wild-type and PAR2^−/− mice had similar amounts (P = 0.19), and time-dependent increases, (P < 0.0001) in calcification (Fig. 2B). Moreover, absence of PAR2 did not protect against development of osteosclerosis in subchondral bone (P = 0.89, Fig. 2C). There were also no changes to trabecular bone microarchitecture (Fig. 2D). Despite the lack of micro-architectural changes between wild-type and PAR2-/- mice, evaluation of the soluble serum bone resorption marker, CTX-I, indicated that whilst there was a significant increase in wild-type mice at the 11- and 12-month time points, no change was observed in PAR2^−/− mice (Fig. 2E). Similarly, serum P1NP (measure of bone formation), showed that whilst there was also a significant increase in wild-type mice at 11 and 12 months, no change was observed in PAR2^−/− mice (Fig. 2F). Interestingly, there was a correlation between the resorption marker CTX-I and the time mice spend with the osteoarthritic leg raised (Fig. 2G), indicating that the increase in resorption and the behavioural change to avoid loading the affected leg occurred simultaneously.

Combined, this suggests that bone turnover dynamics are different between wild-type and PAR2^−/− mice towards the end stage of the disease, although this does not lead to measurable changes in bone volume or microarchitecture, indicating that despite the increase in remodelling in the wild-type samples, a balance between resorption and formation is maintained.

PAR2 absence in osteoblasts protects against late-stage OA

Based on prior data¹³, it is unclear what relative contribution PAR2-mediated mechanisms deriving from the bone or cartilage make in driving OA pathology. Previous studies in PAR2^−/− mice demonstrated that PAR2 is involved in cartilage damage and endochondral ossification^13,16. However, the bone pathology observed prior to any cartilage damage means that the contribution of bone or bone-associated cells cannot be dismissed. To investigate whether PAR2 exerts an effect in the joint from the cartilage itself, or from the bone, we generated tissue-specific knock out mice where we deleted PAR2 in cells expressing either Collagen type 2 (chondrocytes) or Osteocalcin (osteoblasts and osteocytes). Unexpectedly, deletion of PAR2 in chondrocytes (PAR2^ch/ch) did not lead to a significantly lower cartilage damage in the medial compartment of the joint (Fig. 3). However, 12 months after surgical intervention, animals with PAR2-specific disruption in osteoblasts (PAR2^ob/ob) displayed a significant protection against cartilage damage in comparison to the PAR2^f/f controls (P < 0.01, Fig. 3).

Deletion of PAR2 in chondrocytes or osteoblasts display opposing bone phenotypes

Further assessment of bone in tissue-specific knock outs showed that joint calcifications were apparent in all groups, yet the PAR2^ch/ch mice showed significantly reduced total ectopic calcification volume in the medial side of the joint (Figs. 4A and B). Subchondral bone osteosclerosis was also reduced in PAR2^ch/ch (Fig. 4C). Neither bone resorption nor formation markers were significantly different between any of the genotypes (Figs. 4D and E), whilst tibial trabecular bone was significantly increased in the PAR2^ob/ob osteoarthritic leg (Fig. 4F). In comparison to the contralateral leg there was a significant reduction in bone density in the metaphyseal trabecular bone of the PAR2^f/f mice osteoarthritic tibia (Fig. 4G).

The examination of the bone phenotype of the osteoblast and chondrocyte tissue specific PAR2 knock out mice indicated there are striking differences when compared to the control. To understand this observation, the phenotype and its evolution from developmental stages to maturity and old age was further investigated. To achieve this the tibia and femur bone phenotype of naïve 5-week-old male mice were studied, as well as the contralateral leg of 14- and 62-week-old DMM and sham operated mice of the different genotypes (Fig. 5 and Supplemental Figs. 3 and 4). Cortical bone in the femur of these mice was also interrogated (Supplemental Fig. 5).

The PAR2^ob/ob shows reduced trabecular bone density (Figs. 5A and B) during development, characterised by reduced trabecular number and thickness in tibia and femur (Supplemental Figs. 3 and 4), as well as reduced cortical thickness, porosity, perimeter, and area, when comparing to the PAR2^f/f controls (Supplemental Fig. 5). These changes are normalised by the time the mice reach skeletal maturity (14 weeks) where all bone measurements are equal to the controls. In old age the PAR2^f/f mice show a decrease in trabecular and cortical parameters, yet the PAR2^ob/ob mice maintain bone more than the PAR2^f/f controls, displaying increased trabecular bone density and number in the tibia as well as increased cortical thickness and perimeter in the femur (Supplemental Figs. 2, 3 and 4). This indicates PAR2^ob/ob mice are slower during the initial bone development, but once skeletal maturity is reached this improves and maintains the bone quality for longer.

