Thomas C. Pesacreta

The synergistic effect of a hybrid graphene oxide–chitosan system and biomimetic mineralization on osteoblast functions

Graphene oxide and chitosan are promising materials for tissue regeneration. The present study explores a novel biomimetic mineralization route employing a graphene oxide (GO)-chitosan (CS) conjugate as a template material for the biomineralization of hydroxyapatite (HAP). Structural and morphological studies involving X-ray diffraction, Fourier transform infrared spectroscopy, and electron microscopy indicated that extensive mineralization occurred in the CS-GO conjugate system because of strong electrostatic interactions between the functional groups (carboxyl groups of GO and amino groups of CS) and calcium ions in the simulated body fluid (SBF). The combination of chitosan-graphene oxide conjugate and biomineralization was advantageous in favorably modulating cellular activity (osteoblast functions: cell attachment, proliferation, actin, vinculin and fibronectin expression). It is concluded that biomineralized hydroxyapatite in the HAP-CS-GO system induced homogeneous spatial osteoblastic cell growth and quantitatively (e.g. area) and qualitatively (e.g. mineral-to-matrix ratio) increased mineralization in relation to the HAP-GO system. The data underscore that covalent linkage of HAP to chitosan influences osteoblastic cell differentiation, mineralization, and cell growth. The proposed system and the revelation of fundamental insights merit consideration in tissue engineering.

Contractile proteins in Drosophila development

In summary, we have used a multidisciplinary approach to the analysis of actomyosin-based motility during Drosophila embryogenesis. We have documented the movements of early embryogenesis with modern, video methods. We have characterized the cytoplasmic myosin polypeptide, made specific polyclonal antisera to the molecule, studied its distribution during early embryogenesis, cloned and partially characterized the gene that encodes it, and have recently completed the nucleotide sequence of a nearly full length cDNA that encodes the entire protein-coding region. We have initiated studies on myosin function in living embryos both by direct microinjection of antibodies and through classical genetics. To better understand how myosin function is regulated, we have begun analysis of its light chains. Finally, to investigate the molecular mechanism by which its function is integrated into a labile cytoskeleton, whose architecture is constantly changing, we have also investigated Drosophila spectrins. Together, these studies are designed to shed light on the dynamics of biologic form at the cellular level, with current focus on such complex processes as cytokinesis and morphogenesis.

Cellular response of preosteoblasts to nanograined/ultrafine-grained structures

Metallic materials with submicron- to nanometer-sized grains provide surfaces that are different from conventional polycrystalline materials because of the large proportion of grain boundaries with high free energy. In the study described here, the combination of cellular and molecular biology, materials science and engineering advances our understanding of cell-substrate interactions, especially the cellular activity between preosteoblasts and nanostructured metallic surfaces. Experiments on the effect of nano-/ultrafine grains have shown that cell attachment, proliferation, viability, morphology and spread are favorably modulated and significantly different from conventional coarse-grained structures. Additionally, immunofluorescence studies demonstrated stronger vinculin signals associated with actin stress fibers in the outer regions of the cells and cellular extensions on nanograined/ultrafine-grained substrate. These observations suggest enhanced cell-substrate interaction and activity. The differences in the cellular response on nanograined/ultrafine-grained and coarse-grained substrates are attributed to grain size and degree of hydrophilicity. The outcomes of the study are expected to reduce challenges to engineer bulk nanostructured materials with specific physical and surface properties for medical devices with improved cellular attachment and response. The data lay the foundation for a new branch of nanostructured materials for biomedical applications.

Maize Myosins: Diversity, Localization, and Function

This first analysis of monocotyledon myosin genes showed that at least five genes, one of which was probably spliced to yield two isoforms, were expressed in maize (Zea mays L.). The complete coding sequence of ZMM1 was determined, as were most of the sequences of two other myosin cDNAs (ZMM2 and ZMM3). ZMM1 and ZMM2 belonged to myosin class XI while ZMM3 was in class VIII. ZMM1 was abundantly expressed in leaves, roots, coleoptiles, and stems. ZMM3 showed a similar distribution but was expressed poorly in pollen. ZMM2 was predominantly expressed in seeds and may be part of a suite of cytoskeletal proteins in reproductive tissues. Phylogenetic analysis suggested that the origin of myosin classes VIII and XI predated that of angiosperms. Immunofluorescence studies using M11L1, a myosin XI antibody specific for the exposed loop 1 head region of myosin, indicated that myosin XI occurred in the cytoplasm of all root tip cells. The highest concentration of myosin XI was in the differentiating epidermal cells. In dividing cells, myosin XI was present near the cytokinetic apparatus at approximately the same concentration seen in other portions of the cytoplasm. Western blot analysis of subcellular fractions indicated that myosin XI was concentrated in mitochondria and low-density membranes.

Biochemical analysis of elastic and rigid cuticles of Cirsium horridulum

Abstract. The cuticle is a complex structure of soluble lipids, lipid polymers and polysaccharides. In addition to its functions to reduce water loss and provide a protective barrier, its mechanical properties may be significant to plant growth and development. We investigated the cuticle of Cirsium horridulum Michx. because of its involvement in the thigmonastic contraction of staminal filaments. The staminal filaments and portions of the style are surrounded by a highly elastic cuticle in contrast to the rigid cuticle of the corolla and leaves. Our aim was to determine if the biochemical composition affected the elasticity of the cuticle. We discovered that the ratio of carbohydrates to lipids is 1:7 in floral parts but 2:1 in leaf cuticle. Esterified cutin components represented about 80% of the cuticle and di-hydroxyhexadecanoic acids were the major monomers of cutin, regardless of origin. The cutin of elastic tissues is characterized by a higher content of tri-hydroxy monomers than the cutin of rigid tissues. The data suggest that hydroxyl groups enhance the hydrophilic character of the cuticle and contribute to cuticular elasticity.

Atomic force microscopy of cotton fiber cell wall surfaces in air and water: quantitative and qualitative aspects

Abstract. Cotton (Gossypium hirsutum cv. MD51) fiber cell walls were analyzed with an atomic force microscope to determine the effect of chemical treatments on cell wall organization and topography. Analysis of fibers in either air or water and without any staining or coating produced high-resolution images of cell wall microstructure which could be used for detailed quantitative analysis. Treatment of fibers with 1% H2O2 had little effect on surface morphology. Alkali removed much of the cuticle, some primary wall components, and revealed mostly thin-diameter microfibrils. Acidic Updegraff reagent fragmented the fibers, removed much of the cuticle, and revealed mostly thick microfibrils. The surface roughness of fibers treated sequentially with alkali and acid was quantitatively distinguishable from all other fiber types based on the standard deviation of the height data, amplification of surface area, and integration of the scan line data. Analysis of the fractal dimension enabled untreated and peroxide-treated fibers to be clearly distinguished from the other fiber types. Segmentation of the fractal data revealed specific portions of the fractal dimension which were especially useful for defining the size of structures that differentiated fiber types. Areas containing microfibrils could be quantitatively differentiated from non-microfibrillar areas. In water, some alkali-treated fibers had microfibrils that were relatively small in diameter while others appeared to consist of crystalline arrays of smaller fibrils.

Biological significance of nanograined/ultrafine-grained structures: Interaction with fibroblasts.

