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The effects of autologous platelet gel on wound healing.

Abstract

Laser resurfocing techniques have become a popular means of achieving rejuvenation of damaged skin. Interest is great in attempting to speed re-epithelialization and healing so that patients can return to their normal activities as quickly as possible. Previous studies have demonstrated that wounds heal more quickly when they are covered and keptmoist than when they are left open to the air. Until now, no study has been conducted to investigate whether the healing process of a superficial skin burn might be accelerated by the use of an autologous platelet gel us a biologic dressing. Our study of five pigs showed that autologous platelet gel can influence wound healing by stimulating an intense inflammatory process that leads to highly significant increases in the production of extra-cellular matrices and granulation tissue. The platelet gel accelerated vascular ingrowth, increased fibroblastic proliferation, and accelerated collagen production. However, the gel did not appear to accelerate re-epithelialization. The aggressive production of granulation tissue and the acceleration of collagen production might mean that autologous platelet gel will have a future role in the treatment of burns because the highly vascularized bed it helps create should promote the success of skin grafting in patients with deep partial-thickness and full-thickness burns.

Introduction

Over the past several years, carbon dioxide (C[O.sub.2] laser ablation has been widely promoted as a means of smoothing skin irregularities by removing the epidermis and superficial dermis. The clinical result is the tightening of lax skin and the removal of superficial blemishes. Healing of skin and soft-tissue burn injuries has been a focus of increased interest in view of the popularity of these laser resurfacing techniques.

Investigations are under way to test ways of accelerating the healing process, speeding re epithelialization, and decreasing the degree and duration of erythema, which would allow patients to resume normal daily activities in a timely fashion. (1) Previous studies have demonstrated that simply covering and keeping a wound site moist improves the rate of healing to a greater degree than does leaving the site open to the air. (2) Most attempts to speed re-vepithelialization have focused on variations of the moist dressing. (3) Several new dressing materials have been marketed and are frequently used. These dressings are passive in nature in that they produce an environment conducive to healing, but they do not actively alter the healing process.

Growth factors have also attracted interest as potential accelerators of growth. Danilenko et al investigated recombinant epidermal growth factor, keratinocyte growth factor, platelet-derived growth factor-BB, and neu differentiation factor. (4) They found that the use of each of these growth factors generated marginal acceleration of re-epithelialization.

Platelet gel, a concentration of platelet-rich plasma derived from whole blood, has been anecdotally reported by Timothy Hannon, MD, (oral communication, February 1997) to expedite the healing process. Platelets are believed to play an important role in primary hemostasis, wound healing, and re-epithelialization. Platelets contain is a number of cytokines that are important in angiogenesis promoting vascular ingrowth and in fibroblast proliferation, which in turn leads to increased collagen production. These cytokines include platelet-derived growth factor, transforming growth factor B1, platelet-derived epidermal growth factor, platelet-derived angiogenesis factor platelet factor 4, and platelet-activating factor.

In this article, we describe our study to determine if the healing of superficial skin burns is expedited by a platelet gel held in place by a standard OpSite dressing.

Materials and methods

Processing of the platelet gel. Our study was conducted on five Yorkshire pigs. A baseline hematocrit and platelet count was obtained from each. Then 300 ml of whole blood was drawn from each pig via a jugular venous, cutdown with a large-bore needle. The blood was drained into a sterile citrated collection bag. The pigs' average blood volume was 69.4 ml/kg of body weight, and each pig weighed approximately 25 kg, so the 300 ml of whole blood that was removed represented no more than 18% of each animal's blood volume, and the loss of blood was well tolerated by each animal. The vein was suture ligated, and the neck incision was closed.

Each blood sample was processed by a Sequestra 100 blood-processing machine (Medtronic; Parker, Colo.) in a 125-ml Latham processing bowl. Initial processing was conducted at 5,600 revolutions per minute (rpm) to separate the plasma from the cellular elements, then at 2,400 rpm to separate the platelets from the red blood cells Processing of each sample yielded approximately 30 ml of platelet concentrate suspended in plasma. A platelet count was obtained and recorded for each sample of platelet concentrate, Platelet counts were typically twice the baseline value in each pig. The remainder of each blood sample--approximately 270 ml of red cells and plasma--was returned to each pig via a peripheral intravenous line within 30 minutes of processing.

