Skin Graft Donor Site and Use of Polydeoxyribonucleotide as a Treatment for Skin Regeneration: A Randomized, Controlled, Double-

Abstract: The effect of polydeoxyribonucleotide (PDRN) vs. placebo on the wound healing process in patients undergoing skin grafts during reconstructive surgery was evaluated. In this double-blind, placebo-controlled study, 58 patients of both sexes subjected to skin grafts were studied and randomly allocated in two homogeneous groups treated with standard therapy plus PDRN (3mL ampoule contains PDRN mg. 5.625) or placebo. PDRN or placebo were administered by intramuscular injection once daily for 10 days and by perilesional infiltration at Days 1 and 5, postoperatively. Checks were carried out at the end of surgery and after 5, 10, 14, and 21 days. The primary end point of the trial was the rate of repair of the graft donor site. At the beginning, the two groups were homogeneous in terms of age, sex, and size of the graft donor site. On Day 10, the surface of the reepithelized area was 26.7±6.2cm² (mean± SD) in the PDRN group and 20.7±6.7cm² in the placebo group (pDisclosure: This study was supported by a grant from Mastelli Srl, Sanremo, Italy. P olydeoxyribonucleotide (PDRN) (Placentex Integro, Mastelli Srl-Sanremo, Italy) is a preparation used in therapy as a tissue repair stimulating agent in diseases characterized by a loss of substance, such as chronic wounds^1–3 and burns⁴. It consists of low molecular weight deoxyribonucleic acid (DNA) fractions that can be defined as deoxyribonucleotide linear polymers that are combined by phosphodiester bonds whose monomeric units are the purine and pyrimidine nucleotides. The compound is extracted from the sperm of trout bred for human food purposes and is then purified and sterilized to obtain an over 95-percent pure active principle without pharmacologically active proteins and peptides (Registration Dossier, Ministry of Health). The PDRN trophic effect is attributed to the nucleotides by which it is formed⁵. A link between nucleotides and wound healing had already been established at the beginning of the 1990s^6–9. The stimulating effect of the extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) on cell multiplication, DNA synthesis, and wound healing has also been specified⁶. It has been demonstrated that the nucleic acids of damaged cells, released in the extracellular environment, are rapidly lysated into nucleotides that trigger a repair response by modulating its development⁷. The same nucleotides are able to activate different cell types8 and to promote the repair of the solutions of continuity^8–10. There is evidence that the stimulation of type A2 purinergic receptors accelerates wound healing,^11–14 and it is well documented that PDRN and nucleotides stimulate cell regeneration and cell metabolic activity by acting as A2 receptors agonists^15,16. Nucleotides promote cell regeneration also stimulating the synthesis of new nucleic acids necessary for cell duplication. The preformed nucleotides, in fact, activate the “salvage” metabolic pathways (reuse of nucleotides as such with a considerable energy saving) as an alternative to the traditional metabolic pathways of the ex–novo synthesis^17,18 (nucleotide neosynthesis starting from amino acids with a high-energy consumption). The activation of “salvage” pathways enables a quicker synthesis of nucleic acids with a lower energy consumption¹⁹. In-vitro studies have shown that PDRN can stimulate the growth of human fibroblast primary cultures at concentrations of 20–100mg/mL and that cell proliferation is at least partly mediated by stimulating type A2 purinergic receptors^5,15 and by activating “salvage” pathways. A recent study documented the stimulation effect of PDRN on the osteoblasts in culture, and it also showed that this effect is dramatically reduced by pretreating the cells with ^3,7-dimethyl-1-propargylxanthine (DMPX)²⁰, and A2, a selective receptor antagonist. In one of our previous pilot study²¹, it was observed that the administration of PDRN by intramuscular and perilesional route in patients who underwent skin grafts was associated with a significant increase of the reepithelization rate of the skin graft donor site. The purpose of this randomized, double-blind, placebo-controlled clinical study was to evaluate the activity of PDRN on the wound healing process in patients undergoing skin graft for reconstructive surgery. Patients and Methods Sixty patients of both sexes, with ages ranging from 18 to 90 years, were enrolled in the trial, and they were candidates for skin explant to be used as autologous graft for reconstructive surgery. Other inclusion criteria included five or more years of school and informed consent. Exclusion criteria included participation in other clinical trials still underway or within 30 days from enrollment in the trial; treatment with scarring preparations still underway or followed in the 10 previous days; hypoalbuminemia; alcoholism; nontherapeutic use of substances with a pharmacological action; smoking (>10 cigarettes a day); pregnancy or breast-feeding; therapy with neuroleptic drugs and/or antidepressants; severe hepatic/renal/cardiac failure; severe concomitant diseases; and hypersensitivity to the medicine under study. A dermatome was used to remove the skin graft, and the graft donor site was used as an experimental model to study the effects of PDRN on the wound repair process. An electric dermatome was used (Colibrì Intramatic 4000, 100 Watt) in order to obtain a 0.5mm thick and 4cm wide donor skin flap. Length was the only variable parameter that depended upon the type of reconstructive