Rupak M. Rajachar

Regulation of Vascular Calcification Roles of Phosphate and Osteopontin

Vascular calcification is prevalent in aging as well as a number of pathological conditions, and it is now recognized as a strong predictor of cardiovascular events in the general population as well as diabetic and end-stage renal disease patients. Vascular calcification is a highly regulated process involving inductive and inhibitory mechanisms. This article focuses on two molecules, phosphate and osteopontin, that have been implicated in the induction or inhibition of vascular calcification, respectively. Elevated phosphate is of interest because hyperphosphatemia is recognized as a major nonconventional risk factor for cardiovascular disease mortality in end-stage renal disease patients. Studies to date suggest that elevated phosphate stimulates smooth muscle cell phenotypic transition and mineralization via the activity of a sodium-dependent phosphate cotransporter. Osteopontin, however, appears to block vascular calcification most likely by preventing calcium phosphate crystal growth and inducing cellular mineral resorption.

Ultrastructural changes accompanying the mechanical deformation of bone tissue: a Raman imaging study.

Raman spectroscopy and imaging are known to be valuable tools for the analysis of bone, the determination of protein secondary structure, and the study of the composition of crystalline materials. We have utilized all of these attributes to examine how mechanical loading and the resulting deformation affects bone ultrastructure, addressing the hypothesis that bone spectra are altered, in both the organic and inorganic regions, in response to mechanical loading/deformation. Using a cylindrical indenter, we have permanently deformed bovine cortical bone specimens and investigated the ultrastructure in and around the deformed areas using hyperspectral Raman imaging coupled with multivariate analysis techniques. Indent morphology was further examined using scanning electron microscopy. Raman images taken at the edge of the indents show increases in the low-frequency component of the amide III band and high-frequency component of the amide I band. These changes are indicative of the rupture of collagen crosslinks due to shear forces exerted by the indenter passing through the bone. However, within the indent itself no evidence was seen of crosslink rupture, indicating that only compression of the organic matrix takes place in this region. We also present evidence of what is possibly a pressure-induced structural transformation occurring in the bone mineral within the indents, as indicated by the appearance of additional mineral factors in Raman image data from indented areas. These results give new insight into the mechanisms and causes of bone failure at the ultrastructural level.

Microporous Nanofibrous Fibrin-based Scaffolds for Bone Tissue Engineering

The fibrotic response of the body to synthetic polymers limits their success in tissue engineering and other applications. Though porous polymers have demonstrated improved healing, difficulty in controlling their pore sizes and pore interconnections has clouded the understanding of this phenomenon. In this study, a novel method to fabricate natural polymer/calcium phosphate composite scaffolds with tightly controllable pore size, pore interconnection, and calcium phosphate deposition was developed. Microporous, nanofibrous fibrin scaffolds were fabricated using sphere-templating methods. Composite scaffolds were created by solution deposition of calcium phosphate on fibrin surfaces or by direct incorporation of nanocrystalline hydroxyapatite (nHA). The SEM results showed that fibrin scaffolds exhibited a highly porous and interconnected structure. Osteoblast-like cells, obtained from murine calvaria, attached, spread and showed a polygonal morphology on the surface of the biomaterial. Multiple cell layers and fibrillar matrix deposition were observed. Moreover, cells seeded on mineralized fibrin scaffolds exhibited significantly higher alkaline phosphatase activity as well as osteoblast marker gene expression compared to fibrin scaffolds and nHA incorporated fibrin scaffolds (0.25 and 0.5g). All types of scaffolds were degraded both in vitro and in vivo. Furthermore, these scaffolds promoted bone formation in a mouse calvarial defect model and the bone formation was enhanced by addition of rhBMP-2.

Injectable Dopamine-Modified Poly(ethylene glycol) Nanocomposite Hydrogel with Enhanced Adhesive Property and Bioactivity

A synthetic mimic of mussel adhesive protein, dopamine-modified four-armed poly(ethylene glycol) (PEG-D4), was combined with a synthetic nanosilicate, Laponite (Na0.7+(Mg5.5Li0.3Si8)O20(OH)4)0.7–), to form an injectable naoncomposite tissue adhesive hydrogel. Incorporation of up to 2 wt % Laponite significantly reduced the cure time while enhancing the bulk mechanical and adhesive properties of the adhesive due to strong interfacial binding between dopamine and Laponite. The addition of Laponite did not alter the degradation rate and cytocompatibility of PEG-D4 adhesive. On the basis of subcutaneous implantation in rat, PEG-D4 nanocomposite hydrogels elicited minimal inflammatory response and exhibited an enhanced level of cellular infiltration as compared to Laponite-free samples. The addition of Laponite is potentially a simple and effective method for promoting bioactivity in a bioinert, synthetic PEG-based adhesive while simultaneously enhancing its mechanical and adhesive properties.

Bone Chemical Structure Response to Mechanical Stress Studied by High Pressure Raman Spectroscopy

While the biomechanical properties of bone are reasonably well understood at many levels of structural hierarchy, surprisingly little is known about the response of bone to loading at the ultrastructural and crystal lattice levels. In this study, our aim was to examine the response (i.e., rate of change of the vibrational frequency of mineral and matrix bands as a function of applied pressure) of murine cortical bone subjected to hydrostatic compression. We determined the relative response during loading and unloading of mineral vs. matrix, and within the mineral, phosphate vs. carbonate, as well as proteinated vs. deproteinated bone. For all mineral species, shifts to higher wave numbers were observed as pressure increased. However, the change in vibrational frequency with pressure for the more rigid carbonate was less than for phosphate, and caused primarily by movement of ions within the unit cell. Deformation of phosphate on the other hand, results from both ionic movement as well as distortion. Changes in vibrational frequencies of organic species with pressure are greater than for mineral species, and are consistent with changes in protein secondary structures such as alterations in interfibril cross-links and helix pitch. Changes in vibrational frequency with pressure are similar between loading and unloading, implying reversibility, as a result of the inability to permanently move water out of the lattice. The use of high pressure Raman microspectroscopy enables a deeper understanding of the response of tissue to mechanical stress and demonstrates that individual mineral and matrix constituents respond differently to pressure.

Bone tissue ultrastructural response to elastic deformation probed by Raman spectroscopy.

Raman spectroscopy is used as a probe of ultrastructural (molecular) changes in both the mineral and matrix (protein and glycoprotein, predominantly type I collagen) components in real time of murine cortical bone as it responds to elastic deformation. Because bone is ia composite material, its mechanical properties are dependent on the structure and composition at a variety of dimensional scales. At the ultrastructural level, crystal structure and protein secondary structure distort as the tissue is loaded. These structural changes are followed as perturbations to tissue spectra. We load murine femora in a custom-made mechanical tester that fits on the stage of a Raman microprobe and can accept hydrated tissue specimens. As the specimen is loaded in tension, the shifts in mineral P-O4 v1 are followed with the microprobe. Average load and strain are measured using a load cell. These devices ensure that specimens are not loaded to or beyond the yield point. Changes occur in the mineral component of bone as a response to loading in the elastic regime. We propose that the mineral apatitic crystal lattice is deformed by movement of calcium and other ions. Raman microspectroscopy shows that bone mineral is not a passive contributor to tissue strength. The mineral active response to loading may function as a local energy storage and dissipation mechanism, thus helping to protect tissue from catastrophic damage.

Mitigation of Ectopic Calcification in Osteopontin-Deficient Mice by Exogenous Osteopontin

Ectopic calcification is a major cause of bioprosthetic heart valve failure. New therapeutic opportunities are offered by the growing understanding that ectopic calcification is an actively regulated process involving several key gene products. One of these products, osteopontin (OPN), is a glycosylated phosphoprotein previously shown to inhibit apatite crystal formation, induce carbonic anhydrase II, and promote mineral resorption. In this study, OPN-deficient mice (OPN−/−) were utilized as an in vivo model to stimulate the ectopic calcification of glutaraldehyde-fixed bovine pericardium (GFBP) tissue and to examine OPN delivery and structure-function relationships with respect to its anti-calcific activity. Significant calcification of GFBP tissue was obtained within 7 days of subcutaneous implantation in OPN−/− mice. Direct rescue of the calcification phenotype was achieved by the administration of exogenous recombinant rat, histidine-fused OPN (rat His-OPN) to the implant site via soluble injection (up to 72% mitigation achieved) or adsorption onto the implant materials (up to 91% mitigation achieved). Effects were specific, since neither fibronectin nor polyhistidine alone could mitigate calcification of GFBP. The maximum anti-calcific effect was achieved only when rat His-OPN was adequately phosphorylated and contained a functional arginine-glycine-aspartate (RGD) cell adhesive domain. Furthermore, CAII levels in host cells surrounding GFBP were greatest when phosphorylated, RGD-containing rat His-OPN was adsorbed. These data suggest that both physical inhibition, mediated by phosphorylation sites in OPN, as well as the induction of CAII and mineral regression, mediated by the RGD domain, contribute to the unique ability of OPN to mitigate ectopic calcification of bioprosthetic valve tissue.

