Maxillofacial Bone Regeneration Using Tissue Engineering Concepts - Engineered Bone: Tissue Engineering Intelligence Unit - page 190

Figure 9. Schema of experimental protocol by using MSCs/fibrin glue- β -TCP admixtures.
MSCs isolation and culture expansion was performed according to previously described meth-
ods. 16 Pasteurized fibrin glue (Bolheal, the ChemoSero-Therapeutic Reseach Institute, Kumamoto,
Japan) was formed by mixing two separate solutions, A and B. Solution A consisted of fibrinogen
(80 mg/ml) and fibrin-stabilizing factor XIII (75 units/ml) dissolved in 1 ml of plasmin inhibitor
aprotinin (1000 kIE/ml). 17 Solution B contained thrombin (250 units) dissolved in 1 ml of 40É M
Cacl 2 . Solution A and B were mixed in a 1:1 (volume/volume) ratio. The clotting reaction between
A and B produced a semirigid three-dimensional network at room temparature. The β -TCP loaded
with MSCs after 20 days of subculture (MSCs/ β -TCP composite) was reduced to small lumps in
sterilized 5ml syringe and was resuspended in a solution A. Solution A containing MSCs/ β -TCP
composite and solution B [1:1 (volume/volume) ratio] were placed in the barrel of the sterilized
5ml syringe and mixed by inverting the syringe repeatedly. The MSCs concentration in the admix-
ture prepared was approximately 10 x 10 6 cells/ml. Syngenic 7-week-old male Fischer rats were
anesthetized by intramascular injection of pentobarbital (nembutal 3.5 mg/100 g body weight)
following light ether inhalation. The skin was prepared with povidone-iodine. An 18 gauge needle
was inserted into the loose connective space between the carnosum layer and muscle fascia. 0.5 ml
of the MSCs/fibrin glue- β -TCP admixture was injected in the subcutaneous space (Fig. 10A).
After injection of MSCs/fibrin glue- β -TCP admixtures into rat, subcutaneous nodules formed
by 8 weeks and these were hard, resisted compression and had well-defined margins upon dissec-
tion from subcutaneous tissue (Fig. 10B). Injection of fibrin glue- β -TCP alone failed to form
nodules. Vascularization had increased throughout the implants at 8 weeks. At no time point was
evidence of malignant growth found in any of the specimens from the MSCs/fibrin glue- β -TCP
admixture groups. On the other hand, control implants of injected fibrin glue- β -TCP admix-
tures had only a shiny appearance and an elastic consistency at 2 weeks, which persisted until 8
weeks. The implant margin was not detectable at 8 weeks and its dimensions remained flattened.
Histologic examination at 2, 4, and 8 weeks showed calcified bone matrix with occasional
small remnants of biodegraded fibrin glue- β -TCP in the implants. At 2 weeks after implanta-
tion, we could observe the osteoblast lining and at 4 weeks, a few mature bone together with
cuboidal active osteoblasts was observed. At 8 weeks after implantation when osteocalcin con-
tent was significantly higher than before, bone formation was still progressing and an increase
in the mature lamellar bone areas could be observed (Fig. 11). We confirmed that the bone