SURGICAL ONCOLOGY AND RECONSTRUCTION
Use of Platelet-Rich Plasma Solution Applied With Composite Chondrocutaneous Graft Technique: An Experimental Study in Rabbit Model Kamuran Zeynep Sevim, MD,* Memet Yazar, MD,y Fatih Irmak, MD,z Merva Soluk Tekkes¸in, MD,x Kemalettin Yildiz, MD,k and Selami Serhat Sirvan, MD{ Purpose:
Composite chondrocutaneous grafts have been used widely for patients with cleft lip nasal deformity, alar defects, and septal perforations; however, the graft viability can be easily compromised. The aim of the present study was to extend the safe length of the composite chondrocutaneous grafts by enhancement of angiogenesis and re-epithelialization through platelet-rich plasma (PRP) and to investigate the changes that occur when PRP is administered to the graft and the recipient site.
Materials and Methods:
Composite grafts of critical sizes (1.5, 2.0, and 2.5 cm) were planned on the rabbit ears on 1 side. Group A consisted of grafts pretreated with PRP, group B consisted of recipient beds pretreated with PRP, and group C was the control group in which defects 1.5, 2.0, and 2.5 cm in size were formed on the right ears of the rabbits. On postoperative day 7, matching size chondrocutaneous grafts were adapted to the defect areas without PRP. In all groups, graft viability was evaluated 7 days after graft adaptation in group C and 14 days after PRP administration in groups A and B. Wound healing was scored histopathologically and immunohistologically using hematoxylin and eosin, CD34, and smooth muscle actin staining. The terminal transferase fluorescein-dUTP nick end labeling assay was performed to quantitatively demonstrate the apoptosis ratio among the groups.
Results:
In groups A, B, and C, the mean graft survival of the 2.0-cm equilateral triangle-shaped composite grafts was 65.43% 15.7%, 78.12% 12.8%, and 41.31% 37.4%, respectively (P = .0364).
Conclusions: PRP pretreatment accelerated composite graft survival in the 2.0-cm equilateral triangle grafts by increasing epithelial regeneration and fibrosis, inducing neovascularization, and ameliorating apoptosis rates. Ó 2014 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg -:1-13, 2014
Reconstruction of full-thickness defects, including cartilage loss, presents several challenges. Composite chondrocutaneous grafts have been a useful step in the reconstructive ladder in patients with cleft lip nasal deformity for elongation of the columella and augmentation of the nostril sill and alar base, alar
reconstruction after tumor resection, septal perforations after aggressive septoplasty, nipple areola reconstruction, and restoration of the eyelids.1-4 It is a relatively easy, malleable technique that has been especially useful for the reconstruction of tissue that has a free border, such as the alar rim or eyelids. In
*Attending Surgeon, Department of Plastic and Reconstructive
{Attending Surgeon, Department of Plastic and Reconstructive
Surgery, Sisli Etfal Research and Training Hospital, Istanbul, Turkey.
Surgery, Sisli Etfal Research and Training Hospital, Istanbul, Turkey.
yAttending Surgeon, Department of Plastic and Reconstructive Surgery, Sisli Etfal Research and Training Hospital, Istanbul, Turkey.
Address correspondence and reprint requests to Dr Sevim: Ornek Mahallesi Ercument Batanay Caddesi Ikon Bloklari A3 Blok D:11,
zAttending Surgeon, Department of Plastic and Reconstructive
Atas¸ehir, Istanbul, Turkey; e-mail: [email protected]
Surgery, Sisli Etfal Research and Training Hospital, Istanbul, Turkey. xAssistant
Professor,
Department
of
Pathology,
Received October 23 2013
Istanbul
Accepted January 4 2014 Ó 2014 American Association of Oral and Maxillofacial Surgeons
University Oncology Institute, Istanbul, Turkey. kAssistant Professor, Department of Plastic and Reconstructive
0278-2391/14/00071-8$36.00/0
Surgery, Bezm-i Alem Vakıf University, Istanbul, Turkey.
http://dx.doi.org/10.1016/j.joms.2014.01.001
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our current practice, its use has been limited by the dimensions of the viable composite graft, 1 cm, because the graft will be nourished by the surrounding tissues and requires a well-vascularized recipient bed. Cartilage is an important supporting tissue that is avascular, and its perfusion is dependent on diffusion from the peripheral tissues. The composite chondrocutaneous graft’s vascularity will be determined by the vascular anastomosis established at the subdermal plexus level between the graft and recipient bed.5,6 The survival of these composite grafts has been limited by the imbibition and inosculation rates from the narrow wound edges and the recipient bed. The largest size of composite grafts defined in published studies has been a 2.5-cm equilateral triangle graft; however, in routine practice, the size has not exceeded 1 to 1.5 cm.7 To enhance the survival length of these grafts, a number of surgical and pharmacologic techniques have been developed. Among some of the surgical techniques there has been increasing the contact surface by de-epithelialization of the recipient bed, adapting the graft in a ‘ tongue-in-groove’’ fashion, and enhancing the vascularity of the recipient bed with a dermal ‘ turnover’’ flap or graft cooling.8-12 The reported expedient pharmacologic survival enhancement methods for composite grafts have included corticosteroids, dimethyl sulfoxide, dimethylurea, melatonin, indometacin, fibroblast growth factor, chlorpromazine, and hyperbaric oxygen therapy.13 Platelet-rich plasma (PRP) is rich in vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-a and -b), transforming growth factor (TGF)-a and TGF-b, epidermal growth factor, fibroblast growth factor, and insulin-like growth factor (IGF), which has been depleted from the erythrocytes and leukocytes by centrifugation as a biologic product.14 PRP has a high concentration of growth factors and cytokines initially secreted by platelets and leukocytes and later amplified by macrophages, including a large number in endothelial cells and migrating fibroblasts. PRP has been proved to be effective in wound and fracture healing, accelerating the proliferative phase in tendon healing, tendon regeneration, and tension strength of the tendons.15-21 Moreover, Anitua et al22 and Blanton et al23 demonstrated that the growth factors in PRP most efficiently enhanced fibroblast chemotaxis, neovascularization, collagen synthesis, and vessel densities after 2 weeks of administration, in accordance with elevated VEGF secretion. PRP is an autologous, safe, easily prepared bioproduct that is more cost-effective than other recombinant technique products.14,19 The present experimental study aimed at extending the safe length of composite chondrocutaneous grafts by enhancement of angiogenesis and re-epithelialization through the myriad of factors in PRP. We also investigated
the changes that occurred after PRP administration to the graft. In addition, the influence of the recipient site of the graft was examined. Determining the effect of PRP on attenuating programmed cell death was also targeted using the terminal transferase fluorescein-dUTP nick end labeling (TUNEL) assay.
