Local intra-articular injection of rapamycin delays articular cartilage degeneration in a murine model of osteoarthritis

All studies were performed according to protocols approved by the University of Pittsburgh’s Institutional Animal Care and Use Committee. Forty, 10-week-old, male C57Bl/6 J mice were used in this study. The animals were anesthetized with 3% isoflurane in O₂ gas (1.5 liter/minute). Experimental osteoarthritis was induced by destabilizing the medial meniscus (DMM) in the right knee [13]. Briefly, the joint capsule was opened with an incision just medial to the patellar tendon and the medial meniscotibial ligament was sectioned with microsurgical scissors. As a control, surgery was performed on left knee joints but the ligaments were left intact and used as sham joints. All mice were allowed to move freely within their cages after surgery. After surgical induction of OA, the animals were divided into two groups, namely the rapamycin treatment group and the dimethyl sulphoxide (DMSO) (control) group. Mice were sacrificed at 8 and 12 weeks after DMM surgery and subjected to histological and gene expression analyses.

To perform the intra-articular injection, the animals were first anesthetized and the skin was subsequently incised longitudinally over the center of the knee joint. The capsule and patellar tendon were exposed to clarify the anatomy of the knee to ensure reproducible intra-articular injection of either rapamycin or DMSO (control). Rapamycin was obtained from LC Laboratories (Woburn, MA, USA) and was dissolved in dimethyl sulphoxide (DMSO) to make a 50 mg/ml stock solution. For injection, the stock solution was diluted in PBS: 10 μl of rapamycin (10 μM) with DMSO (0.02%) was administered twice a week for 8 weeks in both knees in the rapamycin group. The dosage and frequency of rapamycin was selected based on previous studies [8],[14]. These studies have demonstrated that autophagy activation by 10 μM rapamycin regulated the changes in the expression of OA-related genes through the modulation of apoptosis and reactive oxygen species (ROS) in human chondrocytes in vitro[8], and the effect of rapamycin on mechanical-injury-induced cell death continued up to 96 hours after treatment in ex vivo study [14]. The control group received intra-articular injections of 10 μl of DMSO (0.02%) in both knees according to the same schedule as the rapamycin group.

Six knee joints from each group were fixed in 10% neutral buffered formalin, decalcified with 10% formic acid, and embedded in paraffin. Coronal histological sections were performed through the joint at 80-μm intervals and stained with toluidine blue, and articular cartilage damage was scored by two observers blinded to sample identity using a scoring system reported by Glasson [15]. In this system, histological scores were measured in four quadrants (medial femoral condyle, medial tibial plateau, lateral femoral condyle, and lateral tibial plateau). The scores are defined as follows: 0: normal; 0.5: loss of toluidine blue without structural changes; 1: small fibrillations without loss of cartilage; 2: vertical clefts extending from the articular surface down to the layer immediately below the superficial tangential zone with some loss of surface lamina; 3: vertical clefts/erosion extending down to the calcified articular cartilage comprising <25% of the quadrant width; 4: vertical clefts/erosion extending down to the articular calcified cartilage comprising 25 to 50% of the quadrant width; 5: vertical clefts/erosion extending down to the calcified articular cartilage comprising 50 to 75% of the quadrant; and 6: vertical clefts/erosion extending down to the calcified articular cartilage comprising >75% of the quadrant width. Ten sections of each knee joint were scored and the final score was expressed as the summed histologic score for each joint. The summed score represents the additive score of each quadrant of the joint on each histologic section through the joint. This method of analysis enabled assessment of the severity of the lesions while also reflecting the surface area of articular cartilage affected with OA lesions.

We performed immunohistochemical staining to detect the expression of phosphor-mammalian target of rapamycin (p-mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) in the articular cartilage. Rabbit polyclonal antibodies to p-mTOR, LC3 (Cell Signaling, Beverly, MA, USA), VEGF, MMP13 (Abcam, Cambridge, MA, USA), and COL10A1 (Abbiotec, San Diego, CA, USA) were used at a 1:100 dilution and incubated overnight at 4°C. Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Molecular Probes, Grand Island, NY, USA) was used at 1:200 dilution as the secondary antibody against the primary antibodies at room temperature for 2 hours. After staining, we evaluated the expression levels in the articular cartilage using Northern Eclipse software (Empix Imaging Inc, Cheektowaga, NY, USA).

