Biomedical Engineering Reference
In-Depth Information
thebasalcellsofthecellsheets.Immunostainingpatternsofkeratin
3 and p63 were highly similar to those in native limbal epithelium.
Theseobservationsimplythatepithelialstemcellswerelocalizedin
the basal layer of the harvested cell sheets and that differentiated
epithelial cells migrated to the surface layers as seen
in vivo
.
Conjunctival scar tissue on the cornea was surgically removed to
exposethenative,transparentcornealstroma.Then,cultured,autol-
ogous, multilayered corneal epithelial cell sheets were harvested by
reducing the temperature and using a poly(vinylidene difluoride)
(PVDF) membrane with an outer diameter of 30 mm and a 12-mm-
diameter hole punched out of its center. The intact multilayered
cornealsheetwasplaceddirectlyoverthebarecornealstroma.After
fiveminutesofcontactafterplacement,thesheetreadilyadheredto
theunderlyingcornealstroma,self-stabilizingwithoutanysuturing.
The PVDF membrane was then excised. Immediately after surgery,
fluorescein staining showed that the corneal epithelial sheet cov-
eredtheentirecornealsurfacehomogeneously,reflectingasuccess-
fulprocedure.Thenextday,thegraftremainedfirmlyattachedtothe
corneal surface, with some ocular surface inflammation seen. Dur-
ingpostoperativehealing,theocularsurfaceinflammationsubsided,
and corneal transparency was restored. During observation periods
of up to 180 days, all bioengineered corneal sheet grafts remained
stable at the initial placement site, covering the entire corneal sur-
face. The corneal epithelium regenerated by corneal sheet graft
healing exhibited a normal appearance, with slightly thin corneal
epithelium, cuboidal basal cells, and flattened medial and superfi-
cial cells. All of the epithelial cell layers expressed keratin 3 in each
of the two regenerated corneas examined by immunohistochem-
istry. To follow the transplanted corneal epithelial cells after graft-
ing, transplanted cells were stained with a cell-tracer red dye one
day before surgery. Later, the entire thickness of the regenerated
corneal epithelium exhibited red fluorescence, indicating that the
regeneration originated withthe transplanted cells.
Clinical results have shown that the corneal surface remains
clear with significantly improved visual acuity more than one year
after the corneal epithelial cell sheet transplantation. Transplan-
tation of corneal epithelial cell sheets also prevents the develop-
ment of corneal haze after excimer laser keratectomy. We have also
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