Gene expression in osteoblast cells treated with submicron to nanometer hydroxyapatite-mullite eluate particles

The present research focused on determining the effect of hydroxyapatite-20 wt% mullite (H20M) particle eluates on apoptosis and differentiation of human fetal osteoblast (hFOB) cells. The H20M particles (257 +/- 37 nm) were prepared, starting with the production of a nanocomposite using a unique route of spark plasma sintering, followed by a repeated grinding-cryo treatment and elution process. Tetrazolium based cytotoxicity assay results showed a time-and dose-dependent effect of H20M particle eluates on hFOB cytotoxicity. In particular, the results revealed statistically reduced cell viability after hFOB were exposed to the above 10% H20M (257 +/- 37 nm) eluates for 48 h. The apoptotic cell death triggered by H20M treatment was proven by the analysis of molecular markers of apoptosis, that is, the Bcl-2 family of genes. hFOB expression of Bcl-xL and Bcl-xS significantly increased 25.6- and 25.2-fold for 50% of H20M concentrations, respectively. The ratio of Bcl-xL/Bax (4.01) decreased 2-fold for hFOB exposed to 100% of H20M eluates than that for 10% H20M eluate (7.94) treated hFOB cells. On the other hand, the Bcl-xS/Bax ratio for the 10% H20M eluate was 4.15-fold, whereas for 100% H20M eluates, it was 11.55-fold. Specifically, the anti-apoptotic effect of the H20M particle eluates was corroborated by the up-regulation of bone cell differentiation marker genes such as, collagen type I, cbfa, and osteocalcin. In summary, the present work clearly demonstrated that H20M submicron to nanometer composite particle eluates have a minimal effect on hFOB apoptosis and can even up-regulate the expression of bone cell markers at the molecular level.

Development, in vitro and in vivo characterization of zoledronic acid functionalized hydroxyapatite nanoparticle based formulation for treatment of osteoporosis in animal model

We investigated the potential of using novel zoledronic acid (ZOL)-hydroxyapatite (HA) nanoparticle based drug formulation in a rat model of postmenopausal osteoporosis. By a classical adsorption method, nanoparticles of HA loaded with ZOL (HNLZ) drug formulation with a size range of 100-130 nm were prepared. 56 female Wistar rats were ovariectomized (OVX) or sham-operated at 3 months of age. Twelve weeks post surgery, rats were randomized into seven groups and treated with various doses of HNLZ (100, 50 and 25 mu g/kg, intravenous single dose), ZOL (100 mu g/kg, intravenous single dose) and HA nanoparticle (100 mu g/kg, intravenous single dose). Untreated OVX and sham OVX served as controls. After three months treatment period, we evaluated the mechanical properties of the lumbar vertebra and femoral mid-shaft. Femurs were also tested for trabecular microarchitecture. Sensitive biochemical markers of bone formation and bone resorption in serum were also determined. With respect to improvement in the mechanical strength of the lumbar spine and the femoral mid-shaft, the therapy with HNLZ drug formulation was more effective than ZOL therapy in OVX rats. Moreover, HNLZ drug therapy preserved the trabecular microarchitecture better than ZOL therapy in OVX rats. Furthermore, the HNLZ drug formulation corrected increase in serum levels of bone-specific alkaline phosphatase, procollagen type I N-terminal propeptide, osteocalcin, tartrate-resistant acid phosphatase 5b and C-telopeptide of type 1 collagen better than ZOL therapy in OVX rats. The results strongly suggest that HNLZ novel drug formulation appears to be more effective approach for treating severe osteoporosis in humans. (C) 2014 Elsevier B.V. All rights reserved.

Interplay of Substrate Conductivity, Cellular Microenvironment, and Pulsatile Electrical Stimulation toward Osteogenesis of Human Mesenchymal Stem Cells in Vitro

The influences of physical stimuli such as surface elasticity, topography, and chemistry over mesenchymal stem cell proliferation and differentiation are well investigated. In this context, a fundamentally different approach was adopted, and we have demonstrated the interplay of inherent substrate conductivity, defined chemical composition of cellular microenvironment, and intermittent delivery of electric pulses to drive mesenchymal stem cell differentiation toward osteogenesis. For this, conducting polyaniline (PANI) substrates were coated with collagen type 1 (Coll) alone or in association with sulfated hyaluronan (sHya) to form artificial extracellular matrix (aECM), which mimics the native microenvironment of bone tissue. Further, bone marrow derived human mesenchymal stem cells (hMSCs) were cultured on these moderately conductive (10(-4)10(-3) S/cm) aECM coated PANI substrates and exposed intermittently to pulsed electric field (PEF) generated through transformer-like coupling (TLC) approach over 28 days. On the basis of critical analysis over an array of end points, it was inferred that Coll/sHya coated PANI (PANI/Coll/sHya) substrates had enhanced proliferative capacity of hMSCs up to 28 days in culture, even in the absence of PEF stimulation. On the contrary, the adopted PEF stimulation protocol (7 ms rectangular pulses, 3.6 mV/cm, 10 Hz) is shown to enhance osteogenic differentiation potential of hMSCs. Additionally, PEF stimulated hMSCs had also displayed different morphological characteristics as their nonstimulated counterparts. Concomitantly, earlier onset of ALP activity was also observed on PANI/Coll/sHya substrates and resulted in more calcium deposition. Moreover, real-time polymerase chain reaction results indicated higher mRNA levels of alkaline phosphatase and osteocalcin, whereas the expression of other osteogenic markers such as Runt-related transcription factor 2, Col1A, and osteopontin exhibited a dynamic pattern similar to control cells that are cultured in osteogenic medium. Taken together, our experimental results illustrate the interplay of multiple parameters such as substrate conductivity, electric field stimulation, and aECM coating on the modulation of hMSC proliferation and differentiation in vitro.