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.
Highlights
Osteomyelitis is a severe and often debilitating disease characterized by inflammatory destruction of bone
As shown in the representative Scanning electron microscopy (SEM) images, mature biofilms of both strains were characterized by large heterogeneous structures composed of accumulations of aggregated bacteria, with few visible planktonic bacteria, confirming that the majority of cells using this model were in a biofilm state (Figure 1A)
Proteomic analysis of the biofilm conditioned media (BCM) from S. aureus SAMMC-700, demonstrated the presence of a heterogeneous mixture of proteins encompassing all aspects of bacterial physiology, including transcription, translation, energy metabolism, pathogenesis, and proteins of unknown function (Figure 1C; Additional file 1)
Summary
Osteomyelitis is a severe and often debilitating disease characterized by inflammatory destruction of bone. Chronic infection often develops which is associated with increased rates of treatment failure, delayed osseous-union, and extremity amputation. Osteomyelitis is a debilitating disease, characterized by the inflammatory destruction of bone and surrounding tissues. Disease is most commonly preceded by hematogenous spread of microorganisms to the bone from either a contiguous infection or directly following trauma. Staphylococcus aureus is the microorganism most commonly associated with hematogenous and post-traumatic osteomyelitis, accounting for more than half of all cases [1]. Despite treatment and surgical intervention up to 30% of osteomyelitis cases progress into a chronic infection [2]. Chronic osteomyelitis is associated with high rates of antimicrobial treatment failure, increased rates of non-osseous union and extremity amputation [1,3]. Over the past decade clinicians have adopted the ‘biofilm theory’ to explain the chronicity of bone infections, recalcitrance to conventional antimicrobial treatment, and incidence of infectious relapse [4,5]
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