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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., (Copyright 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Our recent electron microscopy study on biomimetic nanostructured coatings on nanograined/ultrafine-grained (NG/UFG) substrates [Mater Sci Eng C 2009;29:2417-27] indicated that electrocrystallized nanohydroxyapatite (nHA) on phase-reversion-induced NG/UFG substrates exhibited a vein-type interconnected and fibrillar structure that closely mimicked the hierarchical structure of bone. The fibrillar structure on NG/UFG substrate is expected to be more favorable for cellular response than a planar surface. In contrast, hydroxyapatite (HA) coating on coarse-grained (CG) substrate more closely resembled a film rather than a fibrillar structure. Inspired by the differences in the structure of HA coating, we describe here the cell-substrate interactions of pre-osteoblasts (MC 3T3-E1) on bioactive NG/UFG and CG austenitic stainless steel substrates. NG/UFG austenitic stainless steel was obtained by a novel controlled phase-reversion annealing of cold-deformed austenite. This example provides an illustration of how a combination of cellular and molecular biology, materials science and engineering can advance our understanding of cell-substrate interactions. Interestingly, the cellular response of nanohydroxyapatite (nHA)-coated NG/UFG substrate demonstrated superior cytocompatibility, improved initial cell attachment, higher viability and proliferation, and well-spread morphology in relation to HA-coated CG substrate and their respective uncoated (bare) counterparts as implied by fluorescence and electron microscopy and MTT assay. Similar conclusions were derived from an immunofluorescence study that involved examination of the expression levels of vinculin focal adhesion contacts associated with dense actin stress fibers and fibronectin, protein analysis through protein bands in SDS-PAGE, and quantitative total protein assay. The enhancement of cellular response followed the sequence: nHA-coated NG/UFG>nHA-coated CG>NG/UFG>CG substrates. The outcomes of the study are expected to counter the challenges associated with the engineering of nanostructured surfaces with specific physical and surface properties for medical devices with significantly improved cellular response., (Copyright 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The favorable cellular response of newly developed cell line, buffalo embryonic stem (ES) cells to three-dimensional biodegradable chitosan-gelatin composite scaffolds with regard to stem-cell-based tissue engineering is described. Chitosan-gelatin composites were characterized by a highly porous structure with interconnected pores, and the mechanical properties were significantly enhanced. Furthermore, X-ray diffraction study indicated increased amorphous content in the scaffold on the addition of gelatin to chitosan. To develop a transfectant of green fluorescence protein (GFP)-buffalo ES cell, transfection of GFP plasmid to the cell was carried out via the electroporation procedure. In comparison with pure chitosan, cell spreading and proliferation were greater in highly visualized GFP-expressing cell-chitosan-gelatin scaffold constructs. The relative comparison of biological response involving cell proliferation and viability on the scaffolds suggests that blending of gelatin in chitosan improved cellular efficiency. Studies involving scanning electron and fluorescence microscopy, histological observations and flow cytometer analysis of the constructs implied that the polygonal cells attached to and penetrated the pores, and proliferated well, while maintaining their pluripotency during the culture period for 28days. Chitosan-gelatin scaffolds were cytocompatible with respect to buffalo ES cells. The study underscores for the first time that chitosan-gelatin scaffolds are promising candidates for ES-cell-based tissue engineering.

A potential approach to achieving the objective of favorably modulating the biological response of implantable biopolymers combined with good mechanical properties is to consider compounding the biopolymer with a bioactive nanocrystalline ceramic biomimetic material with high surface area. The processing of silicone rubber (SR)-nanohydroxyapatite (nHA) composite involved uniform dispersion of nHA via shear mixing and ultrasonication, followed by compounding at sub-ambient temperature, and high-pressure solidification when the final curing reaction occurs. The high-pressure solidification approach enabled the elastomer to retain the high elongation of SR even in the presence of the reinforcement material, nHA. The biological response of the nanostructured composite in terms of initial cell attachment, cell viability and proliferation was consistently greater on SR-5wt.% nHA composite surface compared to pure SR. Furthermore, in the nanocomposite, cell spreading, morphology and density were distinctly different from that of pure SR. Pre-osteoblasts grown on SR-nHA were well spread, flat, large in size with a rough cell surface, and appeared as a group. In contrast, these features were less pronounced in SR (e.g. smooth cell surface, not well spread). Interestingly, an immunofluorescence study illustrated distinct fibronectin expression level, and stronger vinculin focal adhesion contacts associated with abundant actin stress fibers in pre-osteoblasts grown on the nanocomposite compared to SR, implying enhanced cell-substrate interaction. This finding was consistent with the total protein content and SDS-PAGE analysis. The study leads us to believe that further increase in nHA content in the SR matrix beyond 5wt.% will encourage even greater cellular response. The integration of cellular and molecular biology with materials science and engineering described herein provides a direction for the development of a new generation of nanostructured materials.

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.

We describe a comparative assessment of the structure-property-process relationship of three-dimensional chitosan-nanohydroxyapatite (nHA) and pure chitosan scaffolds in conjunction with their respective biological response with the aim of advancing our insight into aspects that concern bone tissue engineering. High- and medium-molecular-weight (MW) chitosan scaffolds with 0.5, 1 and 2 wt.% fraction of nHA were fabricated by freezing and lyophilization. The nanocomposites were characterized by a highly porous structure and the pore size (approximately 50 to 120 microm) was in a similar range for the scaffolds with different content of nHA. A combination of X-ray diffraction, Fourier transform infrared spectroscopy and electron microscopy indicated that nHA particles were uniformly dispersed in chitosan matrix and there was a chemical interaction between chitosan and nHA. The compression modulus of hydrated chitosan scaffolds was increased on the addition of 1 wt.% nHA from 6.0 to 9.2 kPa in high-MW scaffold. The water uptake ability of composites decreased with an increase in the amount of nHA, while the water retention ability was similar to pure chitosan scaffold. After 28 days in physiological condition, nanocomposites indicated about 10% lower degree of degradation in comparison to chitosan scaffold. The biological response of pre-osteoblasts (MC 3T3-E1) on nanocomposite scaffolds was superior in terms of improved cell attachment, higher proliferation, and well-spread morphology in relation to chitosan scaffold. In composite scaffolds, cell proliferation was about 1.5 times greater than pure chitosan after 7 days of culture and beyond, as implied by qualitative analysis via fluorescence microscopy and quantitative study through MTT assay. The observations related to well-developed structure morphology, physicochemical properties and superior cytocompatibility suggest that chitosan-nHA porous scaffolds are potential candidate materials for bone regeneration although it is necessary to further enhance the mechanical properties of the nanocomposite.