Acceleration of Full-thickness Wound Healing in Porcine Model by Autologous Platelet Gel

Platelets contain many growth factors that have been postulated to play an important role in the wound healing process and re-epithelization. Autologous platelet-rich plasma gel (APG) was developed in the early 1970s as a by-product of multicomponent pheresis. Techniques and equipment have dramatically improved in the last decade. APG contains supraphysiologic amounts of various platelet growth factors. It is produced from platelet concentrate plus thrombin/calcium mixture. APG is a fibrin tissue adhesive with hemostatic and tissue sealing properties, which can be used to improve wound healing and enhance osteogenesis.¹
Autologous platelet-rich plasma gel is a viscous coagulum formed when platelet-rich plasma (PRP) is combined with a thrombin solution. Platelet-rich plasma is a rich resource that contains many growth factors. The thrombin solution assists in gel formation and the calcium chloride acts to reverse the anticoagulant effect of citrate dextrose solution A (ACD-A). Calcium/thrombin also activates platelets and clotting factors (eg, primary hemostasis, serotonin, ADP, thromboxane A).
For 30 years, APG has been used in many clinical areas including cardiovascular surgery, chronic nonhealing wounds, the closure of dural tears, various orthopedic applications (eg, spinal fusions, nonunionizing fractures and total joint replacements), oral surgery, and various plastic surgery procedures (eg, face-lifts, laser resurfacing, breast lifts, breast reconstruction, trans-rectus abdominis muscle [TRAM] flaps, and abdominoplasty).
However, controversy over the efficacy of APG is common, with many clinical practitioners still confused as to the real value of this agent. Some are concerned about hypersensitivity to bovine thrombin, which is a crucial component of APG.
Surgeons often face the challenge of full-thickness cutaneous wounds, (eg, in full-thickness skin grafts or full-thickness cutaneous wounds resulting from trauma). A study of accelerating the healing process in such wounds is especially valuable.^2,3
To address these issues, a porcine model was used to evaluate if APG can accelerate the healing process of full-thickness cutaneous wounds.

