Material and methods
Patients
The clinical trial DRKS00023283 (http://apps.who.int/trialsearch/) aimed to clarify whether different patterns of storage of explanted skull flaps after DC, in particular storage at different degrees of ℃, may prognosticate late complications after cranioplasty, among others infection of the skull flaps implants. To this end, we analyzed explanted skull flaps of only deceased adult patients, who underwent a DC in our Department between June 2019 and October 2020 for an acute malignant brain swelling. The trial has been approved by the local ethic committee of University of Muenster (ethic votum: 2020-340-f-S). Informed consent for inclusion in the research and publication was obtained by their legal representatives.
We included in our assays explanted skull flaps of 17 patients, 8 skulls were preserved at − 23 °C (group A), whereas 9 at − 80 °C. In addition, we assessed another group C, which comprised 2 sterile CAD (1 vancomycin-soaked). Specific parameters of demographics, surgical procedure and storage, i.e. age (years), sex (m/w), cause of malignant brain swelling (stroke vs. sTBI), additional skull fracture (yes/no), infection prior to DC (yes/no), duration of DC (minutes), duration (months) and temperature of cryostorage (23 °C vs. − 80 °C) have been included as potential prognostic factors in our statistical analysis (Fig. 1). As endpoints of our study were defined (1) the identification of microorganisms in the bacterial cultures (yes/no) and (2) in case of growth, the patterns of the colonies (Fig. 2) of microorganisms, f.i. concentration of microorganism.
Material collection, storage and microbiological analysis
The bone flaps were collected during DC, sterile packed in triple plastic bags and stored in a medical freezer at a temperature of either − 23 °C (DCs between June and November 2019) or − 80 °C (DCs between December 2019 and October 2020). For the purposes of the microbiological assays, the skulls were thawed at room temperature for 2 h using strictly aseptic technology. The bone flaps were then crushed with a hammer and bone rongeur forceps; five of the resulting centrally located bone fragments (cortex and cancellous bone, sized approximately 0.8 × 2 cm) of each skull flap, have been processed for further microbiological investigations. Two of these fragments were used for aerobic and anaerobic cultures, respectively, according to quality standards of microbiological diagnostic [16].
For the purposes of the aerobic microbiological analysis each bone fragment was rolled out onto chocolate agar (Chocolate PolyViteX Agar, bioMérieux, Marcy l’Étoile, France) and blood agar (Columbia agar with 5% sheep blood, BD, Heidelberg) and at least put in liquid brain heart infusion (BBL™ Brain Heart Infusion, BD, Heidelberg) for enrichment. By analogy, anaerobic culture was performed with an anaerobic blood agar plate (Schaedler agar with 5% sheep blood, BD, Heidelberg) and liquid thioglycolate medium (BBL™ Enriched Thioglycollate Medium with vitamin K1 & Hemin, BD, Heidelberg) for enrichment. The inoculated media were incubated for 14 days at 35 °C ambient air plus 5% CO2. All media were assessed for growth at 48 h, 7 and 14 days. In case of positive cultures, extent of growth was quantified in a semiquantitative manner by categorizing as light, moderate or heavy growth. If solid media showed no growth after 14 days of incubation, subcultures from the liquid enrichment media were performed on blood agar or anaerobic blood agar, respectively, followed by an incubation period of three days under conditions described above. MALDI-TOF MS was employed for bacterial identification.
In a second step, contamination experiments were performed with the remaining three of the five fragments of each skull flap (group A and B) as well as the three fragments of each of the CAD skulls (group C). Each of the bone and CAD fragments were transferred to a suspension of the reference strains Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC12228, and Cutibacterium acnes ATCC 6919 at a concentration of 103 colony forming unit (CFU)/ml in PBS and then stored in a refrigerator overnight at 5 °C.
The following morning, the contaminated fragments were wiped dry on a sterile gauze paid. Subsequently, S. aureus- and S. epidermidis-contaminated fragments were rolled out onto chocolate blood agar plates, whereas C. acnes-contaminated fragment onto anaerobic blood agar plate. Each fragment was then placed in thioglycolate medium. Culture conditions were chosen and procedures were conducted as described above.
Standard statistical methods were used for the comparisons between the subgroup. Additionally, we used a linear mixed model to account for repeated measurements. P-values < 0.05 were considered as statistically significant.