Single injection of PTH improves osteoclastic parameters of remodeling at a stress fracture site in rats.

Stress fractures (SFx) result from repetitive cyclical loading of bone. They are frequent athletic injuries and underlie atypical femoral fractures following long-term bisphosphonate (BP) therapy. We investigated the effect of a single PTH injection on the healing of SFx in the rat ulna. SFx was induced in 120 female Wistar rats (300 ± 15 g) during a single loading session. A single PTH (8 µg.100g-1 ) or vehicle (VEH) saline injection was administered 24 h after loading. Rats were divided into four groups (n = 15) and ulnae were examined 1, 2, 6, or 10 weeks following SFx. Two Toluidine Blue and TRAP-stained sections of the SFx were examined for histomorphometric analysis using Osteomeasure™ software. An increase in osteoclast number (N.Oc) and perimeter (Oc.Pm) was observed two weeks following PTH treatment (p < 0.01). At 6 weeks, bone formation was the main activity in BMUs. At 10 weeks, the proportion of healing along the SFx line remained 50% greater in PTH groups (p = 0.839), leading to a 43% reduction in the porosity area of BMU (p = 0.703). The main effect of time was a significant variable along the entire SFx remodeling cycle, with significant interactions between time and treatment type affecting (N.Oc) (p = 0.047) and (Oc.Pm) (p = 0.002). We conclude that a single PTH injection increases osteoclastogenesis by the second week of the remodeling cycle in a SFx in vivo. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Optimized transfection of diced siRNA into mature primary human osteoclasts: inhibition of cathepsin K mediated bone resorption by siRNA.

Osteoclasts are large multinucleated cells responsible for bone resorption. Bone resorption is dependent on the liberation of calcium by acid and protease destruction of the bone matrix by proteinases. The key proteinase produced by the osteoclast is cathepsin K. Targeted knock-down of cathepsin K was performed using small inhibitory RNA (siRNA). siRNA is a method that introduces short double-stranded RNA molecules that instruct the RNA-induced silencing complex (RISC) to degrade mRNA species complementary to the siRNA. Transfection of siRNA by lipid cations allows for short-term inhibition of expression of the targeted gene. We show that transfection of primary human osteoclasts with siRNA to cathepsin K reduces expression by > or = 60% and significantly inhibits bone resorption with a reduction of both resorption pit numbers (P = 0.018) and resorbed area (P = 0.013). We also show that FuGENE 6 is an effective lipid transfection reagent with which to transfect primary human osteoclasts, that does not produce off-target effects.

Vitamin D receptor genotypes influence the success of calcitriol therapy for recurrent vertebral fracture in osteoporosis.

Osteoporosis is a complex multi-factorial disease where environment, diet and genetics play a role in determining susceptibility. Patients with existing vertebral fracture have a heightened risk of further recurrent vertebral fracture. The efficacy of new osteoporosis therapies is often compared to calcium supplementation. 1,25-dihydroxyvitamin D3 (calcitriol) acts through the vitamin D receptor (VDR) and is effective at reducing recurrent vertebral fracture risk. Because the VDR controls calcium metabolism, we hypothesized that genetic variation at the VDR locus may influence response to both calcium and calcitriol therapy. Postmenopausal women with osteoporosis from a 3-year study comparing calcitriol versus calcium for prevention of vertebral fractures were genotyped for VDR alleles detected by FokI, BsmI, ApaI and TaqI. Data were analysed by hierarchical log-linear analysis and robust analysis of variance for relationships to fracture outcomes. Significant differences in the vertebral fracture rate in response to calcium therapy were observed between VDR genotypes (P<0.001). Calcium appeared to be equally effective as calcitriol in particular genotypes. The response to calcitriol therapy was most pronounced in patients carrying the TaqI t allele in combination with the FokI f initiation codon variant: f+t+ carriers were 11.3-fold less likely to sustain recurrent vertebral fracture in the last 2 years of the trial while on calcitriol therapy compared to calcium (P=1.4x10(-5)). Response to both calcium and calcitriol therapy is dependent on genetic variation at the VDR locus and two loci in the VDR gene may contribute to this effect.