Materials and Methods The animal ethics committee provided approval before the study began, and the animals were cared for in accordance with the protocols approved by the institutional animal care and use committee (reference no. 21.02.2012/74). This experiment was conducted using 31 New Zealand white rabbits aged 14 to 18 weeks (mean 14.5 0.3) and weighing 1.8 to 2.2 kg (mean 2.0 0.1). All the rabbits were screened for common diseases and had not been subjected to any experiments before the present study. EXPERIMENTAL MODEL
The animal model used in the present study was adapted from an experimental study conducted by Karaca and Gursu.8 The rabbits were divided into 3 groups, with 10 rabbits in each group: 1.5-, 2.0-, and 2.5-cm size triangle-shaped composite chondrocutaneous grafts or defects were created. One rabbit was isolated to provide a normal pathologic control view of the rabbit ear model (Fig 1). In group A (graft pretreated with PRP, n = 10 rabbits), a ‘ prepared’’ graft 1.5, 2.0, and 2.5 cm in size was planned for transfer to fresh beds. A ‘ prepared’’ graft was a graft treated with PRP gel on all 3 sides of the 1.5-, 2.0-, and 2.5-cm equilateral triangle of the graft (0.5 mL on each side of the triangle). In group B (recipient bed pretreated with PRP, n = 10 rabbits), a ‘‘prepared’’ recipient bed 1.5, 2.0, and 2.5 cm in size was planned for reconstruction by the fresh grafts. A prepared recipient bed was one that had been treated with PRP gel on all 3 sides (0.5 mL on each side of the triangle), after forming an equilateral triangle-shaped defect. On the day of grafting, the epithelium, scar, or granulation tissue was excised from the sides. In group C (control group, n = 10 rabbits), fullthickness triangle-shaped defects, 1.5, 2.0, and 2.5 cm in size, were created on the right ears of the rabbits. Seven days later, matching size chondrocutaneous grafts were adapted to the defect areas without PRP. EXPERIMENTAL PROTOCOL
Ketamine hydrochloride (50 mg/kg; Ketalar, Pfizer, Istanbul, Turkey) and xylazine hydrochloride (3 mg/kg; Rompun, Bayer, Leverkusen, Germany) were administered intramuscularly for anesthesia. After shaving the rabbit ear with an electric shaver, the surgical field
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FIGURE 1. A, Application of platelet-rich plasma gel to the central part of the planned composite chondrocutaneous grafts in group A. B, Equilateral 1.5-, 2.0-, and 2.5-cm triangle defects carved on rabbit ears and platelet-rich plasma (PRP) solution injected to the sides 7 days before grafting in group B. C, Group C (control group) with 1.5-, 2.0-, 2.5-cm fresh defects and 7 days after grafting with grafts from the opposite ear. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
was cleaned with povidone-iodine solution and draped. The experiment consisted of 3 stages (Fig 2). In the first stage for groups A and B, the critical size defects were created on the rabbit’s ears, and PRP gel was administered to the graft or recipient bed, followed by a 7-day waiting period for activation of platelets, observing wound healing, and angiogenesis. Mupirocin-based ointment (Bactroban 2%, GlaxoSmithKline, Istanbul, Turkey) was used to cover the ears of the rabbits, and the dressings were changed daily. For group C, the defects were created, and the wounds were covered with mupirocin and the dressings changed daily. In the second stage of the experiment (7 days after the first stage), for group A, the ‘‘prepared’’ composite grafts were transferred to the fresh beds and sutured with 5-0 Vicryl (Ethicon, Novartis, Germany) from the dorsal and ventral sides. Inflammation, angiogenesis, and graft take were observed for another 7 days. For group B, the 1.5-, 2.0-, and 2.5-cm size fresh grafts were transferred to the ‘‘prepared’’ recipient beds. Before transfer, the prepared wound edges were
scraped gently with the back of a no. 15 blade to move some of the granulation tissue away such that the transferred graft could adequately attach to the recipient site. For group C, the defect edges were scraped gently to enhance graft take, and untreated grafts were harvested from the left ears of the rabbits and adapted to the defects. These grafts were observed for 7 more days to analyze graft viability. In the third stage of the experiment, graft viability was observed macroscopically in groups A and B. Next, with the rabbits under anesthesia, biopsy samples were taken, including a strip of normal tissue from the sides of the graft for immunohistologic assessment and TUNEL assay. The animals were sacrificed using an overdose of anesthetic. PREPARATION OF PRP
PRP was prepared from each rabbit separately. A total of 10 mL of blood was drawn from the arteria carotis communis of the rabbits in groups A and B and placed
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FIGURE 2. Flow chart of the experimental stages. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
into citrate-phosphate-dextrose (CPD) solution in a 10 mL of blood to 2 mL of CPD ratio. Centrifugation of the blood to separate the plasma from the cellular products was performed for 20 minutes at 150g (1,200 rpm; Eppendorf AG 22331 centrifuge, Hamburg, Germany). The supernatant was aspirated and centrifuged for another 10 minutes at 450g (2,000 rpm). The formed bilayer of plasma, containing the upper platelet-poor plasma and a lower layer of PRP, was then separated using an aspirator. Only the lower layer was used for our experiment. Each vial of prepared PRP was sampled and sent to the laboratory for a platelet count to ensure that a high concentration of platelets was present in the prepared plasma. The number of platelets was 250,000 to 550,000 platelets/mL (mean 342,000 10,000) approximately, 4 times greater than that of the peripheral blood. The PRP obtained from the rabbits was strictly administered to the corresponding rabbit from which the peripheral blood had been obtained. Before injection into the defect site, calcium chloride and bovine thrombin were mixed with the PRP at a ratio of 1:6 for platelet activation. The calcium chloride and bovine thrombin were obtained from Tisseal, which also provided the kit (reference no. VNT 1 J008) for mixing the materials. For the procedure, a viscous material formed within 50 to 60 seconds. With the applicator syringe, the gel was injected onto the sides of the composite grafts.
