The significance of phase reversion-induced nanograined/ultrafine-grained (NG/UFG) structure on the strain hardening behavior and deformation mechanism in copper-bearing antimicrobial austenitic stainless steel.

The unique concept of phase reversion involving severe deformation of parent austenite into martensite, followed by annealing for a short duration, whereby the strain-induced martensite reverts to austenite, was adopted to obtain nano-grained/ultrafine-grained (NG/UFG) structure in a Cu-bearing biomedical austenitic stainless steel resulting in high strength-high ductility combination. Work hardening and accompanying deformation mechanism are two important aspects that govern the mechanical behavior of biomedical devices. Thus, post-mortem electron microscopy of the strained region was carried out to explore the differences in the deformation mechanisms induced by grain refinement, while the strain hardening behavior was analyzed by Crussard-Jaoul (C-J) analysis of the tensile stress-strain data. The strain hardening behavior consisted of four stages and was strongly affected by grain structure. Twinning-induced plasticity (TWIP) was the governing deformation mechanism in the NG/UFG structure and contributed to good ductility. In striking contrast, transformation-induced plasticity (TRIP) contributed to high ductility in the coarse-grained (CG) counterpart and was the governing strain hardening mechanism. When the grain size is less than ~1 µm, the increase in the strain energy and the austenite stability significantly reduce the possibility of strain-induced martensite transformation such that there is a distinct transition in deformation mechanism from nanoscale twinning in the NG/UFG structure to strain-induced martensite in CG structure. The differences in the deformation mechanisms are explained in terms of austenite stability - strain energy relationship.

The significance of phase reversion-induced nanograined/ultrafine-grained structure on the load-controlled deformation response and related mechanism in copper-bearing austenitic stainless steel.

The ingenious concept of phase reversion annealing involving cold deformation of parent austenite to strain-induced martensite, followed by annealing was used to obtain nano-grained/ultrafine-grained (NG/UFG) structure in a Cu-bearing biomedical austenitic stainless steel resulting in high strength-high ductility combination. Having employed the concept effectively, the primary objective of this study is to critically analyze the interplay between the load-controlled deformation response, strain-rate sensitivity and deformation mechanism of NG/UFG austenitic stainless steel via nanoscale deformation experiments and compare with its coarse-grained (CG) counterpart. The study demonstrated that the strain-rate sensitivity of NG/UFG was ~1.5 times that of the CG structure. Post-mortem electron microscopy of plastic zone surrounding the indents indicated that the active deformation mechanism was nanoscale twinning with typical characteristics of a network of intersecting twins in the NG/UFG structure, while strain-induced martensite transformation was the effective deformation mechanism for the CG structure. The fracture morphology was also different for the two steels, essentially ductile in nature, and was characterized by striations marking the line-up of voids in NG/UFG steel and microvoid coalescence in CG counterpart. The differences in deformation mechanisms between the NG/UFG and CG structure are attributed to the austenite stability - strain energy relationship. Furthermore, the presence of ~3 wt % Cu in austenitic stainless steel had somewhat moderate effect on strain-rate sensitivity and activation volume at similar level of grain size in its Cu-free counterpart. Specifically, in the NG/UFG structure, the nanoscale twin density was noticeably higher in Cu-bearing austenitic stainless steel as compared to Cu-free counterpart, as Cu is known to increase the stacking fault energy.

The determining role of nanoscale mechanical twinning on cellular functions of nanostructured materials.

Considering that micromotions generated at the bone-implant interface under physiological loading introduce mechanical strain on the tissue and surface of the implant and that strain can be introduced during processing of the biomedical device, we elucidate here the interplay between mechanically-induced nanoscale twinning in austenitic stainless steel on osteoblast functions. Mechanically-induced nanoscale twinning significantly impacted cell attachment, cell-substrate interactions, proliferation, and subsequent synthesis of prominent proteins (fibronectin, actin, and vinculin). Twinning was beneficial in favorably modulating cellular activity and contributed to small differences in hydrophilicity and nanoscale roughness in relation to the untwinned surface.

The significance of deformation mechanisms on the fracture behavior of phase reversion-induced nanostructured austenitic stainless steel.

