Staphylococcus aureus biofilms decrease osteoblast viability, inhibits osteogenic differentiation, and increases bone resorption in vitro

Abstract

BackgroundOsteomyelitis is a severe and often debilitating disease characterized by inflammatory destruction of bone. Despite treatment, chronic infection often develops which is associated with increased rates of treatment failure, delayed osseous-union, and extremity amputation. Within affected bone, bacteria exist as biofilms, however the impact of biofilms on osteoblasts during disease are unknown. Herein, we evaluated the effect of S. aureus biofilms on osteoblast viability, osteogenic potential, and the expression of the pro-osteoclast factor, receptor activator of NF-kB ligand (RANK-L).MethodsOsteoblasts were exposed to biofilm conditioned media (BCM) from clinical wound isolates of Staphylococcus aureus under normal growth and osteogenic conditions to assess cellular viability and osteoblast differentiation, respectively. Cell viability was evaluated using a live/dead assay and by quantifying total cellular DNA at days 0, 1, 3, 5, and 7. Apoptosis following treatment with BCM was measured by flow-cytometry using the annexin V-FITC/PI apoptosis kit. Osteogenic differentiation was assessed by measuring alkaline phosphatase activity and intracellular accumulation of calcium and osteocalcin for up to 21 days following exposure to BCM. Expression of genes involved in osteogenic differentiation and osteoclast regulation, were also evaluated by quantitative real-time PCR.ResultsBCM from clinical strains of S. aureus reduced osteoblast viability which was accompanied by an increase in apoptosis. Osteogenic differentiation was significantly inhibited following treatment with BCM as indicated by decreased alkaline phosphatase activity, decreased intracellular accumulation of calcium and inorganic phosphate, as well as reduced expression of transcription factors and genes involved in bone mineralization in viable cells. Importantly, exposure of osteoblasts to BCM resulted in up-regulated expression of RANK-L and increase in the RANK-L/OPG ratio compared to the untreated controls.ConclusionsTogether these studies suggest that soluble factors produced by S. aureus biofilms may contribute to bone loss during chronic osteomyelitis simultaneously by: (1) reducing osteoblast viability and osteogenic potential thereby limiting new bone growth and (2) promoting bone resorption through increased expression of RANK-L by osteoblasts. To our knowledge these are the first studies to demonstrate the impact of staphylococcal biofilms on osteoblast function, and provide an enhanced understanding of the pathogenic role of staphylococcal biofilms during osteomyelitis.

Inflammatory osteolysis: a conspiracy against bone

“…The cell wall component peptidoglycan stimulates TLR2 in a NOD2-dependent manner, thereby inducing RANKL expression by osteoblasts. Concordant OPG remains unchanged (159,162,163) or decreases (164), contributing to osteoclast recruitment and osteolysis. Bone loss likely also occurs through reduced osteoblast viability as well as reduced activity induced by substances released from staphylococcal biofilms (163,164).…”

Effects of Local Antibiotic Delivery from Porous Space Maintainers on Infection Clearance and Induction of an Osteogenic Membrane in an Infected Bone Defect

“…35,36 However, it is also possible that analysis of the membrane does not fully represent the profile of RANKL expression. Previous in vitro work in human and murine osteoblasts has shown that RANKL is upregulated in response to S. aureus infection, 18,19,37 and other studies have indicated that osteocytes, as opposed to osteoblasts or bone marrow stromal cells, are the dominant source of RANKL. 38 Because the objective of this study was to analyze mRNA expression profiles of cells in the induced membrane, the study primarily measures RANKL and OPG production by lymphocytes and stromal cells present in the induced membrane.…”

“…This corroborates the finding in several in vitro studies that have shown that the presence of infection reduces, but does not completely inhibit, the differentiation of MSCs, likely through the effects of soluble factors secreted by the S. aureus biofilm. 17,18 Main effect analysis for BMP-5 expression indicates that infection is an independent negative factor in the production of BMP-5, suggesting that prophylactic treatment with clindamycin could be beneficial from the perspective of infection treatment and restoration of growth factor expression. In addition, clindamycin treated defects, with or without infection, also retain similar VEGF expression to the material control, further demonstrating that defects treated at the time of initial surgery are able to maintain the ability to generate membranes with appropriate growth factor expression.…”

“…14,15 Staphylococcus aureus is the most common infecting agent in long bone osteomyelitis and is known to have deleterious effects on local cell populations. [16][17][18] In vitro studies have shown the effects of S. aureus on local cells to be far reaching, such as decreasing osteoblast viability and differentiation, inhibiting the multilineage potential of mesenchymal stem cells (MSCs), increasing the production of resorptive factors and increasing the production of proinflammatory cytokines. [17][18][19] These effects are not conducive to the regeneration of bone, and thus, local antibiotic delivery is intended to eradicate bacteria to restore the regenerative capacity of local cell types.…”

“…[16][17][18] In vitro studies have shown the effects of S. aureus on local cells to be far reaching, such as decreasing osteoblast viability and differentiation, inhibiting the multilineage potential of mesenchymal stem cells (MSCs), increasing the production of resorptive factors and increasing the production of proinflammatory cytokines. [17][18][19] These effects are not conducive to the regeneration of bone, and thus, local antibiotic delivery is intended to eradicate bacteria to restore the regenerative capacity of local cell types. A study of the characteristics of an induced membrane generated in the presence of antibioticreleasing solid PMMA has demonstrated that antibiotics may also affect the thickness and vascularization of induced membranes in the absence of infection.…”