The PAR2^ch/ch mice show normal bone development at 5 weeks of age when compared to PAR2^f/f controls, displaying similar trabecular and cortical bone microarchitecture. Yet, by the time these mice reach skeletal maturity at 14 weeks of age, there is a distinctive loss of trabecular bone (Fig. 5A and B), with reduced trabecular number in the tibia and thickness in the femur, and a significant reduction in femoral cortical perimeter, indicating a narrower bone (Supplemental Fig. 5). These changes are accentuated in old age (62 weeks) with an even more significant loss of trabecular bone number and thickness in both femur and tibia, as well as increased porosity in the cortical bone (Supplemental Figs. 3, 4 and 5).

PAR2 induces osteoblast differentiation and maturation in vitro

Given the previously described bone phenotype of the osteoblast specific deletion of PAR2, we interrogated the in vitro differentiation of bone marrow adherent cells to osteoblasts (alkaline phosphatase expressing cells). This confirmed that PAR2^−/− cells have attenuated osteogenic differentiation (Figs. 6A and B). In comparison, whilst the in vitro growth of primary immature (neonatal calvarial) osteoblasts was unaffected (Fig. 6C), the ability of these cells to further mature and mineralise was substantially reduced by loss of PAR2 (Figs. 6D and E). Moreover, these cells were also unable to deposit the same level of extracellular matrix observed in wild-type cells (Figs. 6F and G). This was verified at the transcriptional level in day 21 cells, where analysis revealed that PAR2^−/− osteoblasts expressed lower levels of osteoblast and mineralisation markers (Fig. 6H). Combined, these data indicate a lower differentiation potential towards the osteoblastic phenotype in the absence of PAR2, as well as lower further maturation and therefore osteoblastic activity within pre-osteoblasts.

Osteoblasts regulate osteoclast differentiation via PAR2

Bone formation and resorption are coupled, meaning that for bone homeostasis, changes in bone formation must be followed by changes in bone resorption. Prior data (Figs. 2E and F) demonstrated that PAR2^−/− mice lacked the increased bone remodelling showed in wild-type osteoarthritic mice. Thus, we examined the role of PAR2 in in vitro osteoclastogenesis and resorption. Despite equal numbers of osteoclast precursors (i.e., bone marrow (BM) monocytes, Fig. 7A), the absence of PAR2 resulted in increased osteoclastogenic potential within the compartment (Fig. 7B). In addition to increased numbers, the differentiated and mature osteoclasts were larger and contained more nuclei (Fig. 7C). However, this did not result in increased functionality, but rather a decreased capacity to resorb matrix (Fig. 7D). We hypothesised that this effect on osteoclasts may depend upon their environment and the interaction with osteoblasts. To examine this, bone marrow monocytes were co-cultured with matured neonatal calvarial primary OBs (pOBs). These co-cultures revealed that absence of PAR2 in the osteoblast compartment led to enhanced osteoclastogenesis in both wild-type and PAR2^−/− monocytes (Fig. 7E). Moreover, the co-culture of PAR2^−/− monocytes with wild-type pOBs did not lead to an enhancement of osteoclastogensis compared to wild-type only co-cultures. This suggests that active PAR2 signalling from osteoblasts results in the regulation of osteoclast formation. However, this occurs without substantial changes to the classical pathway controlling osteoclast differentiation, RANKL / OPG as shown by gene expression in matured osteoblast cultures (Fig. 7F).

PAR2 downregulates mature osteoblast mineralization

The bone phenotype data on PAR2^ob/ob indicated a difference in the bone architecture depending on skeletal maturity. We therefore isolated primary osteoblasts from long bones of 8-month old wild-type and PAR2^−/− mice. The PAR2^−/− osteoblasts stopped growing at a lower confluency (Fig. 8A) yet, unlike immature osteoblasts, these ‘old-age’ mature osteoblasts mineralised faster than the wild-type control osteoblasts (Fig. 8B) and produced more matrix (Fig. 8C and D). Unlike artificially matured osteoblasts (Fig. 7E), long bone osteoblasts did not significantly increase osteoclastogenesis of wild-type bone marrow monocytes (Fig. 8E). PAR2^−/− osteoblasts express significantly higher osteoclast inhibitor OPG whilst in mineralisation culture (Fig. 8D).

