Mechanical stimulation-induced purinome priming fosters osteogenic differentiation and osteointegration of mesenchymal stem cells from the bone marrow of post-menopausal women.

BACKGROUND: Mechanical stimulation (MS) significantly increases the release of adenine and uracil nucleotides from bone marrow-derived mesenchymal stem cells (BM-MSCs) undergoing osteogenic differentiation. Released nucleotides acting via ionotropic P2X7 and metabotropic P2Y6 purinoceptors sensitive to ATP and UDP, respectively, control the osteogenic commitment of BM-MSCs and, thus, bone growth and remodelling. Yet, this mechanism is impaired in post-menopausal (Pm)-derived BM-MSCs, mostly because NTPDase3 overexpression decreases the extracellular accumulation of nucleotides below the levels required to activate plasma membrane-bound P2 purinoceptors. This prompted us to investigate whether in vitro MS of BM-MSCs from Pm women could rehabilitate their osteogenic commitment and whether xenotransplantation of MS purinome-primed Pm cells promote repair of critical bone defects in an in vivo animal model. METHODS: BM-MSCs were harvested from the neck of femora of Pm women (70 ± 3 years old) undergoing total hip replacement. The cells grew, for 35 days, in an osteogenic-inducing medium either submitted (SS) or not (CTR) to MS (90 r.p.m. for 30 min) twice a week. Increases in alkaline phosphatase activity and in the amount of osteogenic transcription factors, osterix and osteopontin, denoted osteogenic cells differentiation, while bone nodules formation was ascertain by the alizarin red-staining assay. The luciferin-luciferase bioluminescence assay was used to quantify extracellular ATP. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC. The density of P2Y6 and P2X7 purinoceptors in the cells was assessed by immunofluorescence confocal microscopy. MS-stimulated BM-MSCs from Pm women were xenotransplanted into critical bone defects drilled in the great trochanter of femora of one-year female Wistar rats; bone repair was assessed by histological analysis 10 days after xenotransplantation. RESULTS: MS-stimulated Pm BM-MSCs in culture (i) release 1.6-fold higher ATP amounts, (ii) overexpress P2X7 and P2Y6 purinoceptors, (iii) exhibit higher alkaline phosphatase activity and overexpress the osteogenic transcription factors, osterix and osteopontin, and (iv) form larger bone nodules, than CTR cells. Selective blockage of P2X7 and P2Y6 purinoceptors with A438079 (3 µM) and MRS 2578 (0.1 µM), respectively, prevented the osteogenic commitment of cultured Pm BM-MSCs. Xenotransplanted MS purinome-primed Pm BM-MSCs accelerated the repair of critical bone defects in the in vivo rat model. CONCLUSIONS: Data suggest that in vitro MS restores the purinergic cell-to-cell communication fostering the osteogenic differentiation and osteointegration of BM-MSCs from Pm women, a strategy that may be used in bone regeneration and repair tactics.

Could hypoxia rehabilitate the osteochondral diseased interface? Lessons from the interplay of hypoxia and purinergic signals elsewhere.

The osteochondral unit comprises the articular cartilage (90%), subchondral bone (5%) and calcified cartilage (5%). All cells present at the osteochondral unit that is ultimately responsible for matrix production and osteochondral homeostasis, such as chondrocytes, osteoblasts, osteoclasts and osteocytes, can release adenine and/or uracil nucleotides to the local microenvironment. Nucleotides are released by these cells either constitutively or upon plasma membrane damage, mechanical stress or hypoxia conditions. Once in the extracellular space, endogenously released nucleotides can activate membrane-bound purinoceptors. Activation of these receptors is fine-tuning regulated by nucleotides' breakdown by enzymes of the ecto-nucleotidase cascade. Depending on the pathophysiological conditions, both the avascular cartilage and the subchondral bone subsist to significant changes in oxygen tension, which has a tremendous impact on tissue homeostasis. Cell stress due to hypoxic conditions directly influences the expression and activity of several purinergic signalling players, namely nucleotide release channels (e.g. Cx43), NTPDase enzymes and purinoceptors. This review gathers experimental evidence concerning the interplay between hypoxia and the purinergic signalling cascade contributing to osteochondral unit homeostasis. Reporting deviations to this relationship resulting from pathological alterations of articular joints may ultimately unravel novel therapeutic targets for osteochondral rehabilitation. At this point, one can only hypothesize how hypoxia mimetic conditions can be beneficial to the ex vivo expansion and differentiation of osteo- and chondro-progenitors for auto-transplantation and tissue regenerative purposes.

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.

Adenosinergic signalling in chondrogenesis and cartilage homeostasis: Friend or foe?

Chondrocytes and their mesenchymal cell progenitors secrete a variety of bioactive molecules, including adenine nucleotides and nucleosides, but these molecules are not usually highlighted in review papers about the secretome of these cells. Ageing and inflammatory insults compromise chondrocytes ability to keep ATP/adenosine synthesis, release and turnover. Cartilage homeostasis depends on extracellular adenosine levels, which acting via four P1 purinoceptor subtypes modulates the release of pro-inflammatory mediators, including NO, PGE2 and several cytokines. Native articular cartilage is challenged by synovial fluid flow during normal joint motion transiently increasing ATP release and adenosine formation in the joint microenvironment. Excessive joint motion and shockwave trauma are deleterious to cartilage homeostasis due to HIF-1α overexpression, resulting in disproportionate ecto-5'-nucleotidase/CD73 production, adenosine accumulation and superfluous A2B receptors activation. Scarcity of data however exists on the putative interplay between coexistent high affinity (A2A and A3) and low affinity (A2B) adenosine receptors activation affecting stem cells fate towards preferential chondrogenic or osteogenic lineages in the human cartilage. Hints gathered in this commentary result mainly from studies using human immortalized cell lines and animal (e.g. rodent, equine, bovine) tissue samples. The available data point towards adenosine A2A and A3 receptors having cartilage protective roles, while excessive adenosine accumulation may be detrimental via low affinity A2B receptors activation, with little reference to the putative role of the adenosine forming enzyme ecto-5'-nucleotidase/CD73. Thus, emphasizing the multiple pathways responsible for controlling adenosine signalling in cartilage will certainly impact on the search for novel therapeutic targets for highly disabling articular disorders.