Justin Bundy

Decreased neural crest stem cell expansion is responsible for the conotruncal heart defects within the Splotch (Sp2H)/Pax3 mouse mutant

OBJECTIVE Several mouse models of cardiac neural crest cell (NCC)-associated conotruncal heart defects exist, but the specific cellular and molecular defects within cardiac NCC morphogenesis remain largely unknown. Our objective was to investigate the underlying mechanisms resulting in outflow tract defects and why insufficient cardiac NCC reach the heart of the Splotch (Sp(2H)) mouse mutant embryo. METHODS For this study we used in vitro cell culture techniques, in vivo mouse-chick chimeras, BrdU cell proliferation labeling, TUNEL labeling to visualize apoptosis and the molecular markers AP-2, Wnt-1 and Wnt-3a to characterize NCC morphogenesis in vivo. RESULTS Expression of the NCC marker AP-2 revealed an extensive reduction in migratory NCC, however the rates of cell proliferation and apoptosis were unaffected, and do not account for the Sp(2H) NCC-associated heart defects. Further expression analysis revealed that Wnt-1, but not Wnt-3a, is expressed at decreased levels within Sp(2H) and that the cardiac NCC fail to undergo normal NC stem cell proliferative expansion prior to migration while still in the neural folds. However, when placed into a wild-type matrix or a tissue culture environment, the Sp(2H) cardiac NCC could migrate normally. Additionally, this reduced population of Sp(2H) NC stem cells do migrate properly within the Sp(2H) environment, as observed by neurofilament expression and cardiac innervation. CONCLUSIONS Taken together, all these data indicate that the Sp(2H) defect is intrinsic to the NC stem cells themselves and that there is a decrease in the number of pre-migratory cardiac NCC that form. It appears that this decrease in NCC number is the primary defect that ultimately leads to a lack of a cardiac NCC-derived Sp(2H) outflow tract septum.

Failure of Orthofix eight-Plate for the treatment of Blount disease.

Background: Hemiepiphysiodesis is a well-established treatment option for angular deformities of the knee. Recently, our institution began using the eight-Plate tension band device by Orthofix (McKinney, Tex) as an alternative to staples. However, several patients have returned with broken screws necessitating revision surgery. Methods: Charts and radiographs of all patients who were treated with the eight-Plate (Orthofix) at our institution were reviewed. The diagnosis, age, amount of angular deformity, weight, and body mass index were analyzed with respect to eventual implant failure. Results: Implant failure occurred in 8 (26%) of 31 proximal tibia constructs. All 8 failures occurred in patients with Blount disease and involved breakage of the tibial metaphyseal screw. The mean time to failure was 13.6 months. Eight hardware failures in 18 Blount disease extremities represent a failure rate of 44%. No implant failures occurred in the remaining diagnoses. Neither age nor degree of deformity correlated with implant failure. The failure group was significantly heavier than the nonfailure group, and the patients with Blount disease were found to be heavier than the other patients. However, no significant difference in weight was found within the Blount group regarding implant failure. In all patients whose plates did not fail, rate of correction was equal to or better than previously reported hemiepiphysiodesis studies. Conclusions: The eight-Plate (Orthofix) is a reasonable option for hemiepiphysiodesis but has an unacceptable failure rate in Blount disease (44%). There were no instances of failure in patients with other diagnoses. In Blount disease, stronger implants should be considered. Future implant designs should include stronger screws to decrease implant failure complications. Level of Evidence: Level 3

Sodium-calcium exchanger is initially expressed in a heart-restricted pattern within the early mouse embryo.

Although the sodium-calcium exchanger (NCX1) is encoded by a single gene, it is widely expressed in both fetal and adult tissues and functions in many diverse physiological processes to maintain intracellular calcium homeostasis. In order to determine whether NCX1 is also ubiquitously expressed in the early mouse embryo, in situ hybridization and RT-PCR were used to determine the spacio-temporal expression of NCX1. Our results indicate that NCX1 expression is present within the 7.75-8.0 dpc cardiogenic plate before the first heartbeat, and that NCX1 is initially expressed in a heart-restricted pattern within the early mouse embryo. However, in more developed embryos (11.0 dpc and older) NCX1 is expressed in other tissues.

Segmental expression of aggrecan in the non-segmented perinotochordal sheath underlies normal segmentation of the vertebral column.

The embryonic vertebral column is derived from the unsegmented axial mesenchyme surrounding the notochord, and its development and differentiation are influenced by the notochord. The role of cartilage in determining the ultimate pattern of the segmental skeleton has been well documented, but a gene whose segmental expression corresponds to the pattern of the developing skeleton has yet to be identified. We show that chick aggrecan is initially expressed within the entire length of the notochord, and as development proceeds, aggrecan expression becomes restricted to the surrounding perinotochordal sheath in a segmental pattern, mirroring the differentiated somite pattern.