Post-traumatic osteoarthritis development is not modified by postnatal chondrocyte deletion of CCN2

CCN2 is a matricellular protein involved in several critical biological processes. In particular, CCN2 is involved in cartilage development and in osteoarthritis. CCN2 null mice exhibit a range of skeletal dysmorphisms, highlighting its importance in regulating matrix formation during development, however its role in adult cartilage remains unclear. The aim of this study was to determine the role of CCN2 in postnatal chondrocytes in models of post-traumatic osteoarthritis (PTOA). CCN2 deletion was induced in articular chondrocytes of male transgenic mice at 8 weeks of age. PTOA was induced in knees either surgically or non-invasively by repetitive mechanical loading at 10 weeks of age. Knee joints were harvested, scanned with micro-CT, and processed for histology. Sections were stained with toluidine blue and scored using the OARSI grading system. In the non-invasive model cartilage lesions were present in the lateral femur but no significant differences were observed between wildtype (WT) and CCN2 knockout (KO) mice 6 weeks post-loading. In the surgical model, severe cartilage degeneration was observed in the medial compartments but no significant differences were observed between WT and CCN2 KO mice at 2, 4, and 8 weeks post-surgery. We conclude that CCN2 deletion in chondrocytes did not modify the development of PTOA in mice, suggesting that chondrocyte expression of CCN2 in adults is not a critical player in protecting cartilage from the degeneration associated with PTOA. Summary Statement Post-natal deletion of CCN2 in chondrocytes does not affect the development of post-traumatic osteoarthritis in mice.


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All work was carried out in accordance with the UK Home Office guidelines and regulations under the 80 Animals (Scientific Procedures) Act 1986. All mice (C57CBA background) were housed in the specific 81 pathogen free biological services unit at the University of Liverpool, UK and housed in cages of up to 82 5 mice, with 12h light/dark cycle, and ad libitum food and drink. Using chondrocyte specific aggrecan 83 (Acan) enhancers, two conditional CCN2 KO mouse models were generated. The first contained an 84 Acan -10kb CreER T2 enhancer as described by Han and Lefebvre (Han and Lefebvre, 2008). This 85 produced Acan -10kb CreER T2 x CCN2 fl/fl mice in which CCN2 was deleted from articular chondrocytes. 86 The second contained an Acan -30kb CreER T2 enhancer described by Li et al (Li et al., 2018). This 87 produced Acan -30kb CreER T2 x CCN2 fl/fl mice where CCN2 was deleted from all chondrocytes. Deletion 88 of CCN2 was regulated using a tamoxifen inducible CreER T2 . 89

Tamoxifen induction of Acan CreER T2 90
Prior to the start of all in vivo experiments, deletion of CCN2 or STOP in reporter td Tomato using Acan 91 CreER T2 was induced at 8 weeks of age using tamoxifen (Sigma Aldrich, UK). All mice, whether WT or 92 CCN2 fl/fl , were administered tamoxifen intraperitoneally at a dose of 1mg/10g body weight on days 1, 93 3, and 5, and were weighed prior to injection on each day. Following tamoxifen injections, mice were 94 left for 1 week before any experimental work commenced. Td tomato mice were sacrificed four weeks 95 after last tamoxifen injection. 96

Non-invasive mechanical loading model of PTOA. 97
Right knees of 10 week-old male Acan -30kb CreER T2 x CCN2 fl/fl (n = 13 (Cre WT ), n = 14 (Cre +/o )) mice 98 were loaded non-invasively, using a model previously described (Poulet et al., 2011) to induce PTOA. 99 Briefly, mice were anaesthetised and the right leg placed in custom-made cups with the knee in 100 flexion. A peak load of 9N was applied for 0.05 seconds, with a rise and fall time of 0.025 seconds, and 101 a baseline hold time of 9.9 seconds for 40 cycles. A baseline load of 2N was employed to keep the tibia 102 in place during peak loading. All mice were subjected to this pattern 3 times per week for 2 weeks. 103 Loading was performed using an ElectroForce 3100 (TA Instruments, USA). Mice were weighed after 104 each loading episode, and on a weekly basis following completion of the loading regimen. All mice 105 were sacrificed 6 weeks post-loading and samples prepared for micro-CT and histological analysis. 106 107 Surgical model of PTOA 108 Left knee joints of 10-week-old Acan -10kb CreER T2 x CCN2 fl/fl mice underwent surgical transection of 109 the medial meniscus (MM) and the medial meniscotibial ligament (MMTL). Mice were induced and 110 maintained under a plane of general anaesthesia using isoflurane during the surgical procedure.. A 111 5 small incision was made over the medial aspect of the patella tendon and the joint capsule incised. 112 Using blunt dissection small amounts of fat were removed allowing for visualisation of the MM and 113 MMTL. Using a scalpel, the MM and MMTL were transected using an upwards motion from the cranial 114 horn of the MM on the proximal tibial plateau. Once transected the joint capsule and the skin were 115 sutured. Mice were immediately transferred to a heated post-operative recovery room. They were 116 monitored daily to ensure they were in good health. Mice were sacrificed 2 weeks post-op (n = 4 117 (Cre WT ), n = 14 (Cre +/o )), 4 weeks post-op (n = 7 (Cre WT ), n = 7 (Cre +/o )), and 8 weeks post-op (n = 7 118 (Cre WT ), n = 8 (Cre +/o )). Samples were prepared for micro-CT and histological analysis. 119