MACROSCOPIC ASSESSMENT
On postoperative day 14, for all groups, each composite graft was examined macroscopically. Nonviable tissue was debrided. The amount of graft was traced on acetate paper and recorded using Image J software (National Institutes of Health, Bethesda, MD). The surviving graft area was then recorded as a percentage of the initial graft harvested. HISTOLOGIC ASSESSMENT
The harvested specimens were fixed in 10% buffered formalin solution and embedded in paraffin blocks, and 3-mm-thick sections were obtained. The mounted histologic sections were stained using hematoxylin and eosin (H&E). The histologic grade of the healed sites in terms of epithelial regeneration, inflammation, and fibrosis was scored for each specimen as none (), mild (+), moderate (++), and severe (+++), according to a scale previously described by Myers et al,24 in which the cellular elements of the wound were counted on the basis of the number per high power field to obtain a value from none () to severe (+++). Immunohistochemically, CD34 and smooth muscle actin (SMA) primer antibodies were used to determine neovascularization. For immunohistochemistry, paraffin blocks prepared from routinely processed specimens were cut into 5-mm-thick
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sections on the charged slides and deparaffinized. For antigen retrieval, the sections were microwaved and endogenous peroxidase activity was blocked by incubating the sections with hydrogen peroxide. CD34 antibody (ready to use, qbeND/10, Thermo Scientific, Cheshire, UK) and SMA (ready to use, 1A4, Thermo Scientific) were applied. After this process, biotinylated secondary antibody (HRP Sc – 2030, Lot no. D1504, Santa Cruz Biotechnology, Santa Cruz, CA), streptavidin peroxidase, and substrate-chromogen solution were applied. Nuclear staining was conducted with hematoxylin. The staining intensity was defined as a percentage and given a score ranging from 1 to 100%: 0 (), 10 to 30% (+), 30 to 60% (++), and more than 60% (+++). The examination was performed by a pathologist who was unaware of the study groups. Using the TUNEL assay, analysis of DNA-fragmented cells from the sections harvested from the rabbits was performed using a commercially available kit (11684795910, Roche USA, New York, NY). In these sections, DNA-fragmented cells were counted in a blinded manner in 4 randomly defined regions (each 62,500 mm2). Apoptotic cells were identified by the characteristics of nuclear condensation and margination. STATISTICAL ANALYSIS
The results were analyzed using nonparametric analysis methods (Kruskal-Wallis and Mann-Whitney U tests) because of the small sample size. The Statistical Package for Social Sciences, version 20.0 (SPSS, Chicago, IL), was used for the analyses. Significant differences were considered present for P < .05.
Results No rabbit deaths or complications were observed during the entire study period. One of the composite grafts, 1.5 cm in size, presented with wound dehiscence followed by graft failure. No signs of autophagy of the ears were observed among the rabbits because the wounds had been dressed with mupirocin and covered with sponges. The platelet counts derived from the whole blood samples were elevated from 157,000 43,000 platelets/mL to an average of 425,000 37,000 platelets/mL. Regarding the composite graft survival ratios, no significant difference was found for the 1.5-cm grafts. Thus, in group A (n = 6), with the grafts prepared with PRP, the survival rate of the 1.5-cm grafts was 82.8% 15.4%, and in group B (n = 6), with the recipient bed prepared with PRP, the graft survival rate was 79.8% 12.1%. In group C, with the composite grafts transferred without any PRP treatment to the recipient beds, the survival rate was 74.4% 10.3%.
For the 2-cm composite grafts, a significant increase was seen in the percentage of survival in group A (65.43% 15.7%). In group B, the graft survival rate was 78.12% 12.8%. In the control group, group C, however, the survival rate was a dismal 41.31% 37.4% (Figs 3, 4). These results have shown that preparing the grafts or recipient bed with PRP solution 7 days before elevating the grafts increased the survival length of the composite grafts 2 cm in size. However, no significant difference was found between groups A and B (Fig 5). For the composite 2.5-cm grafts, significant graft survival was observed in group B (fresh graft to PRP-prepared recipient bed) compared with groups A and C. In group B, 65.4% 11% of the composite grafts survived. However, in group A, the survival rate was 43% 10.3%, and in group C, it was only 10.5% 5.8%. HISTOLOGIC ASSESSMENT RESULTS
The histologic assessment results revealed that groups A and B displayed a significantly increased rate of epithelial regeneration (P < .005) compared with the control group. However, when comparing groups A and B with each other, no significant difference was observed in epithelial regeneration (P > .05). The inflammation rates for the groups were also not significantly different (P > .05). In groups A and B, fibroblast proliferation was significantly different (P < .05). The inflammation, fibrosis, and epithelial regeneration rates for the 1.5-, 2.0- and 2.5-cm composite chondrocutaneous grafts are shown in Figure 6. In groups A and B, the 1.5-cm grafts histologically presented with regular wound healing and elastic cartilage formation and fibrosis. The grafts 2.0 cm in size displayed a thin epithelium with regenerative properties, and the 2.5-cm size grafts displayed partial necrotic debris
FIGURE 3. Immunohistochemical CD34 staining of the endothelial cells and precursors in group A. Original magnification 400. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
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FIGURE 4. Microscopic view of control group 2.0-cm composite grafts 7 days after grafting. Asterisk indicating ulcerated epithelium and granulation tissue and disintegrated cartilage tissue. Hematoxylin and eosin, original magnification x100. Microscopic view of group A 2.0-cm composite grafts 7 days after grafting. Asterisk shows mild inflammation in the dermis, fibrosis, and epithelial regeneration. Hematoxylin and eosin, original magnification x100. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
along the center part of the graft. In the control group, however, the 2.0- and 2.5-cm grafts presented with an ulcerated epithelium and granulation tissue, minimal fibrosis, and large amounts of debris (Figs 7, 8). To assess how inflammation contributes to wound healing, the H&E-stained slides were evaluated by the characteristics of the inflammatory cells in the grafts and the quality of the granulation tissue and fibrosis. A loss of integrity was seen from the epidermis to the
hypodermis, with inflammatory cellular infiltration and increased polymorphonuclear leukocytes in the control group specimens. In groups A and B, moderate histiocytic and neutrophilic infiltration, with notably increased amount of fibroblasts and macrophages, was seen. The density of the macrophage-type cells was significantly greater in groups A and B compared with that in the control group. Interstitial edema was seen more frequently in the control group slides.
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FIGURE 5. Microscopic view of group A 2.5-cm composite grafts, 7 days after grafting showing ulcerated epithelium and mounting debris with disintegrated cartilage tissue. Hematoxylin and eosin, original magnification x100. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
The control group 2.0- and 2.5-cm grafts displayed a viability percentage of 52% 8.1% and 22% 8.0%, respectively. IMMUNOHISTOCHEMICAL RESULTS
The CD34 and SMA-stained slides were scored in terms of angiogenesis, and the results confirmed that
the neovascularization scores in the PRP-prepared groups (groups A and B) displayed 30 to 60% (++) staining, which was a significant result compared with the control group (P < .001; Figs 6, 7). In the angiogenesis comparison of the 1.5-, 2.0-, and 2.5-cm composite grafts prepared with PRP, it was observed that in 2.0-cm composite graft, angiogenesis was
FIGURE 6. Immunohistochemical SMA presentation of neovascularization in groups A and B, 7 days after grafting. Original magnification, x400. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
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FIGURE 7. Immunohistochemical CD 34 staining of the endothelial cells and precursors in group A. Original magnification 400. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
occurring (++) in 60% of the grafts. This result was also significant compared with the angiogenesis in the other sizes of composite grafts (P < .001). Microscopic slides of the 2.0-cm composite grafts (in groups A and B) displayed endothelial cell progenitors stained with CD34- and SMA-stained neovascularization. Also, pronounced granulation reaction was present around the vessels and was accompanied by fibroblastic proliferation, edema, and reparative processes in the dermis of groups A and B. TUNEL ASSAY RESULTS
Positive TUNEL-stained cells were detected and quantified in the composite chondrocutaneous grafts during wound healing in all groups. The TUNELpositive cell number was significantly decreased by PRP treatment in the 2.0-cm chondrocutaneous grafts from groups A and B compared with that in the control group (Table 1; P < .05). However, no significant
difference was noted between groups A and B (P > .05). TUNEL-positive cells were stained using the commercially used kit, revealed an increase in nuclear fluorescence intensity by changing their fluorescence from dark blue (normal cells) to a bright blue, white color, indicating chromatin condensation (Figs 9, 10).
