Minimum essential medium (mem)

MDV reporter viruses expressing the green fluorescent protein (GFP) under the control of the herpes simplex virus 1 (HSV-1) thymidine kinase promotor were generated based on the very virulent RB-1B field strain (GenBank accession number EF523390) and the vaccine strain CVI988 (DQ530348). GFP was inserted into the bacterial artificial chromosome (BAC) backbone replacing the Eco-gpt gene [60 (link)]. BACs were confirmed by Illumina MiSeq sequencing to verify sequence integrity.
Chicken embryo cells (CEC) were isolated from 11-day-old VALO specific-pathogen-free (SPF) embryos (VALO Biomedia; Osterholz-Scharmbeck, Germany) as described previously [61 ]. CEC were maintained in minimal essential medium (MEM, PAN Biotech; Aidenbach, Germany) supplemented with 1% to 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (AppliChem; Darmstadt, Germany) at 37°C and 5% CO₂.
All recombinant GFP reporter viruses were reconstituted by transfection of fresh CEC with purified BAC DNA using CaPO₄ transfection [62 (link)]. The viruses were propagated on CEC for up to 9 passages, and infected cells were stored in liquid nitrogen.

The virus isolate BetaCoV/Germany/BavPat1/2020 [15 (link)] T-cell culture passage 3 was used for SARS-CoV-2 animal challenge. The virus was propagated and titrated on Vero E6 cells (ATCC CRL-1586) in minimal essential medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (PAN Biotech, Aidenbach, Germany), 100 IU/mL penicillin G and 100 µg/mL streptomycin (Carl Roth, Karlsruhe, Germany), and stored at −80 °C prior to experimental infections. Genome integrity, specifically the presence of the furin cleavage site, was confirmed by NGS sequencing of virus stocks used for animal experimentation.

CEC were prepared from 11-day old specific-pathogen-free (SPF) chicken embryos (VALO BioMedia, Germany) as described previously [55 (link)]. CEC were cultured in Eagle’s minimal essential medium (MEM; PAN Biotech, Germany) supplemented with 10% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). Reticuloendotheliosis virus-transformed T cells (CU91) were propagated in RPMI 1640 media (PAN Biotech, Germany) supplemented with 1% sodium pyruvate, 1% nonessential amino acids, 10% FBS, and penicillin–streptomycin, and maintained at 41°C in a 5% CO2 atmosphere. Viruses were reconstituted by transfecting bacterial artificial chromosome (BAC) DNA into CEC as described previously [55 (link)]. Viruses were propagated on CEC for four passages thereafter virus stocks were frozen in liquid nitrogen and titrated on CEC as described previously [56 (link),57 (link)].

For the direct in vitro test, the samples were prepared in the form of discs. Glass samples with a diameter of 6 mm and a height of ca. 3 mm were prepared using a PYTE model uniaxial press at a pressure of 5 MPa. Biocomposite hydrogel–bioglass samples with a diameter of 12 mm and a height of 2 mm were prepared by casting in PTFE moulds.
The MG-63 osteoblast-like cells (European Collection of Cell Cultures, Salisbury) were cultured in modified Eagle medium (MEM, PAN BIOTECH, Aidenbach, Germany) supplemented with 10% foetal bovine serum (FBS) (Biowest, Nuaillé, France), 5% amino acids and sodium pyruvate, and 1% penicillium/streptomycin (all PAN BIOTECH, Germany). A total of 2 × 10⁴ cells per sample were seeded and cultured at 37 °C, 5% CO₂, with increased humidity. The surface of tissue culture polystyrene (TCPS, 24-well culture plate, Nunclon) was used as a control.

Virus stocks were prepared from a previously published SARS-CoV-2 isolate (BetaCoV/Germany/BavPat1/2020) [29 (link)], which was kindly provided by Drs. Daniela Niemeyer und Christian Drosten, Charité Berlin, Germany. The isolate, referred to as SARS-CoV-2 München (SARS-CoV-2M) [30 (link)], was handled under the appropriate safety precautions in a BSL-3 facility (Freie Universität Berlin, Institut für Virologie) and propagated on Vero E6 cells (ATCC CRL-1586) in minimal essential medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (PAN Biotech), 100 IU/mL penicillin G and 100 µg/mL streptomycin (Carl Roth, Karlsruhe, Germany).

Embryonated SPF Valo chicken eggs (VALO BioMedia GmbH,) were used for the preparation of chicken embryo cells (CEC). CEC were maintained in minimal essential medium (MEM, PAN Biotech; Aidenbach, Germany) supplemented with 1-10% fetal bovine serum (FBS) and penicillin/streptomycin as previously described [20 ]. B cells were obtained from the bursa of Fabricius by dissociation of the organ and subsequent isolation of the cells by density gradient centrifugation as previously described [21 (link)]. Briefly, the bursa of Fabricius was homogenized through a 40 μm cell filter to obtain a uniform single cell suspension. Suspension cells were carefully applied on a Biocoll separating solution (Biochrom; Berlin, Germany), centrifuged for 12 min at 650 × g with slow acceleration, and deactivated deceleration. Lymphocytes at the interphase were carefully transferred to a new tube, washed with PBS, and maintained in RPMI 1640 (PAN Biotech) supplemented with 10% FBS and penicillin [100 U/mL]/streptomycin [100 µg/mL] at 41 °C under a 5% CO₂ atmosphere. B cells were activated using recombinant soluble chicken CD40 ligand (chCD40L) [22 (link)], which was expressed in HEK293 cells and purified using a Vivacell 250 ultrafiltration concentrator (Sartorius; Göttingen, Germany).