Title: Juvenile Versus Adult Osteoblast Gene Regulation: Implications for Calvarial

Title: Juvenile Versus Adult Osteoblast Gene Regulation: Implications for Calvarial Re-Ossification

Authors: Stephen M. Warren, MD, Catherine M. Cowan, MA, Natalino Quarto, PhD, Kenton D. Fong, MD, Jonathan A. Mathy, MD, and Michael T. Longaker, MD

Successful calvarial re-ossification is a characteristic generally restricted to juvenile animals and infants younger than 2 years-of-age.1-4 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.5, 6 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).7, 8 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.

The calvaria of young (2 day-old) and adult (60 day-old) Sprague-Dawley rats were harvested and stripped of dura mater and pericranium. The calvaria were subjected to sequential 15-minute digestions in a 0.1% collagenase/ 0.2% dispase II solution. The first fraction was discarded; fractions two through five were collected, and centrifuged at 1,500 rpm for 5 minutes. The cells were resuspended in 5 mls of osteoblast media (-Modified Eagle’s Medium supplemented with 10% FCS, 100 IU/ml penicillin/streptomycin and 100 g/ml amphotericin, all from Life Technologies; Gaithersburg, MD) and plated on a T-25 flask (Falcon, Franklin Lakes, NJ). 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.

Juvenile osteoblasts proliferated significantly faster than adult osteoblasts (Student’s t-Test: *p<0.02 and **p<0.007, Figure 1). 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 (data not shown). Furthermore, juvenile osteoblasts produced more VEGF mRNA in response to FGF-2 stimulation than adult osteoblasts (data not shown). At baseline, adult Obs expressed 2-fold more FGFR1 mRNA; however, juvenile Obs expressed 5-fold more FGFR2 mRNA (Figures 2 and 3). 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 (Figure 4). Adult Obs expressed 2.5-fold more Tob and 11-fold more PC3 compared to juvenile Obs (data not shown).

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.

Refernces

1.Gross RJ. Roentgenologic aspects of head trauma. Amer J Roentgenol. 1950;64:399-408.

2.Ingraham IJ, Matson DD. Neurosurgery of Infancy and childhood. . Springfiled: C. C. Thomas; 1954.

3.Rowbotham GF. Acute injuries of the head. . Edinburgh: E. & S. Livingstone Ltd.; 1949.

4.Vance RG. The healing of linear fractures of the skull. Amer J Roentgenol. 1936;36:744-746.

5.Mulliken JB, Glowacki J. Induced osteogenesis for repair and construction in the craniofacial region. Plast Reconstr Surg. 1980;65:553-60.

6.Bostrom R, Mikos A. Tissue engineering of bone. In: Atala A, Mooney DJ, Vacanti JP, Langer R, eds. Synthetic Biodegradable Polymer Scaffolds. Boston: Birkhauser; 1997:215-234.

7.Greenwald JA, Mehrara BJ, Spector JA, et al. Biomolecular mechanisms of calvarial bone induction: immature vs. mature dura mater. Plast Reconstr Surg. 2000;105:1382-1392.

8.Greenwald JA, Mehrara BJ, Spector JA, et al. Immature vs. mature dura mater II: Differential expression of genes critical to calvarial re-ossification. Plast Reconstr Surg. 2000;106:630-638.

Figure 1: Juvenile vs. adult osetoblast (Ob) growth curve. Juvenile Obs proliferated significantly faster than adult Obs after 3 days in culture (Student’s t-Test: *p<0.02 and **p<0.007).

Figure 2: Northern analysis demonstrating that adult osteoblasts (Ob) express 2-fold more FGF-R1 mRNA at baseline.

Figure 3: Northern analysis demonstrating that juvenile osteoblasts (Ob) express 5-fold more FGF-R2 mRNA at baseline. rhFGF-2 stimulation results in a 22-fold induction in juvenile Ob FGF-R2 expression compared to a 3-fold induction in adult Ob at 6 hours.

Figure 4: Northern analysis demonstrating that juvenile and adult osteoblasts (Ob) express similar levels of Osteopontin (OP) at baseline, but juvenile Ob upregulate OP mRNA expression 13-fold at 24 hours.

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