Anouck Louise Sarah Burzava

Background: A nanostructured titanium surface that promotes antimicrobial activity and osseointegration would provide the opportunity to create medical implants that can prevent orthopaedic infection and improve bone integration. Although nanostructured surfaces can exhibit antimicrobial activity, it is not known whether these surfaces are safe and conducive to osseointegration. Questions/purposes: Using a sheep animal model, we sought to determine whether the bony integration of medical-grade, titanium, porous-coated implants with a unique nanostructured surface modification (alkaline heat treatment [AHT]) previously shown to kill bacteria was better than that for a clinically accepted control surface of porous-coated titanium covered with hydroxyapatite (PCHA) after 12 weeks in vivo. The null hypothesis was that there would be no difference between implants with respect to the primary outcomes: interfacial shear strength and percent intersection surface (the percentage of implant surface with bone contact, as defined by a micro-CT protocol), and the secondary outcomes: stiffness, peak load, energy to failure, and micro-CT (bone volume/total volume [BV/TV], trabecular thickness [Tb.Th], and trabecular number [Tb.N]) and histomorphometric (bone-implant contact [BIC]) parameters. Methods: Implants of each material (alkaline heat-treated and hydroxyapatite-coated titanium) were surgically inserted into femoral and tibial metaphyseal cancellous bone (16 per implant type; interference fit) and in tibial cortices at three diaphyseal locations (24 per implant type; line-to-line fit) in eight skeletally mature sheep. At 12 weeks postoperatively, bones were excised to assess osseointegration of AHT and PCHA implants via biomechanical push-through tests, micro-CT, and histomorphometry. Bone composition and remodeling patterns in adult sheep are similar to that of humans, and this model enables comparison of implants with ex vivo outcomes that are not permissible with humans. Comparisons of primary and secondary outcomes were undertaken with linear mixed-effects models that were developed for the cortical and cancellous groups separately and that included a random effect of animals, covariates to adjust for preoperative bodyweight, and implant location (left/right limb, femoral/tibial cancellous, cortical diaphyseal region, and medial/lateral cortex) as appropriate. Significance was set at an alpha of 0.05. Results: The estimated marginal mean interfacial shear strength for cancellous bone, adjusted for covariates, was 1.6 MPa greater for AHT implants (9.3 MPa) than for PCHA implants (7.7 MPa) (95% CI 0.5 to 2.8; p = 0.006). Similarly, the estimated marginal mean interfacial shear strength for cortical bone, adjusted for covariates, was 6.6 MPa greater for AHT implants (25.5 MPa) than for PCHA implants (18.9 MPa) (95% CI 5.0 to 8.1; p < 0.001). No difference in the implant-bone percent intersection surface was detected for cancellous sites (cancellous AHT 55.1% and PCHA 58.7%; adjusted difference of estimated marginal mean -3.6% [95% CI -8.1% to 0.9%]; p = 0.11). In cortical bone, the estimated marginal mean percent intersection surface at the medial site, adjusted for covariates, was 11.8% higher for AHT implants (58.1%) than for PCHA (46.2% [95% CI 7.1% to 16.6%]; p < 0.001) and was not different at the lateral site (AHT 75.8% and PCHA 74.9%; adjusted difference of estimated marginal mean 0.9% [95% CI -3.8% to 5.7%]; p = 0.70). Conclusion: These data suggest there is stronger integration of bone on the AHT surface than on the PCHA surface at 12 weeks postimplantation in this sheep model. Clinical Relevance: Given that the AHT implants formed a more robust interface with cortical and cancellous bone than the PCHA implants, a clinical noninferiority study using hip stems with identical geometries can now be performed to compare the same surfaces used in this study. The results of this preclinical study provide an ethical baseline to proceed with such a clinical study given the potential of the alkaline heat-treated surface to reduce periprosthetic joint infection and enhance implant osseointegration.