Material and Methods

An 8-week-old, 180-lb, neutered adult Yorkshire and Landrace cross pig was used in this study. A 4-day acclimation period was conducted between the pig and the authors’ animal facility prior to surgery.
The environment was controlled to remain within a temperature range of 70˚F to 75˚F, 65% humidity, and a light cycle of 12 hours on/off. The animal was fed 1.25- to 2.2 lb of standard swine diet and received an uninterrupted supply of clean, fresh water.
On the surgery day, the pig was given a combination of tiletamine-zolazepam (4–6 mg/kg IM) and xylazine (2.2 mg/kg) as a sedative, and placed on 3L of oxygen. Intubation followed, and a mixture of oxygen (3L) and isoflurane (1%–3%) was administered as a general anesthetic. Pulse oximetry, rectal temperature, and respiratory rate were monitored throughout the surgery.
The skin was surgically prepared with successive applications of 7.5% povidone iodine scrub, 10% povidone iodine scrub, and 70% isopropanol. Twenty-four 2-cm x 2-cm tattoo squares were evenly outlined with a sterile surgical marker on each flank of the pig using a prepared template (an x-ray film with 2-cm x 2-cm grids).
After covering the pig with a sterilized sheet, 48 full-thickness cutaneous round wounds were created in the center of each marked tattoo square using a 1.7 cm diameter punch. Each wound was 5 mm in depth.⁴
A 5-cm incision was made on the medial aspect of the right thigh of the pig. The long saphenous vein was exposed. An 18-gauge IV cannula (Mediflon, Eastern Medikit Limited, Delhi, India) with a 3.8-cm (1.5-in) needle was inserted into the vein, and blood was obtained.
First, 10 cc of blood was withdrawn from the saphenous vein by two 5 cc syringes. One was disposed and the other was sent to the lab for platelet counting in order to assure that the pig blood was successfully drawn and processed without activation. The result (198,000)indicated a normal platelet range. Next, 52 cc of blood for each syringe (total of 208 cc) was collected from the same saphenous vein of the pig using 4 60-mL syringes, each containing 8 mL of acid-citrate-dextrose (ACD). The 208 cc blood was then centrifuged to produce 24 cc of PRP by using the platelet concentrate system (Magellan™ Platelet Separator, Medtronic Inc, Minneapolis, Minn) 6 cc for each syringe. The platelet separator separates the platelets in 2 stages. In the first stage, the red blood cells (RBCs) are separated from the plasma at 2800 rpm for 4 min (for a 60-mL sample of input anticoagulated blood). Upon withdrawal of most of the packed RBCs, in the second stage, the platelets and white blood cells (WBCs) are packed against the RBCs at 3800 rpm for 6 min (for a 60 mL sample of input anticoagulated blood). The PRP product is harvested after clearing the remaining RBCs from the line.
One 6 cc PRP syringe drawn from the pig was used to make autologous thrombin-serum; 1 cc was used for PRP platelet counting; and the remaining 5 cc was used to produce 4 cc of autologous thrombin-rich serum with the autologous thrombin preparation syringe, which contained 1 cc calcium chloride to reverse the anticoagulant in the PRP, and glass wool that was used to trigger clot formation. When all the PRP clotted, the resulting thrombin-rich serum was expressed. Ten cc of PRP was evenly sprayed onto the left side wounds with 4 cc autologous thrombin-rich serum by using a dual syringe dispenser (Magellan™ Autologous Serum Dispenser, Medtronic, Minneapolis, Minn) while 10% saline was used on the other side as a control (Figure 1).
After surgery, a transparent, vapor-permeable film (Tegaderm™ Transparent Dressings, 3M™ Health Care, St. Paul, Minn) was sealed around the edges of all wounds as a dressing (Figure 2). Fish net was used in addition to the transparent film covering in order to better protect the wounds. After recovering from anesthesia, the pig was penned in a custom-made, smooth-sided stainless steel cage. Post surgery intensive care was provided, and the pig was examined for signs of wound infection (elevated body temperature, elevated respirations, putrid or cloudy wound fluid), or other illness. Staff veterinarians examined the animal daily for illness or discomfort. This maintenance schedule was followed until Day 28 post-surgery, at which time the 8-month-old pig was euthanized via IV administration of 5 g of thiopental sodium.
On Days 14 and 28 after surgery, all 48 wounds on each experimental and control side of the pig were visually measured in order to do the qualitative analysis of the wound. All wounds were photographed for quantitative assessment using a digital camera.
A constant 3.9 zoom at 12.5 cm distance was used while taking each photo. The contour of each wound was copied onto a transparent film with a marking pen and digitally transferred to a computer in .JPEG format. The image was loaded into the digital imaging measurement software (Metavue, Carl Zeiss Inc, Thornwood, NY), which allowed us to measure the extent of the wound using pixels as units. Each healing wound was measured in square millimeters. These parameters were expressed as a percent ratio of the APG-treated wound on the left side of the pig compared with that of the saline-treated wound at the conclusion of the study.
For the histological study, on Days 14 and 28, biopsy specimens from all 48 wounds on the experimental and control sides were obtained. On Day 14, specimens were taken from 9- and 3 o’clock at the edge of the round wounds. On Day 28, specimens were taken from 12- and 6 o’clock of the round wounds. All specimens were fixed in 10% neutral buffered formalin, embedded in methacrylate, and sectioned (2–5 µm thick). The sections were stained with hematoxylin and eosin stain. Finally, all sections were scanned into the computer by digital imaging system (Carl Zeiss microplate reader software, Carl Zeiss Inc, Thornwood, New York.) and the image was loaded into imaging measurement software (Metavue software, Carl Zeiss Inc, Thornwood, New York.). Under the direction of a dermatopathologist, the authors performed blinded histologic review of the biopsies. Histometric analyses of the biopsy specimens were performed with the digital imaging measurement software (Metavue software, Carl Zeiss Inc, Thornwood, New York). The healing status and dermal thickness of the wounds from each side of the pig were measured and compared.

