The microbiome does not influence cartilage degradation following joint destabilisation in mice when performed under stringently controlled conditions

Abstract

Purpose: The influence of gut bacteria in the modulation of disease severity of OA is poorly understood. Reports to date on the role of the microbiome in germ free (GF) models of OA are conflicting and haven’t always been stringently controlled. In the present study, surgical induction of OA by destabilisation of the medial meniscus (DMM) was used to examine the potential influence of the microbiome on cartilage damage severity. Methods: Male C57BL/6J mice used in the present study were bred and housed in the gnotobiotic facility at the Kennedy Institute. Microbiome analysis by 16S rRNA amplicon sequencing was performed on stool samples from mice prior to DMM surgery. At 10 weeks of age, mice were randomly selected for sham (n=6-8) or DMM (n=17-20) surgery, which was performed in sterile Class II cabinets. Knees from operated mice were harvested at 8 or 12wks post DMM. Faecal samples were collected post surgery and at the end of the experiment to test GF status. Joints were sectioned and severity of cartilage degradation was assessed in a blinded method according to OARSI guidelines. No mice were excluded from data analysis. Differences were analysed by ANOVA with Tukey post test correction and P values less than 0.05 were considered statistically significant. Control (specific pathogen free, SPF) male C57BL/6J mice (originally derived from the GF founders) were bred under SPF conditions but housed in an environment to mimic the GF conditions in terms of bedding, food (irradiated) and water, and underwent sham (n=4-6) or DMM (n=11-13) surgery at 10 weeks for analysis after 8 and 12wks. Results: All mice were confirmed GF at the time of surgery but 50% tested positive for microbes at the end of the surgical procedure suggesting contamination at the time of surgery (indicated in red dots). Proteoglycan loss and cartilage erosion were evident in SPF and GF mice 12 wks after surgery. Cartilage damage following DMM was significantly higher than sham in both groups, but was not different between GF and SPF mice. Cartilage damage seen at 8wks was modest and only reached significance over sham-operated in GF mice. No significant differences in scores between the GF and SPF DMM groups were demonstrated. No apparent relationship between those animals that became contaminated at the time of surgery and disease scores was seen. Conclusions: The present study shows that significant cartilage degradation occurs in GF mice undergoing DMM at both timepoints. The disease severity was not altered by the absence of a microbiome, nor apparently when mice lost their GF status during the experimental course. When SPF mice were housed under tightly matched cage conditions to the GF mice, disease appeared indistinguishable. The absence of a gut microbiome does not appear to protect the cartilage from erosion after joint destabilisation.