Bone and Periodontal Tissue Regeneration by the Secretomes from Bone Marrow Derived Mesenchymal Stem Cells - New Trends in Tissue Engineering and Regenerative Medicine - page 103

Figure 6. Immunohistochemical analysis of newly generated bone area 2 weeks after implantation.
2.7. MSC−CM enhanced periodontal tissue regeneration in dog periodontal defect model
All animal experiments were approved by the Nagoya University animal experiment com‐
mittee. After a period of acclimatization of 30 days, five hybrid dogs were operated on under
general anesthesia by intravenous injection of pentobarbital (Somnopentyl ® ) (20mg/kg body
weight), and under local anesthesia with 2% lidocaine (with 1:80,000 epinephrine, ORA ® Inj.
Dental Cartridge; Showa Yakuhin Kako, Tokyo, Japan). Before the experimental surgery, the
mandibular first and third or fourth premolars were extracted, and the extraction sites were
allowed to heal for 8 weeks. For the experimental surgery, buccal and lingual mucoperiosteal
flaps were elevated, and critical-size, box-type, one-wall intrabony defects (width, 4mm;
height, 5 mm) were created at the distal aspect of the second, and the mesial aspect of the fourth
premolars in the right and left jaw quadrants (Kim, 2005). Following root planing to remove
the root cementum, a reference notch indicating a 5-mm distance from the cement-enamel
junction to the bottom of the defect was made with a burr into the root surface at the base of
the defects. With no differences in bone regeneration in the various grafted areas in terms of
bone healing, two defects were created and implanted with two materials at random sites. An
absorbable atelo-collagen sponge (TERUPLUG ® ; OLYMPUS TERUMO BIOMATERIALS,
Tokyo, Japan) was used as a scaffold and contained 300μl MSC-CM or PBS. The dogs with
defects were randomly divided into three groups (n=6 each) and implanted with graft
materials: MSC-CM plus scaffold, PBS plus scaffold, or unfilled defect. The mucogingival flaps
were advanced, adapted, and completely closed. Post-surgical management involved antibi‐
otics (Azithromycin, 250 mg; Pfizer, Tokyo, Japan) daily for 3 days, a soft diet, and topical
application of 2% chlorhexidine (Hibitane concentrate; Dainippon Sumitomo Pharma, Osaka,
Japan) twice a week. After 4 weeks, the dogs were given general anesthesia and sacrificed by
exsanguination after injection of heparin sodium (400 U/kg).
Standardized radiographic images of the defect sites were obtained with an X-ray apparatus
(Dent navi Hands; Yoshida Co., Ltd., Tokyo, Japan) and dental X-ray films (BW-100; Hanshin
Technical Laboratory, Nishinomiya, Japan) immediately, and 4 weeks after, transplantation.