(704c) Selenium Nanocluster Coatings for Anti-Cancer, Anti-Bacterial Orthopedic Applications

Abstract - Two common causes leading to bone implant failure are: an insufficient bonding between the implant and surrounding bone and infection. Patients with implant failure often undergo revision surgeries which are costly and painful. Moreover, for patients who receive orthopedic implants after cancerous tissue is removed, it would be beneficial to implant an anti-cancer material that can impede the return of cancerous tissue growth that may develop from cancer cells not removed during surgery. Therefore, the objective of this study was to create a coating material that can: (i) promote healthy, normal bone growth, (ii) inhibit bacterial attachment and (iii) impede cancer growth. To achieve that objective, conventional orthopedic implant materials (such as titanium (Ti), stainless steel and ultra high molecular weight polyethylene) were coated with selenium (Se) nanoclusters. Different coating densities were achieved by varying Se concentration in the reaction mixture. For the first time, Se nanocluster coatings were shown to enhance healthy osteoblast (bone-forming cell) and inhibit cancerous osteoblast proliferation in either separate-culture experiments or co-culture experiments. S. epidermidis (one of the leading bacteria that infect implants) functions were inhibited on materials coated with Se-nanoclusters compared to uncoated materials. Therefore, a new orthopedic implant coating material is introduced here that may be ideal for promoting bone growth and inhibiting cancer and bacteria growth.

I. INTRODUCTION

 Cancer can return to an implant site after surgical resection and implant insertion in bone cancer patients due to the fact that cancer cells are not always completely removed. However, currently used materials for bone implants such as Ti, Ti alloys, stainless steel or ultra high molecular weight polyethylene do not have any mechanisms to prevent cancer reoccurrence. Therefore, the objective of this study was to create an implant material which can inhibit cancer growth at the implant surface but still promote healthy bone growth. For this dual purpose, Ti was coated with nanoclusters of Se (which is an anti-cancer element [1,2]). For the first time, this composite material has been shown to improve healthy osteoblast (bone-forming cell) functions and inhibit cancerous osteoblast  functions in co-culture experiments.

II. MATERIALS AND METHODS

Clean Ti substrates (Alfa Aesar) were for Se nanocluster coatings. The substrates were placed in a 4:1 molar mixture of glutathione and sodium selenite solutions. Glutathione reduces sodium selenite to form seleno-diglutathione which further disintegrates as the pH is increased above 7 releasing elemental Se into solution. Various concentrations of the reactants were used in the reaction mixture to achieve variable amounts of Se nanocluster density on the Ti surfaces. The substrates were exposed to UV light for 24 hrs on each side to sterilize them. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to characterize the substrates. Since previous results [3] showed increased healthy osteoblast density and decreased cancerous osteblast density with increasing Se coating density, in this study both types of cells were co-cultured on either uncoated Ti or Ti coated with the highest Se density. Healthy primary osteoblasts (HCO, ScienCell Research Laboratories) and cancerous osteoblasts (CLR-2837, ATCC) were pre-stained with fluorescent dyes Vybrant DiO and Vybrant DiD (Molecular Probes), respectively before seeding. Cells were seeded at 750 cells/cm² of each types in media consisted of 50% Dulbecco's Modified Eagle Media (DMEM) (supplemented with 10% fetal bovine serum (Hyclone) and 1% Penicillin / Streptomycin (Hyclone)) and 50% osteoblast medium (ObM, ScienCell Research Laboratories) in an incubator under standard cell culture conditions (37^oC, 5% CO₂, 95% humidified air).  Media was exchanged and cells were rinsed with PBS and observed under fluorescence microscopes at 16, 28, 40, 52 and 64 hrs. Cells were counted under fluorescence microscopy in five random fields and were averaged for each substrate.

III. RESULTS

SEM images demonstrated that Se nanoclusters were clearly present on the substrates when coated at low, medium and high dose Se concentrations (Fig. 1).

Fig. 1. SEM images of uncoated Ti (A) and Ti coated with low (B), medium (C) and high (D) doses of Se. Bars = 500nm.

The substrates will be referred to as uTi, Low-nSe-Ti, Medium-nSe-Ti and High-nSe-Ti for uncoated Ti, and low, medium and high Se nanocluster density coated Ti, respectively. AFM analysis (Fig. 2) showed increasing nano-scale surface roughness of the composites with increased Se density.

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Fig. 2. Normalized RMS of the substrates (normalized to unit scanned area). Data = mean ± SEM; N=3; * p<0.01 compared to uTi; ** p<0.01 compared to Low-nSe-Ti; # p<0.01 compared to Medium-nSe-Ti. AFM scan size was 5µm x 5µm.

When co-cultured on the uncoated Ti substrates, cancer cell density increased with time while healthy cell density did not change significantly among different time points (Fig. 3).

Fig. 3. Increased cancerous osteoblast density after 53 and 65 hrs of co-culturing on uTi. Healthy osteoblast density showed no significant change. Data = mean ± SEM; N=3.

In a contrast, significantly increased healthy cell densities after 53 and 64 hrs were observed when co-culturing on High-nSe-Ti substrates. Importantly, there was no significant change in cancerous cell densities among different time points (Fig. 4).

Fig. 4. Increased healthy osteoblast density after 53 and 65 hrs of co-culturing on High-nSe-Ti. Cancerous osteoblasts did not show any significant growth in density. Data = mean ± SEM; N=3.

IV. DICUSSION

The co-culture results on uncoated Ti showed an increasing trend in cancerous osteoblast density. This trend was expected since the uncoated substrates do not posses any properties to stop cancer growth. However, healthy osteoblast densities did not significantly change over time on uncoated Ti. Although this needs more investigation, this could be due to the competition for nutrients and implant space occupied by the cancerous cells that inhibited the proliferation of healthy osteoblasts.

On the other hand, the results from co-culturing cells on High-nSe-Ti (Fig. 4) showed an opposite trend: healthy cell proliferation was enhanced after 53 and 64 hrs while the proliferation of cancerous cell was inhibited during those time periods. More in-depth studies are needed to understand the role of Se nanoclusters in these cancerous cell-inhibitory and healthy cell-promoting effects. However, studies have shown increased healthy osteoblast functions on nano-structured surfaces [4,5]. Therefore, it is hypothesized that nano-structures on Ti surfaces brought about by the presence of Se nanoclusters are a factor that promoted healthy osteoblast proliferation.

V. CONCLUSIONS

This study showed that Se nanocluster coatings might be an ideal candidate for anti-cancer applications where one not only needs to inhibit cancerous growth but also promotes healthy bone growth.

ACKNOWLEDGEMENT

The authors thank Anthony W. McCormick for help with AFM and the Hermann Foundation for funding.

REFERENCES

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[3] P.A. Tran, L. Sarin, R.H. Hurt, T.J. Webster. Titanium surfaces with adherent selenium nanoclusters as a novel anti-cancer orthopedic material. J Biomed Mater Res A. 2009 in press.

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[5]  K.L. Elias, R.L. Price, T.J. Webster. Enhanced functions of osteoblasts on nanometer diameter carbon fibers. Biomaterials 2002;23(15):3279-87.