EXPERIMENTAL The Effect of Platelet-Rich Plasma on Composite Graft Survival Yeo Reum Jeon, M.D. Eun Hye Kang, B.S. Chae Eun Yang, M.D. In Sik Yun, M.D., Ph.D. Won Jai Lee, M.D., Ph.D. Dae Hyun Lew, M.D., Ph.D. Seoul, Republic of Korea
Background: Composite grafts are suitable for facial reconstruction because of good color matching, low donor-site morbidity, acceptable texture, and easy surgical techniques. However, their use is limited to small defects and by unpredictable survival rates. As platelet-rich plasma contains large numbers of growth factors and has been widely used for tissue regeneration, this study aimed to investigate platelet-rich plasma as an adjuvant to enhance composite graft survival. Methods: Twenty New Zealand White rabbits were used, and chondrocutaneous composite grafts were applied to their ears. The grafts were then returned to their original positions after rotation to block the original circulation from the base of the graft. Each of the individual ears was assigned randomly into one of two groups: experimental (n = 20; platelet-rich plasma group) or control (n = 20; control group). The surrounding skin of the composite graft was injected with either 1.0 ml of platelet-rich plasma derived from autologous whole blood in the platelet-rich plasma group or normal saline in the control group. Graft survival, cutaneous blood flow, CD31-stained vessels, and vascular endothelial growth factor protein levels were examined. Results: Twelve days after surgery, graft viability in the platelet-rich plasma group was higher than in the control group. Blood perfusion was also higher in the platelet-rich plasma group. Compared with the control group, the number of CD31+ blood vessels and vascular endothelial growth factor expression levels were significantly increased in the platelet-rich plasma group. Conclusions: The authors’ results suggest that platelet-rich plasma restores the perfusion of composite grafts by enhancing revascularization and may exert therapeutic effects on the survival of composite grafts. (Plast. Reconstr. Surg. 134: 239, 2014.)
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lastic surgery aims to reconstruct various types of physical abnormalities.1 Functional and aesthetically satisfactory restoration of defects or deformities of the face is challenging for many plastic and reconstructive surgeons. To repair defects derived from trauma, burns, cancer surgery, and other causes, various types of surgical procedures have been used, such as primary repair, split-thickness skin grafts, full-thickness skin grafts, local flaps, and composite grafts.2,3 Local flaps using nearby tissues show good texture and color matching for facial reconstruction. Nevertheless, donor-site scars and resulting contour deformities are major drawbacks to local From the Institute for Human Tissue Restoration, Department of Plastic and Reconstructive Surgery, Yonsei University College of Medicine. Received for publication July 22, 2013; accepted October 10, 2013. Copyright © 2014 by the American Society of Plastic Surgeons DOI: 10.1097/PRS.0000000000000392
flap surgery. Composite grafts are more resistant to contractile forces than are skin grafts; thus, secondary contraction of composite grafts can be minimized.4 In addition, composite grafts show less melanin pigmentation than other types of grafts,5 offering results that better match the surrounding skin. Furthermore, chondrocutaneous composite grafts containing supporting cartilage can be used for ear or nasal reconstruction, which requires three-dimensional structures to restore functional and aesthetic deficits.6 Although composite grafts show excellent results for facial reconstruction, they are limited by size and unpredictable survival rates. Generally, the most reliable graft size is considered to be less than 1.5 cm,6–8 with larger grafts showing less predictable results. The major causes of graft Disclosure: The authors have no financial interests or commercial associations to declare in relation to the content of this article.
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Plastic and Reconstructive Surgery • August 2014 failure are likely inadequate nutrient supply and ischemia-reperfusion injury. Therefore, numerous studies have been conducted to maximize the survival of composite grafts. Ice-cold compression, corticosteroids, and hyperbaric oxygen therapy have been used to maximize graft survival; however, their clinical significance is questionable.8 Recently, the potential of platelet-rich plasma, which is defined as an autologous concentration of platelets,9 has garnered the interest of many reconstructive surgeons. Because platelets contain large numbers of growth factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor, platelet-derived growth factor (PDGF), transforming growth factor, and insulin-like growth factor, platelet-rich plasma is considered a reservoir of growth factors for wound healing, tissue regeneration, and neovascularization. Platelet-rich plasma has been widely used clinically for tissue regeneration, not only for burns or problematic wounds, but also for cosmetic and orthopedic operations, periodontics, and others.10–14 Therefore, we hypothesized that platelet-rich plasma may also exert a protective effect on composite grafts. In this study, we investigated the effect of plateletrich plasma on chondrocutaneous composite graft survival in a rabbit ear model.
