Surface finish of Additively Manufactured Metals: biofilm formation and cellular attachment

Paola Ginestra. Department of Mechanical and Industrial Engineering, University of Brescia, Via Branze 38, 25123 Brescia, Italy Corresponding author: paola.ginestra@unibs.it Leonardo Riva. Department of Mechanical and Industrial Engineering, University of Brescia, Via Branze 38, 25123 Brescia, Italy Elisabetta Ceretti. Department of Mechanical and Industrial Engineering, University of Brescia, Via Branze 38, 25123 Brescia, Italy David Lobo. Department of Mechanical and Industrial Engineering, University of Brescia, Via Branze 38, 25123 Brescia, Italy Sophie Mountcastle. School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Victor Villapun. School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Sophie Cox. School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Liam Grover. School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Moataz Attallah. School of Metallurgy and Materials, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Owen Addison. School of Dentistry, University of Birmingham, 5 Mill Pool Way, Edgbaston, Birmingham B5 7EG, UK Duncan Shepherd. Department of Mechanical Engineering, University of Birmingham, 5 Mill Pool Way, Edgbaston, Birmingham B5 7EG, UK Mark Webber. School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

meet the requirements of the end user and target application. The medical industry relies on these additive manufacturing technologies for the advantages that these methods offer to accurately fit the patients' needs.
Besides the recent improvements, the production process of 3D printed bespoke implants still requires optimization to achieve the optimal properties that can mimic both the chemical and mechanical characteristics of the anatomical region of interest. In particular, the surface properties of an implant device are crucial to obtain a strong interface and connection with the physiological environment. The layer by layer manufacturing processes lead to the production of complex and high-performance substrates but always require surface treatments during post-processing to improve the implant interaction with the natural tissues and promote a shorter assimilation for the fast recovery and wellness of the patient. Although the surface finishing can be tailored to enhance cells adhesion, proliferation and differentiation in contact with a metal implant, the same surface properties can have a different outcome when dealing with bacteria. This work aims to provide a preliminary analysis on how different post-processing techniques have distinct effects on cells and bacteria colonization of 3D printed titanium implants. The goal of the paper is to highlight the importance of the The demand for implantable devices is expected to rise consistently over the next two decades. Longer life expectancy and an increase in population will raise the volume of medical implantable devices, while the higher mobility demanded by younger patients may outliving such devices. This increase in demand is linked to the inherent complexity of the required implant which paired with the differences between patients makes difficult their standardization, originating the challenge to manufacture high quality and vastly different implantable devices to modern engineering [1,2]. Additive manufacturing (AM) techniques allow the production of highly customized implants meeting the final application specifications. Powder bed fusion processes offer the possibility of fabrication of complex and bespoke metal parts usually characterized by scarce surface finishing [3]. The surface quality of 3D printed parts is extremely important for the biological outcomes of both cells and bacteria colonization during in vivo conditions [4,5]. Infection of implantable devices is, still today, a great healthcare concern. Each year 1 to 5 % indwelling prosthetics became infected [6] where an attempt of salvaging the prosthetics through debridement of the infected site and long term antibiotic treatment are preferred over replacement, but literature shows that this procedure success rates range can be as low as 30 to 50% [7,8,9]. Antimicrobial coatings, photocatalysis antibiodies and antibiotics can be applyed to titanium surfaces to add an active mechanism of defence against bacteria colonization [10], but they are time consuming and complicate the part processing and supply chain. Polishing, etching and sandblasting finishing are commercially available treatments implementable with relatively easy application [11]. Their low cost, practicality and simplicity make them ideal candidates to treat additive manufactured parts. Biofilm formation, cell adhesion and differentiation had been shown to be heavily influenced by the aforementioned treatments, with substantial increases in osteogenesis and hindering of biofilm formation. This demonstrates a real possibility of tailoring the healing response of an implantable device with simple post processing techniques, but optimization of both cell adhesion and biofilm hindering remains a challenge while its impact on untreated additive manufacturing parts its mostly unknown. In this paper, AM samples are produced The Ti6Al4V (Ti64) atomized powder was fully characterized before processing as reported in [12]. Briefly, the morphology of the powder particles and particle size distribution have been analyzed to assess the homogeneity of the powder before the printing and the flow properties have been examined to verify the dynamic properties and consequently the flow ability under low stress conditions.
For this research, cubical samples of 10 mm 3 were printed with a M2 Cusing SLM system (Concept Laser, Germany).
An island pattern was chosen and the parameters for the fabrication were set as: 20 μm layer thickness, 75 μm hatch spacing, 1750 mm/s scanning speed and 150 W laser power.
As fabricated (AF) parts were then processed by polishing (PO), passivation (PA) and sandblasting followed by passivation (SP). All the post-processing techniques were applied to both the top and the side surfaces of the specimens and selected as commonly used in industrial standard operations to modify the surface finish of as built parts. The polishing process was performed using a centrifugal disc finishing machine (Finishing Techniques Ltd., FINTEK) with multiple stages. The whole process was carried out firstly using a G240 grinding disk, then a G1200 and finally a G4000 grinding disk followed by a polishing cloth with aluminum oxide balls (6−10 mm) to deburr and polish the parts, with total process duration of 8 h. All parts were cleaned using compressed air, an ultrasonic bath, and isopropyl alcohol.
Passivated samples were obtained by etching in NHO3 for 30 minutes. Sandblasting was performed in an Air Blast cabinet (CBI Equipment Ltd., UK) for 2 min with a speed of 100 m/s as previously reported [12]. Sandblasted samples were also passivated afterwards. Both the top surface and the side surface have been treated and analyzed. Energy-dispersive X-ray spectroscopy (EDS) was used to evaluate the presence of contaminants due to the postprocessing. The surface roughness of the as built and treated parts was analyzed using a Bruker Contour GT-K 3D Optical Microscope at 20× magnification.
The chemical properties of the surfaces were characterized by measuring the contact angle (CA) using a Attension® Theta tensiometer (Biolin Scientific). A droplet of deionized water (5 μm) was pipetted onto the top and side surfaces of the samples. Three measurements of the roughness and contact angle were obtained on representative areas of the overall surface for three different sample variants.

