Fereshteh Azari

Pyrophosphate Inhibits Mineralization of Osteoblast Cultures by Binding to Mineral, Up-regulating Osteopontin, and Inhibiting Alkaline Phosphatase Activity

Inorganic pyrophosphate (PPi) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent “soft” tissues from mineralizing, whereas its degradation to Pi in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alpl, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PPi are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PPi on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PPi. This inhibition could be reversed by Tnap, but not if PPi was bound to mineral. PPi also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PPi was also insensitive to foscarnet (an inhibitor of phosphate uptake) and levamisole (an inhibitor of Tnap enzymatic activity), suggesting that increased Opn levels did not result from changes in phosphate. Exogenous OPN inhibited mineralization, but dephosphorylation by Tnap reversed this effect, suggesting that OPN inhibits mineralization via its negatively charged phosphate residues and that like PPi, hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PPi inhibits Tnap-mediated Pi release from β-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PPi prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity.

Intracellular precipitation of hydroxyapatite mineral and implications for pathologic calcification.

In contrast to physiologic biomineralization occurring in bones, teeth and otoconia in vertebrates, calcification of soft tissues occurs in many pathologic conditions. Although similarities have been noted between the two processes, and despite the important clinical consequences of ectopic calcification, the molecular mechanisms regulating ectopic calcification are poorly understood. Although calcification is mainly extracellular, intracellular calcification has been reported and might indeed contribute to pathologic calcification of soft tissues. To better understand the process of intracellular calcification as a potential origin for pathologic calcification, and to examine the role of proteoglycans in this process, we investigated a pattern of intracellular nucleation and growth of hydroxyapatite in Madin-Darby Canine Kidney (MDCK) epithelial cells using electron microscopy, secondary ion mass spectroscopy (NanoSIMS), cytochemical staining, immunolabeling and biochemical analysis. We report here that under mineralizing cell culture conditions where beta-glycerophosphate (betaGP) was added as an exogenous organic source of phosphate, betaGP-cleaving alkaline phosphatase activity increased and hydroxyapatite crystals subsequently nucleated within intracellular, membrane-bounded compartments. The small, leucine-rich proteoglycan decorin was also upregulated and associated with mineral in these cultures. Such information provides insight into the mechanisms leading to pathologic calcification and describes a process whereby hydroxyapatite deposition in cells might lead to ectopic calcification.

Modulated release of OP-1 and enhanced preosteoblast differentiation using a core-shell nanoparticulate system

A release-controlled OP-1 delivery system consisting of a suspension of core-shell nanoparticles was prepared. The nanoparticles were composed of a core of positively-charged large unilamellar liposomes and a shell constructed through the L-b-L assembly of alternating layers of negatively-charged sodium alginate and positively-charged chitosan. Cytotoxicity was assayed with MC3T3-E1.4 mouse preosteoblast cells and cell viability was determined by colorimetry (CellQuanti-MTT kit). The system was loaded with a range of OP-1 concentrations and the release profiles were obtained and fitted into the Higuchi model to determine release kinetics. Alkaline phosphatase (ALP) activity of preosteoblasts was evaluated using a micro-BCA assay. The resulting monodisperse and nontoxic spherical nanoparticles exhibited high physical stability in simulated physiological media as well as an extended shelf-life allowing for immediate protein loading before future administration. ALP activity increased over time with the OP-1 loaded delivery system when compared with control, protein alone, and nanoparticles alone (p < 0.05). The system offers copious compartments for protein entrapment including the aqueous core and within the polyelectrolyte layers in the shell and demonstrates a sustained triphasic linear release of OP-1 over a prolonged period of 45 days, in vitro. This system offers a great advantage for optimum growth factor performance when applied in different anatomical sites of varying defect sizes and vascularity.

The response of fibrinogen, platelets, endothelial and smooth muscle cells to an electrochemically modified SS316LS surface: Towards the enhanced biocompatibility of coronary stents

Modification of a biomedical-grade stainless steel 316LS surface by electrochemical cyclic potentiodynamic passivation (CPP) and the response of fibrinogen (Fg), platelets, endothelial cells (ECs) and smooth muscles cells (SMCs) to this surface was investigated. Polarization modulation infrared reflection absorption spectroscopy revealed a significant difference between the secondary structure of Fg adsorbed on the unmodified and CPP surface, the latter being closer to that of native Fg. This was postulated as the origin of the significantly lower surface density of attached platelets on the CPP surface. The competitive interaction of ECs and SMCs with the surface showed that the ECs/SMCs surface density ratio is significantly higher on the CPP surface over the first 2h of attachment, suggesting faster initial attachment kinetics of ECs on the CPP surface. The presented results thus clearly demonstrate an increase in biocompatibility of the CPP 316LS surface.

The positive influence of electrochemical cyclic potentiodynamic passivation (CPP) of a SS316LS surface on its response to fibronectin and pre-osteoblasts

The influence of an electrochemical surface passivation technique (cyclic potentiodynamic polarization, CPP) on the physico-chemical surface properties of SS316LS and its subsequent response to fibronectin (Fn) and pre-osteoblasts were investigated. Contact angle and zeta-potential measurements showed that the CPP-modified surface is more hydrophilic and more positively charged than the unmodified surface. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was used to investigate the interaction of Fn with both surfaces. The saturated surface concentration of adsorbed Fn was higher on the CPP-modified surface. As well, significant changes were identified in the secondary structure of Fn adsorbed on both surfaces, in comparison to its native state. This data also indicated a higher degree of Fn unfolding on the CPP-modified surface. Cell studies indicated that the attachment, proliferation and morphology of pre-osteoblasts were significantly improved on the CPP-modified surface, which was attributed to the more open conformation of Fn on the CPP-modified surface. Thus, the CPP surface passivation method was demonstrated to yield a SS316LS surface of enhanced biocompatibility.

