1.

Introduction

Healthcare associated infection (HAI) affects approximately 10% of patients admitted to the hospital, and is responsible for over 5000 deaths in the UK annually (Improving Patient Care by Reducing the Risk of Hospital Acquired Infection: A Progress Report, National Audit Office 2004). HAI can be caused by airborne and environmental bacterial contamination which can be transmitted to surgical site wounds either directly from the environment or indirectly via vectors such as healthcare workers’ hands.^1,2 A 405-nm high-intensity narrow spectrum (HINS) light has antimicrobial activity against HAI-related bacterial pathogens including methicillin-resistant Staphylococcus aureus³ and, due to the use of visible light wavelengths, this does not pose the same health concerns as those associated with using ultraviolet light. This 405-nm light technology has already been used to develop an environmental decontamination system for the disinfection of air and environmental surfaces, which has been successfully clinically evaluated within isolation rooms at Glasgow Royal Infirmary.⁴

Infection rates following orthopedic arthroplasty surgery are as high as 4%, while the infection rates are even higher after revision surgery,⁵ and the environment and surgical devices have been highlighted as sources of bacterial contamination.⁶

The bactericidal properties of 405-nm light suggest that this may aid in reducing the incidence of infections that arise from environmental contamination during arthroplasty surgery. The toxic effect of 405-nm light on bacterial cells is not replicated to the same extent in mammalian cells, and this has been successfully demonstrated in previous studies.^7,8

If 405-nm light technology was to be promoted for localized disinfection during arthroplasty surgery, the bone would likely be exposed to 405-nm light during the course of surgery and any inhibitory effects on the function of osteoblasts may affect the integration of the orthopedic implant into the bone postsurgery. It is therefore essential to consider the duration of an orthopedic surgical procedure and establish the effects of 405-nm light on osteoblast function during this potential exposure. Discussion with surgeons has revealed that the duration of routine arthroplasty surgeries was between 1 and 2 h, although a complex revision may take up to 4 h. Previous studies on fibroblasts for wound healing have shown that 405-nm light had no significant effect on cell viability when exposed to a dose of $18\mathrm{J}/{\mathrm{cm}}^2$ ($5\mathrm{mW}/{\mathrm{cm}}^2$ for 1 h), while a substantial decline in cell viability was observed at a dose of $54\mathrm{J}/{\mathrm{cm}}^2$ ($15\mathrm{mW}/{\mathrm{cm}}^2$ for 1 h).⁷ It has also been found that exposing osteoblasts to 405-nm light with a dose of $54\mathrm{J}/{\mathrm{cm}}^2$ ($5\mathrm{mW}/{\mathrm{cm}}^2$ for 3 h) had a detrimental effect on cell viability.⁹ Hence, the aim of this study was to establish whether the adverse effects of 405-nm light seen on mammalian cells were dose dependent, to determine a damage threshold of exposure of osteoblasts to 405-nm light, and to find out whether doses below the damage threshold can exert a bactericidal effect on clinically relevant pathogenic bacteria. Demonstration of these key parameters would be an essential step toward assessing whether this novel technology could be developed and applied for localized decontamination of the patient environment during arthroplasty surgeries.

2.

Methods and Materials

3.

Results and Discussion

Data in Fig. 1 show that exposure of cells to $18\mathrm{J}/{\mathrm{cm}}^2$ 405-nm light using different irradiance/exposure period regimes caused no significant difference in the crystal violet staining or protein concentration between the control and the treated samples after 48- and 72-h posttreatment periods. In contrast, a significant difference was evident between the control and the 405 nm treated samples exposed to higher doses of $54\mathrm{J}/{\mathrm{cm}}^2$ irrespective of how the dose was administered. These results confirm that the effect of 405-nm light on mammalian cells is dose dependent.

Fig. 1

Crystal violet staining and protein content of osteoblasts exposed to different doses of 405-nm light. (a) Crystal violet absorbance (% control), (b) protein concentration (% control) of rat osteoblasts exposed to 405-nm light at a low dose of $18\mathrm{J}/{\mathrm{cm}}^2$ and (c) crystal violet absorbance (% control), (d) protein concentration (% control) of rat osteoblasts exposed to a high dose of $54\mathrm{J}/{\mathrm{cm}}^2$. Doses were achieved using different irradiance/exposure time regimes, and were followed by either 48 or 72-h posttreatment periods. Results are means of $n=8\pm \mathrm{SEM}$. *Statistically different using unpaired Student’s t-test comparing control and light treated samples, $p<0.05$.

