Influence of physico-chemical material characteristics on staphylococcal biofilm formation – A qualitative and quantitative in vitro analysis of five different calcium phosphate bone grafts

Abstract

Various compositions of synthetic calcium phosphates (CaP) have been proposed and their use has considerably increased over the past decades. Besides differences in physicochemical properties, resorption and osseointegration, artificial CaP bone graft might differ in their resistance against biofilm formation. We investigated standardised cylinders of 5 different CaP bone grafts (cyclOS, chronOS (both β-TCP (tricalcium phosphate)), dicalcium phosphate (DCP), calcium-deficient hydroxyapatite (CDHA) and α-TCP). Various physico-chemical characterisations e.g., geometrical density, porosity, and specific surface area were investigated. Biofilm formation was carried out in tryptic soy broth (TSB) and human serum (SE) using Staphylococcus aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984). The amount of biofilm was analysed by an established protocol using sonication and microcalorimetry. Physico-chemical characterisation showed marked differences concerning macro-and micropore size, specific surface area and porosity accessible to bacteria between the 5 scaffolds. Biofilm formation was found on all scaffolds and was comparable for α-TCP, chronOS, CDHA and DCP at corresponding time points when the scaffolds were incubated with the same germ and/or growth media, but much lower for cyclOS. This is peculiar because cyclOS had an intermediate porosity, mean pore size, specific surface area, and porosity accessible to bacteria. Our results suggest that biofilm formation is not influenced by a single physico-chemical parameter alone but is a multi-step process influenced by several factors in parallel. Transfer from in vitro data to clinical situations is difficult; thus, advocating the use of cyclOS scaffolds over the four other CaP bone grafts in clinical situations with a high risk of infection cannot be clearly supported based on our data.

Microcalorimetric detection of staphylococcal biofilm growth on various prosthetic biomaterials after exposure to daptomycin

“…The biofilm evaluations were performed according to a well‐established protocol including sonication, culture, and microcalorimetric heat flow measurement previously used to analyze the influence of physicochemical biomaterial properties, as well as studies of antimicrobial susceptibility of biofilm bacteria …”

“…TTD‐50 was defined as the time from insertion of an ampoule into a microcalorimeter until exponential growth produced a heat flow value of 50 µW. This initial lag phase exceeds the experimental threshold of 10 µW in order to take into account the heterogeneous starting point of those experiments that already produced high amounts of heat flow when measurements started after the equilibration period …”

“…The time to detection (TTD‐50) was defined as the time from the insertion of the ampoule into the calorimeter until the exponentially rising heat flow signal exceeding 50 μW. This measure indirectly quantifies the amount of biofilm bacteria, with a shorter TTD‐50 representing a larger amount of bacteria . The maximum heat flow during the experiment, heat flow peak (HFP), is specific to the strain and reduced with decreasing biofilm fitness due to growth inhibition factors, such as antibiotic exposure …”

“…Isothermal microcalorimetry and sonication were recently used to analyze the influence of bone graft material properties on the initial adhesion and formation of biofilm under in vitro and in vivo settings . Microcalorimetry is a highly sensitive and accurate method for the detection of slow‐growing microorganisms and reduced bacterial numbers after antibiotic pre‐exposure .…”

Staphylococcal biofilm formation on the surface of three different calcium phosphate bone grafts: a qualitative and quantitative in vivo analysis

“…Their importance and use have considerably increased over the past decades [ 19 ]. Besides differences in physico-chemical properties, resorption and osseointegration, artificial bone grafts differ in vitro in case of staphylococcal colonization and biofilm formation [ 11 , 20 ]. As there is an increasing use of these bone substitutes, infections associated with these devices may also increase.…”