Cytotoxicity assay

Mouse peritoneal macrophages (PM) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Four categories of materials were used to study the cells response. Except the CB and DCB group, the commercial decellularized human bone (Cancellous bone strip, Beijing Xinkangchen Medical Science and Technology Development Co., Ltd., China, named MC1) and commercial biological ceramic artificial bone (DONGBO, Hunan Gongchuang Biofunctional Materials Co., Ltd, China, named MC2) were used as two control group.

To evaluate PM responses to different scaffolds at direct contact, all scaffolds (named D-CB, D-DCB, D-MC1 and D-MC2, respectively) balance 15 min in 24-well ultra-low attachment plates. PM (1 × 10⁶ cells) was inoculated on the scaffolds and allowed to attach under 37°C and 5% CO₂ for 1 h, and the culture medium was adjusted to a final volume of 1 ml, as described previously [26].

In order to evaluate the response of PM to different scaffolds during indirect contact, all scaffolds (named I-CB, I-DCB, I-MC1 and I-MC2) were placed in the apical chamber (micropore) of 24-well ultra-low attachment plate (Millipore) and separated from PM in 250 μl medium. PM (1 × 10⁶ cells) was inoculated in the basolateral chamber and the culture medium was 750 μl. The scaffolds and cells were co-cultured in 37°C and 5% CO₂ for 2 days, and the culture medium was changed on the first day.

PM (1 × 10⁶ cells) was seeded in basolateral chamber in 750 μl of culture medium. M0 controls were obtained by treating PM with culture medium alone. M1 (pro-inflammatory macrophage) controls were obtained by treating cells with 20 ng/ml of γ-IFN and 100 ng/ml of LPS.

After 48 h of incubation, the fresh culture medium 100 μl/well Containing Cell counting Kit 8 (CCK-8, Dojindo, Japan) was added and incubated at 37°C for 1 h. The absorbance of the supernatant was measured by microplate instrument at 450 nm. The formula of relative growth rate (RGR) is: RGR (%) = (OD value of experimental group/OD value of negative control group) × 100. Evaluation of cytotoxicity of samples according to the standards of American Pharmacopoeia [27]. Briefly, (i) RGR ≥ 75%, cytotoxicity grade 0 or 1, qualified; (ii) 74% ≥ RGR ≥ 50%, cytotoxicity grade 2; (iii) RGR ≤ 49%, cytotoxicity grade 3–5, unqualified.