Previously we and others have reported that absence or inhibition of PAR2 provides early protection of cartilage damage in the DMM model at 4 and 8 weeks^12–15. Given the chronic long-term nature of OA pathogenesis, the critical question in terms of targeting PAR2, as a future disease-modifying therapy, then becomes whether this protection can be retained over the long-term. One key finding in this study is that PAR2 is a key driver in the continued development of late-stage pathology in murine OA, and therefore confirms PAR2 as an attractive therapeutic target for OA.

The late-stage DMM model led to several observations. Notably, the evolution of the osteoarthritic disease in the male mouse joint is highly variable in these late stages of the disease, yet linear, due to the accumulation of damage in the different compartments of the joint. PAR2 deletion maintains a lower cartilage degradation phenotype in many of the time points measured, with a steady increase similar to the wild-type mice. What was striking was that this protection against cartilage degradation in late-stage OA was driven by the absence of PAR2 in osteocalcin expressing cells, whilst chondrocyte Col2a1 driven PAR2 deletion did not lead to a substantial reduction.

Another important feature of late-stage OA is the evaluation of pain, which we conducted via a surrogate assay (dynamic weight bearing). This revealed increased levels of pain-associated behaviour at around 11 months post-induction of OA in wild-type mice. In comparison, no pain-associated behaviour was observable in PAR2^−/−, chondrocyte or osteoblast-specific PAR2 knock out mice (Supplemental Fig. 2). The lack of correlation between the disease presentation measured by cartilage damage and pain is a feature that mimics clinical observations. Some patients with radiographic OA do not display pain, whilst others with the same radiographic score show large variation in measured pain^17–20. However, despite the lack of correlation, in the present study it is difficult to distinguish whether the lack of surrogate pain measured is due to the role of PAR2’s in nociception^21–23 or the absence of advanced disease.

Prior studies have shown that alterations in joint loading (i.e., through altered gait) can lead to alterations in resorption²⁴. Conceivably therefore, the observed increase in circulating remodelling markers (Figs. 2E and F) is a result of the pain-induced gait changes in wild-type mice. However, it should be appreciated that the observed changes may not be restricted to modified biomechanics but also to underlying cellular factors. For instance, our in vitro studies demonstrated that PAR2^−/− bone marrow cells and pre-osteoblasts have decreased osteogenic differentiation capacity, whilst also having the ability to drive enhanced osteoclastogenesis, albeit in a state of decreased functionality. Yet mature PAR2^−/− osteoblasts have enhanced mineralisation. This reduced osteogenic differentiation followed by increased mineralisation ability agrees with the in vivo phenotype of the PAR2^ob/ob mice, where there is delayed skeletal development followed by an increase in bone density and thickness in skeletal maturity despite reduced bone turnover. These data therefore suggest that PAR2 and its signalling in osteoblasts enhances the bone remodelling process during the pathological processes of OA. Subchondral bone remodelling is enhanced in late-stage OA ^25–27, and a hindered increase in subchondral bone remodelling could have a positive impact by delaying OA disease development²⁸.

In essence, our data show a fundamentally pathogenic role of PAR2 in osteoblast biology, and that perturbation of this role can lead to maintenance of cartilage integrity. Crosstalk between bone cells and the adjacent articular cartilage is now considered a central factor in OA^29–32, where communication between bone and cartilage is enhanced in late-stage disease³³. This may be due to the development of microcracks³⁴ and increased vascular invasion³⁵ from bone to cartilage, providing additional pathways of communication and favouring the transport of larger soluble molecules. Thus, factors such as cytokines and prostaglandins involved in tissue remodelling can reach the adjacent cartilage, enhancing its catabolism^26,36. We propose that PAR2 in osteoblasts increases bone remodelling in the subchondral bone, enhancing signalling detrimental to the cartilage. Further studies are needed to investigate how osteoblast signalling modulates cartilage maintenance, focusing on the role of PAR2 in this process.

