Delayed fracture healing in growth differentiation factor 5-deficient mice: a pilot study.

BACKGROUND: Growth differentiation factor-5 (GDF-5) is a key regulator of skeletogenesis and bone repair and induces bone formation in spinal fusions and nonunion applications by enhancing chondrocytic and osteocytic differentiation and stimulating angiogenesis. Elucidating the contribution of GDF-5 to fracture repair may support its clinical application in complex fractures. QUESTIONS/PURPOSE: We therefore asked whether the absence of GDF-5 during fracture repair impaired bone healing as assessed radiographically, histologically, and mechanically. METHODS: In this pilot study, we performed tibial osteotomies on 10-week-old male mice, stabilized by intramedullary and extramedullary nailing. Healing was assessed radiographically and histologically on Days 1 (n = 1 wild-type; n = 5 bp [brachopodism]), 5 (n = 3 wild-type; n = 3 bp), 10 (n = 6 wild-type; n = 3 bp), 14 (n = 6 wild-type; n = 6 bp), 21 (n = 6 wild-type; n = 6 bp), 28 (n = 7 wild-type; n = 6 bp), and 56 (n = 6 wild-type; n = 6 bp) after fracture. After 10 (n = 7 wild-type; n = 7 bp contralateral and n = 3 bp fractured tibiae), 14 (n = 6 wild-type; n = 6 bp), 21 (n = 6 wild-type; n = 6 bp), 28 (n = 6 wild-type; n = 3 bp), and 56 (n = 8 wild-type; n = 6 bp) days, the callus cross-sectional area was calculated. We characterized the mechanical integrity of the healing fracture by yield stress and Young's modulus at 28 (n = 6 wild-type; n = 3 bp) and 56 (n = 8 wild-type; n = 6 bp) days postfracture. RESULTS: The absence of GDF-5 impaired cartilaginous matrix deposition in the callus and reduced callus cross-sectional area. After 56 days, the repaired bp fracture was mechanically comparable to that of controls. CONCLUSIONS: Although GDF-5 deficiency did not compromise long-term fracture healing, a delay in cartilage formation and remodeling supports roles for GDF-5 in the early phase of bone repair. CLINICAL RELEVANCE: Local delivery of GDF-5 to clinically difficult fractures may simulate cartilage formation in the callus and support subsequent remodeling.

Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits.

The beneficial effects of physical activity (PA) are well documented, yet the mechanisms by which PA prevents disease and improves health outcomes are poorly understood. To identify major gaps in knowledge and potential strategies for catalyzing progress in the field, the NIH convened a workshop in late October 2014 entitled "Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits." Presentations and discussions emphasized the challenges imposed by the integrative and intermittent nature of PA, the tremendous discovery potential of applying "-omics" technologies to understand interorgan crosstalk and biological networking systems during PA, and the need to establish an infrastructure of clinical trial sites with sufficient expertise to incorporate mechanistic outcome measures into adequately sized human PA trials. Identification of the mechanisms that underlie the link between PA and improved health holds extraordinary promise for discovery of novel therapeutic targets and development of personalized exercise medicine.

Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells.

Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.

Extracellular zinc and ATP restore chloride secretion across cystic fibrosis airway epithelia by triggering calcium entry.

Cystic fibrosis (CF) is caused by defective cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator Cl(-) channels. Thus, CF epithelia fail to transport Cl(-) and water. A postulated therapeutic avenue in CF is activation of alternative Ca(2+)-dependent Cl(-) channels. We hypothesized that stimulation of Ca(2+) entry from the extracellular space could trigger a sustained Ca(2+) signal to activate Ca(2+)-dependent Cl(-) channels. Cytosolic [Ca(2+)](i) was measured in non-polarized human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Primary human CF and non-CF airway epithelial monolayers as well as Calu-3 monolayers were used to assess anion secretion. In vivo nasal potential difference measurements were performed in non-CF and two different CF mouse (DeltaF508 homozygous and bitransgenic gut-corrected but lung-null) models. Zinc and ATP induced a sustained, reversible, and reproducible increase in cytosolic Ca(2+) in CF and non-CF cells with chemistry and pharmacology most consistent with activation of P2X purinergic receptor channels. P2X purinergic receptor channel-mediated Ca(2+) entry stimulated sustained Cl(-) and HCO(3)(-) secretion in CF and non-CF epithelial monolayers. In non-CF mice, zinc and ATP induced a significant Cl(-) secretory response similar to the effects of agonists that increase intracellular cAMP levels. More importantly, in both CF mouse models, Cl(-) permeability of nasal epithelia was restored in a sustained manner by zinc and ATP. These effects were reversible and reacquirable upon removal and readdition of agonists. Our data suggest that activation of P2X calcium entry channels may have profound therapeutic benefit for CF that is independent of cystic fibrosis transmembrane conductance regulator genotype.