Biomedical Engineering Reference
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according to the manufacturer's instructions and 80 μm thick sections cut with a saw
microtome (Leica 1600, Germany) parallel to the piezoelectric film long axis. A mini-
mum of five sections of each resin block was cut. Sections were then appropriately
processed for routine staining (Giemsa Eosin), mounted for fluorescence microscopy.
The prepared slides were evaluated qualitatively. For histomorphometric studies
the interface between the bone and implant was divided in four distinct areas: A1, A2,
A3, and A4, from cortical towards bone surrounding the free extremity of the piezo-
electric film (Figure 5).
Figure 5.
Tibia section the osteotomy where the piezoelectric film was placed. The figure shows
example of bone section prior to inclusion and how the areas for histomorphometry were distributed;
A1 corresponds to the film encastre (clamped) region.
Pictures were taken from the bone surrounding both sides of the film in areas A1
to A3 and A4.
For immunohistochemistry, bone sections were decalcified in formic acid 5% for
3 weeks, dehydrated in ethanol, cleared in xylene and embedded in paraffin wax and 3
µm sections cut. After deparaffinization and rehydration, immunohistochemistry sec-
tions were treated with 3% hydrogen peroxide for 10 min.
Primary antibodies for Proliferating Cell Nuclear Antigen (PCNA) (NeoMarkers,
USA, Mouse Monoclonal Antibody, Ab-1, Clone PC10, Cat. #MS-106-P0), Osteo-
pontin (NeoMarkers, Rabbit Polyclonal Antibody, Cat. #RB-9097) and Osteocalcin
(Abcam, Mouse monoclonal [OC4-30], Cat. ab13418) were diluted to 1:200, 1:50,
and 1:40, respectively. Prior to immunostaining the sections were pretreated for anti-
gen retrieval at 100ºC in 10 mM citrate buffer, pH 6, for 20 min in microwave oven,
followed by cooling for 30 min at room temperature. For double staining of PCNA
and osteopontin, immunohystochemistry was done using a double staining kit Pic
TureTM (Zymed Laboratories Inc, USA.), according with the manufacturer's instruc-
tions. Slides were counterstained with Mayer's hematoxilin and Clearmount used to
mount the slides.
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