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The effects of platelet rich plasma on healing of full thickness burns in swine
JBUR 5557 No. of Pages 8
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ScienceDirect journal homepage: www.elsevier.com/locate/burns
The effects of platelet rich plasma on healing of full thickness burns in swine Adam J. Singer a,b, * , Jimmy Toussaint a,b, Won Taek Chung a,b, Steve McClain a,b, Vivek Raut a,b, Lior Rosenberg a,b a b
Department of Emergency Medicine, Stony Brook University, Stony Brook, NY, United States Department of Plastic and Reconstructive Surgery, Ben-Gurion University, Beer-Sheba, Israel
article info
abstract
Article history:
Introduction: Platelet rich plasma (PRP) is rich in growth factors and has been shown to
Accepted 27 April 2018
improve healing in a variety of wounds. We determined the effects of PRP on healing and
Available online xxx
scarring in full thickness porcine burns with and without tangential excision and grafting (TEG).
Keywords: Burns Platelet rich plasma Tangential excision Grafting Reepithelialization Scarring
Methods: Standardized full thickness 5cm by 5 cm burns were created on each of the backs and flanks of 10 anesthetized female pigs (25 kg) using a validated model. The burns were created with a heating device that emits heat at a temperature of 400 C for a period of 30 s. The burns were randomized to one of six treatments: no TEG or PRP, no TEG +PRP, early (day 2) TEG and no PRP, early TEG + PRP, late (day 14) TEG and no PRP, and late TEG+ PRP. Tangential excision was performed down to viable tissue and autografts were 0.2 mm thick. When used, a thin layer of autologous PRP was applied below the graft. All wounds were then treated with a topical antibiotic ointment 3 times weekly for 42 days. Digital images and full thickness biopsies were taken at 9, 11, 14, 18, 21, 28, 35 and 42 days after injury to determine percentage reepithelialization, scar depth, and scar contraction. Tissue sections were stained with H&E and viewed by a dermatopathologist masked to treatment assignment. Results: There was no reduction in platelet and white blood cell concentrations in PRP and blood samples for the first 14days after-full thickness burns. A total of 120 burns were created on 10 animals evenly distributed between the six treatment groups. Burns undergoing early TEG reepithelialized fastest and with the thinnest scars followed by late TEG. Burns that did not undergo TEG had the slowest reepithelialization and greatest amount of scarring. Application of PRP had no additional effects on reepithelialization, scar depth, or scar contraction in any of the treatment groups. Conclusions: Addition of PRP had similar effects on reepithelialization and scarring of full thickness porcine burns as standard topical antibiotic ointment regardless of whether the burns underwent excision or grafting or the timing of excision and grafting. © 2018 Elsevier Ltd and ISBI. All rights reserved.
* Corresponding author at: Department of Emergency Medicine, Stony Brook University, Stony Brook, NY, 11794-8350, United States. E-mail address: [email protected] (A.J. Singer). https://doi.org/10.1016/j.burns.2018.04.021 0305-4179/© 2018 Elsevier Ltd and ISBI. All rights reserved.
Please cite this article in press as: A.J. Singer, et al., The effects of platelet rich plasma on healing of full thickness burns in swine, Burns (2018), https://doi.org/10.1016/j.burns.2018.04.021
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1.
Introduction
Platelet-rich plasma (PRP) is the plasma fraction of autologous blood in which the platelet concentration is considerably higher than in whole blood due to processing and concentration [1]. Activated platelets release key wound healing factors including platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-b), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), and epidermal growth factor [1,2]. By delivering supra-physiological concentrations of a variety of growth factors and cytokines these preparations are used to enhance wound healing in bone and soft tissue injuries. Unfortunately, studies regarding the potential benefits of PRP in both animals and humans with burns are contradictory and inconclusive [3–11]. While superficial burns generally heal within 2 weeks with minimal if any scarring, deeper burns generally take greater than 3 weeks to heal and result in significant scarring unless excised and grafted with a split thickness autologous skin graft [12–14]. As a result, excision and grafting of full thickness burns has become the standard of care. The goals of the current study were twofold. First, to compare the healing of full thickness burns treated with or without autologous PRP in a swine model. Second, to determine whether addition of PRP to early and delayed excision and grafting of full thickness burns improves healing and reduces scarring in a porcine model.
2.
Methods
2.1.
Study design
We performed a prospective, randomized animal experiment. The study was approved by the Institutional Animal Care and Use Committee and was conducted in the institutional division of laboratory and animal research in accordance with national guidelines [15].
2.2.
Animals
In this study we used 10 female domestic pigs weighing approximately 20–25kg. Pigs were chosen for this in vivo experiment because it has been demonstrated that of all animals, pig skin most closely resembles that of the human [16]. Animals were given a standard diet ad lib several days prior to the investigation and were fasted overnight before any procedures.
2.3.
Experimental protocol
2.3.1.
Animal preparation and sedation
We used a previously validated porcine model of full thickness burns that were tangentially excised and grafted with a split thickness autologous skin graft [17]. Animals were sedated with a combination of acepromazine 0.1mg/kg, atropine 0.02mg/kg, ketamine 20mg/kg, and xylazine 2mg/kg by intramuscular injection. The pigs were then intubated endotracheally and maintained under a surgical plane of
anesthesia with isoflurane 1–3% in O2 USP. The hair on the backs and flanks of each pig was clipped.
2.3.2.
Burn creation
While under general anesthesia, twelve 5cm by 5cm burns were created on each pig’s back and flanks using a specialized radiant heating device. The burns were spaced at least 2cm from each other. We used a heating coil heated to 400 C, which emits infra-red/visible light radiation that heats the skin surface. The burn infliction device was placed in direct contact with the animal’s skin for a period of 30s thus creating full thickness burns on the back and flanks of the animal. In each of the ten animals we created three rows of four burns each (Fig. 1).
