Sinus; Empyema, Maxillary (Chronic) Clinical Trial
Official title:
Phase 1: Study of the Osteogenic Potential of Human Maxillary Sinus Shneiderian Membrane
The objectives of this study are :
1. to test the the osteogenic potential of human maxillary sinus schneiderian membrane
(hMSSM).
2. To investigate the expression of mesenchymal stem cell marker (STRO-1), a marker of
mesenchymal progenitor cells using flow cytometry, and Alkaline phosphate expression,
Red alizarin and Von Kossa staining and quantitive PCR.
1. Isolation and Culture of hMSM-Derived Cells.
The tissue will be washed again with Phosphate Buffered Saline (PBS) that included
antibiotic and antimytotic, cut into small pieces under aseptic conditions, treated
with 0.06% collagenase type II (In vitrogen) and dispase, shaken with an incubator (CO2
incubator, Forma Scientific) containing 5% CO2 at 37°C for 4 hours, and centrifuged at
1,000 (Revolution Per Minute) for 10 minutes (large-capacity tabletop centrifuge).
- The precipitate was suspended in an alpha minimum essential medium (α-MEM)
(Sigma-Aldrich) containing 10% fetal bovine serum and 1% penicillin-streptomycin
and filtered with a 40-μm cell strainer (BD Bioscience), with which all tissues
except for the hMSSM-derived cells were filtered out.
- The solution filtered out was cultured in an incubator. Daily morphologic
characteristics will be observed with an inverted microscope, and the culture
solution will be changed every other day. When the medium will be changed, cells
not adherent to the culture plate will be removed and only the adherent cells will
be cultured. When the culture dishes become nearly confluent, the cells will be
separated with Trypsin-ethylenediaminetetraacetic acid (EDTA) and subsequently
repeated for continued passaging. The cells will be assayed at passage 3 for their
osteogenic potential.
2. Flow Cytometry. Fluorescence-activated cell sorting (FACS) analysis for the expression
of osteoprogenitor cell markers was performed on samples from primary cells (P0),
passage 1 (P1) cultures, and passage 2 (P2) cultures cultured in nonosteogenic medium.
Flow cytometry on the cell surface markers STRO-1, CD105, and CD146 for separation of
Mesenchymal progenitor cells (MPCs) from hMSM will be conducted. The hMSSM-derived
cells at passage 3 will be placed into a test tube (Becton- Dickinson) by 1 x104
cells/mL and washed three times with wash buffer (0.2% bovine serum albumin, 0.1%
sodium azide, 0.5 mmol/L EDTA). The antibodies of CD146 (BD Bioscience) and CD105 (BD
Bioscience) to which fluorescein isothiocyanate and phycoerythrin were adhered will be
treated for 1 hour and washed with wash buffer three times, and the origin of stem cell
was observed with flow cytometry (BD bioscience). For STRO-1 (human antimouse
monoclonal antibody, immunoglobulin M subclass; R&D Systems), antibody was treated for
1 hour and washed three times with wash buffer, and the secondary antibody, to which
fluorescein isothiocyanate was attached, was treated for 30 minutes, washed three times
with wash buffer, and observed with flow cytometry.
3. Osteogenic Differentiation of hMSM
The hMSM-derived cells at passage 3 were incubated in osteogenic media for 1 to 4 weeks
in 12 well plates at a density of 105 cells per well and divided into two groups. The
control group was replated in a normal medium (complete α-MEM), and the experimental
group was replated in osteogenic differentiation medium (α-MEM including 0.1 μm
dexamethasone, 10 μm glycerol phosphate, and 50 μm L-ascorbic acid 2-phosphate). The
media were changed every 3 days.
3.1- Alkaline Phosphatase and Alizarin Red Staining. Control and experimental cultures
were each washed three times with sterile triple-distilled water at 7, 14, 21, and 28
days after the treatment date, fixed with citrate-acetone- formaldehyde fixation
solution (Sigma), and washed again three times with sterile triple-distilled water.
