Human maxillary sinus mucosal membrane (hMSM)-derived stem cells were isolated and cultivated in accordance with the author's previous study13. Maxillary sinus tissue was collected and contained in phosphate-buffered saline (PBS; Gibco BRL, Grand island, NY, USA) including 1% antibiotic and antimycotic (10× antibiotic & antimycotic, Gibco BRL) and transported to the laboratory under aseptic condition. It was then washed with PBS (Gibco BRL) including antibiotic and antimycotic, cut into small pieces with a mesh under aseptic condition, treated with 0.06% collagenase type II (Invitrogen, Carlsbad, CA, USA), shaken with an incubator (CO₂ incubator; Forma Scientific, Marietta, OH, USA) containing 5% CO₂ at 37℃ for 4 hours, and centrifuged at 1,000 rpm for 10 minutes (large-capacity table-top centrifuge; Hanil Science Industrial, Gangneung, Korea). The precipitate was suspended onto α-MEM (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% Penicillin-Streptomycin (Gibco BRL) and filtered with 40 µm cell strainer (BD Bioscience, Bedford, MA, USA). Tissues except hMSM-derived cells were filtered, with the filtered out solution cultured in the incubator. The daily morphological characteristics were observed with inverted microscope, and the culture solution was replaced every other day. When the medium was changed, cells not adhering to the culture plate were removed; only the adhering cells were cultured. When culture dishes became near-confluent, cells were separated with Trypsin-EDTA (Gibco BRL) and subsequently repeated for continued passaging. The cells were cultured at passage 3.

Colony-forming unit-fibroblast assay was used to evaluate MSC presence in hMSM. When the initially cultured cells became near confluent, cells were separated with Trypsin-EDTA, plated at 1×10⁴ cells/mL onto the 100 mm culture plate, stained with hematoxilin (Sigma-Aldrich) after 24 hours, and observed with inverted microscope.

Flow cytometry on cell surface markers STRO-1, CD105, and CD34 for the separation of MSCs from hMSM was conducted. The hMSM-derived cells at passage 3 were plated into a test tube (Bectom Dickinson, Franklin Larks, NJ, USA) by 1×10⁴ cells/mL. The antibodies of CD34 (BD Bioscience) and CD105 (Serotec, Oxford, UK) - to which fluorescein isothicyanate (FITC) and phycoerythrin were adhered - were treated for 1 hour and washed with wash buffer 3 times, and the origin of stem cell was observed with flow cytometry (BD Bioscience). For STRO-1 (human anti-mouse monoclonal antibody, IgM subclass; R&D system, Minneapolis, MA, USA), antibody was treated for 1 hour and washed 3 times with wash buffer; the secondary antibody to which FITC was attached was treated for 30 minutes, washed 3 times with wash buffer, and observed with flow cytometry.

Sinus mucosa-derived stem cells were washed with PBS 72 hours after treatment on normal media and simvastatin (0.1, 0.2, 0.5, 1.0, 2.0 µM)-treated media. Afterward, the cells were collected with cell scraper (Sarsted, Newton, NC, USA). After the total RNA was separated by QIAGEN RNA extraction kit (QIAGEN, Hilden, Germany), it underwent reverse transcription reaction with the separated RNA, sense primer, antisense primer, and reverse transcriptase-polymerase chain reaction (RT-PCR) premix (Bioneer, Daejeon, Korea) for 60 minutes at 42℃ and 5 minutes at 94℃ and denatured for 5 minutes at 94℃ and 1 minute at 94℃, and genes were annealed for 90 seconds at 52℃, elongated for 1 minute at 72℃, and then annealed and elongated repeatedly 30 times and incubated for 5 minutes at 72℃.(Table 1)

Sinus mucosa-derived stem cells were washed with PBS 72 hours after treatment on normal media and simvastatin (0.1, 0.2, 0.5, 1.0, 2.0 µM)-treated media. The cells were then collected with cell scraper (Sarsted) and spun at 12,000 rpm for 20 minutes at 4℃ after being rinsed with RIPA-B buffer (0.5% Nonidet P-40, 20 mM Tris, pH 8.0, 50 mM NaCl, 50 mM NaF, 100 µM Na₃VO₄, 1 mM DTT, 50 µg/mL PMSF) for 1 hour at 4℃. The supernatant was transferred to the nitrocellulose membrane, blocked with TTBS (1 M Tris, 1.5 M NaCl, 500 µL Tween 20) containing 5% skim milk, and treated by BMP-2 primary antibody and secondary antibody. Afterward, the results were detected by the radioactive system.

Sinus mucosa-derived stem cells were cultured 3, 6, 9, and 12 days after treatment on normal media and determined concentrated simvastatin-treated media. After the total RNA was separated with QIAGEN RNA extraction kit, it underwent reverse transcription reaction with the separated RNA, OC sense primer, antisense primer, and RT-PCR premix for 60 minutes at 42℃ and 5 minutes at 94℃ and denatured for 5 minutes at 94℃ and 1 minute at 94℃; genes were annealed for 90 seconds at 52℃, elongated for 1 minute at 72℃, and then annealed and elongated repeatedly 30 times and incubated for 5 minutes at 72℃.(Table 1)

The control and simvastatin-treated group were each washed 3 times with sterile triple-distilled water 3, 6, 9, and 12 days after the treatment date, fixed with citrate-acetone-formaldehyde fixer solution (Sigma-Aldrich), and washed 3 times with sterile triple-distilled water. They were stained with alkaline-dye mix (Nitrite, FRV-alkaline, Naphthol ASBI alkaline solution; Sigma-Aldrich) with light isolated for 15 minutes at normal temperature, washed 3 times with sterile triple-distilled water, counterstained with hematoxylin, washed again 3 times with sterile triple-distilled water, and observed with inverted microscope. The specimens were observed by two separate observers.

All groups were washed 3 times with sterile triple-distilled water 3, 6, 9, and 12 days after the treatment date. All groups were fixed with 4% paraformaldehyde (Merck & Co., Inc., Darmstadt, Germany) at normal temperature for 15 minutes and washed again 3 times with sterile triple-distilled water. They were stained for 30 minutes with light isolated with 1% silver nitrate (Merck) solution, washed 3 times with sterile triple-distilled water, left for 1 hour under ultraviolet, counterstained with 0.1% eosin (Sigma-Aldrich), and observed with inverted microscope. The specimens were observed by two separate observers.