2.3.3.
Treatment allocation
The burns were then randomized to one of the following six treatment groups, equally distributed between the animals, using a computerized random number allocator: 1. A negative control group that did not receive any excision and grafting or PRP. 2. A control group that was treated with topical PRP two days after burn injury with no additional excision and grafting. 3. A treatment group that was excised and grafted two days (early) after burn injury without PRP. 4. A treatment group that was excised and grafted two days (early) after burn injury with PRP applied topically beneath the graft. 5. A treatment group that was excised and grafted 14 days (late) after burn injury without PRP. 6. A treatment group that was excised and grafted 14days (late) after injury with PRP applied topically beneath the graft. Each animal had an equal number of each treatment group randomly assigned to the different anatomical locations (i.e., 12 burns, 6 treatment groups, 2 of each treatment group per pig). The allocation of treatment groups to various anatomical locations (i.e., cranial or caudal) was balanced to account for any anatomical variation in healing.
2.3.4.
Preparation of autologous PRP
Immediately prior to the application of PRP to the burn wounds, anticoagulated whole blood was drawn from the pigs’ ear veins in three 60 ml sterile syringes, with each 60ml blood comprising of 52 ml whole blood and 8ml anticoagulant citrate dextrose formula A (ACD-A; Arteriocyte Medical Systems, Hopkinton, MA). Thus, PRP was prepared freshly, from blood collected immidiately prior to the application. The blood was then processed by a commercially available 1 device (Magellan , Arteriocyte, Hopkinton, MA) for 15 min to yield 7ml of PRP from each 60 ml batch. The PRP was mixed with a combination of Ca+/thrombin in a ratio of 10:1 forming a gel in sterile 5cm by 5cm molds and transferred to the wounds using a sterile spatula. Approximately 1.5ml of PRP+ calcified thrombin was applied to cover each wound. Hematology analysis was performed for each whole blood and PRP sample prepared at each designated time point to quantify the concentrations of platelets and white blood cells.
Please cite this article in press as: A.J. Singer, et al., The effects of platelet rich plasma on healing of full thickness burns in swine, Burns (2018), https://doi.org/10.1016/j.burns.2018.04.021
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2.4.
3
Tissue biopsies
Full thickness 4mm punch biopsies were taken from all wounds immediately after burn creation and at 9, 11, 14, 18, 21, 28 and 35 days, leaving a perimeter of 5 mm from the burn edge to eliminate any edge effect or reepithelialization arising from adjacent surrounding uninjured skin rather than from epithelial elements deep to the burn. Biopsies at different time points were taken from corresponding areas from the different burns to reduce sampling bias. Full thickness 8mm punch biopsies were also taken on day 42 prior to euthanasia with a pentobarbital-based solution (Fatal Plus, Vortech Pharmaceuticals, Michigan). The biopsies were marked for alignment, bisected, then placed in labeled cassettes and fixed in formalin for 24 h. Processed by alcohol-dehydration, xylene-clearing, and paraffin-embedding, tissue samples were sectioned at 5 mm and stained with hematoxylin and eosin (H&E) and Masson’s trichrome (day 42 biopsies only). Histomorphometric analyses were done by a board certified dermatopathologist, who was blinded to treatment allocation.
2.5.
Fig. 1 – Gross appearance of burns immediately after injury.
Hematology analysis was performed using a HematrueTM hematology analyzer (Heska, Loveland, CO). The HematrueTM provided counts of platelets, and white blood cells, which is important for confirming the ability of the device to consistently concentrate these components, and to determine whether the creation of full thickness burns changes the platelets and white blood cells concentration as a function of time after burn injury.
2.3.5.
Dressings and topical antibiotics
After the allocated treatment was received, the burns were treated with a thin layer of a topical petrolatum based triple antibiotic (neosporin, polysporin, bacitracin) and a nonadherent dressing (Telfa, Kendall Healthcare Products Company, Mansfield, MA) covered with polyurethane film (Tegaderm, 3M Health Care, St. Paul, MN) three times weekly. All wounds were covered with a gauze bandage roll (Sof-Form, Medline Industries, Inc., Mandekein, IL), and an adhesive elastic bandage (Tensoplast, BSN Medical S.A.S., Vibraye, France). The wounds were photographed and dressings were changed three times a week for the remainder of the study, which lasted 42 days. Sedation and anesthesia was performed as above, prior to any dressing changes or treatments.
2.3.6.
Pain management
All animals were treated with a transdermal patch of fentanyl 50mcg/kg every 72h and intramuscular buprenorphine 0.005– 0.02mg/kg every 8–12h as needed.
Outcomes
The following outcomes were measured in all wounds. Reepithelialization was determined using H&E-stained sections using a calibrated ocular micrometer to measure the total length in cross-section and the length of the neoepidermis. The percentage of reepithelialization was calculated by dividing the epithelialized length by the length of the specimen and multiplying by 100. Scar contraction was measured by tracing the perimeter of the discolored (red or purple) scar with no hair present using digital images of the wounds at day 42 with ImageJ software (National Institute of Health, Bethesda, MD). The surface area of the scar was subtracted from the original burn size and divided by the original burns size and multiplied by 100 to calculate percentage contraction. Scar depth was determined at 28, 35, and 42 days after injury using histological sections stained with Masson’s trichrome. An ocular lens with a grid was used to make straight-line measurements from the epidermal–dermal junction to the bottom of the scar. Three measurements were made per bisected half of the sample; one measurement was made at the center of the section, and two more were made 1mm left and right of the center. A total of six measurements were taken per sample and the mean depth of the scar was reported in millimeters. Scar tissue was identified by the presence of thin bundles of collagen oriented mostly in a single horizontal plain on H&E or trichrome staining.
2.6.