Creation of the experimental burns. The platelet concentrates were stored at room temperature while the pigs were prepared for laser burning. Following the administration of general endotracheal tube anesthesia, the hair or each pig's back was trimmed back by electric shears and then shaved off with a hand-held razor. Chlorhexidin solution was applied to the skin. Each pig's back was marked with a grid of 22 equally sized squares, 11 on each side of the dorsal midline (figure 1). One superficial laser burn was created in each square with an UltraPulse C[O.sub.2] laser (Coherent, Inc.; Santa Clara, Calif.). The laser was set at pattern 3, density 9, 60 W, 500 mJ, and 120 pulses/ sec. Three passes were made, and mechanical debridement of any eschar was performed between passes. There was approximately a 60% overlap between adjacent laser impacts. Each burn site measured 2 x 2 cm. After creation of the burns, a punch biopsy was taken from both sides of the dorsal midline to determine the depth of each burn.

[FIGURE 1 OMITTED]

Administration of the platelet gel. For each animal, the 11 burn sites on one side of the dorsal midline were designated as treatment sites and the 11 on the other side were designated as control sites. Each treatment site 0 received approximately 2 ml of activated platelet gel and was then covered with OpSite dressing. The control sites were covered with OpSite dressing alone. In preparing the platelet gel, 6 ml of platelet concentrate was drawn into a 10-ml syringe along with 2 ml of air. A 14-gauge Angiocath plastic catheter was attached, and 1 ml of bovine thrombin resuspended in 10% calcium chloride (1,000 U/ml of thrombin plus 100 mg/ml of calcium chloride) was drawn into the syringe with the platelet concentrate. The syringe was quickly inverted three times, and platelet concentrate was applied to each wound in 3 to 5 seconds.

Antibiotics were administered to each animal at the onset of the study and continued for 5 days following the procedure. Clinical and histologic evaluations were performed on postprocedure days 2, 4, 7, and 17. The timing of the biopsies was based on data obtained in wound healing studies by Ross et al. (5) Prior to each biopsy, each animal was anesthetized with an inhalation agent. Then two 3.5-mm punch biopsies were taken from each, one from a control site and the other from a treated site. Afterward, each biopsy site was covered with a new application of OpSite dressing. After clinical assessment and biopsy were completed, each animal was redressed with Kerlex gauze and Coban wrap, and a specially designed "pig jacket" was placed over each animal's back to protect the burn sites from further injury. The punch biopsy specimens were preserved in formalin and sectioned and examined by hematoxylin and eosin staining by a pathologist, who was blinded to the site from which each specimen was taken. Each slide was evaluated for four dependent variables: epidermal thickness, the depth of tissue necrosis, the thickness of granulation tissue, and the presence or absence of a stratum corneum.

Statistical analysis. The four dependent variable measures were compared statistically among pigs, between treatment and control burns, and for time in days by using single-factor analysis of variance (ANOVA) and two-way ANOVA with repeated measures. The two-way ANOVA also tested for any interaction effects between treatment and time. The alpha level was set at 0.05 for ANOVA comparisons.

Results

Days 0 through 6. For the first 6 days post-procedural both the treated and control sites were similar with respect to epidermal thickness (figure 2), the depth of necrosis (figure 3), and the thickness of granulation tissue (figure 4).

[FIGURE 2-4 OMITTED]

Day 7 evaluation. On day 7, the sites that had been treated with the platelet gel exhibited robust beds of granulation tissue and an intense inflammatory response (figure 5). At the control sites, no inflammatory phase was noted clinically, and the formation of granulation tissue was not detected histologically until an average of 11.25 day post-treatment. The difference in thickness between the treated and control site was statistically significant (F [1,31] = 1.04; p = 0.008).

[FIGURE 5 OMITTED]

On day 7, no statistically significant differences were seen with regard to epidermal thickness (F [1,31] = 0.13; p = 0.7198), the depth of tissue necrosis (F [1, 31] = 2.17; p = 0.1507), and the presence or absent of a stratum corneum (F [1, 31] = 1.3; p = 0.2608). The average depth of tissue necrosis was 0.135 mm in the treated sites and 0.127 mm in the control sites. There was no evidence of progression of tissue necrosis on either side over time.

The pace of re-epithelialization was slower on the treated side. In fact, both re-epithelialization and the reforming of the stratum corneum occurred twice as fast the control side. The mean times for re-epithelialization were 14.5 days on the treated side and 7.25 days on the control side.

Day 17 evaluation. On the final day of the experiment, the treated and control sites exhibited markedly different clinical appearances. The treated sites maintained a robust bed of granulation tissue and minimal surrounding erythema, while the untreated sites appeared to be well healed with minimal erythema. There was no evidence of infection in any of the animals.