Discussion In the case of a severe tissue deficiency with a secondary cleft lip nasal deformity, it has not been easy to correct the deficiency completely using only nasal tissue. Stenosis of the nasal vestibule and septal deviation intruding in the contralateral airway will result in severe nasal obstruction. Asymmetry of the nasal base has been the most striking esthetic problem, involving the alar rim and anterior nares region. The nostril sill will often be absent in these patients. Composite
FIGURE 8. Microscopic view of group A 2.5-cm composite grafts, 7 days after grafting showing ulcerated epithelium and mounting debris with disintegrated cartilage tissue. Hematoxylin and eosin, original magnification 100. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
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Table 1. APOPTOTIC CELLS IN 2.0-CM CHONDROCUTANEOUS GRAFTS, QUANTIFIED BY TUNEL ASSAY (CELLS/62.500 2 mM )
Group Group A (PRP prepared graft; n = 10) Group B (PRP prepared recipient site; n = 10) Group C (control; n = 10)
Region 1
Region 2
Region 3
Region 4
10 3 51 16 2
32 5 18 3 45 1
45 6 50 1 125 5
60 1 37 5 65 1
P < .05, groups A and B (PRP pretreatment applied) were compared with the control group. P > .05, groups A and B were compared with each other. Abbreviations: PRP, platelet-rich plasma; TUNEL, terminal transferase fluorescein-dUTP nick end labeling. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
chondrocutaneous grafts might be placed to reconstruct all these areas. Additionally, it is well known that if a more than 5-mm disparity of columellar length is present compared with the normal side, composite grafting could be useful.25 However, patients with cleft lip nasal deformity undergo numerous operation, and the scars on the vestibular floors and alar base from the previous operations disturb the normal anatomy, making composite graft take more challenging.26 The aim of our study was to describe a surgical enhancement technique that increases the size of the available composite chondrocutaneous grafts by either preparing the grafts or the nonideal recipient beds to enhance angiogenesis and fibrosis using a PRP solution. Composite grafts, by definition, are composed of 2 or more germ layers of tissue or 2 separate tissues (ie, skin + cartilage, mucosa + cartilage, skin + fat, skin + mucosa + nail, skin + fat + periosteum). The principles of reconstruction with composite grafts were defined by Konig in 1902.27 Interest in composite grafts was revitalized in 1946 by Dupertuis,28 when 15 nasal reconstructions were successfully performed with composite grafts prepared from the ear lobule. Composite grafts can be harvested from the ear lobule, helical rim, root of helix, nipple areola, or nail incorporated into skin and mucosa.29-31 Composite tissue grafts have been used as a convenient and esthetic method of head and neck reconstruction, nipple areola reconstruction, anophthalmic socket reconstruction, and finger pulp reconstruction for more than 1 century.4,7 Anastomosis of the recipient and donor vascular tissue on the subdermal plexus level is an essential aspect of the success of composite grafts.32,33 McLaughlin34 defined the life cycle of a composite graft and stated that they initially present with a dead white color when harvested, followed by a pale pink color after 6 hours, cyanotic color until 5 days after transplant, and healthy pink within approximately 10 days. The disadvantages of this useful, relatively easy, reconstructive option is that the available size of composite grafts has been limited to 1 cm, color discrepancies occur, composite grafts tend to shrink 10 to 20%, and it is challenging to the inexperienced sur-
geon to properly evaluate the life cycle of the graft. Within the published data, multiple surgical treatment modalities have been proposed to increase the viability of composite grafts. In 1977, MacCollum and Grabb5 applied the concept of the advanced preparation of the recipient bed to composite grafts with Rhesus monkeys. They demonstrated 88% survival in auricular composite transfers when the recipient bed was ‘‘prepared’’ 10 days in advance versus 22% in fresh composite transfers. In our experiment, the preparation period was 7 days, and this period was shorter than the 10- to 11-day delay period in the study by MacCollum and Grabb.5 Park et al35 in 1996 studied the survival rate of composite grafts relative to the graft size in a rabbit ear model. They reported that from 5 to 15 mm, the percentage of survival area did not display any decrease relative to the increased graft size, although it did decrease with a graft size of 17 mm.35 Our study challenged the results of that study because we compared the graft survival of 1.5-, 2.0-, and 2.5-cm size composite grafts with PRP solution enhancement. Chandawarkar et al36 advocated harvesting a larger skin island relative to cartilage, in the belief that more perichondrium of the cartilage extension will be in contact with the overlying undermined dermis. Keck et al12 and Pilanci et al11 suggested enhancing the healing of composite grafts by locoregional skin flaps or dermal turnover flaps. Eley et al37 and Rashid et al38 sculptured skin tags to restore a nasal deformity using local flaps or transposition flaps. Tiengo et al39 proposed the use of dermal substitutes such as Integra (Integra Lifesciences, Plainsboro, NJ) for the reconstruction of nose tip avulsions. All these techniques, however, require a morbidity of donor site plus excessive scarring. The technique we investigated in our study is superior to these techniques, because it is less invasive and does not involve the use of neighboring tissue or expensive biomaterials, such as dermal substitutes. In the design of the experiment, realizing that the ear’s vascularity might differ from the proximal to distal ends, the 1.5-cm size defects were placed closer
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FIGURE 9. A, Terminal transferase fluorescein-dUTP nick end labeling (TUNEL) assay micrograph of group A grafts. Arrow shows the nuclear decomposition and chromatin condensation. B, TUNEL assay micrograph of group B grafts. Arrow shows the nuclear decomposition and chromatin condensation. (Fig 9 continued on next page.) Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
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FIGURE 9 (cont’d). C, TUNEL assay micrograph of group C grafts. Arrow shows the increased amount of nuclear decomposition and chromatin condensation (P < .05). Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
to the helical root. The largest size defect, 2.5 cm, was placed in the middle, and 2.0-cm size defect was placed at the distal end. The order and placement of the created defects were the same for all rabbits and all composite grafts among the groups.