Data analysis
All data were reported as the meanstandard deviation (SD). Data analysis was performed using SPSS for Windows 22.0 software (SPPS Inc., Chicago, IL). For parametric data sets, one-way analysis of variance (ANOVA) followed by the Tukey post-hoc test was used to detect statistical significance with p<0.05. For nonparametric data sets, the Kruskal–Wallis test followed by the Dunn’s post-hoc test was used to detect statistical significance with p<0.05. A sample of 20 wounds in
Please cite this article in press as: A.J. Singer, et al., The effects of platelet rich plasma on healing of full thickness burns in swine, Burns (2018), https://doi.org/10.1016/j.burns.2018.04.021
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each group had 90% power to detect a 2mm difference in scar depth among the groups.
3.
Results
We created a total of 120 full thickness burns, 12 on each of the ten animals, evenly distributed between the two sides of the animals and the six treatment groups. There were no wound infections or any systemic adverse events in any of the animals or treatment groups. The outcomes are summarized in Table 1.
3.1.
Wound reepithelialization
At day 9, reepithelialization was approximately 50–60% in the early excision and grafting group, while there was no evidence of any reepithelialization in the delayed excision and conservatively managed groups. The effects of PRP on reepithelialization were similar to those of standard topical antibiotics. The application of PRP had no additional effect on reepithelialization regardless of whether the burns were excised or the timing of excision and grafting. Reepithelialization was also highest in the early excision and grafting groups at days 11, 14 and 18, and the application of PRP had no additional effects beyond those of a topical antibiotic in any of the sub-groups (Table 1 and Fig. 2). By day 21 most burns were completely reepithelialized (Fig. 3).
3.2.
Scar contraction
At day 42, scar contraction was greatest for burns treated conservatively without any excision and grafting (Fig. 4). The effects of PRP on scar contraction were similar to those of topical antibiotics alone. The application of PRP had no additional effects on scar contraction regardless of whether excision and grafting were performed or their timing (Table 1).
3.3.
Scar depth
Scars were most superficial at days 28, 35, and 42 in burns treated with early excision and grafting with no differences noted between those burns treated with or without PRP. The deepest scars were noted at all time points in burns treated conservatively. Scar depth did not differ based on whether PRP was applied or not (Table 1, Fig. 5).
3.4. PRP
Effect of burns on the composition of whole blood and
As shown in Fig. 6, full thickness burns did not reduce platelet and white blood cell concentrations in PRP and blood for the first 14 days after burn.
4.
Discussion
The results of the current study suggest that topically applied PRP performed comparably to topical antibiotics alone and its application had no additional beneficial effects on healing and scarring of full thickness porcine burns treated conservatively without excision and grafting. Similarly, application of PRP below the skin graft after excision of the burn eschar had comparable effects on healing and scarring as topical antibiotics and the addition of PRP after early or delayed excision and grafting of full thickness porcine burns also did not further improve reepithelialization or scarring. Whether or not injection of the PRP directly into or beneath the burns would have led to different results and outcomes is unclear. However, intra-lesional injection would be more clinically challenging, and was not investigated in this study. Our results confirm that early excision and grafting is more effective than delayed excision and grafting, and that delayed excision and grafting is more effective than conservative management of full thickness porcine burns [17]. This is evidenced by the fact that
Table 1 – Outcomes. TEG/ PRP Reepithelialization day 9 (SD), % Reepithelialization day 11 (SD), % Reepithelialization day 14 (SD), % Reepithelialization day 18 (SD), % Reepithelialization day 21 (SD), % Reepithelialization day 28 (SD), % Scar depth day 28 (SD), % Scar depth day 35 (SD), mm Scar depth day 42, (SD), mm Scar contraction day 42 (SD), %
TEG/ +PRP
Early TEG/ PRP
Early TEG/ +PRP
Late TEG/ PRP
Late TEG/ +PRP
P value
0 (0) 2 (7)
0 (0) 4 (13)
50 (50) 72 (44)
61 (46) 60 (48)
0 (0) 2 (6)
0 (0) 0 (0)
<0.001 <0.001
23 (41)
17 (33)
79 (41)
70 (47)
–
–
<0.001
68 (37)
43 (46)
89 (26)
95 (15)
NA
NA
<0.001
100 (0)
87 (33)
100 (0)
100 (0)
100 (0)
100 (0)
0.011
100 (0)
90 (0)
100 (0)
100 (0)
100 (0)
100 (0)
0.035
7.5 (1.4) 4.9 (1.7) 6.3 (1.3) 62 (14)
7.8 (1.6) 4.6 (2.3) 6.3 (1.4) 59 (15)
4.7 (2.8) 3.7 (2.1) 3.9 (2.1) 36 (22)
4.7 (2.7) 3.5 (1.7) 4.4 (1.9) 44 (25)
5.8 (1.4) 4.4 (1.8) 4.7 (1.6) 48 (17)
5.6 (1.6) 4.0 (1.6) 4.8 (1.2) 46 (17)
<0.001 0.133 <0.001 <0.001
TEG: tangential excision and grafting; PRP: platelet rich plasma; NA: not applicable. Biopsies were not taken at this time since the graft was still very fragile.