Specimen preparation 120
All animals were sacrificed by cervical dislocation. Experimental and contralateral joints were 121 dissected, immediately fixed in 10% neutral buffered formalin for 24hrs and transferred to 70% 122 ethanol for storage. 123

Micro-CT analysis 124
Experimental and contralateral joints were scanned at a resolution of 4.5µm using a 0.25mm 125 aluminium filter, with a rotation step of 0.6 o (Skyscan 1272, Bruker microCT, Belgium). Image 126 reconstruction was performed using NRecon software (Bruker microCT, Belgium), followed by manual 127 selection of regions of interest for tibial and femoral epiphysis, and joint space (including menisci). 128 Bone volume/tissue volume (BV/TV) and tissue volume (TV) were determined. Data were tested for 129 normality and Student's t-test was used for statistical evaluation with significance set at P<0.05. 130

Histological analysis 131
Samples were decalcified in either 10% ethylenediaminetetraacetic acid (EDTA) (Sigma Aldrich, UK) 132 for 2 weeks or 10% formic acid (Sigma Aldrich, UK) for 1 week. Once decalcified, samples were given 133 a processing number independent of their genotype, processed, paraffin embedded in either the 134 coronal (surgical model) or sagittal plane (loading model), and 6µm sections were taken throughout 135 the entire joint. Sections across the joint at 120µm intervals were stained with toluidine blue/fast 136 green (0.04% in 0.1M sodium acetate buffer, pH 4.0) and cartilage lesion severity graded using the 137 OARSI histopathology initiative scoring method (Glasson et al., 2010). Grading each of the four 138 compartments of the tibio-femoral joint (lateral and medial tibia and femur) throughout the entire 139 joint allowed for the determination of a maximum lesion grade (most severe lesion) for the whole 140 joint and each individual compartment. The mean score, which involved determining the average 141 grade across multiple slides was calculated for each joint and each compartment (Poulet et al., 2011). 142 The summed score was determined by adding together the maximum score of each compartment per 143 6 joint. Osteophyte formation was graded histologically using the scale described by Kamekura  Osteophyte formation was observed at 2 weeks post-surgery in WT and CCN2 KO mice (Fig. 4) and 229 advanced to fully ossified osteophytes at 4-and 8-weeks post-surgery (Fig. 4) This study aimed to determine the postnatal role of CCN2 in articular cartilage during PTOA in two 242 different models. We have found that despite its importance during skeletal development and its 243 increased expression by OA chondrocytes, CCN2 in articular cartilage chondrocytes in adult joints did 244 not play a significant role in preventing PTOA development. 245 A number of studies have focused on the regenerative ability of CCN2 due to its role in cellular 246 proliferation and differentiation (Takigawa et al., 2003, Shimo et al., 2000. Its role in chondrogenesis 247 highlighted its potential as a possible regenerative therapy for the treatment of OA, particularly as 248 exogenously added rCCN2 did not stimulate hypertrophy of chondrocytes in vitro (Nishida et al., 2002). 249 Moreover, treatment of articular cartilage defects with rCCN2 showed repaired cartilage to be 250 structurally similar to healthy articular cartilage (Nishida et al., 2004). A recent study by Tang  new Acan specific CreER T2 system, Acan -30kb CreER T2 with CCN2 fl/fl mice were crossed with tdTomato 276 reporter mouse, which allowed for detection of Cre recombinase activity following tamoxifen injection 277 by visualisation of the tomato fluorescence compared to oil injected control, and confirmed 278 recombination and hence CCN2 deletion, from all articular cartilage chondrocytes. Furthermore, the 279 use of two different Acan-Cre systems to drive Cre-recombinase expression in chondrocytes ensured 280 any responses were independent of Cre activity targeting and were most likely a direct result of the 281 action of CCN2 deletion. 282 In this study, we used two models of PTOA, with different severities of OA progression. The non-283 invasive loading of the knee has previously been shown to induce moderate OA lesions on the lateral 284 femur (Poulet et al., 2011), whereas surgical intervention is known to lead to a higher degree of OA 285 severity. The importance of using different models pertain to the fact that, although similar 286 pathologies can be seen in both models, both may trigger different cellular responses linked to the 287 severity of the disease. For example, the surgical model might trigger more severe inflammatory 288 responses. In addition, the mechanical environment is severely affected in the surgical model 289 throughout the whole study, whereas the traumatic loads are applied at specific and controlled times, 290 with a maximum of 6 episodes of 7 minutes; the rest of the time, the mechanical environment is 291 relatively normal compared to that engendered by surgical instability. In future studies, the lack of 292 effects of chondrocyte specific CCN2 expression in adults could be tested in other models of OA, 293 including ageing and obesity-induced OA. 294 In conclusion, this study showed through the use of two models of trauma-induced OA that CCN2 295 expression by chondrocytes is not required for maintenance of cartilage in adults and that CCN2 296 expression by other tissues within the joint may be more important for any effect to be observed.