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Sane et al. Microbiome Res Rep 2023;2:18 https://dx.doi.org/10.20517/mrr.2023.12 Page 3 of 12

using a 100 kDa cut-off membrane to remove the broth proteins. The resulting retentate was collected and
diafiltrated extensively against sterile water. The residual broth proteins were looked for by subjecting the
lyophilized retentate to gel filtration chromatography on a Superdex200® column (Sigma-Aldrich, St
[3]
Quentin Fallavier, France) . The bifidobacterial Lpps peak corresponded to 90% of the lyophilized powder.

Medium Aggregate Extract (MAE) used as a control was produced as follows: the unfermented broth was
acidified at pH 4.8 by adding a mix of 2M Lactic/ 3M Acetic acid, then incubated 48 h at 37 °C in anaerobic
conditions. The acidified broth supernatant was collected by high-speed centrifugation, and then
concentrated as described above.

OA induction
Experiments were carried out in accordance with the EU directive 2010/63/EU and approved by the Ethical
Committee N°120 (n° 4160-201602171657248). Male Crl:CD (SD) rats were purchased from Charles River
(Saint Germain Nuelles, France). The colony of rats was originally started from foundation colony rats that
had only eight species of bacteria, the Charles River Altered Schaedler Flora and later bred in SPF housing
[14]
facility . The 8 species are: Clostridium sp (ASF 356), Lactobacillus intestinalis (ASF360), Ligilactobacillus
murinus (ASF 361), Mucispirillum schaedlerii (ASF 457), Eubacterium plexicaudatum (ASF492),
Pseudoflavonifractor sp (ASF 500), Schaedlerella arabinosiphila (ASF502), Parabacteroides goldsteinii
(ASF519).

Experimental OA was induced in rats weighing 125-150g with a single intra-articular injection of MIA
(3 mg/50 uL, Day 0) into the right hind knee joint. The sham group received 50 μL of sterile PBS into the
right hind knee joint. Lameness recovery occurred within 7-8 days. Rats were then transferred to the SPF
housing facility. They were acclimated to the new environment seven days before the study’s
commencement. They were housed in type IV cages (2/cage), with ambient temperature 20-25 °C, a 12:12
light/dark cycle (lights on at 0800 h) and access to food (sterile RO3-40 diet-Safe-Diets, Augy, France) and
sterile water during the dark period.

To monitor Lpps or water intake, rats were isolated at 1800 h and placed individually into a new
experimental Plexiglas cage with a background shelf. Bottles with a certain volume of 0.3 mg/L or water
were placed each evening in the feeders, the remaining volumes being recorded the next morning. The
treated group received the Lpps solution 5 nights per week for 6 weeks. The procedure was applied from
D15 after surgery until the end of the study.

Rats were weighed daily. Energy intake was estimated daily by weighing the pellets left in the feeders each
morning. Animals were euthanized at the end of the study (8-9 weeks post-surgery), with intraperitoneal
injections of sodium pentobarbital (CEVA santé animal, Libourne, France).

Macroscopic and histopathological scoring
Whole knee joints from both hind paws of rats were dissected. Cutaneous and subcutaneous tissues were
removed to expose the musculotendinous structure and patella for macroscopic staging. Macroscopic
scoring was performed as follows: 0 no damage (no morphological differences as compared with the left
untreated paw), 1 moderate damage, 2 severe damages (obvious joint degeneration and inflammation
characterized by hyperplasia of synovial membrane and swelling of articular joint). Specimens were fixed
immediately in 7.5 % formaldehyde and then decalcified by immersion in Osteomoll® solution (Merck,
Molsheim, France) for 7 days. After dehydration by a series of ethanol immersions, samples were embedded
in paraffin and then frontally sectioned. Glass slides were mounted with 10 µm tissue sections.