Please cite this article in press as: A.J. Singer, et al., The effects of platelet rich plasma on healing of full thickness burns in swine, Burns (2018), https://doi.org/10.1016/j.burns.2018.04.021
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Fig. 2 – Appearance of wounds on day 14. Left column (labeled 2) from top to bottom: early TEG+PRP, early TEG PRP, no TEG +PRP. Right column (labeled 3) from top to bottom: late TEG PRP, late TEG+PRP, no TEG PRP.
wound reepithelialization was greatest and fastest after early excision followed by late excision. In contrast, wound reepithelialization was slower and less complete when no excision was performed. Similarly, scar depth and contraction were least after early excision, followed by delayed excision. Despite its theoretical effects on wound healing, it is unclear why PRP did not improve reepithelialization or scarring of burns in this study. Other investigators have studied the effects of PRP on burns both in animals and humans with mixed results. Venter et al. studied the effects of PRP in deep second degree burns, deep second degree burns with diabetes, and third degree burns in rats [3]. PRP was associated with faster wound closure in deep second degree burns in normal and diabetic rats but not in third degree burns. Another small study of 4 horses with burns found that PRP accelerated repair and induced fibrosis [4]. A randomized double blind study of 52 patients with deep dermal or full thickness burns in which the patients served as
5
Fig. 3 – Appearance of wounds on day 21. Left column (labeled 2) from top to bottom: early TEG+PRP, early TEG PRP, no TEG +PRP. Right column (labeled 3) from top to bottom: late TEG PRP, late TEG+PRP, no TEG PRP.
their own control found no differences in graft take rate, reepithelialization, or scar quality [5]. A non-controlled study of 18 patients with second and third degree burns from the Czech Republic demonstrated earlier pain relief, decreased use of analgesics and anti-pruritic agents, earlier hospital discharge, and reduced costs when PRP was added to excision and grafting [6]. Another non-randomized study of 23 patients with deep second or third degree burns found that addition of PRP reduced the time required to recover the viscoelastic properties of the scars [7]. A side by side comparison of split thickness donor sites treated with PRP or a control in a 67-year-old man with third degree burns demonstrated improved epithelialization and angiogenesis with PRP [8]. A review of the role of PRP in burns conducted in 2009 was inconclusive [9]. A more recent systematic review of PRP as an adjunct therapy for acute
Please cite this article in press as: A.J. Singer, et al., The effects of platelet rich plasma on healing of full thickness burns in swine, Burns (2018), https://doi.org/10.1016/j.burns.2018.04.021
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Fig. 4 – Appearance of wounds on day 42. Left column (labeled 2) from top to bottom: early TEG+PRP, early TEG PRP, no TEG +PRP. Right column (labeled 3) from top to bottom: late TEG PRP, late TEG+PRP, no TEG PRP.
wounds and burns also concluded that there is not enough data to make any conclusions regarding the effects of PRP on burns [10]. Variability in PRP products, methods of application, frequency of application, and placement of PRP with relation to the graft further complicates interpretation of the evidence and may help explain the contradictory results [11]. It is possible that altering the treatment regimen by optimizing the number of PRP applications, concentration of platelets in PRP or the dilution factor of the PRP may improve the outcomes.
with and without PRP. Second, we only evaluated a single application of PRP administered 2 days after injury. It is possible that additional applications, different methods of application, or different timing of PRP application would have yielded different results than those reported in our study. Third, our results in a porcine model may not generalize to humans due to differences in anatomy and physiology.
6. 5.
Conclusions
Limitations
Our study has several notable limitations. First, the sample size was small including ten animals and only 20 wounds in each treatment group. Thus, it may have been underpowered to detect significant differences between wounds treated
In conclusion, a single application of PRP two days after injury performed similarly to topical antibiotics alone and its addition did not improve reepithelialization or scarring in full thickness porcine burns whether or not they were excised or grafted, regardless of timing of excision and grafting.
Please cite this article in press as: A.J. Singer, et al., The effects of platelet rich plasma on healing of full thickness burns in swine, Burns (2018), https://doi.org/10.1016/j.burns.2018.04.021
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Fig. 5 – Histological images 42 days after injury demonstrating scar depth. Black dots mark lower limit of purple hypercellular scar. Normal pink dermis noted below some, but not all scars.
Fig. 6 – Changes in platelet and white blood cell (WBC) counts in whole blood and platelet rich plasma (PRP) obtained at various time points before and after injury.
Please cite this article in press as: A.J. Singer, et al., The effects of platelet rich plasma on healing of full thickness burns in swine, Burns (2018), https://doi.org/10.1016/j.burns.2018.04.021
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Acknowledgment The authors would like to acknowledge Yiwei Ma for the contribution in performing hematology analysis on the blood and PRP samples.
Funding This project has been funded in whole or in part with Federal funds from the Department of Health and Human Services; Office of the Assistant Secretary for Preparedness and Response; Biomedical Advanced Research and Development Authority, under Contract No. HHSO100201300020C.
Conflicts of interest None of the authors have any conflict of interest to declare. REFERENCES
[1] Lubkowska A, Dolegowska B, Banfi G. Growth factor content in PRP and their applicability in medicine. J Biol Regul Homeost Agents 2012;26(2 Suppl. 1):3S–22S. [2] Everts PA, Knape JT, Weibrich G, Schonberger JP, Hoffmann J, Overdevest EP, et al. Platelet-rich plasma and platelet gel: a review. J Extra Corpor Technol 2006;38:174–87. [3] Venter NG, Marques RC, dos Santos JS, Monte-Alto-Csota A. Use of platelet-rich plasma in deep second- and third-degree burns. Burns 2016;42:807–14. [4] Maciel FB, DeRossi R, Módolo TJ, Pagliosa RC, Leal CR, Delben AA. Scanning electron microscopy and microbiological evaluation of equine burn wound repair after platelet-rich plasma gel treatment. Burns 2012;38:1058–65. [5] Marck RE, Gardien KL, Stekelenburg CM, Vehmeijer M, Baas D, Tuinebreijer WE, et al. The application of platelet-rich plasma in the treatment of deep dermal burns: a randomized, doubleblind, intra-patient controlled study. Wound Repair Regen 2016;24:712–20.
