Enhanced labelling was performed with streptavidin-peroxidase (VECTASTAIN Top notch ABC kit, Vector Laboratories, Peterborough, UK) and visualised with diaminobenzidine (DAB, ImmPACT, Vector Laboratories, Peterborough, UK), after the sections were dehydrated. vitro and hence could represent a potential cell source to consider for meniscus tissue engineering. Gabapentin enacarbil for 8?min), frozen in liquid N2, and stored at ??80?C temporarily before mRNA extraction. mRNA was extracted using a RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers instructions. A High-Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Warrington UK) was used for reverse transcription. qRT-PCR was performed on the Quant Studio 3 Real-Time Quantitative PCR System (Applied Biosystems) using a SYBR Green Reaction Mix. Gene expression levels of collagen type I (COL1A2), collagen type II (COLIIA1), aggrecan (ACAN), SOX-9 and matrix metalloproteinase-1 (MMP-1) were normalised against the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Qiagen, QuantiTect Primer Assay). The relative gene expression level of each gene was determined by using the comparative CT method42. Chondrogenic differentiation The chondrogenic potency of the three donor-matched cell populations were assessed at passage 2 using a well-established 3D pellet culture system in 6 donors43. Briefly, 2??105 cells were centrifuged into a cell pellet with DMEM F12, P/S (1%), ITS (1%), ascorbic acid (0.1?mM) (Sigma-Aldrich), dexamethasone (10?nM), sodium pyruvate (Sigma-Aldrich) and transforming growth factor -1 (TFG–1, PeproTech, London, UK) (10?ng/ml). After 28?days in culture, n?=?3 pellets were used for biochemical GAG/DNA quantitation, n?=?3 pellets were snap frozen in liquid nitrogen-cooled hexane and stored at ??80?C prior to histological analysis. GAG/DNA analysis Pellets were digested in papain to release GAG and DNA. The papain digestion buffer was composed of 50?mM sodium phosphate (BDH), 20?mM EDTA (Sigma-Aldrich), 20?mM?N-acetyl cysteine (BDH) and adjusted to pH 6.0. Papain was added to the buffer to reach the final concentration of 125?g/ml. Each pellet was digested in 200?l of the papain solution at 60?C for 3?h. Samples were centrifuged at 1000?g for 5?min and stored at -20?C prior to use. The GAG content in pellets was measured by 1,9-dimethylmethylene blue (DMMB) assay44, with chondroitin sulphate (C9819, Sigma-Aldrich) from bovine trachea used to construct Gabapentin enacarbil a standard curve. Briefly, 50?l of each sample was added in duplicate wells of a 96 well plate, with 200?l of DMMB dye. The results were read immediately at A530nm and A590nm. The standard curve was plotted using the following equation: (A530nm/A590nm)???(A530nm blank/A590nm bank). The total GAG content of each pellet was calculated using the standard curve equation. The DNA content was measured spectrofluorometrically using the PicoGreen dsDNA Assay kit (Invitrogen) according to the manufacturers instructions. Finally, the amount of GAG measured in the chondrogenic pellet was normalised to its DNA content. Histological and immuno-histochemical analyses of pellets Three pellets from each cell population were snap frozen in liquid nitrogen and stored at???80?C prior to use. Pellets were sectioned at a 7?m thickness and collected onto poly-l-lysineCcoated slides. Cryosections were stained with TB (BDH) to assess the general tissue morphology and GAG composition of the extracellular matrix. In addition, immunohistochemistry for collagens type I and II Gabapentin enacarbil was undertaken. In brief, sections were incubated with ovine hyaluronidase (4800U/ml, Sigma, UK) prior to fixing in 10% formalin. Primary antibodies raised against collagens type I (1:500, clone I-8H5, MP Biomedicals, Cambridge, UK) or type II (1:50, clone CIIC1, DHSB, University of Iowa, USA) were incubated with sections for 1 h. After washing with PBS, sections were incubated with the secondary biotinylated antibody at 50?g/ml (goat anti-mouse, VECTASTAIN ABC kit, Vector Laboratories, Peterborough, UK), which was added for 30?min. 0.3% hydrogen peroxide in methanol was used to block endogenous peroxidase activity. Enhanced labelling was performed with streptavidin-peroxidase (VECTASTAIN Elite ABC kit, Vector Laboratories, Peterborough, UK) and visualised with diaminobenzidine (DAB, Gabapentin enacarbil ImmPACT, Vector Laboratories, Peterborough, UK), after the sections were dehydrated. The immunochemistry staining intensity of collagen type I and type II Gabapentin enacarbil was quantified using ImageJ Fiji Software (version 1.2; WS Rasband, National Institute of Health, Bethesda, MD)45. Statistical analyses All data were inputted into GraphPad Prism (Version 7.04, Gipc1 USA) and Jamovi (Version 126.96.36.199) for statistical analysis. Differences between cell types were assessed.