Combination of ADMSCs and chondrocytes reduces hypertrophy and improves the functional properties of osteoarthritic cartilage M.R. Ahmed, A. Mehmood,

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Combination of ADMSCs and chondrocytes reduces hypertrophy and improves the functional properties of osteoarthritic cartilage M.R. Ahmed, A. Mehmood, F.-ur-R. Bhatti, S.N. Khan, S. Riazuddin Osteoarthritis and Cartilage Volume 22, Issue 11, Pages 1894-1901 (November 2014) DOI: 10.1016/j.joca.2014.07.028 Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Assessment of Acan and Col2a1 in various groups at 36 h of treatment. (A) Immunoflourescence analysis of Acan and Col2a1 (green). Blue nuclei indicate 4′,6 diamidino-2-phenylindole (DAPI). Bar ∼100 μm. (B) Gene expression analysis of Acan and Col2a1. (C) Western blot analysis of Acan and Col2a1 protein expression. Osteoarthritis and Cartilage 2014 22, 1894-1901DOI: (10.1016/j.joca.2014.07.028) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Assessment of apoptosis, viability, cell damage and hypertrophy in various groups of oxidative stress induced chondrocytes at 36 h of treatment. (A) Annexin-V staining (red). Blue nuclei indicate DAPI. Bar ∼100 μm. (B) Quantification of percentage of annexin-V positive cells. (C) Viability assay. (D) Analysis of relative expression of PCNA gene. (E) LDH release assay. Semi quantitative gene expression analysis of: (F) Casp3; (G) Col1a1; (H) Col10a1; and (I) Mmp13. Data is presented as mean ± SD. *P ≤ 0.05, **P < 0.01, ***P < 0.001 vs group treated with H2O2. •P ≤ 0.05, ••P < 0.01 vs group ADMSCs treated. ωP ≤ 0.05 vs group treated with chondrocytes. Osteoarthritis and Cartilage 2014 22, 1894-1901DOI: (10.1016/j.joca.2014.07.028) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Assessment of homing of transplanted cells in OA cartilage at 42 days of transplantation. (A) PKH26 positive cells (red) in various groups. Blue indicates DAPI labeled nuclei. Bar ∼100 μm. (B) Percentage of PKH26 positive cells (determined by counting the positive cells from randomly selected 30 images per group). Data is presented as mean ± SD. *P ≤ 0.05, **P < 0.01 vs ADMSCs + Chondrocytes-treated group. Osteoarthritis and Cartilage 2014 22, 1894-1901DOI: (10.1016/j.joca.2014.07.028) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Assessment of proteoglycan contents in various groups at 42 days of transplantation. (A) Safranin-O/Fast Green staining. Bar ∼100 μm. (B) Quantification of percentage of safranin staining. (C) Quantification of OA severity with Mankin's score. Osteoarthritis and Cartilage 2014 22, 1894-1901DOI: (10.1016/j.joca.2014.07.028) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Immunoflourescence expression of Acan and Col2a1 in various groups at 42 days of transplantation. (A) Acan and Col2a1 staining (green) in various groups. Bar ∼100 μm. (B) Quantification of Acan and Col2a1 positive cells. Data is presented as mean ± SD. *P ≤ 0.05, **P < 0.01, ***P < 0.001 vs osteoarthritic group. ••P < 0.01 vs group ADMSCs transplanted. ωP ≤ 0.05, ωωP < 0.01 vs chondrocytes-transplanted group. Osteoarthritis and Cartilage 2014 22, 1894-1901DOI: (10.1016/j.joca.2014.07.028) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Semi-quantitative gene expression analysis of cartilage tissue in different groups at 42 days of transplantation. (A) Acan. (B) Col2a1. (C) Col1a1. (D) Col10a1. (E) Mmp13. (F) Casp3. (G) PCNA. Data is presented as mean ± SD. *P ≤ 0.05, **P < 0.01, ***P < 0.001 vs group osteoarthritic group. •P ≤ 0.05, ••P < 0.01, •••P < 0.001 vs group ADMSCs transplanted. ωP ≤ 0.05, ωωP < 0.01 vs chondrocytes-transplanted group. Osteoarthritis and Cartilage 2014 22, 1894-1901DOI: (10.1016/j.joca.2014.07.028) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions