Elizabeth L. Hedberg-Dirk

Surface chemistry regulates valvular interstitial cell differentiation in vitro.

UNLABELLED The primary driver for valvular calcification is the differentiation of valvular interstitial cells (VICs) into a diseased phenotype. However, the factors leading to the onset of osteoblastic-like VICs (obVICs) and resulting calcification are not fully understood. This study isolates the effect of substrate surface chemistry on in vitro VIC differentiation and calcified tissue formation. Using ω-functionalized alkanethiol self-assembled monolayers (SAMs) on gold [CH3 (hydrophobic), OH (hydrophilic), COOH (COO(-), negative at physiological pH), and NH2 (NH3(+), positive at physiological pH)], we have demonstrated that surface chemistry modulates VIC phenotype and calcified tissue deposition independent of osteoblastic-inducing media additives. Over seven days VICs exhibited surface-dependent differences in cell proliferation (COO(-)=NH3(+)>OH>CH3), morphology, and osteoblastic potential. Both NH3(+)and CH3-terminated SAMs promoted calcified tissue formation while COO(-)-terminated SAMs showed no calcification. VICs on NH3(+)-SAMs exhibited the most osteoblastic phenotypic markers through robust nodule formation, up-regulated osteocalcin and α-smooth muscle actin expression, and adoption of a round/rhomboid morphology indicative of osteoblastic differentiation. With the slowest proliferation, VICs on CH3-SAMs promoted calcified aggregate formation through cell detachment and increased cell death indicative of dystrophic calcification. Furthermore, induction of calcified tissue deposition on NH3(+) and CH3-SAMs was distinctly different than that of media induced osteoblastic VICs. These results demonstrate that substrate surface chemistry alters VIC behavior and plays an important role in calcified tissue formation. In addition, we have identified two novel methods of calcified VIC induction in vitro. Further study of these environments may yield new models for in vitro testing of therapeutics for calcified valve stenosis, although additional studies need to be conducted to correlate results to in vivo models. STATEMENT OF SIGNIFICANCE Valvular interstitial cell (VIC) differentiation and aortic valve calcification is associated with increased risk of mortality and onset of other cardiovascular disorders. This research examines effects of in vitro substrate surface chemistry on VIC differentiation and has led to the identification of two materials-based initiation mechanisms of osteoblastic-like calcified tissue formation independent of soluble signaling methods. Such findings are important for their potential to study signaling cascades responsible for valvular heart disease initiation and progression as well providing in vitro disease models for drug development. We have also identified a VIC activating in vitro environment that does not exhibit confluence induced nodule formation with promise for the development of tissue regenerating scaffolds.

Large-scale protein arrays generated with interferometric lithography for spatial control of cell-material interactions

Understanding cellular interactions with material surfaces at the micro- and nanometer scale is essential for the development of the next generation of biomaterials. Several techniques have been used to create micro- and nanopatterned surfaces as a means of studying cellular interactions with a surface. Herein, we report the novel use of interference lithography to create a large (4 cm2) array of 33nm deep channels in a gold surface, to expose an antireflective coating on a silicon wafer at the bottom of the gold channels. The fabricated pores had a diameter of 140-350nm separated by an average pitch of 304-750 nm, depending on the fabrication conditions. The gold surface was treated with 2-(2-(2-(11-mercaptoundecyloxy)ethoxy)ethoxy)ethanol to create protein-resistant areas. Fibronectin was selectively adsorbed onto the exposed antireflective coating creating nanometer-scale cell adhesive domains. A murine osteoblast cell line (MC3T3-E1) was seeded onto the surfaces and was shown to attach to the fibronectin domains and spread across the material surface.

Isolated effect of material stiffness on valvular interstitial cell differentiation

Previous methods for investigating material stiffness on cell behavior have focused on the use of substrates with limited ranges of stiffness and/or fluctuating surface chemistries. Using the co-polymer system of n-octyl methacrylate crosslinked with diethylene glycol dimethacrylate (DEGDMA/nOM), we developed a new cell culture platform to analyze the isolated effects of stiffness independent from changes in surface chemistry. Materials ranging from 25 kPa to 4,700 kPa were fabricated. Surface analysis including goiniometry and X-ray photoelectron spectroscopy (XPS) confirmed consistent surface chemistry across all formulations examined. The mechanosensitive cell type valvular interstitial cell (VIC) was cultured DEGDMA/nOM substrates of differing stiffness. Results indicate that order of magnitude changes in stiffness do not increase gene expression of VIC alpha-smooth muscle actin (αSMA). However, structural organization of αSMA is altered on stiffer substrates, corresponding with the appearance of the osteoblastic marker osteocalcin and nodule formation. This research presents the co-polymer DEGDMA/nOM as ideal substrate to investigate the influence of stiffness on VIC differentiation without the confounding effects of changing material surface chemistry.

Esters of Maleic Anhydride as Both a New and Old Material for Tissue Engineering

Many publications have examined the biodegradable polymer poly(propylene fumerate) (PPF) for use in tissue engineering applications. We have examined a similar crosslinkable polymer system, poly(propylene fumerate)- co -(propylene maleate) (PPFcPM), derived from maleic anhydride (MA) and 1,2-propanediol (PD). Two methods were examined in order to synthesis the copolymer. In the first case, the reaction was carried out at high temperature (250°C) under nitrogen using tosic acid as the catalyst. Only PPF was identified due to the thermal isomerization of the maleate groups to the more stable fumerate group. In the second case, toluene was used as the solvent to azeotropically (85 °C) remove water and drive the acid catalyzed esterification reaction. In the lower temperature case, a small amount of fumerate ( in situ to form porous micro- and nano fiber mats. Initial biocompatibility studies have also been preformed.

Biotechnology development for biomedical applications.

Sandias scientific and engineering expertise in the fields of computational biology, high-performance prosthetic limbs, biodetection, and bioinformatics has been applied to specific problems at the forefront of cancer research. Molecular modeling was employed to design stable mutations of the enzyme L-asparaginase with improved selectivity for asparagine over other amino acids with the potential for improved cancer chemotherapy. New electrospun polymer composites with improved electrical conductivity and mechanical compliance have been demonstrated with the promise of direct interfacing between the peripheral nervous system and the control electronics of advanced prosthetics. The capture of rare circulating tumor cells has been demonstrated on a microfluidic chip produced with a versatile fabrication processes capable of integration with existing lab-on-a-chip and biosensor technology. And software tools have been developed to increase the calculation speed of clustered heat maps for the display of relationships in large arrays of protein data. All these projects were carried out in collaboration with researchers at the University of Texas M. D. Anderson Cancer Center in Houston, TX.