MATERIALS AND METHODS Preparation of Platelet-Rich Plasma Blood collection was carried out before surgery. Peripheral blood (8.5 ml) was collected from
each rabbit’s marginal ear vein and mixed with 1.5 ml of anticoagulant citrate dextrose solution formula A.15 The collected blood was centrifuged at 160 g for 10 minutes at room temperature.16 The supernatant including the buffy coat was separated from the bottommost red blood cell layer. The supernatant was transferred into another tube for a second centrifugation at 400 g for 10 minutes at room temperature.16 This process allowed the platelets to settle to the bottom of the tube. All but 1 ml of the supernatant, called platelet-poor plasma, was removed, and the remaining 1 ml of platelet-poor plasma was added to the platelet pellet to form platelet-rich plasma (platelet count, 8 × 105 cells/μl). Throughout the procedure, platelet-rich plasma was never activated. Surgical Procedure Twenty New Zealand White rabbits (average, 3 kg each) were used for the chondrocutaneous composite graft model.17 The day before surgery, all rabbits were fasted overnight. On the day of surgery, the rabbits were anesthetized intramuscularly with Zoletil 50 (Virbac, Carros, France) and 2% Rompun solution (Bayer, Germany) (1:2 ratio, 1 ml/kg). Bilateral ears were shaved and disinfected with povidone-iodine solution. Approximately 4 cm from the external meatus of the ear, 2 cm diameter, circular, full-thickness chondrocutaneous composite tissue was harvested. The graft was then laid onto the defect site and repaired with 6-0 nylon after being rotated 90 degrees (Fig. 1). The operation was carried out on both ears. An
Fig. 1. Chondrocutaneous composite graft. Approximately 4 cm from the external meatus of the ear, 2 cm-diameter, circular, full-thickness, chondrocutaneous composite tissue was harvested. The graft was then laid in the defect site and repaired with 6-0 nylon after being rotated 90 degrees.
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Volume 134, Number 2 • Platelet-Rich Plasma and Graft Survival independent assistant randomized the ears to the platelet-rich plasma and control groups for each rabbit. Immediately after the operation, 1.0 ml of nonactivated platelet-rich plasma was injected evenly into the surrounding skin of the composite graft with a 28-gauge insulin syringe (Becton, Dickinson & Company, Franklin Lakes, N.J.) on the platelet-rich plasma side, and 1.0 ml of normal saline was injected around the graft in the same manner on the control side. After surgery, 5 mg/ kg of enrofloxacin (Baytril; Bayer HealthCare, Shawnee Mission, Kan.) was administered intramuscularly for 3 days to prevent infection. The wound was treated with compression dressing for 3 days and maintained with open dressing thereafter. The Animal Care and Experiment Committee of Yonsei University approved the experimental protocol. Assessment of Composite Graft Survival Rates On days 1, 3, 6, 9, and 12 after surgery, graft survival was examined, and the grafts were photographed under the same conditions. Digital images were quantitatively analyzed by two independent reviewers, using Image-Pro Plus Software (Media Cybernetics, Silver Spring, Md.). Using digital photographs taken on day 12, flap survival was calculated as the ratio (percentage) of the pixel number of the viable surface area to the pixel number of the total graft surface area. Assessment of Microcirculation in Composite Grafts To assess relative changes in blood perfusion over time, percutaneous blood flow was measured by laser Doppler flowmetry (Peri-Flux System 5000; Perimed Inc., Stockholm, Sweden)18 on days 1, 3, 6, 9, and 12 after repositioning of the graft. For at least 1 minute, a stable signal (blood flow) was traced and recorded at three different viable portions on each graft. Blood flow was analyzed using Perisoft programs (Perimed) and quantified as perfusion units. Immunohistochemical Analysis The animals were killed 12 days after surgery for histologic examination. The surviving part of the grafted tissues was harvested from the same position in each model. Tissue specimens were embedded in paraffin after fixation in 10% formalin. Immunohistochemical staining of endothelial cells was carried out using anti-CD31 antibody [anti-mouse platelet endothelial cell adhesion molecule-1 (PECAM/CD31) polyclonal
antibody (M20; Santa Cruz Biotechnology, Santa Cruz, Calif.)] and anti-VEGF antibody (rabbit anti-VEGF, RB-222-P; Laboratory Vision, Fremont, Calif.). To quantify the amount of neovascularization, CD31+ vessels were counted by two blinded observers under a high-power field (200× magnification). Using computer-assisted planimetry (Metamorph; Universal Image Corp., Buckinghamshire, United Kingdom), expression levels of VEGF were analyzed quantitatively. Statistical Analysis The results were expressed as means ± SEM. Statistical analysis was performed with a two-tailed, paired t test. Differences were considered statistically significant at p < 0.05. SAS Version 9.1.3 (SAS Institute, Inc., Cary, N.C.) was used for statistical analysis.