There are limitations of this study. The power of the study was based on a pilot study, and we increased the minimum number per group anticipating high variability, yet the temporal analysis from 6 to 12 months after induction would have benefited from increased group sizes. Deletion of target proteins utilising the Cre-loxP system is often not as specific as desired³⁷. Using a Rosa tdTomato reporter, Col2-cre expression has been shown mainly in articular cartilage yet also in growth plate, meniscus, endosteum, ligament, and synovium³⁸. Osteocalcin-driven cre recombinase expression has been shown to be more specific to the osteoblast lineage, towards the late osteoblast and osteocyte differentiated cells³⁹, as well as cells in the periosteum bone lining ^40,41, CXCL12-abundant reticular (CAR) cells and arteriolar pericytes⁴², which could potentially give rise to osteoblasts^43,44. Deletion of PAR2 in undifferentiated cells reduced the osteogenic differentiation potential (Figs. 6A and B), and therefore possibly results in a lower availability of osteoblasts from this small mesenchymal niche. Osteocalcin is also expressed during the differentiation of chondrocytes to the hypertrophic phenotype⁴⁵ and thus, utilising an osteocalcin driven cre-recombinase could potentially have deleted PAR2 at the crucial hypertrophy differentiation, which would have an impact on the development of OA. The Tg(BGLAP-Cre) allele expresses catalytically active Cre-recombinase in multiple non-skeletal tissues, such as the central nervous system⁴⁶, albeit to a lower degree. In addition to this, the choice of control was the PAR2 loxP mice, which did not control for any effect the Col2a1 or Osteocalcin driven cre-recombinase would have in the skeletal phenotype. Lastly, the study was performed in males and in the future the use of females should be included.

The study highlights the complexity of PAR2-mediated effects, with age-dependent and cell-specific roles. These data also highlight the importance of bone remodelling as a mechanism which influences joint degradation, where a reduction of both bone formation and resorption can lead to a slower cartilage degradation. Future research should explore the molecular mechanisms through which PAR2 influences osteoblast differentiation, osteoclast regulation, and the overall balance between bone resorption and formation. Potential therapeutic interventions targeting PAR2 have been developed, yet they have not been as specific as initially thought⁴⁷. In absence of successful inhibitors, PAR2 downstream signalling or management of bone remodelling and/or skeletal health could offer new avenues for delaying OA progression.

Generation of PAR2 deficient animals

Generation of PAR2-deficient mice were previously described⁴⁸. Breeding pairs were set up as heterozygous or homozygous pairs that were derived from heterozygous mattings, thus animals in the study were either litter mates or, at most, 1 generation removed. F2rl1^flox (PAR2^F/F) mice were generated in collaboration with Prof Neil Dear (Leeds University) from ES cells (C57Bl6 background) obtained from the Mouse Genetics Project (Sanger Centre, Cambridge). The L1L2_Bact_P cassette, consisting of an FRT site followed by lacZ sequence and a loxP site, was inserted at position 95514758 of Chromosome 13 upstream of the critical exon. Under the control of the human beta-actin promoter, neomycin resistance gene follows the first loxP site, further followed by SV40 polyA, a second FRT site and a second loxP site. Downstream of the target exon, a third loxP site is inserted at position 95511446. Thus, the target exon is flanked by loxP sites. A floxed allele is created by flp recombinase expression in mice carrying this allele to remove the lacZ sequence and neo selection cassette, leaving loxP sites flanking the critical exon 2. Exon 2 was targeted because it contains most of the coding sequence of the PAR2 protein. PAR2^F/F mice were crossed with Tg(Col2a1-cre)1Bhr/J or Tg(BGLAP-cre)1Clem/J mice (Jackson laboratories) to knock out PAR2 specifically in chondrocytes (PAR2^ch/ch) or in osteoblasts (PAR2^ob/ob). Both tissue specific colonies were bred as homozygous PAR2^F/F with a heterozygous cre. Thus, PAR2^F/F controls are littermates. Background of tissue specific knockout mice was tested with MiniMUGA (Transnetyx, USA). Diagnostic SNPs indicated an outbred C57BL/6 background with sub-strains C57BL/6J (~ 53% of possible alleles), C57BL/6JOlaHsd (~ 14% of possible alleles).

