D. Viability and inflammatory marker expression of chondrocytes

It is important to evaluate the viability of the cells under induced stresses. Assessment in our study revealed ratios of live to dead cells of 77% to 23% for NC, 74% to 26% for IL1β, and 84% to 16% for LPS, respectively [Figs. 6(a)–6(c)]. This result was also confirmed with the presto blue viability assay where induced groups had similar viability as NC, though nonsignificant, LPS-treated cells have the highest quantified viability [Fig. 6(d)].Pro-inflammatory expressions of PGE2 and NO are seen to be major contributors to OA.49,5049. S. E. Martinez, Y. Chen, E. A. Ho, S. A. Martinez, and N. M. Davies, Can J. Vet. Res. 79, 241 (2015).50. S. B. Abramson, M. Attur, A. R. Amin, and R. Clancy, Curr. Rheumatol. Rep. 3, 535 (2001). https://doi.org/10.1007/s11926-001-0069-3 To evaluate the effects of our induced inflammatory mediators on cells’ production of key inflammatory markers, we measured NO, ROS, PGE2, and MMP3 contents. Nitric oxide (NO) is an inflammatory mediator formed from inducible NO synthase enzymes, which gets stimulated by inflammatory cytokines.51,5251. Z. Zhuang, G. Ye, and B. Huang, Med. Sci. Monit. 23, 3925 (2017). https://doi.org/10.12659/MSM.90249152. J. N. Sharma, A. Al-Omran, and S. S. Parvathy, Inflammopharmacology 15, 252 (2007). https://doi.org/10.1007/s10787-007-0013-x Our results indicated that the NO content was slightly insignificantly higher in induced groups compared to NC by an average 1.04-fold [Fig. 7(a)]. IL1β group had the slightly highest insignificant NO content by 1.06-fold and 1.03-fold compared to NC and LPS, respectively [Fig. 7(a)]. LPS induction resulted in slightly insignificant higher NO content compared to NC by 1.02-fold. While the values in the induced groups were not considerably higher than those estimated for the NC group, we expect that a longer culture time would have stimulated and shown higher NO content especially as we saw higher PGE2’s expression with induction [Fig. 7(b)]. PGE2’s levels were significantly higher in IL1β-treated cells by 5.7-fold compared to NC and 9.4-fold compared to LPS-treated cells. Interestingly, PGE2’s levels in the NC group were higher than those quantified for the LPS group by 1.65-fold [Fig. 7(b)]. When ROS was concerned, the induced inflammation by LPS resulted in a similar content to that of NC [Fig. 7(c), Table I]. In comparison, the ROS content for the IL1β group was less than that quantified for the NC group by 1.07-fold. Interestingly, it has been found that chondrocytes upon in vitro monolayer culture expressed increasingly higher ROS from day 0 to day 14 under stress-free conditions.5353. H. K. Heywood and D. A. Lee, Biochem. Biophys. Res. Commun. 373, 224 (2008). https://doi.org/10.1016/j.bbrc.2008.06.011 As such and based on our data, we can assume that the chondrocytes’ responses to induction in the form of expressions of inflammatory markers would show up later in culture. MMP3 is an ECM degrading enzyme that reduces the production of proteoglycans, laminin, and collagens (types 3, 4, and 9).5454. K. Nganvongpanit et al., J. Orthop. Surg. Res. 4, 1 (2009). https://doi.org/10.1186/1749-799X-4-45 MMP3 was significantly highest in the NC group by 1.76-fold compared to IL1β and was not detected in the LPS group [Fig. 7(d)]. While the homeostasis cycle of maintaining the tissue consists of the presence of both catabolic proteases such as MMPs and anabolic processes,4,554. J. E. Woodell-May and S. D. Sommerfeld, J. Orthop. Res. 38, 253 (2020). https://doi.org/10.1002/jor.2445755. C. Scotti et al., Tissue Eng. Part B Rev. 22, 149 (2016). https://doi.org/10.1089/ten.teb.2015.0297 the evident MMP3 in the NC group compared to induced inflammation is interesting. Autophagy can be used as a protective mechanism by chondrocytes and has been shown to have an increase in ROS and MMP activity.5656. L.-B. Jiang, S. Lee, Y. Wang, Q.-T. Xu, D.-H. Meng, and J. Zhang, Osteoarthr. Cartil. 24, 1071 (2016). https://doi.org/10.1016/j.joca.2015.12.021 Our data suggest the possibility of LPS having a chondroprotective role in reducing MMP activity. Furthermore, MMPs such as MMP13 are typically upregulated in later stages of osteoarthritis.55,5755. C. Scotti et al., Tissue Eng. Part B Rev. 22, 149 (2016). https://doi.org/10.1089/ten.teb.2015.029757. M. B. Goldring and K. B. Marcu, Arthritis Res. Ther. 11, 224 (2009). https://doi.org/10.1186/ar2592 This can possibly explain why we did not see an increase in MMP 3 in our study as we only induced cells for 24 h [Fig. 7(d)].