Mc170 camera

The migration of native HPCs and ASCs was assessed using the scratch wound healing assay. The cells were seeded onto a 12-well plate and cultured until fully confluent, then a 10 μL pipette tip was used to create the scratch wound. The cells were then cultured for 48 h, and microphotographs of the wells were taken at 4 time points: 0 h, 6 h, 24 h and 48 h using an inverted Leica DMi1 microscope equipped with a MC170 camera (Leica Microsystems, KAWA.SKA Sp. z o. o., Zalesie Gorne, Poland). After 48 h, the cells were fixed with 4% PFA and stained with a pararosaline solution for the purpose of photographic documentation. The scratch closure was calculated based on the Leica software scale bar (Leica Application Suite- LAS EZ, version 3.4.0), using photographs from 4 wells. To assess the clonogenic potential of native ASCs and HPCs, a colony-forming unit assay was performed as described previously [102 (link),103 (link)]. Briefly, 250 cells were seeded onto a 6-well plate and cultured with medium changes every 3 days. After 8 days, the cells were fixed with a 4% PFA solution and stained with a 2% pararosaline solution (Sigma Aldrich/Merck, Poznan, Poland). The number of colonies was counted, and any cluster of 50 cells or more was regarded as a colony.

Sections were deparaffinized in xylene and rehydrated with decreasing concentrations of ethanol. Antigen retrieval was performed at 60 °C with Tris EDTA buffer (pH 9). Hydrogen peroxide (ab64218; Abcam, Cambridge, UK) blocking solution was used followed by BSA protein block (2.5%) in PBS to block non-specific background staining. Immunohistochemical markers of OA including A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTSs) were detected using rabbit anti-ADAMTS4 (1:100; ab185722; Abcam, Cambridge, UK) and rabbit anti-ADAMTS5 (1:100; ab231595; Abcam, Cambridge, UK) polyclonal antibodies. COLII was detected with rabbit Anti-Collagen II polyclonal (IgG) antibody (1:250; ab34712; Abcam, Cambridge, UK). Slides were incubated with Goat Anti-Mouse (IgG) H&L (HRP) secondary antibody (1:2000; ab205719; Abcam, Cambridge, UK) for 1 h at RT followed by DAB (3,3′Diaminobenzidine) chromogen (ab64238; Abcam, Cambridge, UK). Sections were counterstained with Mayer’s Haemaoxylin (MH51; HT110116; Sigma-Aldrich, MA, USA) mounted with DPX (#06522; Sigma-Aldrich, Burlington, MA, USA) and images were taken with a DMi1 light microscope with MC170 camera (Leica microsystems Inc., Wetzlar, Germany).

Intact joints were cut coronally, histologically processed and paraffin wax embedded. Sections (5 μm) were mounted on Superfrost plus slides (Thermofisher, Waltham, MA, USA) and stained with either Hematoxylin and Eosin (H&E) (MH51; HT110116; Sigma-Aldrich, Burlington, MA, USA) or Toluidine Blue (0.04% in 0.2 M sodium acetate buffer, pH 4.2, 198161-5G, Sigma-Aldrich, Burlington, MA, USA) [80 (link)] and mounted with DPX (#06522; Sigma-Aldrich, Burlington, MA, USA). Images were captured using a DMi1 light microscope with an MC170 camera (Leica Microsystems Inc., Wetzlar, Germany) and scored using the semi-quantitative modified Mankin scoring system [81 (link)] by three blinded observers whose scores were averaged to produce a total histologic score.