Effect of topical Klox BioPhotonic System on cutaneous incisional wound healing in dogs: a prospective blinded controlled clinical trial

Introduction Injuries that result in full-thickness skin loss are commonly observed in dogs and the management of these wounds is often laborious and frustrating as not infrequently, secondary complications occur resulting in dehiscence, ulceration or secondary infection. Klox Biophotonic System (KBS) is an innovative treatment that has proven to be very well tolerated and efficacious in the treatment of wounds in humans1. Clinical studies in dogs have also demonstrated an excellent safety profile and efficacy in the treatment of pathologies such as pyoderma and otitis2-3. The aim of this study was to investigate the effects of the KBS on cutaneous incisional wounds to understand, through a histological and immunohistochemical study of treated and control areas, its effects on tissue regeneration and potential mechanism of action. The hypothesis is that KBS can regulate the expression of key biological mediators involved in tissue repair and regeneration and through this improve the quality of the skin healing process. Materials and Methods Healthy client owned dogs undergoing orthopedic surgery at the Veterinary University Teaching Hospital (OVUD) were prospectively recruited. Owners provided an informed consent for the procedure, and the protocol was reviewed and accepted by the local ethical committee. Dogs either remained in the hospital or were discharged to the owners’ care as appropriate, and according to OVUD standard protocols. All non-hospitalized patients met the postoperative instructions given in relation to the type of intervention they had. All subjects had Elizabethan collars applied for 18 days. Inclusion criteria considered dogs undergoing surgery for non-traumatic orthopedic diseases, in general good health and classified as ASA I (ASA physical status classification system), based on physical examination and laboratory blood tests (at Day 0). Exclusion criteria were the presence of skin changes, or other dermatological/metabolic diseases that could confound data interpretation (e.g. immune-mediated disease); oral steroid therapy in the previous 4 weeks; deposit steroid therapy within the previous 8 weeks; dogs receiving photosensitizing molecules. On the first day after orthopedic surgery (T0) and every 3 days until Day 13 (T4), 50% of the length of the surgical wound was cleaned with sterile saline solution and treated with the KBS, while 50% was just cleaned with sterile saline solution (control). The parts of wound treated with KBS or control were randomly assigned (the proximal half or the distal half). In the treated wound, a 2-mm layer of photoconverter gel (KBS) was spread and illuminated with a Light Emitting Diode (LED) lamp at approximately 5 cm distance for 2 minutes. After treatment, any residues of gel were cleaned/removed using gauzes and sterile saline solution. This procedure was carried out 5 times until the removal of stitches on Day 13. The surgical procedure, including the skin suture, was performed by the same orthopedic surgeon. Simple interrupting monofilament not absorbable suture was used to close the wound. At the end of treatment (Day 13) stitches were removed and 2 small biopsies (2 mm in diameter and 3-5 mm deep) were obtained in the median portions of the treated and control parts of the wound. No sutures were required, and the patients continued their follow up program according to the type of intervention they were submitted to. The evaluation protocol consisted of: a visual clinical scale (ASEPSIS scale)4 at T0, T1, T2 T3 and T4; histologic score at 13 days after surgery (see table 1 for scoring system); immunohistochemistry analysis at 13 days after surgery to investigate expression of key cytokines and proteins involved in the wound healing process (TGF, TNF, FVIII, FGF, EGF, Decorin, Collagen III, Ki67). Clinical, histological and immunohistochemical evaluations were blindly performed. Statistical analysis was carried out on the data comparing treatment to control in a paired t-test and Wilcoxon signed rank test. The characteristics of data distribution have been previously evaluated using skeweness and kurtosis. Results Ten dogs of a variety of ages (from 1 y to 10 yrs) and breeds were prospectively recruited as they underwent orthopedic surgeries (5 TPLO, 3 limb alignments, 2 FHO). Ten incisional wounds and 20 biopsy samples (10 from treated part and 10 from control part) were considered. No patient showed any adverse reaction to the treatment. For a valid paired t-test the data passed tests for both skewness and kurtosis and for a valid Wilcoxon test the data passed the test for skewness. Visual clinical assessment of the skin wounds revealed a better wound healing process at the treated area, with reduced scarring and minimal inflammation. No statistically significant differences were found. The KBS-treated part had statistically significant lower histology scores (p=0.001) showing better and more complete re-epithelialization, lesser inflammation of the dermal layer, less neo-angiogenesis and the presence of synthesis activities of the connective matrix (Fig.1). Immunohistochemistry results showed, in the treated wound portions, a greater expression of FVIII (p=0,034), EGF (p=0,008), Decorin (p=0,005), Collagen III (p=0,005), Ki67 (p=0,002) and a lower expression of TGF (p<0,16), TNF (p<0,001), FGF (p<0,098). (Fig.2) Discussion/Conclusion Recent studies have shown KBS technology to have an excellent safety profile in dogs and be efficacious in other skin conditions2-3. These results indicate that KBS could also potentially represent a novel wound care technology, which utilizes fluorescence biomodulation to treat wounds by stimulating critical cellular pathways. These findings encourage new research on using this technology to treat wounds with substance loss, which may potentially become an alternative and fill, still today, unmet medical needs.