Pyrophosphate is a potent mitogen, capable of stimulating proliferation in multiple cell types, and a critical participant in bone mineralization. Pyrophosphate can also affect the resorption rate and bioactivity of orthopedic ceramics. The present study investigated whether calcium pyrophosphate affected proliferation, differentiation and gene expression in early (MC3T3 pre-osteoblast) and late stage (SAOS-2 osteosarcoma) osteoblasts. Pyrophosphate stimulated peak alkaline phosphatase activity by 50% and 150% at 100μM and 0.1μM in MC3T3, and by 40% in SAOS-2. The expression of differentiation markers collagen 1 (COL1), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) were increased by an average of 1.5, 2, 2 and 3 fold, by high concentrations of sodium pyrophosphate (100μM) after 7 days of exposure in MC3T3. COX-2 and ANK expression did not differ significantly from controls in either treatment group. Though both high and low concentrations of pyrophosphate stimulate ALP activity, only high concentrations (100μM) stimulated osteogenic gene expression. Pyrophosphate did not affect proliferation in either cell type. The results of this study confirm that chronic exposure to pyrophosphate exerts a physiological effect upon osteoblast differentiation and ALP activity, specifically by stimulating osteoblast differentiation markers and extracellular matrix gene expression.

In vivo and in vitro evaluation of hydroxyapatite nanoparticle morphology on the acute inflammatory response. AbstractBiomedical implants have been widely used in bone repair applications. However, nanosized degradation products from these implants could elicit an inflammatory reaction, which may lead to implant failure. It is well known that the size, chemistry, and charge of these nanoparticles can modulate this response, but little is known regarding the role that the particle's morphology plays in inducing inflammation. The present study aims to investigate the effect of hydroxyapatite nanoparticle (HANPs) morphology on inflammation, in-vitro and in-vivo. Four distinct HANP morphologies were fabricated and characterized: long rods, dots, sheets, and fibers. Primary human polymorphonuclear cells (PMNCs), mononuclear cells (MNCs), and human dermal fibroblasts (hDFs) were exposed to HANPs and alterations in cell viability, morphology, apoptotic activity, and reactive oxygen species (ROS) production were evaluated, in vitro. PMNCs and hDFs experienced a 2-fold decrease in viability following exposure to fibers, while MNC viability decreased 5-fold after treatment with the dots. Additionally, the fibers stimulated an elevated ROS response in both PMNCs and MNCs, and the largest apoptotic behavior for all cell types. Furthermore, exposure to fibers and dots resulted in greater capsule thickness when implanted subcutaneously in mice. Collectively, these results suggest that nanoparticle morphology can significantly impact the inflammatory response.

Aim Erythropoietin (EPO) is regulated by hypoxia‐inducible factor (HIF)‐2. In the kidney, it is produced by cortico‐medullary perivascular interstitial cells, which transdifferentiate into collagen‐producing myofibroblasts in response to injury. Inhibitors of prolyl hydroxylase domain (PHD) dioxygenases (HIF‐PHIs) activate HIF‐2 and stimulate kidney and liver EPO synthesis in patients with anemia of chronic kidney disease (CKD). We examined whether HIF‐PHIs can reactivate EPO synthesis in interstitial cells that have undergone myofibroblast transdifferentiation in established kidney fibrosis. Methods We investigated Epo transcription in myofibroblasts and characterized the histological distribution of kidney Epo transcripts by RNA in situ hybridization combined with immunofluorescence in mice with adenine nephropathy (AN) treated with HIF‐PHI molidustat.  Lectin absorption chromatography was used to assess liver‐derived EPO.  In addition, we examined kidney Epo transcription in Phd2 knockout mice with obstructive nephropathy. Results In AN, molidustat‐induced Epo transcripts were not found in areas of fibrosis and did not colocalize with interstitial cells that expressed α‐smooth muscle actin, a marker of myofibroblast transdifferentiation. Epo transcription was associated with megalin‐expressing, kidney injury molecule 1‐negative nephron segments and contingent on residual renal function. Liver‐derived EPO did not contribute to serum EPO in molidustat‐treated mice. Epo transcription was not associated with myofibroblasts in Phd2 knockout mice with obstructive nephropathy. Conclusions Our studies suggest that HIF‐PHIs do not reactivate Epo transcription in interstitial myofibroblasts and that their efficacy in inducing kidney EPO in CKD is dependent on the degree of myofibroblast formation, the preservation of renal parenchyma and the level of residual renal function.

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