title: Staphylococcus aureus biofilms decrease osteoblast viability, inhibits osteogenic differentiation, and increases bone resorption in vitro.
abstract: BACKGROUND
  Osteomyelitis 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).
  
  
  METHODS
  Osteoblasts 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.
  
  
  RESULTS
  BCM 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.
  
  
  CONCLUSIONS
  Together 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.
authors.affiliation: Department of Extremity Trauma and Regenerative Medicine, United States Army Institute of Surgical Research, Ft, Sam Houston, San Antonio, TX, USA. carlos.j.sanchez3@us.army.mil
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: Sanchez
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Ward
authors.forename: Desiree R
authors.initials: DR
authors.lastname: Romano
authors.forename: Brady J
authors.initials: BJ
authors.lastname: Hurtgen
authors.forename: Sharanda K
authors.initials: SK
authors.lastname: Hardy
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Woodbury
authors.forename: Alex V
authors.initials: AV
authors.lastname: Trevino
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Rathbone
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Wenke
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D017209:Apoptosis; D018441:Biofilms / Q000254:growth & development*; D015415:Biomarkers / Q000378:metabolism; D001862:Bone Resorption / Q000382:microbiology*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D054884:Host-Pathogen Interactions; D006801:Humans; D008855:Microscopy, Electron, Scanning; D010006:Osteoblasts / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D015675:Osteocalcin / Q000378:metabolism; D010010:Osteoclasts / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D010012:Osteogenesis / Q000502:physiology*; D013211:Staphylococcus aureus / Q000502:physiology* / Q000648:ultrastructure
chemical_list: D015415:Biomarkers; D015675:Osteocalcin; D000469:Alkaline Phosphatase