*4.12. RNA Isolation and Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)*

For gene expression analysis, MSC RNA was extracted using TRizol (Invitrogen, Carlsbad, CA, USA) reagent following manufacturer's instructions, quantified by NanoDrop ND-1000 (ThermoFisher Scientific, Waltham, MA, USA) and integrity assessed by agarose electrophoresis. RNA (900 ng of total RNA) was digested with TURBO DNA-free Kit (Life Technologies, Carlsbad, CA, USA), according to manufacturer's protocol, and complementary DNA (cDNA) was synthesized with random hexamers (Life Technologies, Carlsbad, CA, USA) and dNTPs using SuperScript III Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA). RT-qPCR was performed in an iQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Irvine, CA, USA) using cDNA, gene specific primers for osteogenic and chondrogenic differentiation markers (Table 1), and iQ SYBR Green Supermix (Bio-Rad Laboratories, Irvine, CA, USA). Relative gene expression was calculated using the 2−ΔCT method and normalized with the GAPDH reference gene, considering threshold cycles <35.


**Table 1.** Sequences of primers used for gene expression analysis by RT-qPCR.

### *4.13. Statistical Analysis*

Data analysis were performed using GraphPad Prism v7.0 software. Normality distribution of data was tested by D'Agostino and Pearson normality test. For weight, paw swelling measurements and arthritis score, 2-way ANOVA followed by Tukey's multiple comparisons was used. For non-parametric data, when two groups were compared, Mann–Whitney test was performed. To compare multiple unpaired groups, Kruskal–Wallis test was used, followed by uncorrected Dunn's multiple comparison test. To compare multiple paired groups, Friedman test followed by uncorrected Dunn's multiple comparisons was performed. Statistical significance was considered whenever \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/21/ 5436/s1.

**Author Contributions:** J.H.T., M.A.B. and S.G.S. planned all the experiments. J.H.T., A.M.S., M.I.A., M.B.-G., C.C. and S.G.S. performed animal experiments and sample collection. J.H.T., A.M.S., S.G.S. executed experiments, acquired and analyzed the data. J.H.T. and S.G.S. wrote the main manuscript text. J.H.T., A.M.S., M.I.A., M.B.-G., C.C., M.A.B. and S.G.S. discussed and approved the manuscript.

**Funding:** This research was funded by the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and AO Foundation-Switzerland (project S-15-83S). J.H.T, A.M.S, M.B.G, M.I.A and C.C were supported by FCT-Fundação para a Ciência e a Tecnologia, through the fellowships SFRH/BD/112832/2015, SFRH/BD/85968/2012, PD/BD/135489/2018, DL 57/2016/CP1360/CT0008 and DL 57/2016/CP1360/CT0004, respectively.

**Acknowledgments:** Authors thank to Sofia Lamas for the guidance on the animal welfare and support with animal experiments (Animal facility, i3S); Catarina Meireles for the help on flow cytometry analysis (Translational Cytometry Unit, i3S) and Cláudia Ribeiro-Machado for the support with the histological procedures and analysis. **Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
