Axial strain enhances osteotomy repair with a concomitant increase in connexin43 expression.

The mechanical environment is known to influence fracture healing. We speculated that connexin43 (Cx43) gap junctions, which impact skeletal homeostasis, fracture healing and the osteogenic response to mechanical load, may play a role in mediating the response of the healing bone to mechanical strain. Here, we used an established rat fracture model, which uses a 2 mm osteotomy gap stabilized by an external fixator, to examine the impact of various cyclical axial loading protocols (2%, 10%, and 30% strain) on osteotomy healing. We examined the presence of Cx43 in the osteotomy-healing environment and assessed how mechanical strain modulates Cx43 expression patterns in the callus. We demonstrated that increased cyclical axial strain results in increased radiographic and histologic bone formation. In addition, we show by immunohistochemistry that Cx43 is abundantly expressed in the healing callus, with the expression most robust in samples exposed to increased cyclical axial strain. These data are consistent with the concept that an increase in Cx43 expression by mechanical load may be part of the mechanisms by which mechanical forces enhances fracture healing.

Risk of intracranial hemorrhage associated with autosomal dominant polycystic kidney disease in patients with end stage renal disease.

BACKGROUND: An analysis of intracranial hemorrhage (ICH) in a national sample of autosomal dominant polycystic kidney disease (ADPKD) patients receiving long-term dialysis has not been reported. It is often assumed that patients with ADPKD are not at increased risk of ICH after starting dialysis. We hypothesized that patients with ADPKD would have a higher subsequent risk of ICH even after the start of chronic dialysis. METHODS: Retrospective cohort study of Medicare primary patients with and without ADPKD in the United States Renal Data System (USRDS), initiated on chronic dialysis or transplanted between 1 January 1999 and 3 July 2009, and followed until 31 December 2009. Covariates included age, gender, race, prior stroke, diabetes mellitus, dialysis modality, body mass index, serum albumin and other co-morbid conditions from the Medical Evidence Form. Primary outcome was ICH, based on inpatient and outpatient Medicare claims, and all-cause mortality. Kaplan-Meier analysis was used for unadjusted assessment of time to events. Cox regression was used for assessment of factors associated with ICH and mortality. We performed competing risk regression using kidney transplant and death as competing risks. Kidney transplant was also modeled as a time-dependent covariate in Cox regression. RESULTS: Competing risk regression demonstrated that ADPKD had a subhazard ratio 2.97 for ICH (95% CI 2.27-3.89). Adjusted Cox analysis showed that ADPKD patients had an AHR for death of 0.59 vs. non-ADPKD patients (95% CI 0.57-0.61). CONCLUSIONS: ADPKD is a significant risk factor for ICH among patients on maintenance dialysis. Our Medicare primary cohort was older than in previous studies of intracranial aneurysm rupture among ADPKD patients. There are also limitations inherent to using the USRDS database.

The transcriptional activity of osterix requires the recruitment of Sp1 to the osteocalcin proximal promoter.

The transcription factor osterix (Osx/Sp7) is required for osteogenic differentiation and bone formation in vivo. While Osx can act at canonical Sp1 DNA-binding sites and/or interact with NFATc1 to cooperatively regulate transcription in some osteoblast promoters, little is known about the molecular details by which Osx regulates osteocalcin (OCN) transcription. We previously identified in the OCN proximal promoter a minimal C/T-rich motif, termed OCN-CxRE (connexin-response element) that binds Sp1 and Sp3 in a gap junction-dependent manner. In the present study, we hypothesized that Osx could act via this non-canonical Sp1/Sp3-binding element to regulate OCN transcription. OCN promoter luciferase reporter assays show that Osx alone is an insufficient activator that requires Sp1, but not Sp3, to synergistically stimulate OCN promoter activity. Moreover, promoter deletion analyses demonstrate that both the Sp1/Sp3-binding OCN-CxRE (-70 to -57) and the -92 to -87 region of the OCN proximal promoter are critical for Osx/Sp1 synergistic activities. Our data show that Sp1 influences Osx activity by enhancing Osx occupancy on the OCN promoter, perhaps via physical interactions between the two transcription factors. Finally, alteration of the expression of the gap junction protein connexin43 modulates the recruitment of both Sp1 and Osx to the OCN promoter. In total, our data are strongly in support of Sp1 as an essential transcription factor required for Osx recruitment and transactivation of the OCN promoter. Further, these data lend insight into a mechanism by which alteration of connexin43 impacts osteogenesis in vitro and in vivo.

Induction of an osteocyte-like phenotype by fibroblast growth factor-2.

The purpose of this study was to characterize the molecular phenotype that occurs during the profound morphological shift of cultured osteogenic cells upon treatment with fibroblast growth factor-2 (FGF2). A time course of treatment with FGF2 was performed on an osteoblast cell line, primary bone marrow stromal cells and an osteocyte-like cell line. Morphologic changes were recorded, and gene profiling was carried out by real time PCR. By 8h of FGF2 treatment, there is a striking morphological shift of osteoblast and stromal cells to an elongated dendritic-like morphology that is remindful of osteocytes. In osteoblasts treated with FGF2, this morphologic shift is preceded by an induction of several osteocyte markers, including dentin matrix protein 1 (>20-fold) and E11 (>5-fold). There is a transient increase in the gene expression of sclerostin (3.5-fold) and PHEX (2.5-fold). Sclerostin regulation by FGF2 is complex, as gene expression becomes markedly inhibited by FGF2 at times points after 8h of treatment before rebounding at day 12. Analogous modulation of osteocyte markers is seen in bone marrow stromal cells and MLO-Y4 osteocyte-like cells. In conclusion, this study shows that FGF2 can regulate the transition of osteogenic cells towards the osteocyte lineage, as well as, regulate the expression of critical genes in osteocytes.