FIGURE 10. Quantitative analysis of terminal transferase fluorescein-dUTP nick end labeling (TUNEL)-positive cells per mm2 among all groups. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
Three main factors contribute to angiogenesis: formation of new blood vessel sprouts owing to inflammatory wound healing responses, endogenous release of angiogenic growth factors, and exogenous release of angiogenic growth factors, the preferred method of angiogenesis targeted with PRP therapy.40 It has been established that PRP releases growth factors and contains a high concentration of PDGF-a and PDGF-b. TGF-b, IGF, and fibrillar 3-dimensional scaffolds formed by fibrinogen from activated PRP that easily biodegrades and is a safe autologous biomaterial. The platelet lifespan is approximately 7 to 9 days. Therefore, in our experiment, we waited 7 days after the PRP had been administered. Many discrepancies are present in terms of PRP preparation, and standardization of the techniques has been lacking throughout published studies. Lower platelet concentrations can lead to suboptimal effects, and greater concentrations might have an inhibitory affect by secreting thrombospondin-1, which interferes with mitogenic effects of PRP.39 Platelets modulate inflammation by their ability to secrete a variety of chemokines. These chemokines control the amount of accumulation of leukocytes and monocytes in the injured tissue.18 Remembering this, one can deduce that the
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chemokines secreted from PRP in addition to the myriad of factors released from the prepared recipient site should result in an increased graft take percentage. In our study, the group B composite grafts had the greatest survival rate, although, statistically, nonsignificant results were obtained. The quantification of the concentration of some chemokines that platelets augment such as CXCL-5, monocyte chemoattractant protein 1, and interleukin-8 could be investigated in an additional study using this chondrocutaneous graft model.41 The results obtained from the present study revealed that 2.0-cm equilateral triangle chondrocutaneous grafts prepared with bovine thrombin and calcium chloride-enhanced PRP gel survived at a rate of 65.43% 15.7% in the groups in which PRP was administered to the graft and 78.12% 12.8% in the groups in which it was administered to the recipient bed. Both were significantly greater than that of the control group, for which the graft survival rate decreased to 41.3% 37.4% (P = .0364). Therefore, we deduced that the location of PRP administration (either the graft or recipient bed) is not crucially important, although it was minimally more effective in group B, as long as adequate angiogenesis can be achieved. Immunohistochemical analysis of CD34 and SMA staining revealed that in the PRP-prepared groups (groups A and B), a moderate rate of neovascularization (60%) in the graft specimens was observed and was significantly different from that of the control group (P > .005). In the present study, we demonstrated that the safe length of composite chondrocutaneous grafts can be extended to 2.0 cm with the application of autologous-prepared PRP 7 days before transfer. Furthermore, the TUNEL assay results obtained from our study have clearly shown that PRP pretreatment, to either the recipient site or the graftameliorated apoptosis, such that the available graft size can be increased to 2.0 cm using PRP. PRP contains a high concentration of growth factors and cytokines initially secreted by platelets and leukocytes and later amplified by macrophages, including a rapid increase in endothelial cells and migrating fibroblasts. The evident hypoxic environment within the composite graft will also increase angiogenesis. Secreted growth factors, including TGF-b, PDGF-b, and angiopoietin-1, are important for vessel stabilization.22 A comparison of angiogenesis among the 1.5-, 2.0-, and 2.5-cm composite grafts revealed that in the 2.0-cm composite grafts, angiogenesis was occurring (++) in 60% of the grafts. This result was significantly greater than the angiogenesis rate of the other composite graft sizes (P < .001). The initial in vivo experiments involving PRP were in the field of oral and maxillofacial surgery and in dentistry, in particular, periodontal therapy. As Marx et al42 reported, PRP has been used to allow denser bone regeneration and improving the contact surface of the
implants. Relating this to our study, we can conclude that larger size composite grafts prepared with PRP can be used with our method, to prepare dental implant sites, mandibular defects forming after removal of jaw cysts, anophthalmic sockets, or augmentation of maxillary sinus floors in elderly patients owing to its fibrinforming capacity and enhancing angiogenesis. In a study by Wei et al,43 in 2007, chondrocytes harvested from the auricular root of New Zealand rabbits were cultured, harvested, and prepared with PRP as autologous injectable scaffolds to cartilage defects. However, our technique is more feasible and easily obtained than that method. In conclusion, the results obtained from our study support the report by MacCollum and Grabb5 and indicate an advantage from advanced preparation of the recipient bed and not the graft itself. The repeated application of PRP or any drug delivery system might be practical for clinical application from the perspective of safety and invasiveness. A 0.5-cm increase in the possible graft size, and a possible 1.5-cm increase, is quite significant, because these grafts are mainly used in alar reconstruction or alar base reconstruction. The present study reports a reliable and easy-to-use rabbit model for studying composite graft survival for various sizes of grafts. Our aim was to initiate the clinical use of PRP in patients with cleft lip nasal deformity for whom extended-size composite grafts are in demand.
References 1. Cho BC, Park JW, Baik BS: Correction of severe secondary cleft lip nasal deformity using a composite graft: Current approach and review. Ann Plast Surg 48:131, 2002 2. Douglas B: Successful replacement of completely avulsed portions of fingers as composite grafts. Plast Reconstr Surg 23: 213, 1959 3. Adams WM: Free transplantation of the nipples and areola. Surgery 34:140, 1944 4. Zampar AG, Salomons RL, Dornelles RF, et al: Chondrocutaneous auricular graft for reconstruction of ocular socket in anophthalmic cavities. J Craniofac Surg 22:602, 2011 5. MacCollum M, Grabb W: Increasing the incidence and the size of successful experimental composite ear grafts by advance preparation of the recipient bed. Plast Reconstr Surg 60:759, 1977 6. Musgrave RH, Lehman JA: Composite grafts, in Georgiade GS, Rieffkohl R, Levin LS, (eds). Georgiade Textbook of Plastic, Maxillofacial and Reconstructive Surgery (ed 2). Baltimore, Williams & Wilkins, 1992, pp 37–38 7. Davenport G, Bernard FD: Improving the take of composite grafts. Plast Reconstr Surg 24:175, 1959 8. Karaca C, Gursu G: The effects of delaying composite cartilage grafts and their recipient bed on the graft survival. Eur J Plast Surg 17:247, 1994 9. Fann PC, Hartman DF, Goode RL: Pharmacologic and surgical enhancement of composite graft survival. Arch Otolaryngol Head Neck Surg 119:313, 1993 10. Conley JJ, Von Fraenkel PH: The principle of cooling as applied to the composite graft in the nose. Plast Reconstr Surg 17:444, 1956 € Sac¸ak B, Kuvat SV, et al: Use of outsized composite 11. Pilanci O, chondrocutaneous grafts in conjunction with dermal turnover
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12.
