R.G. Richards

Adhesion formation of primary human osteoblasts and the functional response of mesenchymal stem cells to 330 nm deep microgrooves

The surface microtexture of an orthopaedic device can regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved to include the field of surface modification; in particular, nanotechnology has allowed for the development of experimental nanoscale substrates for investigation into cell nanofeature interactions. Here primary human osteoblasts (HOBs) were cultured on ordered nanoscale groove/ridge arrays fabricated by photolithography. Grooves were 330 nm deep and either 10, 25 or 100 μm in width. Adhesion subtypes in HOBs were quantified by immunofluorescent microscopy and cell–substrate interactions were investigated via immunocytochemistry with scanning electron microscopy. To further investigate the effects of these substrates on cellular function, 1.7 K gene microarray analysis was used to establish gene regulation profiles of mesenchymal stem cells cultured on these nanotopographies. Nanotopographies significantly affected the formation of focal complexes (FXs), focal adhesions (FAs) and supermature adhesions (SMAs). Planar control substrates induced widespread adhesion formation; 100 μm wide groove/ridge arrays did not significantly affect adhesion formation yet induced upregulation of genes involved in skeletal development and increased osteospecific function; 25 μm wide groove/ridge arrays were associated with a reduction in SMA and an increase in FX formation; and 10 μm wide groove/ridge arrays significantly reduced osteoblast adhesion and induced an interplay of up- and downregulation of gene expression. This study indicates that groove/ridge topographies are important modulators of both cellular adhesion and osteospecific function and, critically, that groove/ridge width is important in determining cellular response.

A phenotypic comparison of osteoblast cell lines versus human primary osteoblasts for biomaterials testing

Immortalized cell lines are used more frequently in basic and applied biology research than primary bone-derived cells because of their ease of access and repeatability of results in experiments. It is clear that these cell models do not fully resemble the behavior of primary osteoblast cells. Although the differences will affect the results of biomaterials testing, they are not clearly defined. Here, we focused on comparing proliferation and maturation potential of three osteoblast cell lines, SaOs2, MG-63, and MC3T3-E1 with primary human osteoblast (HOb) cells to assess their suitability as in vitro models for biomaterials testing. We report similarities in cell proliferation and mineralization between primary cells and MC3T3-E1. Both, SaOs2 and MG-63 cells demonstrated a higher proliferation rate than HOb cells. In addition, SaOs2, but not MG-63, cells demonstrated similar ALP activity, mineralization potential and gene regulation to HObs. Our results demonstrate that despite SaOs-2, MG63, and MC3T3 cells being popular choices for emulating osteoblast behavior, none can be considered appropriate replacements for HObs. Nevertheless, these cell lines all demonstrated some distinct similarities with HObs, thus when applied in the correct context are a valuable in vitro pilot model of osteoblast functionality, but should not be used to replace primary cell studies.

Antimicrobial delivery systems for local infection prophylaxis in orthopedic- and trauma surgery

Infectious complications occur in a minor but significant portion of the patients undergoing joint replacement surgery or fracture fixation, particularly those with severe open fractures, those undergoing revision arthroplasty or those at elevated risk because of poor health status. Once established, infections are difficult to eradicate, especially in the case of bacterial biofilm formation on implanted hardware. Local antibiotic carriers offer the prospect of controlled delivery of antibiotics directly in target tissues and implant, without inducing toxicity in non-target organs. Polymeric carriers have been developed to optimize the release and targeting of antibiotics. Passive polymeric carriers release antibiotics by diffusion and/or upon degradation, while active polymeric carriers release their antibiotics upon stimuli provided by bacterial pathogens. Additionally, some polymeric carriers gelate in-situ in response to physiological stimuli to form a depot for antibiotic release. As antibiotic resistance has become a major issue, also other anti-infectives such as silver and antimicrobial peptides have been incorporated in research. Currently, several antibiotic loaded biomaterials for local infection prophylaxis are available for use in the clinic. Here we review their advantages and limitations and provide an overview of new materials emerging that may overcome these limitations.

