ORIGINAL ARTICLE Osteogenesis of Prefabricated Vascularized Periosteal Graft in Rabbits Alvin Chao-Yu Chen, MD, Song-Shu Lin, MS, Yi-Sheng Chan, MD, Mel S. Lee, MD, PhD, and Steve Wen-Neng Ueng, MD Background: The role of vascularized periosteum flap as a graft and the factors stimulating it into osteogenic activity remain obscure. Few articles recognized its osteogenic process without the dependence of stress stimula- tion or bony contact. Methods: We analyzed the osteogenic capacity of the vascularized periosteum in an ectopic site via the method of histomorphologic assess- ment. Pedicled vascularized periosteum was taken from the proximal tibia in rabbits and transferred to the soft tissue pouch in the distal thigh. Specimen was harvested for osteogenesis assessment at 4 weeks, 8 weeks, and 16 weeks. Results: During macroscopic examination, all rabbits were dissected along the previous surgical wound in the distal femur. Dense osseous-like tissue was found in the prefabricated vascularized periosteum. The tissue sections showed red-mineralized matrix of Alizarin Red S staining increased neovascularization. Conclusions: The study results revealed the osteogenic capacity of prefab- ricating vascularized periosteum without the dependence of stress stimula- tion or its proximity to viable bone. Key Words: Prefabrication, Vascularized periosteum, Osteogenesis. (J Trauma. 2009;67: 165–167) R ecent advances in microsurgery have made it possible to maintain blood circulation during tissue transplants. Re- vascularized bone grafts have since become popular in the reconstruction of defects involving major segments of long bones. Researches regarding the osteogenic properties of the vascularized periosteum have been in progress since 1742. 1 The osteogenecity of vascularized periosteum has been clearly established 2,3 ; however, several aspects regarding its role as a graft and the factors underlying its osteogenic activity remain obscure. 4–6 The purpose of this study was to clarify through histomorphologic analyses in the rabbit model whether the vascularized periosteum per se is able to enhance new bone formation in an ectopic site. MATERIALS AND METHODS Animal Surgery A total of six white New Zealand male rabbits weighing 2,500 g to 3,000 g were used. They were purchased from a licensed dealer and humanely handled. The procedures for animal breeding and killing were undertaken according to the guidelines laid down by the animal house; before this, the approval of the ethics committee of our institution was obtained. In three of the six rabbits (these three rabbits served as the vascular group), an incision was made along the medial side of the femur and tibia from the inguinal ligament to the foot. A portion of the tibial periosteum along with a perivas- cular tissue cuff (average size, 1 cm  5 cm) was elevated (Fig. 1) based on the saphenous artery and its venae comi- tantes. The pedicle was dissected to include femoral vessels. The dissection resulted in the formation of a subcutaneous soft pocket at the level of the distal femur. The elevated tibial periosteum flap was then transferred to the pocket (Fig. 2) and fixed with 5-0 nylon (marking the location). Surgery on both legs was performed sequentially. The remaining three rabbits served as the control group. In these rabbits, a piece of free periosteum of size 1 cm  5 cm was harvested and marked with 5-0 nylon sutures. It was then transferred into the soft tissue pocket in the distal thigh and served as a comparison to the vascular group. Before the operation, each animal was anesthetized by an intramuscular injection of 10 mg/kg ketamin. Both hind limbs were shaved and prepared with povidone-iodine. The operations are performed under strict aseptic conditions. Moreover, prophy- lactic antibiotics were administrated perioperatively. Animal Killing and Tissue Processing For osteogenic examination, the rabbits were killed by administering an overdose of phenobarbital at 4 weeks, 8 weeks, and 16 weeks postoperatively. The tissue marked with the 5-0 nylon sutures (previously transferred periosteal flap) in the soft tissue pouch of the distal thigh was extracted for histologic examination. The specimen was decalcified and embedded in paraffin. Sections of 10-m thickness were prepared, stained with hematoxylin and eosin, and examined under a light microscope. Submitted for publication April 2, 2008. Accepted for publication July 25, 2008. Copyright © 2009 by Lippincott Williams & Wilkins Supported in part by the National Science Council grant (NSC 93-2314-B-182A- 034) and Chang Gung Memorial Hospital grant (NMRPG 3089), Taiwan, Republic of China. Presented at the Annual Meeting of Orthopedic Association, Taiwan, October, 2005, titled “Neo-osteogenesis Capacity of Vascular Periosteum (Poster).” None of the authors have received or will receive benefits from a commercial party related directly or indirectly to the subject of this article. From the Department of Orthopaedic Surgery, Chang Gung Memorial Hospital and Chang Gung University, Taiwan, Republic of China. Address for reprints: Alvin Chao-Yu Chen, MD, 5th Fu-Hsin Street, Kweishan, Taoyuan City, Taiwan 333, Republic of China; email: alvinchen@adm. cgmh.org.tw. DOI: 10.1097/TA.0b013e3181881338 The Journal of TRAUMA ® Injury, Infection, and Critical Care • Volume 67, Number 1, July 2009 165