Animals

Animals were housed in cages with enrichment and in a 12 h light cycle. Group sizes are stated in the supplemental table 1. A small pilot study (n = 4) on the cartilage damage comparing WT to PAR2^−/− 12 months post induction, determined a signal to noise ratio of 2.4. The required sample size for 90% power assuming a 5% significance level and a two-sided test is a minimum of 5 mice per group. Considering the need to carry out microCT analysis, for which the PAR2^−/− have only a mild phenotype in early development, we aimed to have 8 mice per time point to also allow for attrition, in addition to the samples on the pilot study. 4 week time point studies on the chondrocyte- and osteoblast-specific knockout did not show any significant changes in cartilage damage, thus we could not estimate a sample size as the long term effect may be different. Given the long-term design of the study, we aimed to have 10 to 12 mice per conditional knock out group. Supplemental table 1 describes the groups utilised in the study. No animals were excluded, although during the study 3 animals needed to be euthanised due to tumours or teeth malformation. Animals were randomly assigned to each group (excel) and assigned an ID which was only matched to the group after scoring and data analysis. Animal health was monitored based on a scoring system. Dynamic weight bearing was measured at end point. Blood was collected for serum assays and knees harvested for microCT followed by histology to determine cartilage score. Bone marrow was extracted from 6- to 8-week-old mice. Immature primary osteoblasts were extracted from 3 to 5-day old neonatal calvariae as previously described⁴⁹. Mature osteoblasts were extracted from 8- to 12-month-old mice from a colleague’s ageing study controls. Osteoclasts were derived from enriched monocyte populations from the bone marrow.

All procedures were in accordance with Home Office regulations and approved by the University of Glasgow Animal Welfare & Ethics Review Body.

Induction of OA

OA was induced by DMM surgery on adult 10-week-old male mice. PAR2^−/− mice were compared to wild type littermates. For tissue specific knockout studies PAR2^f/f was used as a control. As previously described^8,50, medial compartment OA was induced by DMM involving transection of the left medial meniscotibial ligament under aseptic conditions. The surgeon was unaware of the genotype and was told what surgery to conduct (sham or DMM) by the assistant who randomly allocated mice to the different groups. Buprenorphine (Vetergesic; 30 µg intraperitoneally) was administered postoperatively and animals maintained for up to a year. Our lab has optimised this surgery to allow a quick cut of the ligament without any artificial damage to the cartilage during the intervention⁵⁰. This results in a slower progressing model due mainly to the destabilisation of the joint. All data is available upon request.

MicroCT

Knee joints were fixed in 4% paraformaldehyde solution for 24 h and subsequently stored in 70% EtOH, then analysed by µCT to examine the calcified tissues using Skyscan 1272 (Bruker, Belgium; 0.5 aluminium filter, 50 kV, 200 mA, voxel size 4.57 µm, 0.3° rotation angle). Scans were reconstructed in NRecon software (Bruker, Belgium), with stacks analysed as follows: (1) medial subchondral bone was analysed by selecting a volume of interest (450 x 450 x 900 µm) within the loaded region of the tibial epiphysis^13,51 manually eliminating the growth plate and below, (2) a 900 µm thick slice from the metaphysis (225 µm from the growth plate) was selected to analyse trabecular bone parameters with an automated selection of the ROI (CTan, Bruker, Belgium) and (3) ectopic calcifications on the medial side around the knee, including the displaced meniscus, were manually selected to then shrink the ROI to the edge of the existing bone, hence measuring the volume occupied by these calcifications.

Assessment of cartilage damage

Histological analysis of progression and severity of cartilage damage was undertaken on joints previously scanned, then decalcified (Formical 2000; Decal Chemical, New York, USA) overnight. Joints were embedded in paraffin wax and coronal sections (6 µm) cut then stained with haematoxylin, safranin-O/fast green. We used the new cartilage damage score described by Haubruck et al.⁵² applied to each quadrant of the joint. This newly published scoring system gives a more progressive score separating the damage to the uncalcified and calcified regions of the articular cartilage and is more suitable for severe OA. The scoring system is summarised as follows: 0 = Normal cartilage, 1 = Roughened surface AND/OR superficial fibrillation < 10% of cartilage depth (any % of joint surface area),, 2 = Fibrillation extending > 10% of cartilage depth but not reaching the calcified cartilage AND/OR loss of surface lamina (any % or joint surface area), 3 = Horizontal cracks/separations between calcified and non-calcified cartilage OR clefts down to calcified cartilage BUT no loss of non-calcified cartilage, 4 = Fibrillation to the calcified layer OR loss of non-calcified cartilage lesion for 1–25% of the joint surface, 5 = Fibrillation to the calcified layer OR loss of non-calcified cartilage lesion for 25–50% of the joint surface, 6 = Fibrillation to the calcified layer OR loss of non-calcified cartilage lesion for 50–75% of the joint surface, 7 = Fibrillation to the calcified layer OR loss of non-calcified cartilage lesion for > 75% of the joint surface, 8 = lesion extends through the calcified cartilage (1–25% joint surface area), 9 = lesion extends through the calcified cartilage (25–50% joint surface area), 10 = lesion extends through the calcified cartilage (50–75% joint surface area), 11 = lesion extends through the calcified cartilage (> 75% joint surface area). 6 sections from each mouse knee, between 25 and 100 µm apart, were graded by two scorers blinded to the specimens, with scores averaged. Poor histological sections (ripped, folded or wrong orientation) were not scored. The second scorer had no previous experience in scoring histological sections and was unaware of the aims of the study. There was good agreement between scorers with a Spearman correlation coefficient of 0.80 (95% CI 0.68 to 0.89), the mean difference in score being 1.6.

