Original research articleWound healing using plasma modified collagen
Introduction
Plasma techniques have been used in medicine for some time, but have been mainly limited to simple cutting, ablation or coagulation applications [1], [2], [3]. Argon plasma coagulation (APC) treatment offers a pain free, non-contact system to eliminate bleeding from injured tissue. The APC device is effectively a powered monopolar RF electrode which uses the patient as the ground electrode. A recent clinical review (34 patients) showed that using APC to treat pressure ulcers produced positive results and suggested that plasma treatment could reduce inflammatory and infectious complications when compared to traditional treatments in patients with chronic ulcers [4]. APC has also been clinically shown to effectively reduce bleeding and improve the healing of rectal ulcers [5] and results like these have led to the development of an emerging field entitled Plasma Medicine, which offers the potential to treat chronic wounds [6]. Unlike traditional APC techniques which employ high energy plasma discharges to desiccate and coagulate, plasma medicine relies on the application of a low energy, cold plasma discharge to a wound site to induce healing without damaging healthy tissue. Aside from the known coagulation effects, the energetic species within the plasma have also been clinically shown to reduce bacterial load in the wound bed [7]. Studies have also shown that plasma promotes wound healing [8], [9], even on chronic wounds that did not respond to traditional treatments [10]. This has given rise to significant interest in developing a plasma tool to enhance healing of chronic wounds.
Treatment of wounds represent a growing market, with chronic wounds accounting for a disproportionate share of the overall costs, as the wounds are persistent, recurring and require advanced wound therapies. The market is driven primarily by the growth in chronic wounds associated with an ageing population and chronic healthcare conditions such as diabetes. This has driven the development of advanced skin substitutes based on biological materials such as collagen, hyaluronan, or living cells which offer improved healing rates [11], [12]. Despite the availability of these artificial skin approaches, overall patient outcomes are still poor [13], with over 100,000 amputations arising annually in the USA from diabetic foot ulcers alone [14]. Many of these amputations are associated with complications such as infections and poor blood flow. Furthermore, the advanced dressings are complex to manufacture, expensive to purchase and store, difficult to apply over large areas and their removal during redressing procedures often reinjures the wound - undoing some of the healing benefits. They are also prone to infection and many of these chronic wounds are not readily treated with systemic antibiotics due to poor localised blood supply, immune-compromised hosts and other complications. To illustrate this, a meta-analysis of 18 randomized controlled trials evaluating different systemic antibiotics in treating diabetic foot ulcer infections showed a failure rate of 22.7% [15].
Given the global drive to reduce treatment costs, reduce healing times and deliver cost-effective wound management, there is a clear requirement for a low cost advanced dressing and artificial skin type treatment that combines the healing rate of biological dressings with an infection reducing effect. Plasma treatment has been shown to promote wound healing [4], [5], [6], [7], [8], [9] and to kill prokaryotic bacterial cells at doses which do not have any detrimental effect on eukaryotic cells [16].
Furthermore, due to their low discharge energy, non-thermal atmospheric plasma devices are widely known to deposit coatings from liquid precursors with minimal precursor fragmentation [17], [18] and have been shown to be capable of depositing sensitive biomolecules such as enzymes with significant retention of biological activity [19], [20]. Increasing the plasma power leads to increased cross-linking and stability, but at the cost of higher degrees of protein degradation [20]. A plasma based coating derived solely from biomolecules such as collagen would represent a significant step towards a new biological dressing. If such a plasma based treatment was capable of applying a biological coating directly onto a wound, then it could potentially combine the benefits of plasma medicine and those of advanced biological dressings in a single treatment.
To this end, this study seeks to examine if a pure collagen coating can be effectively deposited using a plasma device, to determine if that coating could be applied onto soft tissue using a non-thermal plasma applicator and to examine if the collagen retains biological activity and promotes healing in an in vivo wound healing model. A hyperglycemic animal model of wound healing was chosen as this mimics the compromised healing of diabetic patients.
Section snippets
Materials
Type 1 atellocollagen was isolated using a similar method to that previously described [21]. Briefly, frozen bovine tendons were cut into small pieces and put through a blender with ice. The blended tendon was washed using a buffer solution containing 3.7 mM Na2HPO4, 0.35 Mm KH2PO4 and 0.51 Mm NaCl. The washed tendon was then dissolved in 0.5 M acetic acid, and pepsin added to cleave the telopeptide regions. 0.9 M NaCl was added to the filtered solution to precipitate the collagen. This was
Results
Under the conditions employed in this study, a non-thermal equilibrium plasma was generated within the plasma device. Voltage measurements collected during deposition recorded an applied voltage of 8.2 kV (peak to peak) with an operating frequency of 148 kHz, as shown in Fig. 2. The applied voltage appears as a distorted sinusoidal signal, as reported previously [25]. Temperature sensors placed on the outside of the plasma device and at the exit of the plasma showed that the overall temperature
Discussion
From the data presented above, it can be deduced that exposing a wound to a plasma discharge can induce wound healing. Although the data presented here does not show a statistical difference between the wound closure rates produced by a simple plasma treatment and the untreated control, there are indications that the plasma is effective at promoting wound healing and this is in line with previous studies [5], [6], [7], [8], [9], [10]. Higher levels of average wound closure and angiogenesis were
Conclusions
Spraying a solution of collagen through a non-thermal plasma has been shown to produce a dry, coagulated coating on a surface. The coating was well adhered and did not require the presence of additional polymer layers, cross-linking agents or primer layers which could induce a negative reaction in vivo. Chemical analysis was consistent with the deposition of a protein layer. To confirm the biological viability of the collagen, a number of plasma-collagen coatings were successfully applied onto
Conflict of interest statement
Dr. O'Neill is a shareholder in TheraDep. Both Dr. O'Neill and Mr. Dobbyn are shareholders in PlasMedica Limited and are named inventors on a series of patents associated with the technology outlined in this article.
Dr. Kulkarni and Prof. Pandit have no financial interests or other conflicts of interest to report.
Financial disclosure
Financial support for the conduct of the research and manuscript preparation was provided by PlasMedica Technologies Ltd. and TheraDep Ltd. Employees from both companies were involved in the collection, analysis and interpretation of the in vitro data. All in vivo data was collected, analysed and interpreted by employees of the National University of Ireland, Galway. Employees from both companies and the university were involved in the writing of the report; and in the decision to submit the
Ethical statement
The attached manuscript contains details of an experiment involving living animals. Prior to commencing this research, appropriate ethical approval was sought and received from the governing agency within the University Ethics Committee. All experiments were conducted in accordance with Statutory Instrument No 543 of 2012 (under Directive 2010/63/EU on the Protection of Animals used for Scientific Purposes) and were subject to on site monitoring by the appropriate University staff.
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