They were then stained with an alkaline dye mixture (nitrite, Fast Red Violet-alkaline,
naphthol alkaline solution; Sigma), in the dark for 15 minutes at normal temperature,
washed three times with sterile triple-distilled water, counterstained with hematoxylin
(Sigma), washed again three times with sterile triple-distilled water, and observed
with an inverted microscope.
Alizarin Red Staining. Control and experimental group samples were each washed three
times with sterile triple-distilled water at 7, 14, 21, and 28 days from the treatment
date, fixed with 4% paraformaldehyde (Merck) for 15 minutes, stained while light was
isolated for 30 minutes with 2% alizarin red solution (Sigma), washed again three times
with sterile triple-distilled water, and the staining will be observed with an inverted
microscope.
3.2- Von Kossa Staining. Control and experimental cultures were washed three times with
sterile triple-dis- tilled water, fixed with 4% paraformaldehyde (Merck) at a normal
temperature for 15 minutes and washed again three times with sterile triple-distilled
water. They were stained for 30 minutes while light was isolated with 1% silver nitrate
(Merck) solution, washed three times with sterile triple-distilled water, left under
ultraviolet light for 1 hour, counterstained with 0.1% eosin (Sigma), and the staining
will be observed with an inverted microscope.
3.3- Quantitative PCR (qPCR). To observe osteocalcin expression, the hMSM-derived
cells, differentiated into two groups, were washed with (PBS) at 7, 14, 21, and 28 days
after treatment in osteogenic differentiation medium and collected with a cell scraper.
After the Ribonucleic acid (RNA) was separated with an RNA extraction kit, it underwent
a reverse-transcription reaction with the separated RNA, osteocalcin, or bone
sialoprotein, or dentin matrix protein 1 primers and probes, and reverse-transcriptase
polymerase chain reaction premix (Applied Biosystems). The obtained data will be
normalized using 18 Shake. The amplified products were analyzed using the deal-delta
Computer Tomography analysis.
4. Histochemical staining of alkaline phosphatase activity in frozen sections of sinus
mucosa.
Freshly prepared sinus mucosa was washed in phosphate-buffered saline (PBS), cut into
pieces of approximately 5x 5 mm, embedded in optimum cutting temperature tissue
compound (OCT compound; Miles Laboratories, USA), and stored at - 80 C. Frozen sections
(7 mm) mounted on poly-D-lysine-coated slides were fixed with ice-cold acetone for 1
min and allowed to air dry. The slides were washed with PBS and subsequently incubated
with the substrate solution for alkaline phosphatase activity containing 4 mg of
naphthol phosphate in 0.15 ml of N, N0 -dimethyl- formamide and 12mg of fast blue salt
(Sigma, St Louis, USA) in 15 ml of Tris-Hydrogen Chloride (pH 9.6). After rinsing with
distilled water, slides were counterstained with hematoxylin-eosin, embedded into
water-soluble resin, and photographed.
5. Immunohistochemical determination of STRO-1-positive cells in frozen sections of
mucosa.
Frozen sections (7 mm) were fixed with cold acetone for 1 min and washed in PBS. The slides
were placed into PBS containing 0.3% of hydrogen peroxide for 15 minutes to quench
endogenous peroxidase, washed, and blocked with 2% bovine serum albumin in PBS for 1hour at
room temperature. Sections were incubated with a 1: 50 dilution of the STRO-1 antibody
(mouse monoclonal antibody, IgM subclass; Developmental Studies Hybridoma Bank, University
of Iowa, USA) in blocking solution overnight at 41 degree C. For detection of
STRO-1-positive cells, slides were incubated with a biotinylated goat anti-mouse Immune
globulin M (IgM) (1 : 100, An der Grub, Vienna, Austria) and a streptavidin-peroxidase
conjugate (BioFX Laboratories, Inc., MD, USA) each at room temperature for 45 min. The
peroxidase-containing complex was visualized by Diaminobenzidine substrate (Dako, Glostrup,
Denmark) and counterstained with hematoxylin. The sections were embedded into water-soluble
resin and photographed.
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