INTRODUCTION: Successful calvarial re-ossification is a characteristic generally restricted to juvenile animals and infants younger than 2 years-of-age. Since this osteogenic capacity rapidly diminishes, older animals and adults with non-healing cranial defects due to trauma, craniofacial or neurosurgical operations present a difficult reconstructive challenge and a substantial biomedical burden. Presently, surgeons use a variety of techniques to reconstruct non-healing calvarial defects. While these operations are usually successful, they are complex procedures with numerous limitations. To address these problems, we are investigating the mechanisms mediating successful juvenile calvarial re-ossification. To date, research has focused on the regenerative potential of the dura mater lining the base of the calvarial defect; however, little is known about the differences in cellular biology between juvenile and adult osteoblasts (Obs). In the present study, we compared the growth characteristics, osteogenic and angiogenic potential of juvenile and adult osteoblasts. In addition, we investigated the mechanisms leading to these different phenotypes by analyzing the expression of tyrosine kinase receptors (FGFRs), osteogenic proteins (osteopontin [OP]) and anti-proliferative factors (transducer of ErbB-2.1 [Tob]; B-cell translocation gene [PC3]) at baseline and in response to rhFGF-2.

METHODS: Rat calvarial Ob cultures were established from 2 day- (juvenile) and 60 (adult) day-old animals. Cell counting was used to compare juvenile and adult osteoblast growth rates. In order to compare their osteogenic/angiogenic potential as well as proliferative profiles, juvenile and adult osteoblast cultures were stimulated with rhFGF-2 (10 ng/ml) and total cellular RNA was harvested at 0, 3, 6, 12, and 24 hours. Northern blot analyses were performed for BMP-2, VEGF, FGFR1, FGFR2, OP, Tob, and PC3.

RESULTS: Juvenile osteoblasts proliferated significantly faster than adult osteoblasts at 3 and 5 days (Student's t-Test: *p<0.003 and *p<0.001, respectively). Both juvenile and adult osteoblasts produced BMP-2 mRNA in response to FGF-2; however, the 6-hour BMP-2 mRNA peak in juvenile osteoblasts was markedly increased compared to adult osteoblasts. Furthermore, juvenile osteoblasts produced more VEGF mRNA in response to FGF-2 stimulation than adult osteoblasts. At baseline, adult Obs expressed 2-fold more FGFR1 mRNA; however, juvenile Obs expressed 5-fold more FGFR2 mRNA. Although, juvenile and adult Obs produced equal amounts of baseline OP, juvenile Obs increased their expression 13-fold after 24 hrs of rhFGF-2 stimulation, compared to an adult Obs increase of only 5-fold. Finally, adult Obs expressed 2.5-fold more Tob and 11-fold more PC3 compared to juvenile Obs.

CONCLUSIONS: Collectively, these data suggest that juvenile animals may be able to successfully heal craniotomy defects because juvenile osteoblasts proliferate more rapidly and produce greater amounts of osteogenic and angiogenic proteins when stimulated with dura-derived cytokines, such as FGF-2. Furthermore adult Obs are more differentiated (high FGFR1) and less proliferative (low FGFR2) than juvenile Obs. Even after stimulation with rhFGF-2, adult Obs do not increase production of genes necessary for bone formation (e.g. OP) to the same degree as juvenile Obs. Finally, adult Ob proliferation may be inhibited by anti-proliferative gene expression (i.e. Tob and PC3). These results begin to explain why complete calvarial re-ossification is restricted to juvenile animals.
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