Four knee joint specimens from both the control and experimental group were used for qPCR. Total RNA was extracted from cartilage using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) with QIAshredder homogenizers and the RNeasy Mini kit (Qiagen, Hilden, Germanay) according to the manufacturer’s protocol. One microgram of total RNA was used for random hexamer-primed cDNA synthesis using the SuperScript II pre-amplification system (Invitrogen, Carlsbad, CA,USA). Quantitative RT-PCR reactions were performed in triplicate using iQ5 (Bio-Rad, Hercules, CA, USA) with Maxima SYBR Green/ROX qPCR Master Mix (Thermo, Thermo Scientific, Rockford, IL, USA) and 300 nM of each primer. The primers were designed based on the sequences in the GenBank database. The primer pairs used for this study are shown in Table 1. Specificity of the reactions was confirmed by 2.5% agarose gel electrophoresis.

All the data were expressed as the mean ± SD. The Mann-Whitney U-test was used for direct comparisons between the two groups. One-way analysis of variance (ANOVA) or the Kruskal-Wallis test was applied for multiple comparisons between independent groups. Pairwise, multiple comparisons were performed using the Tukey-Kramer or Scheffé post hoc test. Data analyses were performed using PASW Statistics 21 (SPSS, Chicago, IL, USA). Statistical significance was determined at level of P <0.05.

There were no structural changes at the anterior cruciate ligament or meniscus identified in the sham knees that were treated with rapamycin or DMSO. Side effects such as weight loss, skin rashes, delayed wound healing, or diarrhea were not observed after local intra-articular injection of rapamycin. Histological sections demonstrated significantly less articular cartilage degeneration in the experimental group treated with local intra-articular injections of rapamycin at 8 and 12 weeks after induction of OA with DMM surgery compared to the OA-induced mice treated with DMSO. Histological grading showed that the DMSO-treated mice had a loss of proteoglycan staining with articular fibrillation at 8 weeks and losses of hyaline cartilage, proteoglycan staining, and lesions extending into the calcified cartilage at 12 weeks after surgery (Figure 1A). In contrast, rapamycin-treated mice showed a focal loss of proteoglycan staining without severe articular cartilage loss at 8 weeks and lesions with a loss of proteoglycan staining slightly increased 12 weeks after surgery; however, hyaline cartilage was preserved (Figure 1B). The extent of OA was evaluated by scoring specific parameters of OA, and is presented as summed scores (the higher the score the greater the articular cartilage degeneration) (Figure 1C, Table 2). Using the summed OA score, DMSO-treated mice developed OA in a time-dependent manner and had a significantly higher score than the rapamycin-treated mice at 8 and 12 weeks following DMM surgery (Figure 1C, P = 0.001 at 8 weeks and P <0.001 at 12 weeks). The summed OA score in the rapamycin-treated mice at 12 weeks was increased compared to the score at 8 weeks (Figure 1C, P <0.001). These results suggested that the intra-articular injection of rapamycin did not completely prevent, but delayed articular cartilage degradation in the presence of meniscus injury after DMM surgery.

To determine whether the local intra-articular injection of rapamycin modulates mTOR and autophagy, the effects that rapamycin had on phospho-mTOR (p-mTOR) and LC3 expressions were examined. The expression of p-mTOR was increased in DMSO-treated mice at 8 and 12 weeks post DMM surgery compared to DMSO-treated mice undergoing the sham knee operation. Rapamycin treatment suppressed p-mTOR expression in these mice compared to those treated with DMSO at both the 8- and 12-week time points post DMM injury. The expression of p-mTOR in the rapamycin-treated mice was significantly increased at 12 weeks compared to 8 weeks after DMM injury (at 8 weeks DMSO 108.5 ± 13.3, rapamycin 43.0 ± 8.9; at 12 weeks DMSO 273.8 ± 27.0, rapamycin 76.5 ± 14.7) (Figure 2A, B). In contrast, there were more LC3-positive cells in the articular cartilage maintaining proteoglycan staining but fewer in the degenerated articular cartilage of the DMSO-treated mice after DMM surgery. An increase in LC3-positive cells was observed in the rapamycin-treated mice compared to those treated with DMSO at 8 and 12 weeks post DMM surgery. The number of LC3-positive cells in the rapamycin-treated mice decreased significantly at 12 weeks compared to 8 weeks post DMM injury (at 8 weeks DMSO 88.2 ± 13.1, rapamycin 236.0 ± 56.3; at 12 weeks DMSO 46.2 ± 12.8, rapamycin 106.3 ± 27.1) (Figure 2A, C).

