Silencing NTPDase3 activity rehabilitates the osteogenic commitment of post-menopausal stem cell bone progenitors.

BACKGROUND: Endogenously released adenine and uracil nucleotides favour the osteogenic commitment of bone marrow-derived mesenchymal stromal cells (BM-MSCs) through the activation of ATP-sensitive P2X7 and UDP-sensitive P2Y6 receptors. Yet, these nucleotides have their osteogenic potential compromised in post-menopausal (Pm) women due to overexpression of nucleotide metabolizing enzymes, namely NTPDase3. This prompted us to investigate whether NTPDase3 gene silencing or inhibition of its enzymatic activity could rehabilitate the osteogenic potential of Pm BM-MSCs. METHODS: MSCs were harvested from the bone marrow of Pm women (69 ± 2 years old) and younger female controls (22 ± 4 years old). The cells were allowed to grow for 35 days in an osteogenic-inducing medium in either the absence or the presence of NTPDase3 inhibitors (PSB 06126 and hN3-B3s antibody); pre-treatment with a lentiviral short hairpin RNA (Lenti-shRNA) was used to silence the NTPDase3 gene expression. Immunofluorescence confocal microscopy was used to monitor protein cell densities. The osteogenic commitment of BM-MSCs was assessed by increases in the alkaline phosphatase (ALP) activity. The amount of the osteogenic transcription factor Osterix and the alizarin red-stained bone nodule formation. ATP was measured with the luciferin-luciferase bioluminescence assay. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC RESULTS: The extracellular catabolism of ATP and UDP was faster in BM-MSCs from Pm women compared to younger females. The immunoreactivity against NTPDase3 increased 5.6-fold in BM-MSCs from Pm women vs. younger females. Selective inhibition or transient NTPDase3 gene silencing increased the extracellular accumulation of adenine and uracil nucleotides in cultured Pm BM-MSCs. Downregulation of NTPDase3 expression or activity rehabilitated the osteogenic commitment of Pm BM-MSCs measured as increases in ALP activity, Osterix protein cellular content and bone nodule formation; blockage of P2X7 and P2Y6 purinoceptors prevented this effect. CONCLUSIONS: Data suggest that NTPDase3 overexpression in BM-MSCs may be a clinical surrogate of the osteogenic differentiation impairment in Pm women. Thus, besides P2X7 and P2Y6 receptors activation, targeting NTPDase3 may represent a novel therapeutic strategy to increase bone mass and reduce the osteoporotic risk of fractures in Pm women.

Return to play after conservative and surgical treatment in athletes with spondylolysis: A systematic review.

PURPOSE: Analyze the return to sports rate and timing after conservative and surgical treatment in athletes with spondylolysis. METHODS: Comprehensive search using Pubmed, Cochrane Library and SPORTDiscus databases to identify English language studies that assessed the return to sports after conservative or surgical treatment of symptomatic spondylolysis in athletes. The main outcome of interest was the return to sports rate and timing, as well as, the follow-up clinical and functional outcomes. RESULTS: A total of 14 trials (592 participants) were included. Eight and seven studies reported the outcomes of conservative and surgical approach, respectively. A total of 92% (n = 492) and 88% (n = 100) of athletes return to sports at any level, and 89% (n = 185) and 81% (n = 103) returned to their pre-injury level of sports for conservative and surgical approaches, respectively. The time to return to sports was 4.6 and 6.8 months for conservative and surgical approaches, respectively. CONCLUSIONS: Conservative management (bracing, sports modification and physiotherapy) of athletes with spondylolysis show excellent return to sports rates at any level and at the pre-injury level at a mean of 4.6 months. Those who fail the conservative treatment can be successfully managed with surgical treatment with a high rate of return to sports at 6.8 months. LEVEL OF EVIDENCE: Level IV, Systematic review of level IV studies.

Clinical Management in Early OA.

Knee osteoarthritis affects an important percentage of the population throughout their life. Several factors seem to be related to the development of knee osteoarthritis including genetic predisposition, gender, age, meniscal deficiency, lower limb malalignments, joint instability, cartilage defects, and increasing sports participation. The latter has contributed to a higher prevalence of early onset of knee osteoarthritis at younger ages with this active population demanding more consistent and durable outcomes. The diagnosis is complex and the common signs and symptoms are often cloaked at these early stages. Classification systems have been developed and are based on the presence of knee pain and radiographic findings coupled with magnetic resonance or arthroscopic evidence of early joint degeneration. Nonsurgical treatment is often the first-line option and is mainly based on daily life adaptations, weight loss, and exercise, with pharmacological agents having only a symptomatic role. Surgical treatment shows positive results in relieving the joint symptomatology, increasing the knee function and delaying the development to further degenerative stages. Biologic therapies are an emerging field showing early promising results; however, further high-level research is required.

P2X7-induced zeiosis promotes osteogenic differentiation and mineralization of postmenopausal bone marrow-derived mesenchymal stem cells.

Polymorphisms of the P2X7 receptor have been associated with increased risk of fractures in postmenopausal women. Although both osteoblasts and osteoclasts express P2X7 receptors, their function in osteogenesis remains controversial. Here, we investigated the role of the P2X7 receptor on osteogenic differentiation and mineralization of bone marrow mesenchymal stem cell (BMSC) cultures from postmenopausal women (age 71±3 yr, n=18). We focused on the mechanisms related to intracellular [Ca(2+)]i oscillations and plasma membrane-dynamics. ATP, and the P2X7 agonist BzATP (100 µM), increased [Ca(2+)]i in parallel to the formation of membrane pores permeable to TO-PRO-3 dye uptake. ATP and BzATP elicited reversible membrane blebs (zeiosis) in 38 ± 1 and 70 ± 1% of the cells, respectively. P2X7-induced zeiosis was Ca(2+) independent, but involved phospholipase C, protein kinase C, and Rho-kinase activation. BzATP (100 µM) progressively increased the expression of Runx-2 and Osterix transcription factors by 452 and 226% (at d 21), respectively, alkaline phosphatase activity by 88% (at d 28), and mineralization by 329% (at d 43) of BMSC cultures in a Rho-kinase-dependent manner. In summary, reversible plasma membrane zeiosis involving cytoskeleton rearrangements due to activation of the P2X7-Rho-kinase axis promotes osteogenic differentiation and mineralization of BMSCs, thus providing new therapeutic targets for postmenopausal bone loss.