Towards Development of Novel Phototherapy Technology for Decontamination and Accelerated Wound Healing

Phototherapy has been successfully applied in a range of healthcare fields for therapeutic purposes including photodisinfection (~400nm) and photobiomdulation (~600-1000nm). It is proposed that this technology has the potential to reduce bacterial numbers and promote healing in contaminated battlefield wounds.

Aim: To develop ex vivo infected wound models for the investigation and translation of novel phototherapy techniques using multiple and simultaneous wavelengths of visible and infra-red light for wound healing and bacterial decontamination.

Year 1

- Review of literature and write up of 1st year report (UoB requirement)

- Establishing microbial cultures of wound related pathogens and commensals (in house, ATCC and DSTL isolates) - planktonic single species cultures

- Development and characterisation of multi-well light array devices (emitting wavelengths in the therapeutic window) using the UoB optical analytical laboratory with a suite of relevant spectrometry equipment to measure the following key properties:

(a) Wavelength
(b) Irradiance
(c) Beam profile
(d) Media / cell culture ware absorption characteristics
(e) Temperature increases in cultures exposed to light irradiation
(f) Variability of LEDs of a single wavelength
(g) Effect of irradiation time on light output characteristics
(h) 'Ageing' studies to determine effect on irradiation parameters

- Determining antimicrobial potential of light irradiation using a range of exposure parameters to determine optimal effects.

This will use well established assays including:

(a) Turbidity measurements
(b) Colony counts
(c) Bacterial viability

This will also include investigation of direct light effects on growth media prior to bacterial inoculation.

- Once optimal antimicrobial parameters have been determined for relevant wound related pathogens potential resistance development to light irradiation will be determined.

Years 2 & 3

- Establishing cell cultures using a range of relevant wound related cell lines including epithelium, connective tissue (fibroblast), bone (osteoclast / osteoblast), and vascular (angiogenesis). In addition key inflammatory cells important in host defence and wound healing will be investigated (e.g. neutrophils)

- Cells will be exposed to a wide variety of irradiation parameters in order to identify optimal conditions to promote cell proliferation and maintenance of cell viability using a range of established assays including:

(a) Automated / manual cell counts
(b) Cell viability assay (methylene blue exclusion assay)
(c) MTT assay (mitochondrial activity)
(d) BrDU assay
(e) Growth factor production (SQ RT PCR & ELISA)

In addition the effect of irradiation on inflammatory response will be determined using:

(a) Activation of NF-kB
(b) Pro-inflammatory gene transcription changes
(c) Pro-inflammatory cytokine production (e.g. IL8)
(d) Reactive oxygen species release (neutrophils)
(e) Chemotaxis (neutrophils)

- Light irradiation will be assessed to determine any detrimental effects on cells investigated i.e. loss of viability and DNA damage.

In addition to the well-established photophysics and biological research areas available within the School of Dentistry, UoB, our collaborator will enable access to a wide range of bacterial and fungal clinical isolates. The student's work placement at Dstl will involve testing relevant pathogens held at Dstl, Porton Down (complimentary to those tested at UoB) using the light array technology developed at UoB, in order to understand the effects of multiple wavelengths, dose and irradiation protocol on antimicrobial efficacy.