3D Cocultures of Osteoblasts and Staphylococcus aureus on Biomimetic Bone Scaffolds as a Tool to Investigate the Host-Pathogen Interface in Osteomyelitis.

Mattia Loppini,Alberto Mantovani,Barbara Bottazzi,Raffaella Parente,Andrea Doni,Cristina Sobacchi,Anna Tampieri,Elisabetta Campodoni,Monica Sandri,Ciro Menale,Antonio Inforzato,Valentina Possetti,Maria Lucia Schiavone

doi:10.3390/pathogens10070837

Abstract

Osteomyelitis (OM) is an infectious disease of the bone primarily caused by the opportunistic pathogen Staphylococcus aureus (SA). This Gram-positive bacterium has evolved a number of strategies to evade the immune response and subvert bone homeostasis, yet the underlying mechanisms remain poorly understood. OM has been modeled in vitro to challenge pathogenetic hypotheses in controlled conditions, thus providing guidance and support to animal experimentation. In this regard, traditional 2D models of OM inherently lack the spatial complexity of bone architecture. Three-dimensional models of the disease overcome this limitation; however, they poorly reproduce composition and texture of the natural bone. Here, we developed a new 3D model of OM based on cocultures of SA and murine osteoblastic MC3T3-E1 cells on magnesium-doped hydroxyapatite/collagen I (MgHA/Col) scaffolds that closely recapitulate the bone extracellular matrix. In this model, matrix-dependent effects were observed in proliferation, gene transcription, protein expression, and cell–matrix interactions both of the osteoblastic cell line and of bacterium. Additionally, these had distinct metabolic and gene expression profiles, compared to conventional 2D settings, when grown on MgHA/Col scaffolds in separate monocultures. Our study points to MgHA/Col scaffolds as biocompatible and bioactive matrices and provides a novel and close-to-physiology tool to address the pathogenetic mechanisms of OM at the host–pathogen interface.

Highlights

Osteomyelitis (OM) is an inflammatory disease of the bone caused by a wide range of opportunistic pathogens, including the Gram-positive bacteria Staphylococcus aureus (SA), S. epidermidis, Streptococcus pyogenes, and Pseudomonas aeruginosa and the Gram-negativeEscherichia coli [1]
Cells proliferated from day 3 to 6 and no further expansion was observed after day 6, when a stationary growth phase was achieved (Figure 1A)
We found significantly higher mRNA levels of osteoprotegerin (Opg, which together with Rankl is a key mediator of the OB–OC crosstalk and bone homeostasis [34]), bone morphogenetic protein 2 (Bmp2, which plays an important role in inducing the osteogenic differentiation of mesenchymal stem cells [35]), and secreted phosphoprotein 1 (Spp1, coding for osteopontin, a marker of late osteogenic differentiation involved in infection [26,36]), in 3D- versus 2D-cultured cells

Summary

Introduction

Osteomyelitis (OM) is an inflammatory disease of the bone caused by a wide range of opportunistic pathogens, including the Gram-positive bacteria Staphylococcus aureus (SA), S. epidermidis, Streptococcus pyogenes, and Pseudomonas aeruginosa and the Gram-negativeEscherichia coli [1]. In children OM primarily results from the hematogenous spread of the pathogen from distant sites of infection [2], whereby in adults most cases of OM originate from bone contamination during invasive surgical procedures, fracture fixation and arthroplasty [3]. Regardless of how the infectious agent colonizes the bone, a series of pathogenetic events follows that eventually leads to dramatic alterations and a loss of diverse bone compartments, including trabecular and cortical bone, marrow, periosteum, and the surrounding soft tissue [4]. The ubiquitous commensal SA is responsible for up to 75% of OM cases [7], likely due to a remarkable tropism for bone tissue and the exploitation of several strategies of adaptation and resistance [8,9,10,11]. The bone-invasive properties of SA likely arise from a variety of microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that mediate the recognition of and adherence to bone extracellular matrix (BEM) components, like type I collagen, bone sialoprotein, osteopontin, fibronectin, and laminin [12]

Methods

Results

Conclusion