The effect of surface roughness on fibroblast adhesion in vitro

Adhesion of tissue cells to biomaterial implants is a major factor of their biocompatibility. Quantitative or qualitative adhesion measurements would therefore be useful in the screening of new implant materials. Results from a quantitative method of measuring the total cell adhesion area of cultured cells is presented. It is postulated that the more compatible the surface, the greater the amount of cell adhesion. Fibroblastic cells were cultured on discs of plastic (Thermanox), commercially pure titanium (ISO 5832/2) or steel (ISO 5832/1), as used in AO fixation plates. The cells were fixed, stained, embedded in resin and their discs removed. Backscattered electron (BSE) imaging in a scanning electron microscope displayed the stained cells within the unstained resin. Imaging at high beam energy allowed visualization of the entire cell. Low beam energy displayed the regions of cell contact with the substrate, i.e. the focal adhesion sites. Images were analyzed with an image analysis and measurement system which allowed the percentage of the cell area involved with adhesion to be calculated. Results show that the material roughness, with the materials and cells tested, does not affect the total amount of cell adhesion. Implant surface design to encourage cell adhesion and discourage bacterial adhesion is discussed.

The use of titanium and stainless steel in fracture fixation

The use of metal in fracture fixation has demonstrated unrivalled success for many years owing to its high stiffness, strength, biological toleration and overall reliable function. The most prominent materials used are electropolished stainless steel and commercially pure titanium, along with the more recent emergence of titanium alloys. Despite the many differences between electropolished stainless steel and titanium, both materials provide a relatively predictable clinical outcome, and offer similar success for fulfilling the main biomechanical and biological requirements of fracture fixation despite distinctive differences in implant properties and biological responses. This article explores these differences by highlighting the limitations and advantages of both materials, and addresses how this translates to clinical success.

Influence of material on the development of device‐associated infections

The use of implanted devices in modern orthopaedic surgery has greatly improved the quality of life for an increasing number of patients, by facilitating the rapid and effective healing of bone after traumatic fractures, and restoring mobility after joint replacement. However, the presence of an implanted device results in an increased susceptibility to infection for the patient, owing to the creation of an immunologically compromised zone adjacent to the implant. Within this zone, the ability of the host to clear contaminating bacteria may be compromised, and this can lead to biofilm formation on the surface of the biomaterial. Currently, there are only limited data on the mechanisms behind this increased risk of infection and the role of material choice. The impacts of implant material on bacterial adhesion, immune response and infection susceptibility have been investigated individually in numerous preclinical in vitro and in vivo studies. These data provide an indication that material choice does have an impact on infection susceptibility; however, the clinical implications remain to be clearly determined.

IMMUNOGOLD LABELLING OF FIBROBLAST FOCAL ADHESION SITES VISUALISED IN FIXED MATERIAL USING SCANNING ELECTRON MICROSCOPY, AND LIVING, USING INTERNAL REFLECTION MICROSCOPY

A new immunogold labelling method for the visualisation of vinculin, an integral protein in focal adhesions of cells, is reported. Quantification of vinculin is indicative of substrate cytocompatibility (cytocompatibility is one aspect of biocompatibility; it is the cellular response to a biomaterial). For efficient labelling, most of the cell body above the cell—substrate interface was removed with detergent. The antigen blocking procedure, size of label (5nm) and duration of silver‐enhancement (6min), for visualisation of the labelled sites on the whole cell by scanning electron microscopy (SEM), were determined. Imaging living cells with interference reflection light microscopy, followed by backscattered electron (BSE) imaging of the same fixed and immunolabelled cells confirmed the results. Collecting low voltage BSE images of embedded cells after the substrate had been removed provided ‘sectional’ views through the cell. This enabled visualisation of vinculin exclusively within the cell‐substrate contact zone; the focal adhesions. The method could be of general use in the imaging of protein distribution at biological tissue/substrate interfaces.