[6] Prochazka V, Klosova H, Stetinsky J, Gumolec J, Vitkova K, Dvorackova J, et al. Addition of platelet concentrate to dermoepidermal skin graft in deep burn trauma reduces scarring and need for revision surgeries. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2014;158:242–58. tinský J, Bryjová I, Hledík S, Klein L. Objective [7] Klosová H, Ste evaluation of the effect of autologous platelet concentrate on post-operative scarring in deep burns. Burns 2013;39:1263–76. [8] Kakudo N, Kushida S, Minakata T, Suzuki K, Kusumoto K. Platelet-rich plasma promotes epithelialization and angiogenesis in a splitthickness skin graft donor site. Med Mol Morphol 2011;44:233–6. [9] Pallua N, Wolter T, Markowicz M. Platelet-rich plasma in burns. Burns 2010;36:4–8. [10] Picard F, Hersant B, Bosc R, Meningaud JP. Should we use platelet-rich plasma as an adjunct therapy to treat “acute wounds,” “burns,” and “laser therapies”: a review and a proposal of a quality criteria checklist for further studies. Wound Repair Regen 2015;23:163–70. [11] Marck RE, Middelkoop E, Breederveld RS. Considerations on the use of platelet-rich plasma, specifically for burn treatment. J Burn Care Res 2014;35:219–27. [12] Tompkins RG, Burke JF, Schoenfeld DA, Bondoc CC, Quinby Jr. WC, Nehringer GC, et al. Prompt eschar excision: a treatment system contributing to reduced mortality. A statistical evaluation of burn care at the Massachusetts General Hopsital (1974-1984). Ann Surg 1986;204:272–81. [13] Sheridan RL, Tompkins RG, Burke JF. Management of burn wounds with prompt excision and immediate closure. J Int Care Med 1994;9:6–19. [14] Herndon DN, Parks DH. Comparison of serial debridement and autografting and early massive excision with cadaver skin overlay in the treatment of large burns in children. J Trauma 1986;26:149–52. [15] Guide for the care and use of laboratory animals National Academy of Science. Washington, D.C: National Academy Press; 2014. [16] Sullivan TP, Eaglstein WH, Davis SC, Mertz P. The pig as a model for human wound healing. Wound Repair Regen 2001;9:66–76. [17] Singer AJ, Toussaint J, Chung WT, McClain SA, Raut V, Rosenberg L. Early versus delayed excision and grafting of fullthickness burns in a porcine model: a randomized study. Plast Reconstr Surg 2016;137:972e–9e.
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burns xxx (2018) xxx –xxx
ScienceDirect journal homepage: www.elsevier.com/locate/burns
The effects of platelet rich plasma on healing of full thickness burns in swine Adam J. Singer a,b, * , Jimmy Toussaint a,b, Won Taek Chung a,b, Steve McClain a,b, Vivek Raut a,b, Lior Rosenberg a,b a b
Department of Emergency Medicine, Stony Brook University, Stony Brook, NY, United States Department of Plastic and Reconstructive Surgery, Ben-Gurion University, Beer-Sheba, Israel
article info
abstract
Article history:
Introduction: Platelet rich plasma (PRP) is rich in growth factors and has been shown to
Accepted 27 April 2018
improve healing in a variety of wounds. We determined the effects of PRP on healing and
Available online xxx
scarring in full thickness porcine burns with and without tangential excision and grafting (TEG).
Keywords: Burns Platelet rich plasma Tangential excision Grafting Reepithelialization Scarring
Methods: Standardized full thickness 5cm by 5 cm burns were created on each of the backs and flanks of 10 anesthetized female pigs (25 kg) using a validated model. The burns were created with a heating device that emits heat at a temperature of 400 C for a period of 30 s. The burns were randomized to one of six treatments: no TEG or PRP, no TEG +PRP, early (day 2) TEG and no PRP, early TEG + PRP, late (day 14) TEG and no PRP, and late TEG+ PRP. Tangential excision was performed down to viable tissue and autografts were 0.2 mm thick. When used, a thin layer of autologous PRP was applied below the graft. All wounds were then treated with a topical antibiotic ointment 3 times weekly for 42 days. Digital images and full thickness biopsies were taken at 9, 11, 14, 18, 21, 28, 35 and 42 days after injury to determine percentage reepithelialization, scar depth, and scar contraction. Tissue sections were stained with H&E and viewed by a dermatopathologist masked to treatment assignment. Results: There was no reduction in platelet and white blood cell concentrations in PRP and blood samples for the first 14days after-full thickness burns. A total of 120 burns were created on 10 animals evenly distributed between the six treatment groups. Burns undergoing early TEG reepithelialized fastest and with the thinnest scars followed by late TEG. Burns that did not undergo TEG had the slowest reepithelialization and greatest amount of scarring. Application of PRP had no additional effects on reepithelialization, scar depth, or scar contraction in any of the treatment groups. Conclusions: Addition of PRP had similar effects on reepithelialization and scarring of full thickness porcine burns as standard topical antibiotic ointment regardless of whether the burns underwent excision or grafting or the timing of excision and grafting. © 2018 Elsevier Ltd and ISBI. All rights reserved.
* Corresponding author at: Department of Emergency Medicine, Stony Brook University, Stony Brook, NY, 11794-8350, United States. E-mail address: [email protected] (A.J. Singer). https://doi.org/10.1016/j.burns.2018.04.021 0305-4179/© 2018 Elsevier Ltd and ISBI. All rights reserved.
Please cite this article in press as: A.J. Singer, et al., The effects of platelet rich plasma on healing of full thickness burns in swine, Burns (2018), https://doi.org/10.1016/j.burns.2018.04.021
JBUR 5557 No. of Pages 8
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burns xxx (2018) xxx –xxx
1.