Cardiovascular disease is a leading cause of death worldwide; however, despite substantial advances in medical device surface modifications, no synthetic coatings have so far matched the native endothelium as the optimal hemocompatible surface for blood-contacting implants. A promising strategy for rapid restoration of the endothelium on blood-contacting biomedical devices entails attracting circulating endothelial cells or their progenitors, via immobilized cell-capture molecules; for example, anti-CD34 antibody to attract CD34+ endothelial colony-forming cells (ECFCs). Inherent is the assumption that the cells attracted to the biomaterial surface are bound exclusively via a specific CD34 binding. However, serum proteins might adsorb in-between or on the top of antibody molecules and attract ECFCs via other binding mechanisms. Here, we studied whether a surface with immobilized anti-CD34 antibodies attracts ECFCs via a specific CD34 binding or a nonspecific (non-CD34) binding. To minimize serum protein adsorption, a fouling-resistant layer of hyperbranched polyglycerol (HPG) was used as a “blank slate,” onto which anti-CD34 antibodies were immobilized via aldehyde-amine coupling reaction after oxidation of terminal diols to aldehydes. An isotype antibody, mIgG1, was surface-immobilized analogously and was used as the control for antigen-binding specificity. Cell binding was also measured on the HPG hydrogel layer before and after oxidation. The surface analysis methods, x-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry, were used to verify the intended surface chemistries and revealed that the surface coverage of antibodies was sparse, yet the anti-CD34 antibody grafted surface-bound ECFCs very effectively. Moreover, it still captured the ECFCs after BSA passivation. However, cells also attached to oxidized HPG and immobilized mIgG1, though in much lower amounts. While our results confirm the effectiveness of attracting ECFCs via surface-bound anti-CD34 antibodies, our observation of a nonspecific binding component highlights the importance of considering its consequences in future studies.

Inspired by observations that the natural topography observed on cicada and dragonfly wings may be lethal to bacteria, researchers have sought to reproduce these nanostructures on biomaterials with the goal of reducing implant-associated infections. Titanium and its alloys are widely employed biomaterials with excellent properties but are susceptible to bacterial colonisation. Hydrothermal etching is a simple, cost-effective procedure which fabricates nanoscale protrusions of various dimensions upon titanium, depending on the etching parameters used. We investigated the role of etching time and the choice of cation (sodium and potassium) in the alkaline heat treatment on the topographical, physical, and bactericidal properties of the resulting modified titanium surfaces. Optimal etching times were 4 h for sodium hydroxide (NaOH) and 5 h for potassium hydroxide (KOH). NaOH etching for 4 h produced dense, but somewhat ordered, surface nanofeatures with 75 nanospikes per µm². In comparison, KOH etching for 5 h resulted sparser but nonetheless disordered surface morphology with only 8 spikes per µm². The NaOH surface was more effective at eliminating Gram-negative pathogens, while the KOH surface was more effective against the Gram-positive strains. These findings may guide further research and development of bactericidal titanium surfaces which are optimised for the predominant pathogens associated with the intended application.

The demand for medical implants globally has increased significantly due to an aging population amongst other reasons. Despite the overall increase in the survivorship of Ti6Al4V implants, implant infection rates are increasing due to factors such as diabetes, obesity, and bacterial resistance to antibiotics. Two commonly found bacteria implicated in implant infections are Staphylococcus aureus and Pseudomonas aeruginosa. Based on prior work that showed nanostructured surfaces might have potential in passively killing these bacterial species, we developed a hierarchical, hydrothermally etched, nanostructured titanium surface. To evaluate the antibacterial efficacy of this surface, etched and as-received surfaces were inoculated with S. aureus or P. aeruginosa at concentrations ranging from 10² to 10⁹ colony-forming units per disc. Live/dead staining revealed there was a 60% decrease in viability for S. aureus and greater than a 98% decrease for P. aeruginosa on etched surfaces at the lowest inoculum of 10² CFU/disc, when compared to the control surface. Bactericidal efficiency decreased with increasing bacterial concentrations in a stepwise manner, with decreases in bacterial viability noted for S. aureus above 10⁵ CFU/disc and above 10⁶ CFU/disc for P. aeruginosa. Surprisingly, biofilm depth analysis revealed a decrease in bacterial viability in the 2 μm layer furthest from the nanostructured surface. The nanostructured Ti6Al4V surface developed here holds the potential to reduce the rate of implant infections.

