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Rat Mandibular Distraction Osteogenesis: II. Molecular Analysis of Transforming Growth Factor Beta-1 and Osteocalcin Gene Expression

Mehrara, Babak J. M.D.; Rowe, Norman M. M.D.; Steinbrech, Douglas S. M.D.; Dudziak, Matthew E. B.S.; Saadeh, Pierre B. M.D.; McCarthy, Joseph G. M.D.; Gittes, George K. M.D.; Longaker, Michael T. M.D.

Plastic and Reconstructive Surgery: February 1999 - Volume 103 - Issue 2 - p 536–547
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Distraction osteogenesis is a powerful technique capable of generating viable osseous tissue by the gradual separation of osteotomized bone edges. Although the histologic and ultrastructural changes associated with this process have been extensively delineated, the molecular events governing these changes remain essentially unknown. We have devised a rat model of mandibular distraction osteogenesis that facilitates molecular analysis of this process. Such information has significant clinical implications because it may enable targeted therapeutic manipulations designed to accelerate osseous regeneration. In this study, we have evaluated the expression of transforming growth factor beta-1, a major regulator of osteogenesis during endochondral bone formation and development, and osteocalcin, an abundant noncollagenous extracellular matrix protein implicated in the regulation of mineralization and bone turnover. The right hemimandible of 36 adult male rats was osteotomized, and a customized distraction device was applied. Animals were allowed to recover and, after a 3-day latency period, were distracted at a rate of 0.25 mm twice daily for 6 days followed by a 2- or 4-week consolidation period. Distraction regenerate was harvested after the latency period, days 2, 4, or 6 of distraction, and after 2 or 4 weeks of consolidation and processed for Northern analysis (n = 4 at each time point) and immunohistochemical localization of TGF-β1 (n = 2 at each time point). Six shamoperated animals (i.e., skin incision without osteotomy) were also killed (immediately postoperatively), and the mandibles were harvested and prepared in a similar fashion. Equal loading and transfer of RNA for Northern analysis was ensured by stripping and probing membranes with a probe against GAPDH (a housekeeping gene). Our results demonstrate that the spatial and temporal patterns of TGF-β1 mRNA expression and protein production coincide with osteoblast migration, differentiation, and extracellular matrix synthesis. In addition, we demonstrate that TGF-β1 production may be an important regulator of vasculogenesis during mandibular distraction osteogenesis. Finally, we have shown that osteocalcin gene expression coincides temporally with mineralization during rat mandibular distraction osteogenesis. (Plast. Reconstr. Surg. 103: 536, 1999.)

New York, N.Y.

Michael T. Longaker, M.D. New York University Medical Center Institute of Reconstructive Plastic Surgery Room H169 New York, N.Y. 10016

From the Laboratory of Developmental Biology and Repair, the Institute of Reconstructive Plastic Surgery, and the Department of Surgery at New York University Medical Center. Received for publication March 6, 1998; revised August 19, 1998.

©1999American Society of Plastic Surgeons