Introduction
Platelet-rich plasma (PRP) is the plasma fraction of autologous blood in which the platelet concentration is considerably higher than in whole blood due to processing and concentration [1]. Activated platelets release key wound healing factors including platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-b), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), and epidermal growth factor [1,2]. By delivering supra-physiological concentrations of a variety of growth factors and cytokines these preparations are used to enhance wound healing in bone and soft tissue injuries. Unfortunately, studies regarding the potential benefits of PRP in both animals and humans with burns are contradictory and inconclusive [3–11]. While superficial burns generally heal within 2 weeks with minimal if any scarring, deeper burns generally take greater than 3 weeks to heal and result in significant scarring unless excised and grafted with a split thickness autologous skin graft [12–14]. As a result, excision and grafting of full thickness burns has become the standard of care. The goals of the current study were twofold. First, to compare the healing of full thickness burns treated with or without autologous PRP in a swine model. Second, to determine whether addition of PRP to early and delayed excision and grafting of full thickness burns improves healing and reduces scarring in a porcine model.
2.
Methods
2.1.
Study design
We performed a prospective, randomized animal experiment. The study was approved by the Institutional Animal Care and Use Committee and was conducted in the institutional division of laboratory and animal research in accordance with national guidelines [15].
2.2.
Animals
In this study we used 10 female domestic pigs weighing approximately 20–25kg. Pigs were chosen for this in vivo experiment because it has been demonstrated that of all animals, pig skin most closely resembles that of the human [16]. Animals were given a standard diet ad lib several days prior to the investigation and were fasted overnight before any procedures.
2.3.
Experimental protocol
2.3.1.
Animal preparation and sedation
We used a previously validated porcine model of full thickness burns that were tangentially excised and grafted with a split thickness autologous skin graft [17]. Animals were sedated with a combination of acepromazine 0.1mg/kg, atropine 0.02mg/kg, ketamine 20mg/kg, and xylazine 2mg/kg by intramuscular injection. The pigs were then intubated endotracheally and maintained under a surgical plane of
anesthesia with isoflurane 1–3% in O2 USP. The hair on the backs and flanks of each pig was clipped.
2.3.2.
Burn creation
While under general anesthesia, twelve 5cm by 5cm burns were created on each pig’s back and flanks using a specialized radiant heating device. The burns were spaced at least 2cm from each other. We used a heating coil heated to 400 C, which emits infra-red/visible light radiation that heats the skin surface. The burn infliction device was placed in direct contact with the animal’s skin for a period of 30s thus creating full thickness burns on the back and flanks of the animal. In each of the ten animals we created three rows of four burns each (Fig. 1).
2.3.3.
Treatment allocation
The burns were then randomized to one of the following six treatment groups, equally distributed between the animals, using a computerized random number allocator: 1. A negative control group that did not receive any excision and grafting or PRP. 2. A control group that was treated with topical PRP two days after burn injury with no additional excision and grafting. 3. A treatment group that was excised and grafted two days (early) after burn injury without PRP. 4. A treatment group that was excised and grafted two days (early) after burn injury with PRP applied topically beneath the graft. 5. A treatment group that was excised and grafted 14 days (late) after burn injury without PRP. 6. A treatment group that was excised and grafted 14days (late) after injury with PRP applied topically beneath the graft. Each animal had an equal number of each treatment group randomly assigned to the different anatomical locations (i.e., 12 burns, 6 treatment groups, 2 of each treatment group per pig). The allocation of treatment groups to various anatomical locations (i.e., cranial or caudal) was balanced to account for any anatomical variation in healing.
2.3.4.
Preparation of autologous PRP
Immediately prior to the application of PRP to the burn wounds, anticoagulated whole blood was drawn from the pigs’ ear veins in three 60 ml sterile syringes, with each 60ml blood comprising of 52 ml whole blood and 8ml anticoagulant citrate dextrose formula A (ACD-A; Arteriocyte Medical Systems, Hopkinton, MA). Thus, PRP was prepared freshly, from blood collected immidiately prior to the application. The blood was then processed by a commercially available 1 device (Magellan , Arteriocyte, Hopkinton, MA) for 15 min to yield 7ml of PRP from each 60 ml batch. The PRP was mixed with a combination of Ca+/thrombin in a ratio of 10:1 forming a gel in sterile 5cm by 5cm molds and transferred to the wounds using a sterile spatula. Approximately 1.5ml of PRP+ calcified thrombin was applied to cover each wound. Hematology analysis was performed for each whole blood and PRP sample prepared at each designated time point to quantify the concentrations of platelets and white blood cells.
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2.4.
3
Tissue biopsies
Full thickness 4mm punch biopsies were taken from all wounds immediately after burn creation and at 9, 11, 14, 18, 21, 28 and 35 days, leaving a perimeter of 5 mm from the burn edge to eliminate any edge effect or reepithelialization arising from adjacent surrounding uninjured skin rather than from epithelial elements deep to the burn. Biopsies at different time points were taken from corresponding areas from the different burns to reduce sampling bias. Full thickness 8mm punch biopsies were also taken on day 42 prior to euthanasia with a pentobarbital-based solution (Fatal Plus, Vortech Pharmaceuticals, Michigan). The biopsies were marked for alignment, bisected, then placed in labeled cassettes and fixed in formalin for 24 h. Processed by alcohol-dehydration, xylene-clearing, and paraffin-embedding, tissue samples were sectioned at 5 mm and stained with hematoxylin and eosin (H&E) and Masson’s trichrome (day 42 biopsies only). Histomorphometric analyses were done by a board certified dermatopathologist, who was blinded to treatment allocation.
2.5.
Fig. 1 – Gross appearance of burns immediately after injury.
Hematology analysis was performed using a HematrueTM hematology analyzer (Heska, Loveland, CO). The HematrueTM provided counts of platelets, and white blood cells, which is important for confirming the ability of the device to consistently concentrate these components, and to determine whether the creation of full thickness burns changes the platelets and white blood cells concentration as a function of time after burn injury.
