Maxillofacial Bone Regeneration Using Tissue Engineering Concepts - Engineered Bone: Tissue Engineering Intelligence Unit - page 188

Figure 7. Schematic drawing of marrow cell preparation.
and the marrow plugs were flushed out using 10 ml of cultured medium expelled from a
syringe through a 23-gauge needle (Fig. 7). The released cells were collected in two TC flasks
(8 × 10 cm 2 ) containing 15 ml medium. The medium was changed after the first 24 h to remove
nonadherent cells. Subsequently, the medium was removed 3 times a week. Cultures were
maintained in a humidified atmosphere of 95% air with 5% CO 2 at 37º. A culture medium
consisted of a minimum essential medium (MEM) containing 15% fetal bovine serum and
antibiotics (100 U/ml penicillin, 100 µ m/ml streptomycin, and 0.25 µ g/ml amphotericine B,
Sigma-Aldrich, Poole, UK). After 10 days in primary culture, bone marrow-derived MSCs
were released from their culture substratum using 0.05% trypsin-EDTA. The cells were con-
centrated by a centrifuge at 900 rpm for 5 min at room temperature, resulting in 10 × 10 6 cells/
ml. The β -TCP blocks were soaked in cell suspension (10 x 10 6 cells/ml) for 2 h in a CO 2
incubator. After 2 h of incubation, each block was transferred into a 24-well plate for subcul-
tures. One block was subcultured in one well with 1 ml of the culture medium suplemented
with 10 mM Na β -glycerophosphate, 80 µ g/ml vitamin C phosphate (L-ascorbic acid phos-
phate magnesium salt n-hydrate, C 6 H 6 O 6 ) and 10 -8 M dexamethasone (Sigma-Aldrich, Poole,
UK). The subcultures were maintained for 20 days.
Syngenic 7-week-old male Fischer rats were anesthetized by intramascular injection of pen-
tobarbital (nemubtal 3.5 mg/100 g body weight) following light ether inhalation. The β -TCP
loaded with MSCs after 20 days of subculture and each empty ceramic were implanted subcu-
taneously in the back of syngenic rats under sterile conditions. For each animal, empty β -TCP,
and cell-loaded β -TCP, were randomly assigned to and implanted at one of four sites, not in
contact with any osseous tissue.
Each implantation was harvested 1, 2, 4 and 8 weeks after implantation. The specimens
were fixed in 10% buffered formalin, decalcified (K-CX, falma Co., Tokyo), and stained with
hematoxylin and eosin. These specimens were examined under light microscopy. At 2 weeks
after implantation, a few mature bone together with cuboidal active osteoblasts was observed
when alkaline phosphatase activity achieved a peak. The osteoblast layer was in close contact
with the vasculature. At 4 weeks after implantation, consistent bone formation was observed in
more pores of β -TCP. At 8 weeks after implantation when osteocalcin content was significantly
higher than before, bone formation was still progressing and an increase in the mature lamellar
bone areas could be observed (Fig. 8). We confirmed that the bone areas were increased