3.3. Co-Culture Experiments

The co-culture experiments were conducted using THP-1-derived macrophages, normal human fetal osteoblast cell line (hFOB 1.19, ATCC-LGC standards, Teddington, UK), and human bone marrow-derived stem cells (BMDSCs, ATCC-LGC standards, Teddington, UK). The THP-1 cells were cultured as described in Section 3.2.1. The hFOB 1.19 cells were maintained in a 1:1 mixture of DMEM/Ham’s F12 medium without phenol red (Sigma-Aldrich Chemicals, Warsaw, Poland) containing 10% FBS, penicillin/streptomycin (100 U/mL and 100 μg/mL, respectively), and incubated at 34 °C in 5% CO₂ in air atmosphere. The BMDSCs were maintained in Mesenchymal Stem Cell Basal Medium (ATCC-LGC Standards, Teddington, UK) containing a Bone Marrow-Mesenchymal Stem Cell Growth Kit (ATCC-LGC Standards, Teddington, UK), penicillin/streptomycin (10 U/mL and 10 μg/mL, respectively) and incubated at 37 °C in 5% CO₂ in air atmosphere.

THP-1-derived macrophages growing in polystyrene wells (M0, M1, and M2 macrophages) and onto the surface of the chit/aga/HA were co-cultured with BMDSC or with hFOB 1.19 cells to confirm the paracrine effect of macrophages on osteogenic differentiation. The differentiation of THP-1 monocytes into adherent THP-1-derived macrophages and their polarization to the M1 and M2 phenotypes was conducted in the same way as described in Section 3.2.1. Briefly, the THP-1 cells were seeded at a concentration of 1 × 10⁶ cells per well into a 24-multiwell plate and onto the biomaterial in 500 μL of a basal culture medium (RPMI-1640 supplemented with 10% FBS, 0.05 mM 2-mercaptoethanol, 100 U/mL penicillin, 100 μg/mL streptomycin) supplemented with 200 nM PMA. After 1 day of culture, adherent THP-1-derived macrophages (M0 phenotype) were polarized to M1 and M2 phenotypes by 3-day exposure to appropriate culture medium (Table 1). Then, the media from the macrophages culture were removed, and cell culture inserts with 1 μm pore size were placed above the macrophage layer. The BMDSCs and hFOB 1.19 cells were seeded into the inserts at a concentration of 1 × 10⁵ cells per sample. The co-cultured cells were maintained in the complete culture medium supplemented with 50 μg/mL ascorbic acid, 10 mM β-glycerophosphate (Sigma-Aldrich Chemicals, Warsaw, Poland), and 0.05 mM 2-mercaptoethanol. Moreover, three different control groups were applied in the experiment: (1) test control—BMDSCs or hFOB 1.19 cells cultured in the inserts without a macrophage layer on the well bottom but with chit/aga/HA biomaterial (assessment of the biomaterial effect on osteogenic differentiation), (2) negative control of osteogenic differentiation (marked as (-)control)—BMDSCs or hFOB 1.19 cells cultured in the inserts without macrophages and biomaterial (assessment of the level of osteogenic markers without induction of bone formation using dexamethasone), (3) positive control of osteogenic differentiation (marked as (+)control)—BMDSCs or hFOB 1.19 cells cultured in the inserts without macrophages and biomaterial and maintained in osteogenic medium additionally supplemented with 10⁻⁷ M dexamethasone to induce bone formation (Sigma-Aldrich Chemicals, Warsaw, Poland) to induce osteogenic differentiation (Figure 4, Table 2). The co-culture experiment was performed for 21 days, and half of the media was exchanged every 3rd day.

Experimental conditions of control groups used in the co-culture experiment.

On the 7th and 21st day of the co-culture system experiment, quantitative evaluation of osteogenic markers in the BMDSC and hFOB 1.19 cell lysates was performed using commercially available human-specific ELISAs: type I collagen (Col I), osteocalcin (OC) (EIAab ELISA kit, Wuhan, China), and bone alkaline phosphatase (bALP) (FineTest ELISA Kit, Wuhan, China). The cell lysates were prepared in accordance with the method described previously [34] via two freeze–thaw cycles and sonification (ultrasonic processor UP100H, Hielscher Ultrasound Technology, Teltow, Germany) for 30 s at 30% amplitude. Additionally, immunofluorescent staining of Col I in ECM of BMDSC and hFOB 1.19 cells was carried out on the 6th day. The immunofluorescent staining was performed in accordance with the procedure described previously [35]. In brief, the cells were incubated overnight at 4 °C with primary human-specific anti-collagen I antibodies (Col1a1/Col1a2, Abnova, Taoyuan City, Taiwan) at a concentration of 10 µg/mL and then incubated for 1 h at room temperature with secondary antibodies Alexa-Fluor^®647 donkey anti-goat IgG antibody (Abcam, Cambridge, UK) at a concentration of 2 µg/mL. Additionally, the cell nuclei were labeled with the DAPI. Stained cells were observed using CLSM.