Engineered Nanotopography on Electrospun PLLA Microfibers Modifies RAW 264.7 Cell Response

In this study, we created a new method of electrospinning capable of controlling the surface structure of individual fibers (fiber nanotopography). The nanotopographical features were created by a phase separation in the fibers as they formed. To control the phase separation, a nonsolvent (a chemical insoluble with the polymer) was added to an electrospinning solution containing poly-l-lactic acid (PLLA) and chloroform. The nanotopography of electrospun fibers in the PLLA/chloroform solution was smooth. However, adding a small weight (<2% of total solution) of a single nonsolvent (water, ethanol, or dimethyl sulfoxide) generated nanoscale depressions on the surface of the fibers unique to the nonsolvent added. Additionally, nanoscale depressions on electrospun fibers were observed to change with dimethyl sulfoxide (DMSO) concentration in the PLLA/chloroform solution. A nonlinear relationship was found between the concentration of DMSO and the number and size of nanotopographical features. The surface depressions did not alter the hydrophobicity of the scaffold or degradation of the scaffold over a two-day period. To determine if fiber nanotopography altered cell behavior, macrophages (RAW 264.7 cells) were cultured on fibers with a smooth nanotopography or fibers with nanoscale depressions. RAW 264.7 cells spread less on fibers with nanoscale depressions than fibers with a smooth topography (p<0.05), but there were no differences between groups with regard to cell metabolism or the number of adherent cells. The results of this study demonstrate the necessity to consider the nanotopography of individual fibers as these features may affect cellular behavior. More importantly, we demonstrate a versatile method of controlling electrospun fiber nanotopography.

Fabrication of Biocompatible, Vibrational Magnetoelastic Materials for Controlling Cellular Adhesion

This paper describes the functionalization of magnetoelastic (ME) materials with Parylene-C coating to improve the surface reactivity to cellular response. Previous study has demonstrated that vibrating ME materials were capable of modulating cellular adhesion when activated by an externally applied AC magnetic field. However, since ME materials are not inherently biocompatible, surface modifications are needed for their implementation in biological settings. Here, the long-term stability of the ME material in an aqueous and biological environment is achieved by chemical-vapor deposition of a conformal Parylene-C layer, and further functionalized by methods of oxygen plasma etching and protein adsorption. In vitro cytotoxicity measurement and characterization of the vibrational behavior of the ME materials showed that Parylene-C coatings of 10 µm or greater could prevent hydrolytic degradation without sacrificing the vibrational behavior of the ME material. This work allows for long-term durability and functionality of ME materials in an aqueous and biological environment and makes the potential use of this technology in monitoring and modulating cellular behavior at the surface of implantable devices feasible.

Role of carbonic anhydrase II in ectopic calcification

INTRODUCTION Osteopontin (OPN) is a potent inhibitor of ectopic calcification. Previous studies suggested that, in addition to blocking apatite crystal growth, OPN promoted regression of ectopic calcification by inducing the expression of acid-generating carbonic anhydrase II (CAR2) in monocyte-derived cells. METHODS To test this hypothesis, OPN and CAR2 expression and calcification of subcutaneously implanted glutaraldehyde-fixed bovine pericardium (GFBP) were studied in CAR2 mutant mice. RESULTS Consistent with previous studies in Black Swiss mice, GFBP calcified to a greater extent in OPN-deficient mice compared to wild types on the C57Bl/6 background. GFBP implanted in CAR2-deficient mice (CAR2(-/-)) were significantly more calcified than those implanted into wild-type mice (CAR2(+/+)) [37+/-5 vs. 20+/-6.5 microg Ca/mg tissue, respectively, at 30 days (P<.001), and 42+/-5 versus 20+/-4 microg Ca/mg tissue at 60 days, respectively (P<.001)]. On the other hand, OPN levels within and surrounding the implants were similar in CAR2(+/+) and CAR2(-/-) mice, suggesting that OPN expression in the absence of CAR2 was not sufficient to mitigate ectopic calcification. CONCLUSIONS These results indicate that CAR2 expression is an important regulator of ectopic calcification, potentially by facilitating OPN mediated mineral regression.