Chronic exposure to methylphenidate impairs appendicular bone quality in young rats.

Methylphenidate (MP) is a psychostimulant widely prescribed to treat Attention Deficit Hyperactivity Disorder (ADHD). Although generally well tolerated, growth deficits have been reported in children and adolescents undergoing MP treatment. This study was designed to elucidate the skeletal effects of chronic MP administration in adolescent rats. Male, 4-week-old rats received one of two doses of MP (MP-Low or MP-High) delivered for 8 h a day via drinking water, or were untreated (water only). After 13 weeks, half were sacrificed (N=12/group) and the remaining rats were left to recover, untreated for 5 additional weeks. Femora, tibiae, and L5 vertebra were analyzed using calipers, DXA, and mechanical testing. Immediately following treatment, MP decreased femoral anterior-posterior diameter (5% and 9% for MP-Low and MP-High, respectively), femoral and tibial bone mineral density (BMD) (6% and 5% for MP-High femora and tibiae, respectively), and bone mineral content (BMC) (9% for MP-High femora and tibiae). In addition, femora from MP treated rats had reduced ultimate force (20% for MP-High) and energy to failure (20% and 33% for MP-Low and MP-High, respectively). However, after recovery, there were no statistically significant differences for any measured parameters. Despite these effects on the appendicular skeleton, no differences were identified between vertebral samples at either time-point. In summary, MP treatment resulted in smaller, less mineralized, and weaker bones at appendicular sites, but did not affect the axial site. Although these effects were ameliorated within 5 weeks, these data suggest that adolescents undergoing MP treatment may be at an increased risk for long bone fractures.

Mechanoreceptors found in a posterior cruciate ligament from a well-functioning total knee arthroplasty retrieval.

Histologic analysis of the posterior cruciate ligament has been reported in the normal and osteoarthritic knee but not after cruciate-retaining (CR) total knee arthroplasty (TKA). Retention of the posterior cruciate ligament during TKA has been debated as to whether it is beneficial in stability and function. If the presence of mechanoreceptors is shown to be maintained in CR TKA, then there may be an argument for retention. This case report used a retrieval of a well-functioning TKA specimen that had a CR TKA. To prove the presence of mechanoreceptors within the ligament, immunohistochemistry techniques using S100 protein and neurofilament protein were used. This specimen had pacini and lamellar type of mechanoreceptors present on immunohistochemistry analysis. The presence or retention of mechanoreceptors and innervations of the ligament may indicate an advantage when retained during TKA.

Modulation of Wnt signaling influences fracture repair.

While the importance of Wnt signaling in skeletal development and homeostasis is well documented, little is known regarding its function in fracture repair. We hypothesized that activation and inactivation of Wnt signaling would enhance and impair fracture repair, respectively. Femoral fractures were generated in Lrp5 knockout mice (Lrp5-/-) and wild-type littermates (Lrp5+/+), as well as C57BL/6 mice. Lrp5-/- and Lrp5+/+ mice were untreated, while C57BL/6 mice were treated 2x/week with vehicle or anti-Dkk1 antibodies (Dkk1 Ab) initiated immediately postoperatively (Day 0) or 4 days postoperatively (Day 4). Fractures were radiographed weekly until sacrifice at day 28, followed by DXA, pQCT, and biomechanical analyses. Lrp5-/- mice showed impaired repair compared to Lrp5+/+ mice, as evidenced by reduced callus area, BMC, BMD, and biomechanical properties. The effects of Dkk1 Ab treatment depended on the timing of initiation. Day 0 initiation enhanced repair, with significant gains seen for callus area, BMC, BMD, and biomechanical properties, whereas Day 4 initiation had no effect. These results validated our hypothesis that Wnt signaling influences fracture repair, with prompt activation enhancing repair and inactivation impairing it. Furthermore, these data suggest that activation of Wnt signaling during fracture repair may have clinical utility in facilitating fracture repair.

Expression of the Arf tumor suppressor gene is controlled by Tgfbeta2 during development.

The Arf tumor suppressor (also known as Cdkn2a) acts as an oncogene sensor induced by ;abnormal' mitogenic signals in incipient cancer cells. It also plays a crucial role in embryonic development: newborn mice lacking Arf are blind due to a pathological process resembling severe persistent hyperplastic primary vitreous (PHPV), a human eye disease. The cell-intrinsic mechanism implied in the oncogene sensor model seems unlikely to explain Arf regulation during embryo development. Instead, transforming growth factor beta2 (Tgfbeta2) might control Arf expression, as we show that mice lacking Tgfbeta2 have primary vitreous hyperplasia similar to Arf(-/-) mice. Consistent with a potential linear pathway, Tgfbeta2 induces Arf transcription and p19(Arf) expression in cultured mouse embryo fibroblasts (MEFs); and Tgfbeta2-dependent cell cycle arrest in MEFs is maintained in an Arf-dependent manner. Using a new model in which Arf expression can be tracked by beta-galactosidase activity in Arf(lacZ/+) mice, we show that Tgfbeta2 is required for Arf transcription in the developing vitreous as well as in the cornea and the umbilical arteries, two previously unrecognized sites of Arf expression. Chemical and genetic strategies show that Arf promoter induction depends on Tgfbeta receptor activation of Smad proteins; the induction correlates with Smad2 phosphorylation in MEFs and Arf-expressing cells in vivo. Chromatin immunoprecipitation shows that Smads bind to genomic DNA proximal to Arf exon 1beta. In summary, Tgfbeta2 and p19(Arf) act in a linear pathway during embryonic development. We present the first evidence that p19(Arf) expression can be coupled to extracellular cues in normal cells and suggest a new mechanism for Arf control in tumor cells.

Persistent hyperplastic primary vitreous due to somatic mosaic deletion of the arf tumor suppressor.

PURPOSE: Mice lacking the Arf tumor-suppressor gene develop eye disease reminiscent of persistent hyperplastic primary vitreous (PHPV). The current work explores mechanisms by which Arf promotes eye development, and its absence causes a PHPV-like disease. METHODS: Chimeric mice were made by fusing wild-type and Arf(-/-) morulae. In these experiments, wild-type cells are identified by transgenic expression of GFP from a constitutive promoter. PCR-based genotyping and quantitative analyses after immunofluorescence staining of tissue and cultured cells documented the relative contribution of wild-type and Arf(-/-) cells to different tissues in the eye and different types of cells in the vitreous. RESULTS: The contributions of the Arf(-/-) lineage to the tail DNA, cornea, retina, and retina pigment epithelium (RPE) correlated with each other in wild-type<-->Arf(-/-) chimeric mice. Newborn chimeras had primary vitreous hyperplasia, evident as a retrolental mass. The mass was usually present when the proportion of Arf(-/-) cells was relatively high and absent when the Arf(-/-) proportion was low. The Pdgfrbeta- and Sma-expressing cells within the mass arose predominantly from the Arf(-/-) population. Ectopic Arf expression induced smooth muscle proteins in cultured pericyte-like cells, and Arf and Sma expression overlapped in hyaloid vessels. CONCLUSIONS: In the mouse model, loss of Arf in only a subset of cells causes a PHPV-like disease. The data indicate that both cell autonomous and non-cell autonomous effects of Arf may contribute to its role in vitreous development.