Titanium crystal orientation as a tool for the improved and regulated cell attachment.

Cell adhesion is a fundamental process that controls cell proliferation, migration, and differentiation and is crucial for biomaterial-tissue integration. Osteoblast attachment on the surfaces of implant materials is, therefore, essential for the proper function of any implant in which osseointegration is required. Although many reports are available on osteoblast attachment using different surface modification, there is no specific report, so far, that investigates the effect of atomic order of specific crystallographic orientation of substrates on cell behavior. A novel coculture system is proposed to show the differential response of preosteoblast and fibroblast cell lines to the titanium single-crystal substrates. Our investigation has shown that surface recognition by the cell is influenced by the atomic structure of the surface leading to cell-type-specific adhesion. The degree of preosteoblast attachment is significantly higher on the Ti-(1120), whereas the fibroblast adhesion is increased on the Ti-(1010). This demonstrates that the three distinct faces of titanium substrates differ greatly in their capacity to serve as cell adhesive substrates. It also provides clear evidence for the role of crystal structure in regulating and improving cell-substrate interactions relevant for the optimal function of bone implant materials.

On the importance of crystallographic texture in the biocompatibility of titanium based substrate

The role of grain size and crystallographic orientation on the biocompatibility of commercially pure titanium was investigated. Samples, with significant differences in crystallographic texture and average grain size (from 0.4 to 40 µm) were produced by equal channel angular pressing (ECAP) and post deformation annealing. X-ray diffraction and electron back scattered diffraction (EBSD) were used to evaluate differences in texture and microstructural characteristics. The titanium oxide film present on the surface of the samples was analyzed to determine the oxidation state of titanium and the chemical bonds between oxygen and titanium using X-ray photoelectron spectroscopy (XPS). Biocompatibility experiments were conducted using MC3T3 preosteoblast cells. Cell attachment was found to be texture-sensitive, where the number of attached cells was higher on the samples with higher number of (0002) planes exposed to the surface, regardless of the grain size. A relationship was also found between the titanium oxide species formed on the surface and the crystallographic texture underneath. The surface texture consisting of more densely packed basal planes promote the formation of Ti-OH on the surface, which in turn, enhances the cell-substrate interactions. These surface characteristics are deemed responsible for the observed difference in cell attachment behaviour of surfaces with different textures. Finally, it is inferred that texture, rather than the grain size, plays the major role in controlling the surface biocompatibility of biomedical devices fabricated from pure metallic titanium.

Self-assembled multifunctional nanoplexes for gene inhibitory therapy

AIM To enhance the stability of siRNA while improving their therapeutic properties and visualization at the target site, a novel nanoplex system was developed. MATERIALS & METHODS The designed nanoplex system involved functionalizing siRNA with near-infrared quantum dots and loading them into histidylated glycol chitosan (GC-His). RESULTS Colocalization studies revealed a twofold increase in siRNA uptake after encapsulation with GC-His and nanoparticles were localized in cytoplasm, suggesting that histidine promoted their dissociation from the endosomal membranes. Furthermore, as opposed to siRNAs treated with commercial transfection reagent, siRNAs loaded within GC-His showed a marked reduction (64%) of MDM2 protein expression 24 h after transfection. CONCLUSION These findings concur that GC-His/siRNA-quantum dot nanoplexes are promising multifunctional vehicles for gene inhibitory therapy.

The Role of Crystallographic Texture of Ti-6Al-4V Alloy on Cell Attachment and Proliferation

Owing to their lower modulus, great corrosion resistance and superior biocompatibility, titanium alloys are increasingly used as artificial joint replacements. However bone bonding capability of these materials needs to be improved. Many studies are currently conducted to improve the osseo-integration of titanium based implants. In the present study, the role of crystallographic texture of titanium alloy Ti-6Al-4V on bone bonding capability was investigated in vitro systems. X-ray diffraction analysis was used to determine preferred orientation in each substrate. These substrates were seeded with preosteoblast cells to examine cell attachment and proliferation. Attachment of cells was assessed by counting the number of adhered cells within 30-240 min. The proliferation rate of cells was measured between the 3rd-11th days of incubation. The results suggest that the substrate with (100) orientation shows better osteoblastic cell adhesion and proliferation rate than the (110).

Effects of Grain Size and Texture on the Biocompatibility of Commercially Pure Titanium

Ultra fine grained (UFG) pure titanium fabricated by severe plastic deformation techniques has been recently considered for biomedical applications. In this study, the effects of grain size and crystallographic orientation on the biocompatibility of commercially pure titanium have been evaluated. Samples having significant differences in terms of average grain size (from 0.4 to 20 mm) and crystallographic textures have been produced using equal channel angular pressing (ECAP) and compared. X-ray diffraction and electron back scattered diffraction (EBSD) were used to document the texture and microstructural properties. Cell attachment tests were done to study the biocompatibility of the samples using MC3T3 pre-osteoblast cells. The number of attached cells was found to be higher on the samples having more (0002) plane parallel to the surface regardless of their grain sizes. It was concluded that the texture plays a more significant role than the grain size in the biocompatibility of pure titanium.