The animal study was approved by the Institutional Animal Care and Use Committee of Chonnam National University (Approval No. CNU IACUC-YB-2023-12) and conducted in accordance with its guidelines. Murine BMSCs and aBMSCs were obtained from C57BL/6 mice aged 7 to 8 weeks (Damool Science, Daejeon, Korea). For the isolation of BMSCs, mice were sacrificed by CO₂ inhalation, and their hind limbs were dissected. The tibias and femurs were meticulously cleaned to remove surrounding soft tissues and were subsequently placed in minimum essential medium-α (MEM-α) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific) and 1% penicillin-streptomycin. The bone marrow was extracted by flushing and passed through a 70 μm strainer (SPL, Daejeon, Korea) to eliminate any remaining bone debris.

The isolated cells from each mouse were suspended in 40 mL of complete culture medium and distributed evenly into four 10 cm culture plates at a density of 2.5×10⁶ cells/mL. The cultures were incubated at 37°C with 5% CO₂, and fresh media was provided every three days to remove unattached cells. After eight days, once the cells reached sub-confluency, adherent cells (passage 0, P0) were rinsed with phosphate-buffered saline (PBS) and detached by treating them with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco; Thermo Fisher Scientific) for 2 min.

For the isolation of aBMSCs, mice were euthanized by carbon dioxide inhalation, and the upper and lower jaws were carefully dissected and transferred to a 60 mm Petri dish. Soft tissues, including gingival tissue, were meticulously removed under sterile conditions. The molars were loosened by inserting a 1 mL needle between the crowns and subsequently extracted. Surrounding soft and non-alveolar bone tissues were carefully excised, leaving only the alveolar bone for further processing.

The isolated alveolar bone fragments were collected and placed in 2 mL of PBS containing 2% FBS. They were then finely minced using sterile scissors before being transferred to a 15 mL centrifuge tube. A solution containing 1 mg/mL type II collagenase (Gibco; Thermo Fisher Scientific) and 0.5 mg/mL DNase I (Sigma-Aldrich, St. Louis, MO, USA) was added for digestion. The sample was then incubated in a shaking incubator at 37°C, 1,500 rpm, for 30 min to break down the tissue enzymatically.

Following digestion, the resulting cell suspension was passed through a 70 μm cell strainer (SPL) to eliminate undigested tissue debris and then transferred into a 50 mL centrifuge tube. The filtered sample was centrifugated, after which the supernatant was discarded. The cell pellet was resuspended in 3 mL of PBS and centrifuged again under identical conditions. This cycle of digestion and centrifugation was repeated three times to enhance cell recovery.

After the final centrifugation step, the supernatant was carefully removed, and the remaining cell pellet was resuspended in 12 mL of complete culture medium (MEM-α supplemented with 10% FBS and 1% penicillin-streptomycin). The cells were then plated in a 10 cm culture dish and incubated at 37°C in a 5% CO₂ environment. The culture medium was replenished every three days until the cells attained confluency.

To assess cell surface marker expression through flow cytometry, BMSCs, and aBMSCs were first detached using 0.25% trypsin-EDTA, washed with pre-chilled PBS, and then resuspended in binding buffer (PBS containing 0.5% FBS and 0.1% sodium azide). The cells were then incubated at room temperature for 20 min with allophycocyanin (APC)-conjugated monoclonal antibody targeting CD11b marker and phycoerythrin (PE)-conjugated monoclonal antibodies targeting the following markers: Sca-1, CD44, CD105 (Endoglin), CD90.2 (Thy1.2), and CD140a (PDGFRα). These antibodies were procured from BioLegend (San Diego, CA, USA), except for CD105, obtained from eBioscience (San Diego, CA, USA). To ensure proper gating, isotype control antibodies were used, including APC-conjugated Rat IgG2b, κ, PE-conjugated Rat IgG2a, κ, and Rat Ig-G2b, κ (BioLegend).

Following incubation, the cells underwent a single wash with binding buffer before being promptly analyzed using an Accuri C6 Plus flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with CSampler software. The specificity of antibody binding to target markers was validated by comparing fluorescence intensity levels with those of the respective isotype controls.

BMSCs and aBMSCs were seeded at a 5×10⁴ cells/mL density in a complete culture medium to induce osteogenic differentiation. The cells were allowed to adhere overnight, after which the culture medium was replaced with an osteogenic induction medium. This specialized medium contained 50 μg/mL ascorbic acid (Sigma-Aldrich), 10 mM β-glycerophosphate (Sigma-Aldrich), and 100 ng/mL recombinant human BMP-2 (Cowellmedi Co., Busan, Korea) in MEM-α. For optimal differentiation conditions, the medium was replenished every three days.

For the control group, cells were cultured in a standard growth medium (GM) without osteogenic supplements. After the differentiation period, cells were fixed with 4% paraformaldehyde and stained with Alizarin Red S (ARS) (Sigma-Aldrich) to evaluate mineralization levels. The staining assessment was conducted on day 14 for BMSCs and day 10 for aBMSCs to monitor calcium deposition.