RESULTS Effect of Platelet-Rich Plasma on Composite Graft Survival On examination of graft survival 12 days after surgery, graft necrosis was observed to extend from nearly every centered area of the grafts in both groups, whereas around the peripheral edge, the viable area was larger in the platelet-rich plasma group. Representative results for each group are shown in Figure 2. On day 12 after surgery, necrotic areas were clearly demarcated in the graft. Survival rates were analyzed by assessing the viable area compared with the total graft area at postoperative day 12. The average surviving area in the composite graft was 46.49 ± 5.50 percent in the platelet-rich plasma group and 30.13 ± 6.65 percent in the control group, which was a statistically significant difference (p < 0.01) (Fig. 3). Effect of Platelet-Rich Plasma on the Microcirculation in Composite Grafts Doppler measurements were carried out before surgery to establish normal blood flow. They were not significantly different between the two groups. Postoperative measurements were performed immediately after the operation and on days 1, 3, 6, 9, and 12 after surgery. Blood flow varied over time, and progressive improvements were observed after surgery in both groups. There were no significant differences in perfusion between the groups until day 3. Interestingly, in the platelet-rich plasma group, blood flow was significantly increased compared with the control group 3 days after the operation;
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Fig. 2. The surviving areas of the grafts in the control group (above) and the platelet-rich plasma group (below). The congestive color of the graft gradually progressed in the control group, whereas congestion improved from days 1 to 3 in the platelet-rich plasma group. By postoperative day 12, necrotic areas were clearly demarcated in the grafts.
Effect of Platelet-Rich Plasma on VEGF Expression in Composite Grafts The MetaMorph imaging analyzer was used for semiquantitative analysis of VEGF expression levels. Injection of platelet-rich plasma around the chondrocutaneous composite graft induced a significant increase in VEGF expression 12 days after surgery (control group, 2849.39 ± 466.97; platelet-rich plasma group, 5003.29 ± 473.30; p
Background: Composite grafts are suitable for facial reconstruction because of good color matching, low donor-site morbidity, acceptable texture, and easy surgical techniques. However, their use is limited to small defects and by unpredictable survival rates. As platelet-rich plasma contains large numbers of growth factors and has been widely used for tissue regeneration, this study aimed to investigate platelet-rich plasma as an adjuvant to enhance composite graft survival. Methods: Twenty New Zealand White rabbits were used, and chondrocutaneous composite grafts were applied to their ears. The grafts were then returned to their original positions after rotation to block the original circulation from the base of the graft. Each of the individual ears was assigned randomly into one of two groups: experimental (n = 20; platelet-rich plasma group) or control (n = 20; control group). The surrounding skin of the composite graft was injected with either 1.0 ml of platelet-rich plasma derived from autologous whole blood in the platelet-rich plasma group or normal saline in the control group. Graft survival, cutaneous blood flow, CD31-stained vessels, and vascular endothelial growth factor protein levels were examined. Results: Twelve days after surgery, graft viability in the platelet-rich plasma group was higher than in the control group. Blood perfusion was also higher in the platelet-rich plasma group. Compared with the control group, the number of CD31+ blood vessels and vascular endothelial growth factor expression levels were significantly increased in the platelet-rich plasma group. Conclusions: The authors’ results suggest that platelet-rich plasma restores the perfusion of composite grafts by enhancing revascularization and may exert therapeutic effects on the survival of composite grafts. (Plast. Reconstr. Surg. 134: 239, 2014.)
