A Human Osteocyte Cell Line Model for Studying Staphylococcus aureus Persistence in Osteomyelitis

Abstract

Infectious osteomyelitis associated with periprosthetic joint infections is often recalcitrant to treatment and has a high rate of recurrence. In the case of Staphylococcus aureus, the most common pathogen in all forms of osteomyelitis, this may be attributed in part to residual intracellular infection of host cells, yet this is not generally considered in the treatment strategy. Osteocytes represent a unique cell type in this context due to their abundance, their formation of a syncytium throughout the bone that could facilitate bacterial spread and their relative inaccessibility to professional immune cells. As such, there is potential value in studying the host-pathogen interactions in the context of this cell type in a replicable and scalable in vitro model. Here, we examined the utility of the human osteosarcoma cell line SaOS2 differentiated to an osteocyte-like stage (SaOS2-OY) as an intracellular infection model for S. aureus. We demonstrate that S. aureus is capable of generating stable intracellular infections in SaOS2-OY cells but not in undifferentiated, osteoblast-like SaOS2 cells (SaOS2-OB). In SaOS2-OY cells, S. aureus transitioned towards a quasi-dormant small colony variant (SCV) growth phenotype over a 15-day post-infection period. The infected cells exhibited changes in the expression of key immunomodulatory mediators that are consistent with the infection response of primary osteocytes. Thus, SaOS2-OY is an appropriate cell line model that may be predictive of the interactions between S. aureus and human osteocytes, and this will be useful for studying mechanisms of persistence and for testing the efficacy of potential antimicrobial strategies.

Osteocytes Serve as a Reservoir for Intracellular Persisting Staphylococcus aureus Due to the Lack of Defense Mechanisms

“…Higher intracellular bacteria counts were found in osteocytes compared to osteoblasts. These results are in line with other studies that propose osteocytes as a niche for staphylococcal persistence (Muthukrishnan et al, 2019;Gunn et al, 2021). Staphylococcal persistence is related to a switch to a slowgrowing, metabolically inactive phenotype called small colony variants (SCVs; Sendi et al, 2006;Tuchscherr et al, 2011).…”

“…However, no differences were observed with low doses. By contrast, a recent publication has shown that osteoblasts were more susceptible to S. aureus infection than osteocytes (Gunn et al, 2021). This possible discrepancy may be related to the staphylococcal strains and cells used in both studies.…”

“…The isolation of human osteocyte from human trabecular bone has been described (Prideaux et al, 2016); but the number of harvested cells is very low and not sufficient to be used in larger experimental series. Recently, a new model of a human osteocyte cell line with SaO-2 cells was described for studying S. aureus persistence (Gunn et al, 2021). In this study, the SaO-2 cells were cultivated for 28 days to obtain osteocytes-like cells.…”

“…In this study, the SaO-2 cells were cultivated for 28 days to obtain osteocytes-like cells. However, these cells lack the expression of E11, one of the osteocytes markers, and express a phenotype more related to the transition from osteoblasts to osteocytes (Prideaux et al, 2014;Gunn et al, 2021). To mimic the human in vivo situation best, 3D cultures systems of human primary osteocytes in combination with osteoblast and osteoclast have been developed (Bernhardt et al, 2020) and these systems might be suitable for further investigations of bacterial persistence in bone cells.…”

“…Although different studies have described that S. aureus can survive for long periods within osteoblasts (Kalinka et al, 2014;Ji et al, 2020;Bongiorno et al, 2021;Gunn et al, 2021;Marro et al, 2021), the mechanisms that may explain the persistence within bone cells are still unknown. In addition to osteoblasts, several studies have revealed osteocytes as possible niche for bacteria within bone tissue (De Mesy Bentley et al, 2018;Yang et al, 2018;Krauss et al, 2019;Masters et al, 2021).…”