Journal: bioRxiv

Article Title: Modelling Osteoarthritis pathogenesis through Mechanical Loading in an Osteochondral Unit-on-Chip

doi: 10.1101/2023.08.29.555292

Figure Lengend Snippet: a , Schematic indicating the experimental procedure used for bmMSCs differentiation. bmMSCs were seeded our previously introduced CoC device and cultured statically for 14 days in either in CHM or OCM b , SMToCs mature over time. Gene expression was quantified through RT-qPCR (n=9 biologically independent samples from n=3 donors were analysed for each condition and each cellular type). Statistical significance was determined by Kruskal-Wallis test with Dunn’s post hoc test for multiple comparisons. All genes expression was referred to GAPDH expression. Values are reported as mean + s.d. c , Brightfield pictures of bmMSCs constructs at day 0 and after 14 days of static maturation. Construct appeared black upon exposure to OCM, consistently with the deposition of calcium salts. (n≥30 biologically independent samples from n≥5 donors for each cellular type were considered in analyses). Scale bar, 100 μm. d , Immunofluorescence images bmMSCs constructs cultured in CHM or OCM for 14 days. Day 0 constructs were adopted as negative controls (n≥.1 biologically independent samples from n≥3 donors were considered in analyses). The OCM consistently led to bmMSCs depositing hydroxyapatite (HA). Hyaline cartilage markers ACAN and COL2A1 are represented in red and white respectively. HA is represented in green. The bone markers ALP and OCN are represented in in yellow and magenta. In all images DAPI (represented in blue) was used as nuclear counterstaining. Scale bar, 100 µm. e , Schematic indicating the experimental procedure used to assess if the OCM impaired hACs chondrogenic differentiation capability. hACs were seeded in our previously introduced CoC device and cultured statically for 14 days in either CHM or OCM. f , Brightfield pictures of hACs constructs at day 0 and after 14 days of static maturation. Constructs appeared similar after culture in CHM and OCM. Scale bar, 100 μm. g , Immunofluorescence images hACs constructs cultured in CHM or OCM for 14 days. Day 0 constructs were adopted as negative controls (n≥.1 biologically independent samples from n≥3 donors were considered in analyses). The OCM formulation does not alter hACs chondrogenic potential. Hyaline cartilage markers ACAN and COL2A1 are represented in red and white respectively. HA is represented in green. The bone markers ALP and OCN are represented in in yellow and magenta. In all images DAPI (represented in blue) was used as nuclear counterstaining. Scale bar, 100 µm. h , Assessment of medium formulation and cell type effects on chondrogenic and osteogenic gene expression signature. Gene expression was quantified through RT-qPCR. (n≥9 biologically independent samples from n≥3 donors were analysed for each condition and each cellular type). Statistical significance was determined by Kruskal-Wallis test with Dunn’s post hoc test for multiple comparisons. All genes expression was referred to GAPDH expression. Values are reported as mean + s.d. Passing from HCM to OCM did not imply alteration in hACs and bmMSCs gene expression which retained distinct and defined profiles. For all graphs, populations’ normality was assumed if both Shapiro-Wilk and Kolmogorov-Smirnov tests resulted positive. (Adjusted) P values < 0.05 are reported on the graph.

Article Snippet: Hydrogels were meshed using eight-node linear hexahedral elements with hybrid formulation and trilinear pore pressure (C3D8PH elements: Abaqus), as required for porous materials.

Techniques: Cell Culture, Gene Expression, Quantitative RT-PCR, Expressing, Construct, Immunofluorescence, Formulation