P
lastic surgery aims to reconstruct various types of physical abnormalities.1 Functional and aesthetically satisfactory restoration of defects or deformities of the face is challenging for many plastic and reconstructive surgeons. To repair defects derived from trauma, burns, cancer surgery, and other causes, various types of surgical procedures have been used, such as primary repair, split-thickness skin grafts, full-thickness skin grafts, local flaps, and composite grafts.2,3 Local flaps using nearby tissues show good texture and color matching for facial reconstruction. Nevertheless, donor-site scars and resulting contour deformities are major drawbacks to local From the Institute for Human Tissue Restoration, Department of Plastic and Reconstructive Surgery, Yonsei University College of Medicine. Received for publication July 22, 2013; accepted October 10, 2013. Copyright © 2014 by the American Society of Plastic Surgeons DOI: 10.1097/PRS.0000000000000392
flap surgery. Composite grafts are more resistant to contractile forces than are skin grafts; thus, secondary contraction of composite grafts can be minimized.4 In addition, composite grafts show less melanin pigmentation than other types of grafts,5 offering results that better match the surrounding skin. Furthermore, chondrocutaneous composite grafts containing supporting cartilage can be used for ear or nasal reconstruction, which requires three-dimensional structures to restore functional and aesthetic deficits.6 Although composite grafts show excellent results for facial reconstruction, they are limited by size and unpredictable survival rates. Generally, the most reliable graft size is considered to be less than 1.5 cm,6–8 with larger grafts showing less predictable results. The major causes of graft Disclosure: The authors have no financial interests or commercial associations to declare in relation to the content of this article.
www.PRSJournal.com
239
Plastic and Reconstructive Surgery • August 2014 failure are likely inadequate nutrient supply and ischemia-reperfusion injury. Therefore, numerous studies have been conducted to maximize the survival of composite grafts. Ice-cold compression, corticosteroids, and hyperbaric oxygen therapy have been used to maximize graft survival; however, their clinical significance is questionable.8 Recently, the potential of platelet-rich plasma, which is defined as an autologous concentration of platelets,9 has garnered the interest of many reconstructive surgeons. Because platelets contain large numbers of growth factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor, platelet-derived growth factor (PDGF), transforming growth factor, and insulin-like growth factor, platelet-rich plasma is considered a reservoir of growth factors for wound healing, tissue regeneration, and neovascularization. Platelet-rich plasma has been widely used clinically for tissue regeneration, not only for burns or problematic wounds, but also for cosmetic and orthopedic operations, periodontics, and others.10–14 Therefore, we hypothesized that platelet-rich plasma may also exert a protective effect on composite grafts. In this study, we investigated the effect of plateletrich plasma on chondrocutaneous composite graft survival in a rabbit ear model.
MATERIALS AND METHODS Preparation of Platelet-Rich Plasma Blood collection was carried out before surgery. Peripheral blood (8.5 ml) was collected from
each rabbit’s marginal ear vein and mixed with 1.5 ml of anticoagulant citrate dextrose solution formula A.15 The collected blood was centrifuged at 160 g for 10 minutes at room temperature.16 The supernatant including the buffy coat was separated from the bottommost red blood cell layer. The supernatant was transferred into another tube for a second centrifugation at 400 g for 10 minutes at room temperature.16 This process allowed the platelets to settle to the bottom of the tube. All but 1 ml of the supernatant, called platelet-poor plasma, was removed, and the remaining 1 ml of platelet-poor plasma was added to the platelet pellet to form platelet-rich plasma (platelet count, 8 × 105 cells/μl). Throughout the procedure, platelet-rich plasma was never activated. Surgical Procedure Twenty New Zealand White rabbits (average, 3 kg each) were used for the chondrocutaneous composite graft model.17 The day before surgery, all rabbits were fasted overnight. On the day of surgery, the rabbits were anesthetized intramuscularly with Zoletil 50 (Virbac, Carros, France) and 2% Rompun solution (Bayer, Germany) (1:2 ratio, 1 ml/kg). Bilateral ears were shaved and disinfected with povidone-iodine solution. Approximately 4 cm from the external meatus of the ear, 2 cm diameter, circular, full-thickness chondrocutaneous composite tissue was harvested. The graft was then laid onto the defect site and repaired with 6-0 nylon after being rotated 90 degrees (Fig. 1). The operation was carried out on both ears. An
Fig. 1. Chondrocutaneous composite graft. Approximately 4 cm from the external meatus of the ear, 2 cm-diameter, circular, full-thickness, chondrocutaneous composite tissue was harvested. The graft was then laid in the defect site and repaired with 6-0 nylon after being rotated 90 degrees.