Discussion

The C[O.sub.2] laser is known to induce secondary tissue reactions, and its use leads to slower wound healing because of several well-known factors, including carbonization, thermal necrosis, inflammatory cell infiltration, lack of platelet aggregation, and loss of biologically active factors. There is also a decrease in the removal of necrotic tissue and ultimately a delay in neovascularization.

Re-epithelialization following laser procedures can be accelerated simply by applying a semi-permeable occlusive dressing.

These dressings help keep the wound moist and allow epithelial migration to take place by limiting the formation of the thick eschar that is found in drier environments. Semipermeable occlusive dressings (several are on the market) are cumbersome to use because they are difficult to apply and keep in place. Some occlusive dressings can even adhere to the wound and strip away newly formed epidermis, thereby creating new wounds and delaying the healing process. (6) Although these dressings appear to marginally accelerate the healing process, they are still far from ideal. Therefore, research continues on developing a better dressing material.

We attempted to develop an autologous platelet gel biologic dressing that would improve the re-epithelialization process following the creation of superficial laser skin burns. We believed that platelet gel would be an ideal biologic dressing because it is autologous, readily available, inexpensive, and easily prepared. Unfortunately, we found that the activated platelet gel did not speed the re-epithelialization process. Instead, it induced an intense inflammatory reaction manifested as the production of robust granulation tissue. This inflammatory phase occurred much earlier in the treated burns than in the control burns.

The increased inflammatory response to the platelet gel might have resulted in higher levels of inflammatory mediators that ultimately delayed the healing process. Although this increased response is not an ideal quality for a laser resurfacing procedure (because it can stimulate the formation of hypertrophic scarring), autologous plate let gel might still play a role in the treatment of deep partial-thickness to full-thickness burns. A highly vascularized bed of granulation tissue might provide better vascular support for a skin graft and ultimately increase the percentage area of graft take. Further studies of platelet gel should include its use in treating deep partial- and full-thickness burns. Such studies might lead to improvements in the application vehicle and in the prevention of hypertrophic scars over the long term.

References

(1.) Chan P. Vincent JW, Wangemann RT. Accelerated healing of carbon dioxide laser burns in rats treated with composite polyurethane dressings. Arch Dermatol 1987;123:1042 5.

(2.) Hinman CD, Maibach H. Effect of air exposure and occlusion on experimental human skin wounds. Nature 1963;200:377-8.

(3.) Katz S, McGinley K, Leyden JJ. Semipermeable occlusive dressings. Effects on growth of pathogenic bacteria and reepithelialization of superficial wounds. Arch Dermatol 1986; 122:58-62.

(4.) Danilenko DM, Ring BD, Tarpley JE, et al. Growth factors in porcine full and partial thickness burn repair. Differing targets and etfiect s of keratinocyte growth factor, platelet-derived growth factor-BB, epidermal growth factor, and neu differentiation factor. Am J Pathol 1995;147:1261-77.

(5.) Ross EV, Barneae DJ, Glatter RD, Grevelink JM. Effects of overlap and pass number in CO2 laser skin resurfacing: A study of residual thermal damage, cell death, and wound healing. Lasers Med Surg 1999:24:103-12.

(6.) Zitelli JA. Delayed wound healing with adhesive wound dressings. J Dermatol Surg Oncol 1984;10:709-10.

From the Department of Otolaryngology, U.S. Naval Hospital, Yokuska Japan (Dr. Henderson), and the Department of Otolaryngology (Dr. Cupp, Dr, Keefe, Dr. Wester, and Mr. McConnell), the Department of Dermatology (Dr. Ross), the Laboratory Department (Dr. Shick), and the Department of Anesthesia (Dr. Harmon), U.S. Naval Medical Center, San Diego.

Reprint requests: Craig L. Cupp, MD, Department of Otolaryngology, Naval Medical Center, 34800 Bob Wilson Dr., San Diego, CA 92134-1005. Phone: (619) 532-9604; fax: (619) 532-6088; e-mail: clcupp@nmcsd.med.navy.mil

The Chief, Bureau of Medicine and Surgery, Navy Department, Washington, D.C., Clinical Investigation Program, sponsored this report #$98-008 as required by NSHSBETHINST 6000.41A. The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Navy, Department of Defense, or the United States Government.
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Title Annotation:Original Article
Author:McConnell, Devin
Publication:Ear, Nose and Throat Journal
Geographic Code:1USA
Date:Aug 1, 2003
Words:2400
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