Thm2 interacts with paralog, Thm1, and sensitizes to Hedgehog signaling in postnatal skeletogenesis.

Mutations in the intraflagellar transport-A (IFT-A) gene, THM1, have been identified in skeletal ciliopathies. Here, we report a genetic interaction between Thm1, and its paralog, Thm2, in postnatal skeletogenesis. THM2 localizes to primary cilia, but Thm2 deficiency does not affect ciliogenesis and Thm2-null mice survive into adulthood. However, by postnatal day 14, Thm2-/-; Thm1aln/+ mice exhibit small stature and small mandible. Radiography and microcomputed tomography reveal Thm2-/-; Thm1aln/+ tibia are less opaque and have reduced cortical and trabecular bone mineral density. In the mutant tibial growth plate, the proliferation zone is expanded and the hypertrophic zone is diminished, indicating impaired chondrocyte differentiation. Additionally, mutant growth plate chondrocytes show increased Hedgehog signaling. Yet deletion of one allele of Gli2, a major transcriptional activator of the Hedgehog pathway, exacerbated the Thm2-/-; Thm1aln/+ small phenotype, and further revealed that Thm2-/-; Gli2+/- mice have small stature. In Thm2-/-; Thm1aln/+ primary osteoblasts, a Hedgehog signaling defect was not detected, but bone nodule formation was markedly impaired. This indicates a signaling pathway is altered, and we propose that this pathway may potentially interact with Gli2. Together, our data reveal that loss of Thm2 with one allele of Thm1, Gli2, or both, present new IFT mouse models of osteochondrodysplasia. Our data also suggest Thm2 as a modifier of Hedgehog signaling in postnatal skeletal development.

Age and gender related differences in load-strain response in C57Bl/6 mice.

We examined the changes in mechanical strain response of male and female mouse tibia and ulna, using axial compression tests, to assess age-related changes in tibiae and ulnae by a non-contact strain measurement technique called the digital image correlation (DIC) and the standard strain gage. A unique aspect of the study was to compare bones from the same animal to study variations in behavior with aging. This study was conducted using male and female C57Bl/6 mice at 6, 12 and 22 months of age (N=6-7 per age and sex) using three load levels. The DIC technique was able to detect a greater number of statistically significant differences in comparison to the strain gaging method. Male ulna showed significantly higher DIC strains compared to strains captured from strain gage at all three levels of load at 6 months and in the lowest load at 12 months. DIC measurements revealed that the ulna becomes stiffer with aging for both males and females, which resulted in 0.4 to 0.8 times reduced strains in the 22-month group compared to the 6 month group. Male tibia showed three-fold increased strains in the 22 months group at 11.5 N load compared to 6 months group (p<.05).

Age-related and sex-specific effects on architectural properties and biomechanical response of the C57BL/6N mouse femur, tibia and ulna.

Aging is known to reduce bone quality and bone strength. We sought to determine how aging affects the biomechanical and architectural properties of various long bones, and if sex influences age related differences/changes. While researchers have extensively studied these changes in individual bones of mice, there is no comprehensive study of the changes in the bones from the same mice to study the changes with aging. We performed three point bending tests and microcomputed tomography (microCT) analysis on femurs, tibiae and ulnae. Three point bending tests were utilized to calculate biomechanical parameters and imaging was also performed using high resolution microCT to reveal both cortical and trabecular microarchitecture C57BL/6N mice were divided into three age groups: 6, 12 and 22 months. Each age and sex group consisted of 6-7 mice. The ultimate load to failure (UL), elastic stiffness (ES), modulus of elasticity (E) and the moment of inertia about bending axis (MOI) for each bone was calculated using three point bending test. MicroCT scans of all the bones were analyzed to determine cortical bone volume per tissue volume (C.BV/TV), trabecular bone volume per tissue volume (Tb.BV/TV), cortical bone area (B.Ar) using CTAn's microCT analysis and tested for correlation with the biomechanical parameters. Mean (standard error) values of UL in femur decreased from 19.8(0.6) N to 12.8(1.1) N (p < .01) and 17.9(0.6) N to 14.6(1.0) N (p = .02) from 6 to 22 months groups in males and females respectively. Similarly, UL in tibia decreased from 19.8(0.5) N to 14.3(0.2) N (p < .01) and 14.4(0.6) N to 9.5(1.0) N (p < .01) from 6 to 22 months group in males and females respectively. ES in femur decreased from 113.2(7) N/mm to 69.6(6.7) N/mm (p < .01) from 6 to 22 months in males only. ES in tibia decreased from 78.6(3.2) N/mm to 65.0(2.3) N/mm (p = .01) and 53.1(2.9) N/mm to 44.0(1.7) N/mm (p = .02) from 6 to 22 months in males and females respectively. Interestingly, ES in ulna increased from 8.2(0.8) N/mm to 10.9(1.0) N/mm (p = .051) from 6 to 22 months of age in females only. E in femur decreased from 4.0(0.4) GPa to 2.8(0.2) GPa (p = .01) and 6.7(0.5) GPa to 4.5(0.4) GPa (p = .01) from 6 to 22 months of age in males and females respectively while tibia showed no change. However, E in ulna increased from 7.0(0.8) GPa to 11.0(1.1) GPa (p = .01) from 6 to 22 months of age in females only. Changes in age and sex-related bone properties were more pronounced in the femur and tibia, while the ulna showed fewer overall differences. Most of the changes were observed in biomechanical compared to architectural properties and female bones are more severely affected by aging. In conclusion, our data demonstrate that care must be taken to describe bone site and sex-specific, rather than making broad generalizations when describing age-related changes on the biomechanical and architectural properties of the skeleton.