Biology Reference
In-Depth Information
cells, even after a month. The reporter dye also allowed assessment of AFSC
proliferative ability and documentation of their contribution to healing mechanisms
by monitoring the expression of tissue-specific molecular markers (i.e., collagen
types I and III).
5.2 Materials and Methods
AFSCs were isolated from sheep slaughtered between 60 and 80 days of pregnancy
(fetal length: 18-25 cm). AFSCs were isolated from the amniotic fluid by centrifuga-
tion and cultured in 100-mm Petri dishes in the presence of
-MEM supplemented
with 20% FCS, 2 mM
L
-glutamine, and 5 ng/ml bFGF2. Incubation at 38
Cin5%
CO
2
continued until cells reached 70% confluence. The cells were then detached after
enzyme treatment (trypsin-EDTA) and replated (2
a
10
4
cells/ml) as described
previously.
AFSC phenotypic stability during culture was monitored by flow cytometry,
studying the expression of hematopoietic (CD14, CD31, CD45, and CD58), adhe-
sion (CD49f, CD29, and CD166), and stem cell (OCT , Sox 2, Nanog, TERT, and
c-Kit) molecular markers.
Before transplantation, the cells were labeled by a 30-min coincubation with the
lipophilic dye PKH26 (4
10
6
M) and then resuspended at a concentration of
10
6
cells in 20
2
l PBS.
The experimental mechanical tendon defect was performed (a cylindrical lesion
of 3-mm diameter
m
3-mm depth) on the intermediate portion (5 cm proximal to
the calcaneal tuberosity; Crovace et al.
2008
) of both externalized calcaneal
tendons after surgical incision. To this end, six 1-year-old Merinizzata sheep,
weighing between 40 and 50 kg, were used; the surgery was performed after
sedation of the animals. In the left tendon, the lesion site was covered with fibrin
glue (Tissucol: Baxter Spa), while in the contralateral tendon, AFSCs were added to
fibrin.
After 15 and 30 days, the animals were sacrificed; both calcaneal tendons were
explanted and then processed for histological and IHC analyses. In particular, the
portions of the tendons corresponding to the experimental lesions were immediately
cryopreserved and completely cut into 10-
m sections. The tissue was analyzed by
evaluating at least five sections in each internal, external, and middle portion of the
lesion. On tissue sections, (1) the tissue architecture (hematoxylin/eosin, EE and
Herovici, HE), (2) the composition of the extracellular matrix (IHC for collagen
types I and III, Chemicon), (3) cell proliferation (IHC for the proliferation marker
Ki-67, Santa Cruz), (4) the density and vascular organization (IHC for the endothe-
lial marker, von Willebrand factor, vWF, Dako), and (5) the leukocyte infiltration
(IHC for CD45, an antigen common to all leukocytes; Abd Serotec) were assessed.
In the tendon graft, the presence and localization of PKH26-stained AFSCs were
also assessed. All IHC staining was visualized using secondary anti-mouse Alexa
Fluor488 (Molecular Probes) antibodies, while cell nuclei were labeled with DAPI
m