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16.
17.
18. 19.
20.
21.
22.
23.
24. 25.
26.
flaps for reconstruction of full-thickness alar defects. J Craniofac Surg 22:864, 2011 Keck T, Lindemann J, K€ uhnemann S, Sigg O: Healing of composite chondrocutaneous auricular grafts covered by skin flaps in nasal reconstructive surgery. Laryngoscope 113:248, 2003 En Li, Menon NG, Rodriguez ED, et al: The effect of hyperbaric oxygen therapy on composite graft survival. Ann Plast Surg 53: 141, 2004 Eppley BL, Pietrzak WS, Blanton M: Platelet-rich plasma: A review of biology and applications in plastic surgery. Plast Reconstr Surg 118:147e, 2006 Eppley BL, Woodell JE, Higgins J: Platelet quantification and growth factor analysis from platelet-rich plasma: Implications for wound healing. Plast Reconstr Surg 114:1502, 2004 Anitua E, Sanchez M, Nurden AT, et al: New insights into and novel applications for platelet-rich fibrin therapies. Trends Biotechnol 24:227, 2006 Lyras D, Kazakos K, Verettas D, et al: Immunohistochemical study of angiogenesis after local administration of plateletrich plasma in a patellar tendon defect. Int Orthop 34:143, 2010 Andia I, Sanchez M, Maffulli N: Tendon healing and platelet rich plasma therapies. Expert Opin Biol Ther 10:1415, 2010 Kanthan SR, Kavitha G, Addi S, et al: Platelet-rich plasma (PRP) enhances bone healing in non-united critical-sized defects: A preliminary study involving rabbit models. Injury Int J Care Injured 42:782, 2011 Findikcioglu K, Findikcioglu F, Yavuzer R, et al: Effect of plateletrich plasma and fibrin glue on healing of critical-size calvarial bone defects. J Craniofac Surg 20:34, 2009 Roldan JC, Jepsen S, Miller J, et al: Bone formation in the presence of platelet-rich plasma vs. bone morphogenetic protein7. Bone 34:80, 2004 Anitua E, Sanchez M, Orive G, Andıa I: The potential impact of the preparation rich in growth factors (PRGF) in different medical fields. Biomaterials 28:4551, 2007 Blanton MW, Hadad I, Johnstone BH, et al: Adipose stromal cells and platelet-rich plasma therapies synergistically increase revascularization during wound healing. Plast Reconstr Surg 123(2 suppl): S64, 2009 Myers AH, Postlethwait RW, Smith AG: Histologic grading of the experimental healing wound. Arch Surg 83:771, 1961 Ayhan M, Gorgu M, Erdogan B, et al: Various applications of chondrocutaneous composite grafts in secondary cleft lip nose patients. J Craniofac Surg 17:1065, 2006 Brown JB, Cannon B: Composite free grafts of the skin and cartilage from the ear. Surg Gynecol Obstet 82:253, 1946
13 27. Koenig F: Zur Deckung von Defekten der Nasenflugel. Berl Klin Wochenschr 39:137, 1902 28. Dupertuis SM: Free ear lobe grafts of skin and fat. Plast Reconstr Surg 1:135, 1946 29. Meade RJ: Composite ear grafts for construction of columella. Plast Reconstr Surg 23:134, 1959 30. Ozek C, Gurler T, Uckan A, Bilkay U: Reconstruction of the distal third of the nose with composite ear-helix free flap. Ann Plast Surg 58:74, 2007 € € Kayalı MU, et al: Preliminary study on 31. Karsidag S, Ozkaya O, durability of nail as xenograft. J Craniofac Surg 21:1843, 2010 32. Ozaki W, Chaffoo RA, Vu KC, Markowitz BL: Comprehensive rhinoplasty technique to correct the bilateral cleft lip nasal deformity using conchal composite grafts. J Craniomaxillofac Surg 34:150, 2006 33. Musgrave RH: Surgery of nasal deformities associated with cleft lip. Plast Reconstr Surg 28:261, 1961 34. McLaughlin CR: Composite ear grafts and their blood supply. Br J Plast Surg 7:274, 1954 35. Park JB, Jang HJ, Hong IP, et al: Survival rate of composite graft according to graft size and microscopic examination: Experimental study on ear of rabbit. J Korean Soc Plast Reconstr Surg 23:1204, 1996 36. Chandawarkar RY, Cervino AL, Wells MD: Reconstruction of nasal deformities using modified composite grafts. Br J Plast Surg 56:26, 2003 37. Eley KA, Pleat JM, Wall SA: Reconstruction of a congenital nasal deformity using skin tags as a chondrocutaneous composite graft. J Craniofac Surg 20:573, 2009 38. Rashid M, Islam MZ, Tamimy MS, et al: Rotation-transposition correction of nasal deformity in Tessier number 1 and 2 clefts. Cleft Palate Craniofac J 46:674, 2009 39. Tiengo C, Amabile A, Azzena B: The contribution of a dermal substitute in the three-layer reconstruction of a nose tip avulsion. J Plast Rec Aesth Surg 65:573, 2012 40. Dong Z, Li B, Liu B, et al: Platelet-rich plasma promotes angiogenesis of prefabricated vascularized bone graft. J Oral Maxillofac Surg 70:2191, 2012 41. Flad H-D, Brandt E: Platelet-derived chemokines: Pathophysiology and therapeutic aspects. Cell Mol Life Sci 67:2363, 2010 42. Marx RE, Carlson ER, Eichstaedt RM, et al: Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85:638, 1998 43. Wu W, Fulin C, Yanpu L, et al: Autologous injectable tissueengineered cartilage by using platelet-rich plasma: Experimental study in a rabbit model. J Oral Maxillofac Surg 65: 1951, 2007
Use of Platelet-Rich Plasma Solution Applied With Composite Chondrocutaneous Graft Technique: An Experimental Study in Rabbit Model Kamuran Zeynep Sevim, MD,* Memet Yazar, MD,y Fatih Irmak, MD,z Merva Soluk Tekkes¸in, MD,x Kemalettin Yildiz, MD,k and Selami Serhat Sirvan, MD{ Purpose:
Composite chondrocutaneous grafts have been used widely for patients with cleft lip nasal deformity, alar defects, and septal perforations; however, the graft viability can be easily compromised. The aim of the present study was to extend the safe length of the composite chondrocutaneous grafts by enhancement of angiogenesis and re-epithelialization through platelet-rich plasma (PRP) and to investigate the changes that occur when PRP is administered to the graft and the recipient site.