Dynamic weight bearing

As an indirect indicator of pain, limb weight bearing in mice was assessed at end point, using a dynamic weight bearing chamber (BioSeb, Marseilles, France). Animals were individually recorded for 5 minutes, of which a minimum of 1 minute was subsequently validated and analysed (Dynamic Weight Bearing 1.0, Bioseb). The parameters examined were the individual paw load in proportion to body weight as well as the % time spent on each paw.

Serum assays

To determine differences in bone remodelling, serum was collected from the mice at experimental end point. A sandwich ELISA for C-terminal telopeptide of type I collagen (CTX-I, RatLaps™; IDS) and N-terminal propeptide of type I procollagen (P1NP, IDS) ELISA assays were used and analyses performed according to the manufacturer’s instructions.

In vitro osteoblastogenesis and osteoblast cultures

Bone marrow was flushed out of the long bones of 6- to 8-week-old PAR2^+/+ and PAR2^−/− mice and resuspended in maintenance medium. 1x10⁶ cells/well were plated in a 12 well plate and medium changed after 24 hours. Cells were then cultured in differentiation media (alphaMEM + 10% FBS + 1% Penicillin/Streptomycin + 10 µM Dexamethasone + 100 µM ascorbic acid + 5 mM β-glycerol phosphate). Immature primary osteoblasts (pOBs) were obtained by sequential digestion of excised 3 to 5 day old PAR2^+/+ and PAR2^−/− neonatal calvariae as previously described⁴⁹. Mature long bone pOBs were extracted by digesting bone chips from femur and tibia of 8 to 12 month old mice (experiments were age and sex matched) in 2mg/ml Collagenase II for 2 hours and plating bone chips. Osteoblasts “climb out” the chips reaching confluency in 10 to 14 days. Assessment of pOBs growth was carried out by plating different concentrations of cells in a 96 well plate and changing maintenance media every 2 or 3 days. 7 days after seeding, Alamar blue was added to the medium and incubated for 4 hours. Medium was then colour measured in a spectrophotometer (540 nm). Prior to co-culture with osteoclasts, immature primary osteoblasts were matured to promote osteoclastogenic functions, culturing in 1µM Prostalgandin E2 (PGE2, Sigma,UK) and 10nM 1,25(OH)2D3 (VitD, Sigma, UK) for 8 days with media changed every 3 days and cells split if reaching 70 to 80% confluency.

Mineralization assay

Primary osteoblasts (immature or mature) were plated at 2×10⁴ cells per well in 24 well plates and cultured in maintenance medium (alphaMEM + 10% FBS + antibiotics) for two days before changing to mineralization medium (maintenance medium + 50 µg/ml ascorbic acid and 5 mM β-glycerol phosphate, βGP; Sigma). Medium was changed every 3–4 days for up to 21 days. Cells were fixed for staining or lysed for mRNA extraction. Initial fixation was with 4% paraformaldehyde for 30 minutes at RT, then cells were stained with 2% alizarin red, pH 4.2. Extracellular matrix was stained for 30 minutes with 1% Alcian blue in 3% acetic acid and washed twice in 3% acetic acid. RNA was extracted with RNeasy kit (Qiagen, UK). RNA was quantified and reverse transcribed (Primer Design, UK). qPCR was carried with Precision PLUS SYBR Green Master mix (Primer Design, UK) on a Step One-Plus machine (Applied Biosystems). All gene expression data were normalized against Atp5B (Primer Design, UK; sequence not disclosed) and B-actin. Primer sequences are shown in supplemental table 2.