Biological t Biological tests ests
Staphylococcus epidermidis (gram-positive) and Pseudomonas aeruginosa (gram-negative) were used to evaluate the adhesion of two bacterial strain on Ti64 surfaces as previously shown [12].

R Results esults 4.1 SEM and EDS r 4.1 SEM and EDS results esults
Scanning electron micrographs of AF samples highlighted the presence of the island scanning as already reported [12].  The results of the roughness analysis on the surface of the samples are presented as mean and standard deviation of the mean (Fig. 2).

Contact ang Contact angle r le results esults
The contact angle of the top and side surfaces of the as fabricated and processed samples is reported in Figure 3 as mean and standard deviation of the mean. The results of the Crystal violet staining are reported in Fig. 6. The same behavior was shown by the bacteria on the polished surfaces that prevented the formation of a homogenous biofilm.

Conclusions Conclusions
The paper reports the effects on bacteria and cells adhesion of modifying the surface chemistry and topography of additively manufactured implants by the most used postprocessing processes: polishing, etching and sandblasting. The presented approach offers the opportunity to analyze the differences of antimicrobial and osseointegration properties of 3D printed implants for orthopedic applications. This work highlighted the necessity of minimizing the presence of particles on Ti6Al4V SLM specimens that could prevent a proper adhesion of the cells and promote the S. epidermidis and P. aeruginosa colonization of the surfaces. The polishing treatment of the parts, typically performed on parts before implantation, has been demonstrated effective for reducing the bacterial presence. Although, the presented preliminary study on cell adhesion and mineralization showed that surface polishing can impede the attachment of the cells.
The proposed methodology highlights the necessity of combining physical, chemical and mechanical approaches to properly promote osseointegration reducing the effect of a short-term bacterial infection.
Surface finish of Additively Manufactured Metals: biofilm formation and cellular attach...