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Volume 134, Number 2 • Platelet-Rich Plasma and Graft Survival independent assistant randomized the ears to the platelet-rich plasma and control groups for each rabbit. Immediately after the operation, 1.0 ml of nonactivated platelet-rich plasma was injected evenly into the surrounding skin of the composite graft with a 28-gauge insulin syringe (Becton, Dickinson & Company, Franklin Lakes, N.J.) on the platelet-rich plasma side, and 1.0 ml of normal saline was injected around the graft in the same manner on the control side. After surgery, 5 mg/ kg of enrofloxacin (Baytril; Bayer HealthCare, Shawnee Mission, Kan.) was administered intramuscularly for 3 days to prevent infection. The wound was treated with compression dressing for 3 days and maintained with open dressing thereafter. The Animal Care and Experiment Committee of Yonsei University approved the experimental protocol. Assessment of Composite Graft Survival Rates On days 1, 3, 6, 9, and 12 after surgery, graft survival was examined, and the grafts were photographed under the same conditions. Digital images were quantitatively analyzed by two independent reviewers, using Image-Pro Plus Software (Media Cybernetics, Silver Spring, Md.). Using digital photographs taken on day 12, flap survival was calculated as the ratio (percentage) of the pixel number of the viable surface area to the pixel number of the total graft surface area. Assessment of Microcirculation in Composite Grafts To assess relative changes in blood perfusion over time, percutaneous blood flow was measured by laser Doppler flowmetry (Peri-Flux System 5000; Perimed Inc., Stockholm, Sweden)18 on days 1, 3, 6, 9, and 12 after repositioning of the graft. For at least 1 minute, a stable signal (blood flow) was traced and recorded at three different viable portions on each graft. Blood flow was analyzed using Perisoft programs (Perimed) and quantified as perfusion units. Immunohistochemical Analysis The animals were killed 12 days after surgery for histologic examination. The surviving part of the grafted tissues was harvested from the same position in each model. Tissue specimens were embedded in paraffin after fixation in 10% formalin. Immunohistochemical staining of endothelial cells was carried out using anti-CD31 antibody [anti-mouse platelet endothelial cell adhesion molecule-1 (PECAM/CD31) polyclonal
antibody (M20; Santa Cruz Biotechnology, Santa Cruz, Calif.)] and anti-VEGF antibody (rabbit anti-VEGF, RB-222-P; Laboratory Vision, Fremont, Calif.). To quantify the amount of neovascularization, CD31+ vessels were counted by two blinded observers under a high-power field (200× magnification). Using computer-assisted planimetry (Metamorph; Universal Image Corp., Buckinghamshire, United Kingdom), expression levels of VEGF were analyzed quantitatively. Statistical Analysis The results were expressed as means ± SEM. Statistical analysis was performed with a two-tailed, paired t test. Differences were considered statistically significant at p < 0.05. SAS Version 9.1.3 (SAS Institute, Inc., Cary, N.C.) was used for statistical analysis.
RESULTS Effect of Platelet-Rich Plasma on Composite Graft Survival On examination of graft survival 12 days after surgery, graft necrosis was observed to extend from nearly every centered area of the grafts in both groups, whereas around the peripheral edge, the viable area was larger in the platelet-rich plasma group. Representative results for each group are shown in Figure 2. On day 12 after surgery, necrotic areas were clearly demarcated in the graft. Survival rates were analyzed by assessing the viable area compared with the total graft area at postoperative day 12. The average surviving area in the composite graft was 46.49 ± 5.50 percent in the platelet-rich plasma group and 30.13 ± 6.65 percent in the control group, which was a statistically significant difference (p < 0.01) (Fig. 3). Effect of Platelet-Rich Plasma on the Microcirculation in Composite Grafts Doppler measurements were carried out before surgery to establish normal blood flow. They were not significantly different between the two groups. Postoperative measurements were performed immediately after the operation and on days 1, 3, 6, 9, and 12 after surgery. Blood flow varied over time, and progressive improvements were observed after surgery in both groups. There were no significant differences in perfusion between the groups until day 3. Interestingly, in the platelet-rich plasma group, blood flow was significantly increased compared with the control group 3 days after the operation;
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Fig. 2. The surviving areas of the grafts in the control group (above) and the platelet-rich plasma group (below). The congestive color of the graft gradually progressed in the control group, whereas congestion improved from days 1 to 3 in the platelet-rich plasma group. By postoperative day 12, necrotic areas were clearly demarcated in the grafts.
Effect of Platelet-Rich Plasma on VEGF Expression in Composite Grafts The MetaMorph imaging analyzer was used for semiquantitative analysis of VEGF expression levels. Injection of platelet-rich plasma around the chondrocutaneous composite graft induced a significant increase in VEGF expression 12 days after surgery (control group, 2849.39 ± 466.97; platelet-rich plasma group, 5003.29 ± 473.30; p