Bone marrow monocyte quantification

Freshly isolated BM cells were washed in PBS and then live dead staining was conducted using CyStain DNA 2 Step DAPI stain (Sysmex; UK) diluted 1:2 in PBS, incubated for 15 minutes at room temperature. The samples were topped up with FACS buffer and centrifuged at 400g for 5 minutes at room temperature. Cells were incubated with 1:10 dilution of CD16/32 block (BD Pharmingen; UK) to prevent non-specific binding of antibodies via Fc regions for 10 minutes at room temperature. Following this, an antibody cocktail against markers used to identify BM cell populations in mice (Supplemental table 3) was added to the samples for 30 minutes at room temperature. Monocytes were identified as Ly6G⁻, CD3⁻, B220⁻, CD11b⁺ and Ly6C⁺ (Supplemental table 3). Quantification was expressed as the percentage of gated populations from total bone marrow cells (from single cells). Data analysis was subsequently completed using FlowJo software.

Osteoclast in vitro cultures

Freshly isolated bone marrow was used to obtain osteoclast precursors for culture. In brief, total BM was cultured in a 75cm² tissue culture flask overnight (12–16 hours) in 37°C and 5% CO2. Post overnight incubation the non-adherent bone-marrow cells (NA-BMCs) were collected; this population should be depleted of adherent stromal cells and enriched for monocyte populations. The NA-BMCs were resuspended at 1x10⁶ cells/ml in complete α-MEM. These cells were cultured in flat bottom 96 well plates, at a concentration of 1x10⁵ cells/well in the presence of 30ng/ml of recombinant murine M-CSF overnight (approximately 18 hours). The following day media was half changed by removing 50µl of media and adding 50µl of a-MEM containing 100ng/ml of both murine M-CSF and RANKL (to give a final concentration of 50ng/ml of each). Cells were checked daily to monitor progress and after 4 days media was refreshed again by half changing. Negative control for OC formation was NA-BM cultured in M-CSF alone, with no RANKL. The following day (day 5 of the culture) media was removed, cells were fixed, and TRAP stained. Analysis of whole well images of TRAP-stained murine cultures was conducted with ImageJ software. Analysis included counting total osteoclast numbers (TRAP+, 3 or more nuclei), as well as quantification of the area of each well that contains TRAP + multinucleated osteoclasts, used as a surrogate of osteoclast size. OC area was investigated by manually outlining OC cells on ImageJ and calculating the percentage of the total well area covered by outlined OCs.

In vitro resorption assay

As well as TRAP staining of murine osteoclasts, activity of these cells was also measured by culturing osteoclasts on osteo-assay surface plates (Corning; UK). Culture was extended to 12 days, with culture media half changed every 3 days. On day 12, the media was removed and 60µl of 10–15% sodium hypochlorite solution (Sigma-aldrich; UK) added to each well for 3 minutes to remove the cells. Wells were subsequently washed 3 times in distilled water and left to dry. Images of wells were taken on the EVOS FL Auto Cell Imaging System (Life Technologies; UK) light microscope. The proportion of the cell culture well with resorbed mineral was calculated on ImageJ software.

In vitro co-culture assays

To determine the osteoblast contribution to osteoclast differentiation, osteoblasts were co-cultured with monocytes. 2.5x10³ osteoblasts were plated per well onto flat bottom 96 well plates. The following day, monocytes were isolated from bone marrow suspensions following EasySep mouse monocyte isolation kit manufacturers instructions (StemCell, UK). Monocytes were plated at 1x10⁵ cells/well onto the osteoblasts. Co-cultures were cultured in maintenance media containing 1µM Prostalgandin E2 (PGE2, Sigma,UK) and 10nM 1,25(OH)2D3 (VitD, Sigma, UK) or 50 µg/ml ascorbic acid and 2 mM β-glycerol phosphate Media was changed every 3–4 days and cultured for 9–10 days. Cells were then TRAP stained and quantified.

Statistical analysis

Data were tested for normality (Graphpad Prism v10, Shapiro-Wilk test) and expressed in graphs as mean ± SD unless stated otherwise. Genotype comparisons were carried out with a one-way analysis of variance (ANOVA), unless otherwise stated. Temporal comparisons dependent on genotype were analysed with a two-way ANOVA. Specifics on statistical tests are included in the figure legends.

