An accelerated buoyancy adhesion assay combined with 3-D morphometric analysis for assessing osteoblast adhesion on microgrooved substrata.

An accelerated negative buoyancy method has been developed to assess cell adhesion strength. This method has been used in conjunction with 3-D morphometric analysis to understand the effects of surface topology on cell response. Aligned micro-grooved surface topographies (with a range of groove depths) were produced on stainless steel 316L substrates by laser ablation. An investigation was carried out on the effect of the micro-grooved surface topography on cell adhesion strength, cell and nucleus volumes, cell phenotypic expression and attachment patterns. Increased hydrophobicity and anisotropic wettability was observed on surfaces with deeper grooves. A reduction was noted in cell volume, projected areas and adhesion sites for deeper grooves, linked to lower cell proliferation and differentiation rates and also to reduced adhesion strength. The results suggest that the centrifugation assay combined with three-dimensional cell morphometric analysis has considerable potential for obtaining improved understanding of the cell/substrate interface.Financial support for this work has been provided by the EPSRC (grant GR/R95364/01) and by the ERC (grant No. 240446). The authors are grateful to Dr. Tim Nunney, of Thermo-Fisher Scientific, for carrying out the XPS measurements.This is the final version of the article. It first appeared from Elsevier via https://doi.org/ /10.1016/j.jmbbm.2015.12.03

In vitro osteoblast response to ferritic stainless steel fiber networks for magneto-active layers on implants

The use of a porous coating on prosthetic components to encourage bone ingrowth is an important way of improving uncemented implant fixation. Enhanced fixation may be achieved by the use of porous magneto-active layers on the surface of prosthetic implants, which would deform elastically on application of a magnetic field, generating internal stresses within the in-growing bone. This approach requires a ferromagnetic material able to support osteoblast attachment, proliferation, differentiation, and mineralization. In this study, the human osteoblast responses to ferromagnetic 444 stainless steel networks were considered alongside those to nonmagnetic 316L (medical grade) stainless steel networks. While both networks had similar porosities, 444 networks were made from coarser fibers, resulting in larger inter-fiber spaces. The networks were analyzed for cell morphology, distribution, proliferation, and differentiation, extracellular matrix production and the formation of mineralized nodules. Cell culture was performed in both the presence of osteogenic supplements, to encourage cell differentiation, and in their absence. It was found that fiber size affected osteoblast morphology, cytoskeleton organization and proliferation at the early stages of culture. The larger inter-fiber spaces in the 444 networks resulted in better spatial distribution of the extracellular matrix. The addition of osteogenic supplements enhanced cell differentiation and reduced cell proliferation thereby preventing the differences in proliferation observed in the absence of osteogenic supplements. The results demonstrated that 444 networks elicited favorable responses from human osteoblasts, and thus show potential for use as magnetically active porous coatings for advanced bone implant applications. © 2012 Wiley Periodicals, Inc

Osteoblast and monocyte responses to 444 ferritic stainless steel intended for a Magneto-Mechanically Actuated Fibrous Scaffold

The rationale behind this work is to design an implant device, based on a ferromagnetic material, with the potential to deform in vivo promoting osseointegration through the growth of a healthy periprosthetic bone structure. One of the primary requirements for such a device is that the material should be non-inflammatory and non-cytotoxic. In the study described here, we assessed the short-term cellular response to 444 ferritic stainless steel; a steel, with a very low interstitial content and a small amount of strong carbide-forming elements to enhance intergranular corrosion resistance. Two different human cell types were used: (i) foetal osteoblasts and (ii) monocytes. Austenitic stainless steel 316L, currently utilised in many commercially available implant designs, and tissue culture plastic were used as the control surfaces. Cell viability, proliferation and alkaline phosphatase activity were measured. In addition, cells were stained with alizarin red and fluorescently-labelled phalloidin and examined using light, fluorescence and scanning electron microscopy. Results showed that the osteoblast cells exhibited a very similar degree of attachment, growth and osteogenic differentiation on all surfaces. Measurement of lactate dehydrogenase activity and tumour necrosis factor alpha protein released from human monocytes indicated that 444 stainless steel did not cause cytotoxic effects or any significant inflammatory response. Collectively, the results suggest that 444 ferritic stainless steel has the potential to be used in advanced bone implant designs. © 2011 Elsevier Ltd

The influence of nanostructured features on bacterial adhesion and bone cell functions on severely shot peened 316L stainless steel

Substrate grain structure and topography play major roles in mediating cell and bacteria activities. Severe plastic deformation techniques, known as efficient metal-forming and grain refining processes, provide the treated material with novel mechanical properties and can be adopted to modify nanoscale surface characteristics, possibly affecting interactions with the biological environment. This in vitro study evaluates the capability of severe shot peening, based on severe plastic deformation, to modulate the interactions of nanocrystallized metallic biomaterials with cells and bacteria. The treated 316L stainless steel surfaces were first investigated in terms of surface topography, grain size, hardness, wettability and residual stresses. The effects of the induced surface modifications were then separately studied in terms of cell morphology, adhesion and proliferation of primary human osteoblasts (bone forming cells) as well as the adhesion of multiple bacteria strains, specifically Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and ampicillin-resistant Escherichia coli. The results indicated a significant enhancement in surface work hardening and compressive residual stresses, maintenance of osteoblast adhesion and proliferation as well as a remarkable decrease in the adhesion and growth of gram-positive bacteria (S. aureus and S. epidermidis) compared to non-treated and conventionally shot peened samples. Impressively, the decrease in bacteria adhesion and growth was achieved without the use of antibiotics, for which bacteria can develop a resistance towards anyway. By slightly grinding the surface of severe shot peened samples to remove differences in nanoscale surface roughness, the effects of varying substrate grain size were separated from those of varying surface roughness. The expression of vinculin focal adhesions from osteoblasts was found to be singularly and inversely related to grain size, whereas the attachment of gram-positive bacteria (S. aureus and S. epidermidis) decreased with increasing nanoscale surface roughness, and was not affected by grain refinement. Ultimately, this study demonstrated the advantages of the proposed shot peening treatment to produce multifunctional 316L stainless steel materials for improved implant functions without necessitating the use of drugs