Total RNA was extracted from cultured cells using TRIzol reagent (Ambion Inc., Austin, TX, USA) according to the manufacturer’s protocol. Approximately 2 μg of RNA was reverse-transcribed into complementary DNA (cDNA) using M-MLV reverse transcriptase (Promega, San Luis Obispo, CA, USA). qRT-PCR was performed using a QuantStudio cycler (Applied Biosystems, Foster City, CA, USA) with Power SYBR Green PCR Master Mix (Applied Biosystems) to analyze the expression levels of target genes. The primer sequences used for each target gene were as follows: Runx2 (Forward: 5′-TTCAAGGATGGAATTTGTGGG-3′, Reverse: 5′-GGATGAGGAATGGGCCCTA-3′); Osterix (Osx, Forward:5′-AGGCCACATTGGTAGCAAA-3′, Reverse: 5′-GCGGCTGATTGGCTCTTCT-3′); Bone sialoprotein (Bsp, Forward: 5′-AAGGCACCGTGTGAGTTAGGT-3′, Reverse: 5′-CCTTGAGTAGTGATTCATCCTC-3′); Osteocalcin (Ocn, Forward: 5′-GCAATAGGTAGTGAAGAACATCC-3′, Reverse: 5′-GTTTGTAGGCGGTCTTCAAGC-3′); Osm (Forward: 5′-TCCCCTCCAAAACCTGACACAC-3′, Reverse: 5′-ATGGTATCCCACTGAAAGGC-3′); FAT4 (Forward: 5′-GTTCCCTAGCAGGACTAACGGG-3′, Reverse: 5′-TAACATGACGTTGCTGGGG-3′); 18S rRNA (Forward: 5′-GGCCCTTCTTAGAGGGACA-3′, Reverse: 5′-CCCGGAACATCTCACGAC-3′).

Relative gene expression levels were calculated using the ΔΔCt method with normalization to 18S rRNA as an internal control. All data were analyzed using QuantStudio Design & Analysis software (Applied Biosystems).

All experiments were conducted in at least three independent replicates. Statistical comparisons between groups were performed using a paired Student’s t-test to assess significance. A P-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 9.0 (GraphPad Software Inc., San Diego, CA, USA).

The aBMSCs were isolated from murine alveolar bone tissue using a collagenase digestion method, as illustrated in Figure 1A. Under phase-contrast microscopy, the isolated aBMSCs exhibited a typical adherent growth pattern and a homogeneous spindle-shaped morphology, consistent with the characteristics of MSCs (Fig. 1B). Flow cytometry analysis indicated that aBMSCs expressed MSC markers, including CD44 (83%), CD105 (33%), CD90.2 (93%), and CD140a (52%), while the expression level of the hematopoietic marker CD11b was below 1% (Fig. 1C). These results suggest that the isolation method effectively yields aBMSCs with defined morphological and phenotypic characteristics, providing a suitable cell source for further studies.

Microscopic observation showed that aBMSCs lacked noticeable round non-adherent cells, which were present in BMSCs and morphologically consistent with macrophages (Fig. 2A). In BMSCs, a large number of these cells were observed. In contrast, the aBMSCs culture system appeared more uniform. Flow cytometry analysis was performed to quantify the proportion of CD11b⁺ cells, revealing that macrophages accounted for 1% of aBMSCs, while in BMSCs, this proportion reached 64% (Fig. 2B). These results provide a comparison of cell composition, highlighting differences in macrophage presence between the two cell types.

To evaluate the osteogenic potential of aBMSCs, qRTPCR and western blot analyses were performed to assess osteogenic marker expression, and ARS staining was conducted to examine matrix mineralization. The qRT-PCR analysis showed a significant increase in the expression levels of osteogenesis-related genes, including Runx2, Osx, Bsp, and Ocn (Fig. 3A). Western blot analysis also showed that Runx2 and Osx expression were markedly upregulated under osteogenic induction conditions (Fig. 3B). Additionally, ARS staining analysis showed substantial calcium deposition in aBMSCs after 12 days of induction, further confirming their strong osteogenic differentiation potential (Fig. 3C). These results collectively suggest that aBMSCs possess stable and enhanced osteogenic potential, making them a promising cell source for bone tissue engineering applications.

To assess the osteogenic differentiation potential of aBMSCs and BMSCs under inflammatory conditions, both cell types were treated with the proinflammatory cytokine interleukin-1β (IL-1β ; 10 ng/mL) in osteogenic medium (OM). The qRT-PCR analysis revealed that under IL-1β treatment, the expression levels of osteogenesis-related genes (Osx, Bsp and Ocn) remained stable in aBMSCs. In contrast, they were significantly downregulated in BMSCs (Fig. 4A). Ocn expression in aBMSCs showed a slight decrease compared to OM conditions. Osx and Bsp expression were maintained, suggesting that aBMSCs retained their osteogenic potential.

Western blot analysis showed that IL-1β treatment did not significantly affect Osx protein expression in aBMSCs. In contrast, it was markedly reduced in BMSCs (Fig. 4B). Runx2 protein expression in aBMSCs exhibited a slight decrease compared to OM conditions but remained higher than in GM conditions. Consistent with the protein-level findings, ARS staining demonstrated that aBMSCs retained high levels of calcium deposition under IL-1β treatment. In contrast, BMSCs exhibited approximately a 60% reduction in mineralization capacity compared to untreated conditions (Fig. 4C).

Gene expression analysis by qRT-PCR showed that aBMSCs maintained low Osm and high FAT4 expression under inflammatory conditions (Fig. 4D). In both aBMSCs and BMSCs, Osm expression was higher in OM and OM with IL-1β compared to GM, with a greater increase observed in BMSCs. In aBMSCs, FAT4 expression was upregulated in OM and OM with IL-1β compared to GM, whereas in BMSCs, FAT4 expression showed no significant increase and was reduced in OM with IL-1β.

These data suggest that aBMSCs maintain robust osteogenic differentiation capacity under IL-1β-induced inflammatory conditions, supported by sustained marker expression, preserved protein levels, stable mineralization, and distinct transcriptional features compared to BMSCs.