|
Sign In to gain access to subscriptions and/or personal tools.
|
The Cranial Base in Craniofacial Development: a Gene Therapy Study
S. Kyrkanides1,2,*,
P. Kambylafkas1,
J.H. Miller1,
R.H. Tallents1 and
J.E. Puzas3
1 Departments of Dentistry,
2 Neurobiology & Anatomy, and
3 Department of Orthopedics and Center for Musculo-skeletal Research, School of Medicine and Dentistry, University of Rochester Medical Center, 625 Elmwood Ave., Rochester NY 14620, USA
View larger version (143K):
[in this window]
[in a new window]
|
Figure 1. Cellular organization and chondrocyte maturation are selectively impaired in the cranial base synchondroses. Comparison of histology sections processed for Alcian blue-orange G histochemistry that were harvested from long bone growth plates (tibia) of (A) wild-type, (B) mutant, and (C) FIV(HEX)-treated mice revealed the absence of any histopathology in the long bones of affected mice. These findings were confirmed by immunohistochemical analysis of (D) wild-type, (E) mutant, and (F) FIV(HEX)-treated mice, with antibodies against type 2 collagen. Conversely, we observed significant histopathological differences between (G) wild-type and (H) mutant mice. Representative images are shown here. Specifically, the HexB–/– mice displayed reduced levels of cartilage content (light blue stain) and ectopic bone formation (red stain). There was also loss of chondrocyte column formation that is normally seen in these areas. (I) Neonatal FIV(HEX) gene therapy normalized the cyto-architecture and ameliorated any histopathology at the cranial base synchondroses. These findings were further confirmed by type 2 collagen immunohistochemistry in (J) wild-type, (K) mutant, and (L) FIV(HEX)-treated synchondroses. (M) PTHrP, a known regulator of chondrocyte differentiation and maturation, was absent (N) in HexB–/– synchondroses and was restored in part in (O) the FIV(HEX)-treated mutant mice. Bar = 100 µm.
|
|
View larger version (62K):
[in this window]
[in a new window]
|
Figure 2. COX-2 activation is implicated in abnormal cranial base development. The stress-activated p38 MAK, a known inducer of COX-2, was induced in (A) the proliferative zone chondrocytes of HexB–/– mice, compared with (B) wild-type mice, as assessed by immunofluorescence. The expression of COX-2, a regulator of chondrocyte differentiation and maturation, was also induced in (C) HexB–/– vs. (D) wild-type mice, as detected by immunofluorescence. Nuclear Hoechst staining displays the cell population present in the aforementioned images (A–E; B–F; C–G; D–H). The pertinent histology is depicted (A–I; B–J; C–K; D–L) as captured by light microscopy. Representative images are shown. Bar = 100 µm.
|
|
View larger version (41K):
[in this window]
[in a new window]
|
Figure 3. Craniofacial morphology was evaluated in HexB–/– (N = 6), HexB+/– heterozygotes (N = 6), wild-type (N = 7), and FIV(HEX)-treated HexB–/– mice (N = 10). Lateral cephalometric radiographs of (A) HexB–/–and (B) wild-type mice are shown (arrow points to cranial base). (C) Summary of cephalometric measurements performed in this study. Cephalometric analysis revealed that (D) the Ba-Rh, (E) Na-Rh, and (F) Na-Ba distances were significantly reduced in the HexB–/– mice, compared with HexB+/– and wild-type controls, as well as FIV(HEX)-treated mice. These measurements demonstrate the presence of cranial base and nasomaxillary deficiencies in mice suffering from β-hexosaminidase deficiency. Analysis of the data also showed that neonatal β-hexosaminidase restitution rescued the HexB–/– mice from developing craniofacial dysplasia. Differences among the 4 groups were evaluated by one-way analysis of variance, followed by post hoc analysis by Dunnett’s method. Ba-Rh differences were statistically different at p = 0.00282 (power = 0.8913); for Na-Rh, p = 0.0020 (power = 0.9212); and for Na-Ba, p = 0.0021 (power = 0.91). Mean ± SD. *p < 0.05.
|
|
View larger version (150K):
[in this window]
[in a new window]
|
Figure 4. COX-2 activation is implicated in abnormal chondrocyte maturation secondary to β-hexosaminidase deficiency.GM2 ganglioside, the pathognomonic by-product of β-hexosaminidase deficiency, is selectively found in the proliferative and hypertrophic zones of HexB–/– cranial base synchondroses, as detected by immunohistochemistry with a monoclonal antibody. (A) Wild-type, (B) HexB–/–, and (C) FIV(HEX)-treated HexB–/– mice. Moreover, COX-2 expression correlated with GM2 storage in (D) wild-type, (E) HexB–/– mutant, and (F) FIV(HEX)- treated mice. To test whether chondrocyte maturation and differentiation are affected by the induction of the cyclo-oxygenase-prostaglandin pathway, we used the C2C12 cell line, an in vitro model of chondrocyte differentiation. To this end, the conversion of immature C2C12 cells to an osteoblastic phenotype was evaluated by assessment of the alkaline phosphatase expression in situ (black stain). (G) Untreated control cells showed no signs of conversion over a four-day period. Conversely, (H) treatment with BMP-2 (300 ng/mL) over 4 days induced the expression of alkaline phosphatase (black stain) in 10% of the cells in culture, suggesting a shift in their differentiation toward osteoblastic cells. Conversely, (I) treatment of C2C12 cells with BMP-2 plus PGE2 (10–8 M) over the same time period increased the number of cells expressing alkaline phosphatase by approximately five-fold, demonstrating the ability of PGE2 to increase the differentiation rate of C2C12 to osteoblastic cells (number of cells converted in a defined period of time). Representative images are shown here. *p < 0.05. Bar = 100 µm.
|
|
Journal of Dental Research, Vol. 86, No. 10,
956-961 (2007)
DOI: 10.1177/154405910708601008
 CiteULike  Complore  Connotea  Del.icio.us  Digg  Reddit  Technorati  Twitter What's this?
|
|
