Assessment and Control of ANTI-Microbial and ANTI-Inflammatory Responses of Macrophages to Different Titanium Nanomodifications

ASSESSMENT AND CONTROL OF ANTI-MICROBIAL AND ANTI-INFLAMMATORY RESPONSES OF MACROPHAGES TO DIFFERENT TITANIUM NANOMODIFICATIONS

Garima Bhardwaj¹, Hilal Yazici ¹,Thomas J. Webster¹.

¹Northeastern University, Boston, MA-02115

Introduction: To monitor the immunological response of implants, the host response to materials must be determined prior to implantation. This response is highly dominated by macrophages which are the primary cells governing the immune response. Macrophages not only fuse to become multinucleated foreign body giant cells, but they also activate T lymphocytes by expressing co-stimulatory molecules (e.g., CD86 and CD80) and surface antigens (e.g., MHC II). T lymphocyte activation and giant cell formation have been observed on macrophages adherent to biomaterials and in retrieved adjacent tissues to failed implants. They are major effectors in the defense against bacterial infection and respond to interactions with gram-negative and gram-positive bacteria with a marked enhancement of their functional activities. Thus, the assessment and control of macrophage responses to various surface nano-modifications on titanium implants is of vital importance. The current study aims at modifying the surface of titanium by coating it with nanoscale hydroxyapatite, in a range of sizes, using electrophoretic deposition with DC current. Macrophages are then seeded onto this surface and their behavior observed concerning how to further improve the modification process.

Materials and Methods: Nanoscale hydroxyapatite was synthesized using a wet chemical synthesis process in 4 different sizes ranging from 110-170 nm, using Ca(NO3)24H2O, KH2PO4, distilled water and ammonia in an acid digestion vessel for hydrothermal treatment followed by drying in the oven. It was then coated onto a titanium mesh purchased from Alpha Aesar (Catalog no.7440-32-6) by electrophoretic deposition (EPD) with a DC current at 151 V for a minute each. Macrophages purchased from ATCC (RAW 264.7 (ATCC^® TIB-71^™)) were cultured using EMEM (ATCC^® 30-2003^™), 10% FBS (ATCC^® SCRR-30-2020^™) and a 1% penicillin-streptomycin solution (ATCC^® 30-2300^™). Cell adhesion and proliferation was observed using MTS assay for 1, 3, 5 and 7 days. Levels of TNF-α, IL-1, IL-6 and nitrite released by the macrophages were studied using PCR.

Bacterial assays were conducted using Staphylococcus aureus (ATCC^® 29740^™), Ampicillin resistant E.coli and Pseudomonas aeruginosa (ATCC^® 39324^™) strains of bacteria. 0.03% tryptic soy broth (TSB) and agar plates (Sigma-Aldrich) were used as the media. A small amount of bacteria was taken from the stock culture, streaked onto an agar plate, and then used as the stock plate for further experiments. Colonies were scraped off from the stock plate, added to 3 mL of 3% TSB and incubated at 37°C in humidified conditions under a 5% carbon dioxide atmosphere for 18 hours. A small amount (0.1 mL) of each sample was transferred to a few wells of a 96-well plate and absorption was measured at 562 nm using a plate reader. A value of 0.52 to 0.54 was obtained, indicating a density of 10⁹ bacteria/mL. A dilution of 10⁸ bacteria/mL was then prepared using 0.03% TSB. The samples were sterilized with 70% ethanol for 20 minutes, transferred into a 12-well plate, and rinsed once with PBS. They were then treated with 2 mL of the 10⁸ bacteria/mL solution and incubated for 24 hours. The bacteria solution was removed and the samples were rinsed twice with PBS. They were transferred into 3 mL of PBS and sonicated for 10 minutes to create a first dilution (10^-1) then three subsequent dilutions (10^-2, 10^-3, and 10^-4) were created. Following this, 0.1 mL of each of the 10^-3 and 10^-4 dilutions were plated and incubated for 18 hours. The number of bacterial colonies formed on each sample was counted and using these values, the number of bacteria/mL was found. All experiments were conducted in triplicate and differences between means were determined using analysis of variance followed by Student's t-tests.

The particle size of the powders was determined using transmission electron microscopy (TEM) and the sample composition was established using x-ray diffraction (XRD). BET was used to measure the porosity of the powders. After coating, samples were characterized using scanning electron microscopy (SEM), atomic force microscopy (AFM) and contact angle analysis to confirm surface roughness and wettability of the coatings, all according to standard procedures.

Results and Discussion: Particle size and distribution was determined using TEM as shown in figure 1. After the samples were coated with the synthesized HA powders, SEM was used for surface characterization as shown in figure 2. This helped establish the presence of nano-crytalline features on the surface. Contact angle measurements were made using distilled water to test the hydrophobicity/ hydrophilicity of the surface, as shown in figure 3. The sample coated with HA by EPD showed complete wetting as compared to the hydrophobic behavior displayed by plain Ti. Figure 4 shows the result of confocal and fluorescence microscopy performed on the samples after seeding them with the 3 strains of bacteria and macrophages and fixing them. There was a decreased proliferation of these cells on the surface with reducing size of HA nanoparticles. Also, the change in the surface topography of the material affected the adhesion and proliferation of the macrophages leading to reduced activation of macrophages with increased nanometer roughness. The level of TNF-α, IL-1, IL-6 and nitrite released decreased with increasing hydrophilicity of surface but increased overall with time^1,2. Bacterial activity decreased with smaller particle size of hydroxyapatite.

Figure 1: TEM image of the 110 nm HA powder. Scale bar - 100 nm.

Figure 2: SEM image of the 110 nm HA powder coated onto the titanium mesh using EPD. Scale bar- 1µm

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B

                                  

Figure 3: A) Contact angle image of the surface having 110 nm HA coated onto Ti using EPD. Image shows complete wetting of the surface establishing its highly hydrophilic nature. B) Contact angle image for the Plain Ti surface. Contact angle = 63.54ş

Figure 4: Confocal and fluorescent  microscopy image showing bacterial and macrophage density on Plain Ti, Plasma Sprayed HA on Ti and Ti coated with 100 nm HA by EPD respectively. (a), (b), (c) represents S.aureus ; (d),(e), (f) represents P.aureginosa ; (g), (h), (i) represents Ampicillin resistant E.coli. The scale bar for these images is 20 µm. (j),(k),(l) represents macrophage density on day 1 of culture. The scale bar for these images is 60 µm.

Conclusions:  Controlling the size of the nanophase hydroxyapatite, the coating procedure and its parameters can help influence the fate of macrophage attachment, activation and functionality at the implant surface by reducing levels of TNF-α, IL-1, IL-6 and nitrite released with increased nanoscale topography. Bacterial activity is influenced by the size of the particles, the roughness, the hydrophobicity/hydrophilicity and the coating procedure used.

Acknowledgements: The authors would like to thank Northeastern University for funding.

References: 1.) Control of macrophage responses on hydrophobic and hydrophilic carbon nanostructures, Y. Chun et al, Carbon 49 (2011) 2092–2103. 2.) Reduced responses of macrophages on nanometer surface features of altered alumina crystalline phases, D. Khang et al, Acta Biomaterialia 6 (2010) 3864–3872.