Statistical analysis. To determine the significance of the results, the repeated measures ANOVA test was used to assess if wound area differed significantly with APG and Saline treatment. MiniTab was used for all calculations. All tests were 2-sided, and a P £ 0.05 was considered statistically significant.
Treatment of the animals used in this study conformed to the standards set forth in the US Public Health Services Policy on Humane Treatment of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals.

Results

Evaluation of healing requires the analysis of qualitative and quantitative wound assessments. For the qualitative purpose, a visual measurement was used to evaluate the appearance of the wounds on Day 14 and Day 28 after surgery. The criteria for a healed wound are: wound closed, dry, intact, with central area covered by fresh pink tissue and crust easy to peel off. By contrast, the criteria for a nonhealed wound were: wound is still open and moist; if gauze is used to wipe the surface of the wound, there is some yellow stain on the gauze; and the crust on the wound is difficult to peel off. Removal of the crust will cause bleeding on the base of the wound, and the central area is still covered by red granulation tissue (Figures 3–6). According to the above criteria the healing ratio was compared between both sides (Table 1). On Day 14 after surgery, 75% of the wounds on the APG side were healed compared to only 25% of the wounds on the control side. On Day 28 after surgery, 91.7% of the wounds on the APG side were healed versus 62.5% on the control side. For quantitative purposes, the size of the healing wounds were measured and analyzed. (Table 2). On Days 14 and 28 post-injury, APG-treated wounds were 23% and 25% smaller in area than the control wounds, respectively.^5,6 The mean size of the wounds on the APG side was smaller than that on the control side, and this difference was statistically significant (P < 0.001) both on Day 14 and on Day 28 (Figure 7).
The histological assessment also included both qualitative and quantitative analysis. Histopathologic review was performed for qualitative purposes. On Day 14 after surgery, the main finding is that the thickness of the healed dermis is greater on the APG side compared to the control side (Figures 8 and 9). On Day 28 post surgery, under the high magnification, there were fewer polymorphonuclear leucocyte (PMN) cells in the healing tissue on the APG side compared to the control side, indicating delayed healing process on the control side due to inflammation (Figure 10). Also, more normal skin anatomic structures including hair follicles, dermis and apocrine glands were observed on the APG side compared to control side (Figure 11).
The thickness of the healing dermis was measured for quantitative purposes (Table 3). On Day 14 and 28 after surgery, the thickness of dermis was greater on the APG side, indicating that the dermis was healed faster on the APG side compared to the control side. The difference was statistically significant (P < 0.001).