2.3.5.
Dressings and topical antibiotics
After the allocated treatment was received, the burns were treated with a thin layer of a topical petrolatum based triple antibiotic (neosporin, polysporin, bacitracin) and a nonadherent dressing (Telfa, Kendall Healthcare Products Company, Mansfield, MA) covered with polyurethane film (Tegaderm, 3M Health Care, St. Paul, MN) three times weekly. All wounds were covered with a gauze bandage roll (Sof-Form, Medline Industries, Inc., Mandekein, IL), and an adhesive elastic bandage (Tensoplast, BSN Medical S.A.S., Vibraye, France). The wounds were photographed and dressings were changed three times a week for the remainder of the study, which lasted 42 days. Sedation and anesthesia was performed as above, prior to any dressing changes or treatments.
2.3.6.
Pain management
All animals were treated with a transdermal patch of fentanyl 50mcg/kg every 72h and intramuscular buprenorphine 0.005– 0.02mg/kg every 8–12h as needed.
Outcomes
The following outcomes were measured in all wounds. Reepithelialization was determined using H&E-stained sections using a calibrated ocular micrometer to measure the total length in cross-section and the length of the neoepidermis. The percentage of reepithelialization was calculated by dividing the epithelialized length by the length of the specimen and multiplying by 100. Scar contraction was measured by tracing the perimeter of the discolored (red or purple) scar with no hair present using digital images of the wounds at day 42 with ImageJ software (National Institute of Health, Bethesda, MD). The surface area of the scar was subtracted from the original burn size and divided by the original burns size and multiplied by 100 to calculate percentage contraction. Scar depth was determined at 28, 35, and 42 days after injury using histological sections stained with Masson’s trichrome. An ocular lens with a grid was used to make straight-line measurements from the epidermal–dermal junction to the bottom of the scar. Three measurements were made per bisected half of the sample; one measurement was made at the center of the section, and two more were made 1mm left and right of the center. A total of six measurements were taken per sample and the mean depth of the scar was reported in millimeters. Scar tissue was identified by the presence of thin bundles of collagen oriented mostly in a single horizontal plain on H&E or trichrome staining.
2.6.
Data analysis
All data were reported as the meanstandard deviation (SD). Data analysis was performed using SPSS for Windows 22.0 software (SPPS Inc., Chicago, IL). For parametric data sets, one-way analysis of variance (ANOVA) followed by the Tukey post-hoc test was used to detect statistical significance with p<0.05. For nonparametric data sets, the Kruskal–Wallis test followed by the Dunn’s post-hoc test was used to detect statistical significance with p<0.05. A sample of 20 wounds in
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each group had 90% power to detect a 2mm difference in scar depth among the groups.
3.
Results
We created a total of 120 full thickness burns, 12 on each of the ten animals, evenly distributed between the two sides of the animals and the six treatment groups. There were no wound infections or any systemic adverse events in any of the animals or treatment groups. The outcomes are summarized in Table 1.
3.1.
Wound reepithelialization
At day 9, reepithelialization was approximately 50–60% in the early excision and grafting group, while there was no evidence of any reepithelialization in the delayed excision and conservatively managed groups. The effects of PRP on reepithelialization were similar to those of standard topical antibiotics. The application of PRP had no additional effect on reepithelialization regardless of whether the burns were excised or the timing of excision and grafting. Reepithelialization was also highest in the early excision and grafting groups at days 11, 14 and 18, and the application of PRP had no additional effects beyond those of a topical antibiotic in any of the sub-groups (Table 1 and Fig. 2). By day 21 most burns were completely reepithelialized (Fig. 3).
3.2.
Scar contraction
At day 42, scar contraction was greatest for burns treated conservatively without any excision and grafting (Fig. 4). The effects of PRP on scar contraction were similar to those of topical antibiotics alone. The application of PRP had no additional effects on scar contraction regardless of whether excision and grafting were performed or their timing (Table 1).
3.3.
Scar depth
Scars were most superficial at days 28, 35, and 42 in burns treated with early excision and grafting with no differences noted between those burns treated with or without PRP. The deepest scars were noted at all time points in burns treated conservatively. Scar depth did not differ based on whether PRP was applied or not (Table 1, Fig. 5).
3.4. PRP
Effect of burns on the composition of whole blood and
As shown in Fig. 6, full thickness burns did not reduce platelet and white blood cell concentrations in PRP and blood for the first 14 days after burn.
4.
Discussion
The results of the current study suggest that topically applied PRP performed comparably to topical antibiotics alone and its application had no additional beneficial effects on healing and scarring of full thickness porcine burns treated conservatively without excision and grafting. Similarly, application of PRP below the skin graft after excision of the burn eschar had comparable effects on healing and scarring as topical antibiotics and the addition of PRP after early or delayed excision and grafting of full thickness porcine burns also did not further improve reepithelialization or scarring. Whether or not injection of the PRP directly into or beneath the burns would have led to different results and outcomes is unclear. However, intra-lesional injection would be more clinically challenging, and was not investigated in this study. Our results confirm that early excision and grafting is more effective than delayed excision and grafting, and that delayed excision and grafting is more effective than conservative management of full thickness porcine burns [17]. This is evidenced by the fact that
Table 1 – Outcomes. TEG/ PRP Reepithelialization day 9 (SD), % Reepithelialization day 11 (SD), % Reepithelialization day 14 (SD), % Reepithelialization day 18 (SD), % Reepithelialization day 21 (SD), % Reepithelialization day 28 (SD), % Scar depth day 28 (SD), % Scar depth day 35 (SD), mm Scar depth day 42, (SD), mm Scar contraction day 42 (SD), %
TEG/ +PRP
Early TEG/ PRP
Early TEG/ +PRP
Late TEG/ PRP
Late TEG/ +PRP
P value
0 (0) 2 (7)
0 (0) 4 (13)
50 (50) 72 (44)
61 (46) 60 (48)
0 (0) 2 (6)
0 (0) 0 (0)
<0.001 <0.001
23 (41)
17 (33)
79 (41)
70 (47)
–
–
<0.001
68 (37)
43 (46)
89 (26)
95 (15)
NA
NA
<0.001
100 (0)
87 (33)
100 (0)
100 (0)
100 (0)
100 (0)
0.011
100 (0)
90 (0)
100 (0)
100 (0)
100 (0)
100 (0)
0.035
7.5 (1.4) 4.9 (1.7) 6.3 (1.3) 62 (14)
7.8 (1.6) 4.6 (2.3) 6.3 (1.4) 59 (15)
4.7 (2.8) 3.7 (2.1) 3.9 (2.1) 36 (22)
4.7 (2.7) 3.5 (1.7) 4.4 (1.9) 44 (25)
5.8 (1.4) 4.4 (1.8) 4.7 (1.6) 48 (17)
5.6 (1.6) 4.0 (1.6) 4.8 (1.2) 46 (17)
<0.001 0.133 <0.001 <0.001
TEG: tangential excision and grafting; PRP: platelet rich plasma; NA: not applicable. Biopsies were not taken at this time since the graft was still very fragile.