As it has been reported that the expression of VEGF is related to the development of OA changes [16], VEGF expression was examined with immunohistochemistry and qPCR (Figure 3). The number of VEGF-positive cells in the articular cartilage was significantly lower in the rapamycin group compared with the DMSO group at 8 and 12 week post DMM injury (at 8 weeks DMSO 143.8 ± 29.6, rapamycin 21.2 ± 8.4, P <0.001 for DMSO versus rapamcyin; at 12 weeks DMSO 275.5 ± 56.9, rapamycin 96.0 ± 27.8, P <0.001 for DMSO versus rapamcyin). The number of VEGF-positive cells in the rapamycin-treated mice increased at 12 weeks compared to 8 weeks post DMM injury (P <0.001 for DMSO at 8 weeks versus 12 weeks) (Figure 3A, B). Similarly, the expression of VEGF by qPCR revealed a significant increase in the DMSO-treated mice at 8 weeks after DMM surgery compared to sham knees (Figure 3C, P <0.001 relative to sham). Rapamycin treatment decreased VEGF expression when compared to the DMSO-treated mice at the 8 and 12 week time points post DMM injury (Figure 3C, P <0.001 at 8 and 12 weeks), suggesting that the local intra-articular injection of rapamycin reduced articular cartilage damage, at least in part, through a reduction in VEGF expression.

To investigate the mechanism behind rapamycin’s ability to prevent experimental OA, subsequent experimentation focused on chondrocyte hypertrophy and consequently the expression of COL10A1 and MMP13 (hypertrophic chondrocyte markers) was examined by immunohistochemistry and qPCR (Figures 4, 5). The number of CoL10A1-positive cells in the articular cartilage was significantly lower in the rapamycin group when compared to the DMSO group at 8 and 12 weeks post DMM injury (at 8 weeks DMSO 88.2 ± 13.1, rapamycin 236.0 ± 56.3, P <0.001 for DMSO versus rapamcyin; at 12 weeks DMSO 46.2 ± 12.8, rapamycin 106.3 ± 27.1, P <0.001 for DMSO versus rapamcyin). The number of CoL10A1-positive cells in the rapamycin-treated mice increased at 12 weeks compared to 8 weeks post DMM injury (P <0.001 for DMSO at 8 weeks versus 12 weeks) (Figure 4A, B). Similarly the expression of COL10A1, as measured by qPCR, was increased in the DMSO-treated mice at 8 and 12 weeks after DMM surgery compared to the sham knees (Figure 4C: P = 0.006 at 8 weeks, P <0.001 at 12 weeks, respectively). Also intra-articular injection of rapamycin significantly decreased the expression of COL10A1 compared to the DMSO-treated mice at 8 and 12 weeks after DMM surgery (Figure 4C: P = 0.001at 8 weeks, P <0.001 at 12 weeks, respectively).

The number of MMP13-positive cells in the cartilage was significantly lower in the rapamycin group compared with the DMSO group at 8 and 12 weeks post DMM injury (at 8 weeks DMSO 198.2 ± 34.6, rapamycin 18.0 ± 5.7, P <0.001 for DMSO versus rapamcyin; at 12 weeks DMSO 249.8 ± 31.1, rapamycin 77.8 ± 10.0, P <0.001 for DMSO versus rapamcyin). The number of MMP13-positive cells in the rapamycin-treated mice increased at 12 weeks compared to 8 weeks post DMM injury (P <0.01 for DMSO at 8 weeks versus 12 weeks) (Figure 5A, B). Rapamycin treatment reduced MMP13 expression, as measured by qPCR, at 8 and 12 weeks after DMM surgery compared to the MMP13 expression in the DMSO-treated mice (Figure 5C, P = 0.026 at 8 weeks, P <0.001 at 12 weeks). These results suggest that mTOR inhibition with rapamycin decreases hypertrophic change in articular cartilage after DMM surgery.