We describe here the relationship between grain structure, deformation mechanism and fracture characteristics in an austenitic stainless steel. This was accomplished using the novel concept of phase reversion that enabled a wide range of grain size from nanograined/ultrafine grained (NG/UFG) to coarse-grained (CG) regime to be obtained in a single material through change in temperature-time annealing sequence. In the NG/UFG structure, a marked increase in abundance of stacking faults (SFs) and twin density with strain was observed that led to a decrease in the average spacing between adjacent SFs, thus converting stacking faults into twins. Twinning in NG/UFG structure involved partial dislocations and their interaction with the grain boundaries, including SF overlapping and the coordinated nucleation of partial dislocations from the grain boundaries. The plastic zone in the NG/UFG structure resembled a network knitted by the intersecting twins and SFs. With SFE ~30 mJ/m2, the minimum stress for twin nucleation was ~250 MPa for the experiment steel and the corresponding optimal grain size (dop) wa ~120 nm. In contrast, in the CG structure, strain induced martensite formation was the deformation mechanism. The difference in the deformation mechanism led to a clear distinction in the fracture behavior from striated fracture in high strength-high ductility NG/UFG alloy to microvoid coalescence in the low strength-high ductility CG counterpart. The underlying reason for the change in fracture behavior was consistent with change in deformation mechanism from nanoscale twinning in NG/UFG alloy to strain-induced martensite in the CG alloy, which is related to change in the stability of austenite with grain size. An analysis of critical shear stress required to initiate twinning partial dislocations in comparison to that required to nucleate shear bands is presented. The appearance of striated fracture in the NG/UFG alloy suggests a quasi-static step wise crack growth process.

Dependence of cellular activity at protein adsorbed biointerfaces with nano- to microscale dimensionality.

Protein adsorption is one of the first-few events that occur when a biomedical device comes in contact with the physiological system. The adsorption process is subsequently followed by communication with cells and formation of tissue. Given the strong interest in nanostructured surfaces, we describe here the impact of grain structure from nanograined (NG) regime to coarse-grained (CG) regime on the self-assembly of proteins (bovine serum albumin) and consequent functional response of osteoblasts. The objective is accomplished using the innovative concept of "phase reversion" that enables a wide range of grain size (from NG to CG regime) to be obtained using an identical set of parameters, besides additional attributes of high strength/weight ratio and wear resistance. Depending on the grain structure a consistent and significant change in the adsorption characteristics of protein was observed at biointerface, such that the cell density was statistically different. The high surface coverage and leaf-like conformation of adsorbed protein on NG surface as compared to bare branch-like structure with low surface coverage on the CG surface, was beneficial in favorably modulating cellular activity (osteoblast functions: cell attachment, proliferation, actin, vinculin, and fibronectin expression). This is the first report that elucidates the impact of grain structure from NG to CG regime on cellular activity.

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.

Understanding the impact of grain structure in austenitic stainless steel from a nanograined regime to a coarse-grained regime on osteoblast functions using a novel metal deformation-annealing sequence.

Metallic biomedical devices with nanometer-sized grains (NGs) provide surfaces that are different from their coarse-grained (CG) (tens of micrometer) counterparts in terms of increased fraction of grain boundaries (NG>50%; CG<2-3%). The novel concept of 'phase-reversion' involving a controlled deformation-annealing sequence is used to obtain a wide range of grain structures, starting from the NG regime to the CG regime, to demonstrate that the grain structure significantly impacts cellular interactions and osteoblast functions. The uniqueness of this concept is the ability to address the critical aspect of cellular activity in nanostructured materials, because a range of grain sizes from NG to CG are obtained in a single material using an identical set of parameters. This is in addition to a high strength/weight ratio and superior wear and corrosion resistance. These multiple attributes are important for the long-term stability of biomedical devices. Experiments on the interplay between grain structure from the NG regime to CG in austenitic stainless steel on osteoblast functions indicated that cell attachment, proliferation, viability, morphology and spread varied with grain size and were favorably modulated on the NG and ultrafine-grain structure. Furthermore, immunofluorescence studies demonstrated stronger vinculin signals associated with actin stress fibers in the outer regions of the cells and cellular extensions on the NG surface. The differences in the cellular response with change in grain structure are attributed to grain structure and degree of hydrophilicity. The study lays the foundation for a new branch of nanostructured materials for biomedical applications.

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.

Cellular activity of bioactive nanograined/ultrafine-grained materials.

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.

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.