Measurement of fibroblast and bacterial detachment from biomaterials using jet impingement.

Fibroblast and Staphylococcus aureus detachment strength from orthopaedic alloys and a tissue culture plastic (Thermanox) have been investigated with jet impingement. For S. aureus, unlike fibroblasts, detachment is caused more by pressure than shear. For these biomaterials, detachment strength is much higher for S. aureus than fibroblasts. Comparing materials under equivalent flow conditions, S. aureus attach to stainless steel and titanium with equal strength and more strongly than to Thermanox. For fibroblasts, detachment strength from all materials was similar. Fibroblast detachment strength from these biomaterials substantially decreases with time at equal flow rates and increases with flow rate at equal exposure times. Detachment strength is very similar for 3T3 and L929 fibroblasts on Thermanox for equivalent flow rate/time combinations, though enhanced adhesion of 3T3 cells was often noted for metals. Time effects are less evident for S. aureus. S. aureus adhesion to metals is more affected by flow rate than fibroblast adhesion.

Bacterial adhesion to orthopaedic implant materials and a novel oxygen plasma modified PEEK surface

Despite extensive use of polyetheretherketone (PEEK) in biomedical applications, information about bacterial adhesion to this biomaterial is limited. This study investigated Staphylococcus aureus and Staphylococcus epidermidis adhesion to injection moulded and machined PEEK OPTIMA(®) using a custom-built adhesion chamber with medical grade titanium and Thermanox for comparison. Additionally, bacterial adhesion to a novel oxygen plasma modified PEEK was also investigated in both a pre-operative model in physiological saline, and additionally in a post-operative model in human blood plasma. In the pre-operative model, the rougher machined PEEK had a significantly greater number of adherent bacteria compared to injection moulded PEEK. Bacterial adhesion to titanium and Thermanox was similar. Oxygen plasma surface modification of PEEK did not lead to a significant change in bacterial adhesion in the pre-operative contamination model, despite observed changes in surface characteristics. In the post-operative contamination model, S. aureus adhesion was increased from 5×10(5) CFU cm(-2) to approximately 1.3×10(7) CFU cm(-2) on the modified surfaces due to differential protein adhesion during the conditioning period. However, S. epidermidis adhesion to modified PEEK was less than to unmodified PEEK in the post-operative model. These results illustrate the importance of testing bacterial adhesion of several strains in both a pre-operative and post-operative, clinically relevant bacterial contamination model.

Potential of polymethylmethacrylate cement-augmented helical proximal femoral nail antirotation blades to improve implant stability--a biomechanical investigation in human cadaveric femoral heads.

Background: Cement augmentation may improve fixation stability and reduce cut-out rate in the treatment of intertrochanteric hip fractures. The aim of this study was to compare the number of cycles to failure of polymethylmethacrylate (PMMA)-augmented helical blades with nonaugmented ones in human cadaveric femoral heads. Methods: Six pairs of cadaveric femoral heads were instrumented with a perforated proximal femoral nail antirotation blade. Within each pair, one blade was augmented using 3 mL of PMMA. All specimens underwent cyclic axial loading under physiologic conditions. Starting at 1,000 N, the load was monotonically increased by 0.1 N/cycle until construct failure occurred. To monitor the migration of the blade, anteroposterior radiographs were taken at 250 cycle increments. Nonparametric test statistics were done to calculate correlations and identify differences between study groups. Results: Inducing failure required a significantly higher number of cycles in the augmented group (p = 0.028). Bone mineral density was significantly related with the number of cycles to failure in nonaugmented specimens (p < 0.001, R2 = 0.97), but not in the augmented group (p = 0.91, R2 = 0.34). Conclusion: Implant augmentation with small amounts of PMMA enhances the cut-out resistance in proximal femoral fractures. Especially in osteoporotic bone, the procedure may improve patient care.