Discussion

Over 5 decades, many studies have considered a variety of treatments for wound healing. Platelet-derived products have proven effective in accelerating wound healing.
Platelet-rich plasma is one of these useful products and has been used in many wound care treatments. The PRP contains concentrated platelets as well as white blood cells, a small amount of red blood cells, and a host of cytokines that act to enhance wound healing. Platelet-rich plasma also contains various coagulation components (eg, fibrinogen) that both help form the gel and act to stop bleeding at the site of the wound. Autologous platelet-rich plasma gel is a viscous coagulum formed when PRP is combined with a thrombin solution. Compared to general PRP, APG was used in the present study because it is a solid coagulum. Thus, it has the advantage of staying firmly on the wound compared to the fluid plasma. Furthermore, APG includes thrombin and calcium chloride, which activate the release of supraphysiologic amounts of various growth factors including platelet-derived growth factor (PDGF), platelet factor 4, transforming growth factor-b, platelet-derived angiogenesis factor, and platelet-derived epidermal growth factor, resulting in more effective healing.
Autologous platelet-rich plasma gel was used instead of just 1 particular growth factor, such as the well known PDGF or TGF-b factors, because all of the growth factors work synergistically during the wound healing process to increase the rate of collagen lay down, angiogenesis, fibroblast proliferation, extracellular matrix synthesis, and overall wound healing.^7,8 Detailed study on the function of each growth factor including APG is beyond the scope of this study; however, these issues need to be addressed in future research.
Only 1 pig was used because the study was designed to test whether local use of APG has effects in improving wound healing rather than effects on the whole body. A total of 48 wounds were used for evaluation to ensure that there were enough samples.
Saline was used on the control side of the pig model instead of leaving wounds open or using other treatment, such as general gel, because the goal was to keep statistical comparisons simple and uninfluenced by other factors. Consequently, a moist, electrolyte-balanced, normal physiological environment was created to match the contralateral APG side.
For the dressing, a transparent vapor film with adjunctive fish net was used in this study, and all edges of the round wounds were tightly sealed. The film was kept intact for 2 weeks without any tearing off. When the 1 dressing change was done after 2 weeks, the dressing was easily peeled off. No re-injury or crust formation was seen during the dressing change. The methods eliminated any man-made effect on those wounds.
Staff veterinarians closely monitored the pig regarding feeding, sleeping, and any change in behavior. The pig was intentionally kept lying down on both sides evenly. Cage cleaning was performed 3 times per day. A plastic mesh floor was used so feces and any other contamination could easily be removed. Since the pig was maintained in a clean and dry environment, minimal body rubbing behavior was found. No wounds had any infections or abnormal exudative oozing.
A pig was chosen for this study based upon anatomic and physiologic similarities that exist between its skin and that of humans. The thickness of the dermis and epidermis, the relative lack of hair, and the presence of a papillary dermal layer, subdermal fat, rete ridges, and apocrine sweat glands are characteristics pig integument shares with humans.⁹
Many studies still focus on partial-thickness skin wound healing. Few studies have been done involving full-thickness skin wound healing. Full-thickness open excisions are more difficult to heal because removal of tissue requires filling the gap with a provisional matrix, replacing this matrix with granulation tissue, and revascularizing, reepithelializing, and remodeling the wounded area to restore the functional equivalence of normal skin. However, surgeons often encounter full-thickness cutaneous wounds in their practice (eg, full-thickness skin grafts, and a variety of traumatic events that involve full-thickness, cutaneous wounds). Studying the healing acceleration of full-thickness cutaneous wounds becomes indispensable.
Autologous thrombin was used in the present study and was directly extracted from the pig’s plasma instead of the traditional bovine thrombin, which is extracted from bovine blood. Currently, to make APG product in humans, only thrombin derived from bovine blood is available in the US as a stand-alone thrombin product. There are important and growing safety concerns related to the use of bovine thrombin. These include a high incidence of immune responses directed against bovine thrombin and other contaminating proteins in the thrombin product. In addition, growing concern exists about the potential for transmitting infectious agents from cows to humans, such as bovine spongiform encephalopathy (BSE, the agent that causes “mad-cow” disease), through the use of bovine-derived products. The present study offers a safer option.¹⁰
In all 48 wounds on each side of the pig the biopsy specimens were taken from the 9- and 3 o’clock positions at the edge of the round wounds on Day 14, and from the 12- and 6 o’clock positions of the round wounds on Day 28 in order to reduce bias from variations in thickness of the pig skin.
The crust of the wound was formed as part of the natural healing process. On the control side, crust formation was due to the slower process of healing compared to the APG side. During evaluation, crust was removed only when it could easily be peeled off wounds. There was no re-injury or elimination of those wounds from further assessment.
For histological assessment, considering the major part of the healing process in a full-thickness cutaneous wound is dermis recovery; only dermis thickness was evaluated, no measurement of epithelialization was done. However, in future studies, epithelization needs to be tested.

Conclusion

The results from the present study suggest that autologous platelet gel (APG) is effective in accelerating the healing process of full-thickness cutaneous wounds in a porcine model. Autologous platelet gel should be clinically useful in accelerating the healing process in a variety of wounds.

Acknowledgment
The authors thank Medtronic for the research grant and the Magellan Platelet Separator.