Data in Fig. 2 show that cells exposed to doses of 18, 27, and $36\mathrm{J}/{\mathrm{cm}}^2$ ($5\mathrm{mW}/{\mathrm{cm}}^2$ for 60, 90, and 120 min, respectively) exhibited no significant effect on the MTT reduction, the ALP activity or the cell proliferation rate as measured by the BrdU method. In contrast, cells exposed to doses above $36\mathrm{J}/{\mathrm{cm}}^2$ ($5\mathrm{mW}/{\mathrm{cm}}^2$ for more than 120 min) showed a significant decrease in all the parameters of cell function.

Fig. 2

MTT reduction, ALP activity, and cell proliferation in osteoblasts exposed to different doses of 405-nm light. (a) and (b) MTT reduction ($\text{mean absorbance}\pm \mathrm{SEM}$, $n=8$), (c) and (d) ALP activity ($\mathrm{IU}/\mathrm{L}\pm \mathrm{SEM}$, $n=8$), and (e) and (f) BrdU absorbance ($\text{mean}\pm \mathrm{SEM}$, $n=8$) of rat osteoblasts exposed to 405-nm light doses of 18, 27, 36, and $45\mathrm{J}/{\mathrm{cm}}^2$ ($5\mathrm{mW}/{\mathrm{cm}}^2$ for 60, 90, 120, and 150 min, respectively) followed by 48- (left) and 72-h (right) posttreatment period. *Statistically different, using unpaired Student’s t-test comparing control and light treated samples, $p<0.05$.

AO and PI dyes were used in this study to differentiate live cells, apoptotic and necrotic cells and study the viability of osteoblasts after 48- and 72-h postlight exposure periods. All live nucleated cells fluoresce green, early apoptotic cells fluoresce bright green, late apoptotic cells fluoresce orange, and all dead nucleated cells fluoresce red. After both 48- and 72-h culture postexposure to light, more apoptotic and dead cells were observed following 150 min ($45\mathrm{J}/{\mathrm{cm}}^2$) exposure compared to 120 min ($36\mathrm{J}/{\mathrm{cm}}^2$) exposure to $5\mathrm{mW}/{\mathrm{cm}}^2$ 405-nm light (Fig. 3), confirming the findings with MTT. Staining of the actin with phalloidin-FITC showed that after exposure to $45\mathrm{J}/{\mathrm{cm}}^2$ light, the actin formed a ring around the cell membrane, and more cells rearranged themselves into a circular shape, when compared with the samples exposed to $36\mathrm{J}/{\mathrm{cm}}^2$ for both 48- and 72-h posttreatment period (Fig. 4). Accumulation of actin around the outer-cell perimeter is indicative of membranes being strengthened to prevent leakage of the cell contents and is a sign of apoptosis. At 48 h in culture postexposure to light, the cells show early apoptosis [Fig. 3(b)], but this damage appears to be repaired at 72 h in culture postexposure to light [Fig. 3(f)]. Exposure to 405-nm light may indeed cause sublethal damage to the cells, and further studies such as assessment of deoxyribose nucleic acid (DNA) integrity will be required.

Fig. 3

The cell viability of osteoblast cells after exposure to $5\mathrm{mW}/{\mathrm{cm}}^2$ of 405-nm light. [(a) and (c) Control ($0\mathrm{J}/{\mathrm{cm}}^2$ for 120 and 150 min, respectively for a 48-h postincubation period), (e) and (g) control ($0\mathrm{J}/{\mathrm{cm}}^2$ for 120 and 150 min, respectively for a 72-h postincubation period). (b) and (d) Exposure for 120 min ($36\mathrm{J}/{\mathrm{cm}}^2$) and 150 min ($45\mathrm{J}/{\mathrm{cm}}^2$), respectively, for a 48-h posttreatment period and (f) and (h) exposure for 120 min ($36\mathrm{J}/{\mathrm{cm}}^2$) and 150 min ($45\mathrm{J}/{\mathrm{cm}}^2$), respectively, for a 72-h posttreatment period).] Acridine orange stains live cells green, early and late apoptotic cells bright green and orange, respectively, and propidium iodide stains dead cells red.

