Biomedical Engineering Reference
In-Depth Information
consisted of patients with facial wrinkles mainly in the perioral
region, such as nasolabial folds and lip wrinkles following implant
treatment.
35.2.1
Tissue Preparation
Fibroblasts were obtained from the patient's buccal gingival tis-
sue. Mucosal epithelium was removed with the aid of a dissecting
microscope. The submucosal tissue sample (5 mm diameter) was
treated for one hour at 37
◦
C with 10 mg/mL collagenase (Wako)
dissolved in Dulbecco's Modified Eagle's Medium (DMEM) (Invitro-
gen),containing2.5
μ
g/mLamphotericin B(Sigma-Aldrich)and50
μ
g/mL gentamicin sulfate (Sigma-Aldrich). After the resulting cell
suspension was washed several times in phosphate buffered saline
(PBS) (Invitrogen), it was resuspended in DMEM containing a 10%
autologousserumpreparedfromthepatient'speripheralblood,and
supplemented with 2.5
μ
g/mL amphotericin B and 50
μ
g/mL gen-
tamicin sulfate. The cells in the medium were then seeded into a 6-
well tissue culture plate (Corning). For the histological evaluation,
a piece of tissue was fixed with 4% paraformaldehyde and then
embedded in para
n. Para
n sections were cut, de-para
nized,
and stained withhematoxylin and eosin.
35.2.2
Cell Culture
CellsweremaintainedinDMEMplus10%autologoushumanserum
and incubated at 37
◦
C with 5% CO
2
. The cells demonstrated a typi-
calspindleshape,whichwasmaintainedthroughoutthecellculture
(Fig. 35.1). When cells attained 80%-100% confluency, they were
subcultured into flasks and expanded from T-75 (Greiner Bio-One)
to T-225 flasks (Sumitomo Bakelite). For subculture, the cells were
first washed in PBS and then treated for five minutes at 37
◦
C with
TrypLEExpress(Invitrogen)beforeresuspensioninafreshmedium.
Cells were seeded at a density of 2-5
×
105 cells/mL and subcul-
tured according to a schedule that accounted for the timing of the
patient's treatments. The cell culture timetable was: P (passage) 0
cellsculturedin6-wellplates(7-10days),P1cellsinaT-75flask(7-
10 days), P2 cells in a T-225 flask (7-10 days), and P3 to P6 cells in
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