Barbieri Thesis - BioMedical Materials program (BMM)

Chapter 2 – The role of gels in instructive putties
The observations were done through visual inspection of the shape changes of the
putties over time. At different time points (0, 30 minutes, 1, 3, 6 hours, 1, 2, 4, 7 and
14 days) the samples were observed and pictures were taken. Then the change in
the surface area occupied by the materials (in the well) over time was evaluated with
Photoshop CS5 software (v12.0, Adobe Systems Benelux BV, Amsterdam, the
Netherlands). Firstly, the surface area of each well was selected and the
corresponding number of pixels was read as W. The materials in each well at the
considered time point t were pseudo–coloured and the pixels were read as Mt. The
percentage (changing) surface area occupied by the material (in the well) over time
was determined as
A = 100 – [100 · (Mt – Mt–1) / W]
where Mt–1 is the area occupied by the sample in the previous time point than t. The
data were finally plotted on a graph A over time.
Table 2. Conditions in which gels and putties were prepared.
Gels Putties
Polymer Solvent
Concentration Temperature pH Gel/BCP
[%wt.] [°C]
[%volume ratio]
CMC Water 5 60±5 7.3–7.8 1
PLU Water 38 40±5 6.8–7.5 0.85
PVA Water 25 80±5 7.2–7.6 0.75
CHI 1.2% acetic acid 1.6 37±2 6.9–7.1 0.95
ALG Water 5.25 60±5 7.1–7.8 1
2.2.4. Intramuscular implantation (sheep model)
To evaluate the osteoinductive potential of the putties, sterile controls (BCP particles,
size 1–2 mm, 1 cc, n=10) and putties (1 cc, n=10 per material) were implanted in the
dorsal muscles of sheep for 12 weeks. With the permission of the local animal care
committee (Animal Care and Ethics Committee, University of New South Wales,
Sydney, Australia; protocol #P07037), surgery was performed on 10 sheep (female,
2–3 years old) under general anaesthesia (Isoflurane, Organon, now Merck,
Whitehouse Station, USA; 2% in oxygen, oxygen flow rate of 4 L min –1 ) and sterile
conditions. The muscles were exposed by longitudinal skin incisions and two lines of
independent muscle pockets were created by blunt dissection (the distance between
the pockets on the same line was about 2.5–3 cm to ensure insulation between the
putties and avoid possible reciprocal side effects between implants). The head of the
syringe containing the putty was cut with scissors and the implant was directly
inserted in each muscle pocket. The pockets were sealed individually with non–
resorbable suture material and the wound was then closed using silk sutures. To
monitor the bone formation during time, calcein (Sigma, St. Louis, USA; 10 mg kg –1
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