Fig. 4

The cell morphology of osteoblast cells after exposure to $5\mathrm{mW}/{\mathrm{cm}}^2$ of 405-nm light. [(a) and (c) Control ($0\mathrm{J}/{\mathrm{cm}}^2$ for 120 and 150 min, respectively, for a 48-h postincubation period), (e) and (g) control ($0\mathrm{J}/{\mathrm{cm}}^2$ for 120 and 150 min, respectively, for a 72-h postincubation period). (b) and (d) exposure for 120 min ($36\mathrm{J}/{\mathrm{cm}}^2$) and 150 min ($45\mathrm{J}/{\mathrm{cm}}^2$), respectively, for a 48-h posttreatment period and (f) and (h) exposure for 120 min ($36\mathrm{J}/{\mathrm{cm}}^2$) and 150 min ($45\mathrm{J}/{\mathrm{cm}}^2$), respectively, for a 72-h posttreatment period).] Phalloidin-FITC stains the cytoskeleton green and DAPI stains the nucleus blue.

The mammalian cell studies suggest that cell exposures to 405-nm HINS light for up to a dose of $36\mathrm{J}/{\mathrm{cm}}^2$ cause no observable effects on the cell viability, function, proliferation rate, and morphology using the techniques employed in this study. When investigating the comparative effects of 405-nm light exposure on bacterial cells, results demonstrate that doses up to $36\mathrm{J}/{\mathrm{cm}}^2$, which were found to have no impact on the mammalian cells, induce significant bactericidal effects. Results in Fig. 5 highlight the inactivation kinetics of a range of Gram-positive (S. aureus, S. epidermidis) and Gram-negative (P. aeruginosa, A. baumannii, E. coli, K. pneumoniae) bacteria. The susceptibility of the organisms varied with A. baumannii proving most susceptible with $>98\%$ kill achieved after exposure to $4.5\mathrm{J}/{\mathrm{cm}}^2$ ($p<0.001$). Significant inactivation of all organisms was achieved by exposure to $18\mathrm{J}/{\mathrm{cm}}^2$ and between 99.5% to 100% inactivation of all species was shown after application of $36\mathrm{J}/{\mathrm{cm}}^2$. A previously published study reported the antibacterial effects of 405-nm light against these clinically relevant organisms, demonstrating their susceptibility at population densities of up to $9\text{-}{\log }_{10}$ CFU/ml in liquid suspension;³ however, the present study uses light doses and surface-seeded bacterial contaminants at population densities ($\sim {10}^2$ CFU/plate) that are more relevant to practical clinical environmental decontamination applications,⁴ as has been demonstrated in a previous work.⁹ The present study confirms, by direct comparison, the selective detrimental effect that light of this wavelength can have on infection-causing organisms in comparison to osteoblasts.

Fig. 5

Survival of a range of surface seeded clinically relevant bacterial pathogens exposed to 405-nm HINS light at doses ranging from 4.5 to $36\mathrm{J}/{\mathrm{cm}}^2$. Results are means of $n=4\pm \mathrm{SEM}$. *Statistically different, using unpaired Student’s t-test comparing control and light treated samples, $p<0.05$.

