Rat mandibular distraction osteogenesis: II. Molecular analysis of transforming growth factor beta-1 and osteocalcin gene expression

Distraction osteogenesis is a powerful technique capable of generating viab le osseous tissue by die gradual separation of osteotomized bone edges. Alt hough the histologic and ultrastructural changes associated with this proce ss have been extensively delineated, the molecular events governing these c hanges remain essentially unknown. We have devised a rat model of mandibula r distraction osteogenesis that facilitates molecular analysis of this proc ess. Such information has significant clinical implications because it may enable targeted therapeutic manipulations designed to accelerate osseous re generation. in this study, we have evaluated the expression of transforming growth factor beta-1, a major regulator of osteogenesis during endochondra l bone formation and development, and osteocalcin, an abundant noncollageno us extracellular matrix protein implicated in the regulation of mineralizat ion and bone turnover. The right hemi-mandible of 36 adult male rats was os teotomized, and a customized distraction device was applied. Animals were a llowed to recover and, after a 3-day latency period, were distracted at a r ate of 0.25 mm twice daily for 6 days followed by a 2- or 4-week consolidat ion 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 immunohisto chemical localization of TGF-beta 1 (n = 2 at each time point). Six sham-op erated animals (i.e., skin incision without osteotomy) were also killed (im mediately postoperatively), and the mandibles were harvested and prepared i n a similar fashion. Equal loading and transfer of RNA for Northern analysi s was ensured by stripping and probing membranes with a probe against GADPH (a housekeeping gene). Our results demonstrate that the spatial and tempor al patterns of TGF-beta 1 mRNA expression and protein production coincide w ith osteoblast migration, differentiation, and extracellular matrix synthes is. In addition, we demonstrate that TGF-beta 1 production may be an import ant regulator of vasculogenesis during mandibular distraction osteogenesis. Finally, we have shown that osteocalcin gene expression coincides temporal ly with mineralization during rat mandibular distraction osteogenesis.