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Prefabricated Bone Flap: An Experimental Study Comparing Deep-Frozen and Lyophilized-Demineralized Allogenic Bones and Tissue Expression of Transforming Growth Factor β

Rodrigues, Leandro MD, Msc*; dos Reis, Luciene Machado PhD; Denadai, Rafael MD; Raposo-Amaral, Cassio Eduardo MD, PhD; Alonso, Nivaldo MD, PhD*; Ferreira, Marcus Castro MD, PhD*; Jorgetti, Vanda MD, PhD

doi: 10.1097/SCS.0b013e3182a41be2
Original Articles

Background Extensive bone defects are still a challenge for reconstructive surgery. Allogenic bones can be an alternative with no donor area morbidity and unlimited amount of tissue. Better results can be achieved after allogenic bone preparation and adding a vascular supply, which can be done along with flap prefabrication. The purpose of this study was to evaluate demineralized/lyophilized and deep-frozen allogenic bones used for flap prefabrication and the tissue expression of transforming growth factor β (TGF-β) in these bone fragments.

Methods Fifty-six Wistar rat bone diaphyses were prepared and distributed in 4 groups: demineralized/lyophilized (experimental group 1 and control group 2) and deep freezing (experimental group 3 and control group 4). Two bone segments (one of each group) were implanted in rats to prefabricate flaps using superficial epigastric vessels (experimental groups) or only transferred as grafts (control groups). These fragments remained in their respective inguinal regions until the death that occurred at 2, 4, and 6 weeks after the operation. Semiquantitative histologic (tetracycline marking, cortical resorption, number of giant cells, and vascularization) and histomorphometrical quantitative (osteoid thickness, cortical thickness, and fibrosis thickness) analyses were performed. Transforming growth factor β immunohistochemistry staining was also performed.

Results Group 1 fragments presented an osteoid matrix on their external surface in all periods. Cartilage formation and mineralization areas were also noticed. These findings were not observed in group 3 fragments. Group 1 had more mineralization and double tetracycline marks, which were almost not seen in group 3. Cortical resorption and the number of giant cells were greater in group 3 in all periods. Vascularization and fibrosis thickness were similar in both experimental groups. Group 1 had more intense TGF-β staining within 2 weeks of study. Nevertheless, from 4 weeks onward, group 3 presented statistically significant stronger staining.

Conclusions Although there are some differences between the preparation methods of allogenic bone, it is possible to prefabricate flaps with demineralized/lyophilized and deep-frozen bones.

From the *Division of Plastic Surgery, Department of Surgery, †Laboratory of Bone Diseases, Division of Nephrology, Department of Internal Medicine, Faculty of Medicine, Universidade de São Paulo (USP); and ‡Institute of Plastic and Craniofacial Surgery, SOBRAPAR Hospital, Campinas, São Paulo, Brazil.

Received May 4, 2013.

Accepted for publication July 5, 2013.

Address correspondence and reprint requests to Dr. Vanda Jorgetti Laboratório de Fisiopatologia Renal (LIM-16), Av. Dr. Arnaldo, 455, sala 3342, 01246-903, São Paulo, SP, Brazil; E-mail:

The authors report no conflicts of interest.

© 2013 by Mutaz B. Habal, MD.