The antibacterial effect of 405-nm light is not thought to be mediated through direct DNA crosslinking, unlike that of UV light.¹² Instead, the 405 nm violet-blue light inactivation effect is considered to be caused by the excitation of intracellular photosensitive porphyrin molecules, which results in the production of reactive oxygen species (ROS), inducing oxidative damage and, consequently, cell death.^3,13–15 Indeed, previous studies have detected endogenous porphyrins within S. aureus,¹⁵ P. aeruginosa,¹⁴ and A. baumannii,¹⁶ and implicated these violet-blue light sensitive molecules in the inactivation mechanism. In addition, a study by Lipovsky et al.¹⁵ compared the sensitivity of two strains of S. aureus and found that the more light-sensitive strain contained 10-fold more endogenous porphyrins than the more resilient strain. Further evidence of the involvement of porphyrins in the inactivation process was provided by Nitzan et al., who demonstrated that bacterial susceptibility to violet-blue light inactivation can be increased when exogenous $\delta$-aminolevulinic acid (ALA), the precursor of the porphyrin biosynthesis pathway, is added in order to amplify the levels of endogenous porphyrins within the bacterial cells.^15–18 Since ROS are considered to cause widespread oxidative damage to bacterial cells and the mechanism of action is not site specific, as is the case with many antibiotics, this explains not only the broad spectrum antimicrobial efficacy of 405-nm light but also why highly antibiotic resistant species such as Acinetobacter baumanii show no greater resistance to 405-nm light than nonantibiotic resistant bacteria. Bacterial cells have also been shown to have much greater sensitivity than mammalian cells to violet-blue light in the region of 405 nm.^9,14 Low levels of antioxidant defence enzymes in certain bacteria have been implicated as a possible reason for this,¹⁹ and as such are more susceptible to ROS mediated oxidative damage. In contrast, most mammalian cells are well equipped to survive in high oxygen conditions, and contain ample cytoprotective mechanisms such as superoxide dismutase, catalase, and glutathione peroxidise.¹⁹

These findings thus highlight the potential for development of 405-nm light for inactivation of bacterial contaminants around surgical sites. Exposure to 405-nm light has also been proven to cause no adverse effects on wound healing,⁷ thus providing further evidence for potential clinical application of this disinfection technology.

Importantly, the dose-dependent effects of 405-nm light on osteoblast cells suggest that the dose of $36\mathrm{J}/{\mathrm{cm}}^2$, which may be achieved by applying different irradiances for varying time periods (e.g., $10\mathrm{mW}/{\mathrm{cm}}^2$ for 1 h, $5\mathrm{mW}/{\mathrm{cm}}^2$ for 2 h, $3.3\mathrm{mW}/{\mathrm{cm}}^2$ for 3 h, and $2.5\mathrm{mW}/{\mathrm{cm}}^2$ for 4 h), does not cause detectable damage to mammalian cells, regardless of how the dose is applied. These findings are significant, and should provide a basis for the development of light sources which could be applied practically within operating theaters at levels appropriate for the typical operating times relevant to arthroplasty surgery.

Further safety advantages of light of this wavelength relate to the photon energy of 405-nm light. The depth of penetration of visible light into tissue increases with wavelength²⁰ and the penetrability of 405-nm light, which although greater than that of UV light, is low and will not penetrate deeply into bone; particularly when compared to light of a longer wavelength, e.g., 690-nm laser light.²¹ Also, the 405-nm intensities suggested for use during surgical exposures would be low; therefore, theater personnel are unlikely to require protection from these visible violet-blue light wavelengths. This is unlike UV light which, due to the high photon energy and associated detrimental health effects, is normally only used in the absence of people. There have been limited attempts to utilize UV light during surgical procedures but these have required operating staff to wear protective clothing during exposure.^22,23

4.

Summary

A 405-nm HINS light has proven to have strong antimicrobial effects against a variety of medically significant bacteria at a dose level of $36\mathrm{J}/{\mathrm{cm}}^2$. Conversely, mammalian cell studies found that the osteoblasts appear healthy when exposed to doses up to $36\mathrm{J}/{\mathrm{cm}}^2$; at higher doses, there is a negative impact on cell viability, function, and proliferation rate. The results obtained from the application of low dose and high dose light using a range of irradiance/exposure time regimes have demonstrated that the effects of 405-nm light on osteoblast cells are dose dependent. This study has established a dose-dependent level at which there is a differential sensitivity to the effects of 405-nm light between osteoblasts and bacteria; exposure of mammalian cells up to a dose of $36\mathrm{J}/{\mathrm{cm}}^2$ does not cause any observable effect on osteoblast viability. At this dose, the 405-nm light is still bactericidal indicating that there is potential to develop the technology for decontamination of the environment around patients during arthroplasty surgery.