Osteogenesis in cranial defects: Reassessment of the concept of critical size and the expression of TGF-beta isoforms

Transforming growth factor-betas (TGF-beta) have been demonstrated to be up regulated during osteoblast function in vitro and during cranial suture fus ion in vivo. The authors hypothesized that spontaneous reossification of ca lvarial defects was also associated with upregulation of TGF-beta. The pres ent study was designed to (1) evaluate the concept of a critical-size defec t within the calvaria in an adult guinea pig model and (2) investigate the association between the reossification of calvarial defects and TGF-beta up regulation. Paired circular parietal defects with diameters of 3 and 5 mm a nd single parietal defects with diameters of 8 or 12 mm were made in 45 six -month-old skeletally mature guinea pigs. Three animals per defect size wer e killed after survival periods of 3 days, 1 week, 4 weeks, 8 weeks, or 12 weeks. New bone ingrowth was evaluated by assessing for linear closure by a traditional linear method and by a modified cross-sectional area method us ing an image analysis system in which the thickness of new bone was taken i nto account. Immunohistochemistry was performed using rabbit polyclonal ant ibodies to localize TGF-beta 1, -beta 2. and -beta 3. All specimens were ph otographed, and the intensity of immunostaining was graded based on subject ive photographic assessment by three independent reviewers. No defect demon strated any measurable bone replacement after a survival period of 3 days. All 3- and 5-mm defects were completely reossified after 12 weeks based on the linear analysis of ne iv bone, indicating these defects to be less than critical size. However er, new bone formation in the 5-mm defects never ex ceeded a mean of 40 percent by cross-sectional area of new bone. Percent of new bone formation by cross-sectional area was significantly higher within 3-mm defects than in all larger defects 4 weeks after the craniotomy, reac hing a mean of 89 percent new bone by 12 weeks. Persistent gaps were noted on linear analysis of the 8- and 12-mm wounds by 12 weeks. and mean percent new bone by cross-sectional area remained below 30 percent. Immunolocaliza tion demonstrated osteogenic fronts at the advancing bone edge and the endo cranial side, in which the osteoblasts stained strongly for ail isoforms of TGF-beta. The intensity of osteoblast expression waned considerably af-ter the majority of the defect had reossified. These data indicate that histom etric analysis based on cross-sectional area more accurately reflects the o steogenic potential of a cranial defect than does linear inspection of defe ct closure. Although the interpretation of immunolocalization studies is hi ghly subjective, independent assessment by three reviewers indicates that i soforms of TGF-beta were upregulated during a limited "window" of time corr esponding to the period of active calvarial reossification, and expression of TGF-beta corresponded to osteoblast activity within osteogenic fronts.