The authors have no potential conflicts of interest, including financial and non-financial.

Role of the Funding Source

This work was supported by an Arthritis Research UK programme grant (20199) and Versus Arthritis Fellowships (CH, 22483, 22858). SM was supported by Medical Research Scotland (PHD-778-2014). KM was supported by a University of the West of Scotland studentship. MLV was supported by EPSRC (EP/S02347X/1).

Animal studies

All animal experiments were approved by the University of Glasgow Ethical Review Committee and following UK Home Office guidelines for the care and use of laboratory animals.

Acknowledgements

The authors would like the acknowledge the invaluable contribution of Gemma Charlesworth and Amanda Prior, who scanned, sectioned and stained the samples.

Author contributions

Conceptualization: CH, CSG, ADR, JCL, RP, RvH, WRF

Methodology: CH, RvH, WRF, CSG

Software: JJC.

Validation: MLV, LD.

Formal analysis: CH

Investigation: CH, SM, LD, MLV, KAM, KM, TB

Data Curation: CH

Writing - Original Draft: CH, JCL, CSG

Writing - Review & Editing: KAM, MLV, ADR, RP, RvH, WRF

Visualization: CH

Supervision: CSG, JCL, WRF

Project administration: CH, CSG, JCL, WRF

Funding acquisition: CH, CSG, ADR, JCL, RP, RvH, WRF

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ARRIVE2checklistNC.pdf
Supplementaltables.docx
Supplfigure1.tif
Supplemental figure 1. Cartilage score by compartment. WT and PAR2-/- mice per joint quadrant from 6 to 12 months after induction Data are expressed as mean + Standard deviation and analysed by 2-way ANOVA with Sidak correction for multiple comparisons. *P<0.05, **P<0.01, *** P<0.001, **** P< 0.0001.
Supplfigure2.tif
Supplemental figure 2. Load bearing as a percentage of total weight in all genotypes presented. Data were analysed by paired 2-way ANOVA to match the legs of each mouse. *P<0.05, **P<0.01.
Supplfigure3.tif
Supplemental figure 3. Analysis of trabecular bone from the naïve tibial metaphysis at 5 weeks and the contralateral leg of Sham and DMM operated mice at 14 and 62 weeks of age. Data were analysed by one-way ANOVA with Bonferroni correction for multiple comparisons. Not normally distributed data were analysed by Kurskall-Wallis test with Dunn’s correction for multiple comparisons to the PAR2f/f control group. *P<0.05, **P<0.01, *** P<0.001.
Supplfigure4.tif
Supplemental figure 4. Analysis of trabecular bone from the naïve femoral metaphysis at 5 weeks of age and the contralateral leg of Sham and DMM operated mice at 14 and 62 weeks of age. Data were analysed by one-way ANOVA with Bonferroni correction for multiple comparisons to the PAR2f/f control group. Not normally distributed data were analysed by Kurskall-Wallis test with Dunn’s correction for multiple comparisons. *P<0.05, **P<0.01, *** P<0.001.
Supplfigure5.tif
Supplemental figure 5. Analysis of trabecular bone from the naïve femoral cortical bone at 5 weeks of age and the contralateral leg of Sham and DMM operated mice at 14 and 62 weeks of age. ROI was selected as a 230 µm slice 2.2 mm from the growth plate. Data were analysed by one-way ANOVA with Bonferroni correction for multiple comparisons. Not normally distributed data were analysed by Kurskall-Wallis test with Dunn’s correction for multiple comparisons to the PAR2f/f control group. *P<0.05, **P<0.01, **** P< 0.0001.

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PAR2 deletion in the osteoblast lineage affords long-term cartilage protection in experimental osteoarthritis

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Abstract

Figures

Introduction

Results

Absence of PAR2 delays evolution of symptomatic/late-stage OA in mice

Upregulation of bone remodelling in murine late-stage OA

PAR2 absence in osteoblasts protects against late-stage OA

Deletion of PAR2 in chondrocytes or osteoblasts display opposing bone phenotypes

Osteoblasts regulate osteoclast differentiation via PAR2

PAR2 downregulates mature osteoblast mineralization

Discussion

Methods

Generation of PAR2 deficient animals

Animals

Induction of OA

MicroCT

Assessment of cartilage damage

Dynamic weight bearing

Serum assays

Mineralization assay

Bone marrow monocyte quantification

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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