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Fig. 2 – Appearance of wounds on day 14. Left column (labeled 2) from top to bottom: early TEG+PRP, early TEG PRP, no TEG +PRP. Right column (labeled 3) from top to bottom: late TEG PRP, late TEG+PRP, no TEG PRP.
wound reepithelialization was greatest and fastest after early excision followed by late excision. In contrast, wound reepithelialization was slower and less complete when no excision was performed. Similarly, scar depth and contraction were least after early excision, followed by delayed excision. Despite its theoretical effects on wound healing, it is unclear why PRP did not improve reepithelialization or scarring of burns in this study. Other investigators have studied the effects of PRP on burns both in animals and humans with mixed results. Venter et al. studied the effects of PRP in deep second degree burns, deep second degree burns with diabetes, and third degree burns in rats [3]. PRP was associated with faster wound closure in deep second degree burns in normal and diabetic rats but not in third degree burns. Another small study of 4 horses with burns found that PRP accelerated repair and induced fibrosis [4]. A randomized double blind study of 52 patients with deep dermal or full thickness burns in which the patients served as
5
Fig. 3 – Appearance of wounds on day 21. Left column (labeled 2) from top to bottom: early TEG+PRP, early TEG PRP, no TEG +PRP. Right column (labeled 3) from top to bottom: late TEG PRP, late TEG+PRP, no TEG PRP.
their own control found no differences in graft take rate, reepithelialization, or scar quality [5]. A non-controlled study of 18 patients with second and third degree burns from the Czech Republic demonstrated earlier pain relief, decreased use of analgesics and anti-pruritic agents, earlier hospital discharge, and reduced costs when PRP was added to excision and grafting [6]. Another non-randomized study of 23 patients with deep second or third degree burns found that addition of PRP reduced the time required to recover the viscoelastic properties of the scars [7]. A side by side comparison of split thickness donor sites treated with PRP or a control in a 67-year-old man with third degree burns demonstrated improved epithelialization and angiogenesis with PRP [8]. A review of the role of PRP in burns conducted in 2009 was inconclusive [9]. A more recent systematic review of PRP as an adjunct therapy for acute
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Fig. 4 – Appearance of wounds on day 42. Left column (labeled 2) from top to bottom: early TEG+PRP, early TEG PRP, no TEG +PRP. Right column (labeled 3) from top to bottom: late TEG PRP, late TEG+PRP, no TEG PRP.
wounds and burns also concluded that there is not enough data to make any conclusions regarding the effects of PRP on burns [10]. Variability in PRP products, methods of application, frequency of application, and placement of PRP with relation to the graft further complicates interpretation of the evidence and may help explain the contradictory results [11]. It is possible that altering the treatment regimen by optimizing the number of PRP applications, concentration of platelets in PRP or the dilution factor of the PRP may improve the outcomes.
with and without PRP. Second, we only evaluated a single application of PRP administered 2 days after injury. It is possible that additional applications, different methods of application, or different timing of PRP application would have yielded different results than those reported in our study. Third, our results in a porcine model may not generalize to humans due to differences in anatomy and physiology.
6. 5.
Conclusions
Limitations
Our study has several notable limitations. First, the sample size was small including ten animals and only 20 wounds in each treatment group. Thus, it may have been underpowered to detect significant differences between wounds treated
In conclusion, a single application of PRP two days after injury performed similarly to topical antibiotics alone and its addition did not improve reepithelialization or scarring in full thickness porcine burns whether or not they were excised or grafted, regardless of timing of excision and grafting.
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Fig. 5 – Histological images 42 days after injury demonstrating scar depth. Black dots mark lower limit of purple hypercellular scar. Normal pink dermis noted below some, but not all scars.
Fig. 6 – Changes in platelet and white blood cell (WBC) counts in whole blood and platelet rich plasma (PRP) obtained at various time points before and after injury.
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Acknowledgment The authors would like to acknowledge Yiwei Ma for the contribution in performing hematology analysis on the blood and PRP samples.
Funding This project has been funded in whole or in part with Federal funds from the Department of Health and Human Services; Office of the Assistant Secretary for Preparedness and Response; Biomedical Advanced Research and Development Authority, under Contract No. HHSO100201300020C.
Conflicts of interest None of the authors have any conflict of interest to declare. REFERENCES
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Please cite this article in press as: A.J. Singer, et al., The effects of platelet rich plasma on healing of full thickness burns in swine, Burns (2018), https://doi.org/10.1016/j.burns.2018.04.021