Given the need to develop high strength/weight ratio bioimplants with enhanced cellular response, we describe here a study focused on the processing-structure-functional property relationship in austenitic stainless steel that was processed using an ingenious phase reversion approach to obtain an nanograined/ultrafine-grained (NG/UFG) structure. The cellular activity between fibroblast and NG/UFG substrate is compared with the coarse-grained (CG) substrate. A comparative investigation of NG/UFG and CG structures illustrated that cell attachment, proliferation, viability, morphology and spread are favorably modulated and significantly different from the conventional CG structure. These observations were further confirmed by expression levels of vinculin and associated actin cytoskeleton. Immunofluorescence studies demonstrated increased vinculin concentrations associated with actin stress fibers in the outer regions of the cells and cellular extensions on NG/UFG substrate. These observations suggest enhanced cell-substrate interaction and activity. The cellular attachment response on NG/UFG substrate is attributed to grain size and hydrophilicity and is related to more open lattice in the positions of high-angle grain boundaries.

The internal cuticle of Cirsium horridulum (Asteraceae) leaves.

Leaf internal cuticle has not previously been studied in detail, and yet its existence has profound implications for the path of water movement. The internal cuticle forms a uniform layer on the inner periclinal epidermal walls that border substomatal cavities. This cuticle is continuous with the external cuticle through the stomatal pores. The thickness of the internal cuticle on nonstomatal epidermal cells is approximately one-third that of the external cuticle on the same cells. On both the abaxial and adaxial sides of the leaf the internal cuticle forms irregularly shaped islands bordered by mesophyll cells. The size of the islands coincides with the epidermal area of the substomatal cavity. The internal cuticle remains intact and connected to the external cuticle after incubation in cellulytic enzymes. After treatment with sulfuric acid or chloroform, both cuticles remain intact. The autofluorescence of both cuticles is increased by staining with auramine O. These results indicate that large portions of the leaf epidermis are covered by both an internal and an external cuticle.

Interplay between grain structure and protein adsorption on functional response of osteoblasts: Ultrafine‐grained versus coarse‐grained substrates

The rapid adsorption of proteins is the starting and primary biological response that occurs when a biomedical device is implanted in the physiological system. The biological response, however, depends on the surface characteristics of the device. Considering the significant interest in nano-/ultrafine surfaces and nanostructured coatings, we describe here, the interplay between grain structure and protein adsorption (bovine serum albumin: BSA) on osteoblasts functions by comparing nanograined/ultrafine-grained (NG/UFG) and coarse-grained (CG: grain size in the micrometer range) substrates by investigating cell-substrate interactions. The protein adsorption on NG/UFG surface was beneficial in favorably modulating biological functions including cell attachment, proliferation, and viability, whereas the effect was less pronounced on protein adsorbed CG surface. Additionally, immunofluorescence studies demonstrated stronger vinculin signals associated with actin stress fibers in the outer regions of the cells and cellular extensions on protein adsorbed NG/UFG surface. The functional response followed the sequence: NG/UFG(BSA) > NG/UFG > CG(BSA) > CG. The differences in the cellular response on bare and protein adsorbed NG/UFG and CG surfaces are attributed to cumulative contribution of grain structure and degree of hydrophilicity. The study underscores the potential advantages of protein adsorption on artificial biomedical devices to enhance the bioactivity and regulate biological functions.

Microfilament bundles in the roots of a conifer,Chamaecyparis obtusa

SummaryThe distribution of microfilament bundles (MFBs) in the primary tissues ofChamaecyparis obtusa roots has been investigated by electron microscopy. Nomarski differential interference-contrast (NDIC) images of MFBs in sections of embedded materials are also presented to complement the ultrastructural observations. The peripheral phloem parenchyma cells, also known as precursory phloem, generally possess greater numbers of MFBs than do any other cell type. MFBs are apparently absent in the cortical, meristematic or root cap tissues. The number of MFBs seen in a transection of a cell varies according to its position in the ontogenetic sequence. While all the MFBs in peripheral phloem parenchyma cells lie within 2.0 μm from and on occasion contact the plasmamembrane, some MFBs in other phloem and xylem cells are located in the central areas of the cytoplasm. The possible three-dimensional distribution of MFBs in a streaming peripheral phlowm parenchyma cell is discussed.