surgery. The patients enrolled in the trial were randomized into two balanced groups. Investigators and subjects were blinded to the treatment method. Subjects were treated with either PDRN (3mL ampoule containing active principle 5.625mg and excipients [sodium chloride and water for injectable preparations]) or with placebo (3mL ampoule containing normal saline). Preparations were administered as follows: one intramuscular ampoule a day for 10 days plus one ampoule administered by perilesional infiltration on Days 1 and 5 of the trial. All the patients were treated for the same period of time with the standard therapy envisaged by the Department (antibiotic prophylaxis and protection of the injured area by means of topic application of gauzes containing emollients). The trial lasted 21 days. Examinations were carried out immediately before (T0) and after the explant surgery (T1), then on Days 5 (T2), 10 (T3), 14 (T4), and 21 (T5) from surgery. The primary parameter of evaluation (primary end-point) was the surface of reepithelized area at each control expressed in mm². It was evaluated by applying transparent graph film on the graft donor sites and by calculating, on the basis of the size of the the sides (side x side) since the lesions were approximately rectangular, the residual disepithelized area and subtracting possible already repaired areas. The difference between the value of the initial graft donor site area and the one recorded at each control has been considered as the expression of the repaired area. The percentage of repair was calculated with the following formula: (repaired area at each control/disepithelized area at T1) x100. The signs and symptoms of pain, burning, itching, and perilesional erythema resulting from the graft donor site were considered as secondary parameters of evaluation. Symptoms were quantified by a 10cm visual analogue scale (VAS) with 0 designated as the absence the symptom and 10 the worst imaginable symptom. The VAS was chosen because it is easily administered and is well accepted by patients^22,23. The objective and subjective tolerability of the used medicines was evaluated at each control. The trial was approved by the Ethics Committee of the University of Siena, and it was carried out in compliance with the Declaration of Helsinki and with Good Clinical Practices (G.C.P.s.) Statistical Analysis Unless otherwise specified, the numerical values are indicated as a mean and relevant standard deviation. The inferential analysis was carried out using the variance analysis as a model and followed by the Tukey-Kramer’s Multiple Comparisons test. The Fisher exact test was carried out to compare the recovered cases on Day 14. The minimum value for statistical significance has been set for p2 in the patients treated with PDRN and 30.5±9.7cm² in the patients treated with placebo (p ns). Also medium length of graft donor sites resulted statistically similar in the two groups: 7.6±2.4cm in patients treated with placebo and 8.2±1.8cm in patients treated with PDRN (p ns). Table 1 shows the data concerning the area of the repaired surface that was measured at the controls following surgery. In the patients of the PDRN group, the repair process proved to be clinically quicker, with a statistically significant difference (p1, anal fissures², explanted skin³, burns⁴, and in the reepithelization of corneal lesions²⁴. The trial under way has verified the effects of PDRN, administered by parenteral route, on the repair process of the harvest areas in patients who underwent skin grafts. The study of this kind of surgically induced lesion allows to minimize the variability that characterizes random lesions and to consequently reduce the variability of the results produced by the investigation. Data collected indicate that by administering PDRN according to the regimen we proposed, the repair time of the graft donor sites in patients who underwent skin graft is reduced in a clinically appreciable and statistically significant way (on Day 10) and consequent pain is more quickly relieved as well. The same data show that PDRN promotes the physiological repair processes, especially at the initial stages of scarring. Evidence of this evolution, consisting in higher wound healing rate at the initial stages of the phenomenon, has already been documented in our previous trial²¹ as well as in a recent study on the reepithelization of the anterior epithelium of the cornea²⁴ These results are also in accordance with the studies of basic pharmacology^5,15,20. The data of this trial suggest that PDRN is a promising therapy in improving wound repair. This trial has been conducted on a relatively healthy population, which could have reduced the clinical efficacy of the product. Trials with healing-impaired individuals might exhibit a greater difference. The trial under way confirms that PDRN is safe to use, as demonstrated by the absence of adverse effects related to medicine administration. The aim of the study was to evaluate the efficacy of PDRN in wound healing, and this end point has been suggested by the results obtained. Clinical study has been performed with the administration of PDRN or placebo by intramuscular and perilesional route. It is important to underline that for the double administration route it is not possible to distinguish between the systemic and local effect of the drug. Whether it is better to use local infiltration or intramuscular injection alone or the two together would require further study. Acknowledgment This study was carried out between June 2001 and November 2003. Statistical analysis was performed by Dr. G. Saponati, Ispharm, Lucca, Italy.