Hyperbranched polyglycerol (HPG) was previously investigated as a non-fouling hydrophilic grafted layer on biomaterials surfaces, analogous to the well-known PEO, but the range of adsorbing cells and proteins tested was limited and at times the assays used were not the most sensitive. Thus the questions arise whether HPG-grafted layers can indeed efficiently resist adsorption of a wider range of adsorbing biological entities, and how would different biological entities interact with such coating. A HPG coating of 25 nm thickness was grafted onto a spin-coated and plasma-treated polystyrene (PS) layer on a silicon wafer substrate; this provided a well-suited system for surface analyses by XPS, ToF-SIMS and AFM, which verified the presence of a uniform, smooth grafted HPG layer. Adsorption of bovine serum albumin, lysozyme, fibrinogen, and EGM2 were reduced by > 90%, with the adsorbed amounts close to the detection limit of XPS but still detectable by ToF-SIMS using Principal Component Analysis. With human serum, however, the reduction in adsorption was slightly less pronounced. Smooth muscle cells and fibroblasts were virtually unable to attach onto the grafted HPG layer, with > 99% reductions at 6 h compared with plasma-treated PS; the few attached cells remaining rounded and unable to spread. Their attachment might have resulted from coating defects. Testing with full blood showed that unlike for the control surface (plasma-treated PS), platelets did not adhere to the HPG surface, but there was attachment of some cells that stained CD11b positive and likely are neutrophils. Cells of the fungal organism Candida albicans were also able to attach onto the HPG surface to a limited extent, but in contrast to the control surface the attached cells on HPG did not form hyphal extensions and thus seem to be compromised in their ability to invade and to form biofilms. Our data suggest that “low-fouling” is a better term than non-fouling for a grafted HPG layer as the resistance to adsorption is not uniform across a range of proteins and cells. It is also important in future work to study whether the cells that do attach can still exert their normal functions; our observation of absence of hyphal extensions for Candida albicans suggests that this may not be so. Hence the potential utility of a grafted HPG layer may be not just a function of adsorbed amounts but also of the functionality of adsorbed proteins and cells.

PolyJet three-dimensional (3D) printing allows for the rapid manufacturing of 3D moulds for the fabrication of cross-linked poly(dimethylsiloxane) microwell arrays (PMAs). As this 3D printing technique has a resolution on the micrometer scale, the moulds exhibit a distinct surface roughness. In this study, the authors demonstrate by optical profilometry that the topography of the 3D printed moulds can be transferred to the PMAs and that this roughness induced cell adhesive properties to the material. In particular, the topography facilitated immobilization of endothelial cells on the internal walls of the microwells. The authors also demonstrate that upon immobilization of endothelial cells to the microwells, a second population of cells, namely, pancreatic islets could be introduced, thus producing a 3D coculture platform.

The propensity of glycosaminoglycans to mediate cell–cell and cell–matrix interactions opens the door to capture cells, including circulating blood cells, onto biomaterial substrates. Chondroitin sulfate (CS)-B is of particular interest, since it interacts with the receptor (EGF)-like module-containing mucin-like hormone receptor-like 2 precursor (EMR2) displayed on the surface of leukocytes and endothelial progenitor cells. Herein, CS-B and its isomer CS-A were covalently immobilized onto heptylamine plasma polymer films via three different binding chemistries to develop platform technology for the capture of EMR2 expressing cells onto solid carriers. Surface characterization verified the successful immobilization of both glycosaminoglycans. The EMR2 expressing human myeloid cell line U937 preferentially bound onto CS-B-modified substrates, and U937 cells preincubated with CS-B in solution exhibited reduced affinity for the substrate. The direct capture of hematopoietic and blood-circulating endothelial cell types via a glycosaminoglycan-binding surface receptor opens an unexplored route for the development of biomaterials targeted at these cell types.

Cell aggregates reproduce many features of the natural architecture of functional tissues, and have therefore become an important in vitro model of tissue function. In this study, we present an efficient and rapid method for the fabrication of site specific functionalised poly(dimethylsiloxane) (PDMS) microwell arrays that promote the formation of insulin-producing beta cell (MIN6) aggregates. Microwells were prepared using an ice templating technique whereby aqueous droplets were frozen on a surface and PDMS was cast on top to form a replica. By employing an aqueous alkali hydroxide solution, we demonstrate exclusive etching and functionalisation of the microwell inner surface, thereby allowing the selective absorption of biological factors within the microwells. Additionally, by manipulating surface wettability of the substrate through plasma polymer coating, the shape and profile of the microwells could be tailored. Microwells coated with antifouling Pluronic 123, bovine serum albumin, collagen type IV or insulin growth factor 2 were employed to investigate the formation and stability of MIN6 aggregates in microwells of different shapes. MIN6 aggregates formed with this technique retained insulin expression. These results demonstrate the potential of this platform for the rapid screening of biological factors influencing the formation and response of insulin-producing cell aggregates without the need for expensive micromachining techniques.