Materials and Methods:
Composite grafts of critical sizes (1.5, 2.0, and 2.5 cm) were planned on the rabbit ears on 1 side. Group A consisted of grafts pretreated with PRP, group B consisted of recipient beds pretreated with PRP, and group C was the control group in which defects 1.5, 2.0, and 2.5 cm in size were formed on the right ears of the rabbits. On postoperative day 7, matching size chondrocutaneous grafts were adapted to the defect areas without PRP. In all groups, graft viability was evaluated 7 days after graft adaptation in group C and 14 days after PRP administration in groups A and B. Wound healing was scored histopathologically and immunohistologically using hematoxylin and eosin, CD34, and smooth muscle actin staining. The terminal transferase fluorescein-dUTP nick end labeling assay was performed to quantitatively demonstrate the apoptosis ratio among the groups.
Results:
In groups A, B, and C, the mean graft survival of the 2.0-cm equilateral triangle-shaped composite grafts was 65.43% 15.7%, 78.12% 12.8%, and 41.31% 37.4%, respectively (P = .0364).
Conclusions: PRP pretreatment accelerated composite graft survival in the 2.0-cm equilateral triangle grafts by increasing epithelial regeneration and fibrosis, inducing neovascularization, and ameliorating apoptosis rates. Ó 2014 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg -:1-13, 2014
Reconstruction of full-thickness defects, including cartilage loss, presents several challenges. Composite chondrocutaneous grafts have been a useful step in the reconstructive ladder in patients with cleft lip nasal deformity for elongation of the columella and augmentation of the nostril sill and alar base, alar
reconstruction after tumor resection, septal perforations after aggressive septoplasty, nipple areola reconstruction, and restoration of the eyelids.1-4 It is a relatively easy, malleable technique that has been especially useful for the reconstruction of tissue that has a free border, such as the alar rim or eyelids. In
*Attending Surgeon, Department of Plastic and Reconstructive
{Attending Surgeon, Department of Plastic and Reconstructive
Surgery, Sisli Etfal Research and Training Hospital, Istanbul, Turkey.
Surgery, Sisli Etfal Research and Training Hospital, Istanbul, Turkey.
yAttending Surgeon, Department of Plastic and Reconstructive Surgery, Sisli Etfal Research and Training Hospital, Istanbul, Turkey.
Address correspondence and reprint requests to Dr Sevim: Ornek Mahallesi Ercument Batanay Caddesi Ikon Bloklari A3 Blok D:11,
zAttending Surgeon, Department of Plastic and Reconstructive
Atas¸ehir, Istanbul, Turkey; e-mail: [email protected]
Surgery, Sisli Etfal Research and Training Hospital, Istanbul, Turkey. xAssistant
Professor,
Department
of
Pathology,
Received October 23 2013
Istanbul
Accepted January 4 2014 Ó 2014 American Association of Oral and Maxillofacial Surgeons
University Oncology Institute, Istanbul, Turkey. kAssistant Professor, Department of Plastic and Reconstructive
0278-2391/14/00071-8$36.00/0
Surgery, Bezm-i Alem Vakıf University, Istanbul, Turkey.
http://dx.doi.org/10.1016/j.joms.2014.01.001
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our current practice, its use has been limited by the dimensions of the viable composite graft, 1 cm, because the graft will be nourished by the surrounding tissues and requires a well-vascularized recipient bed. Cartilage is an important supporting tissue that is avascular, and its perfusion is dependent on diffusion from the peripheral tissues. The composite chondrocutaneous graft’s vascularity will be determined by the vascular anastomosis established at the subdermal plexus level between the graft and recipient bed.5,6 The survival of these composite grafts has been limited by the imbibition and inosculation rates from the narrow wound edges and the recipient bed. The largest size of composite grafts defined in published studies has been a 2.5-cm equilateral triangle graft; however, in routine practice, the size has not exceeded 1 to 1.5 cm.7 To enhance the survival length of these grafts, a number of surgical and pharmacologic techniques have been developed. Among some of the surgical techniques there has been increasing the contact surface by de-epithelialization of the recipient bed, adapting the graft in a ‘ tongue-in-groove’’ fashion, and enhancing the vascularity of the recipient bed with a dermal ‘ turnover’’ flap or graft cooling.8-12 The reported expedient pharmacologic survival enhancement methods for composite grafts have included corticosteroids, dimethyl sulfoxide, dimethylurea, melatonin, indometacin, fibroblast growth factor, chlorpromazine, and hyperbaric oxygen therapy.13 Platelet-rich plasma (PRP) is rich in vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-a and -b), transforming growth factor (TGF)-a and TGF-b, epidermal growth factor, fibroblast growth factor, and insulin-like growth factor (IGF), which has been depleted from the erythrocytes and leukocytes by centrifugation as a biologic product.14 PRP has a high concentration of growth factors and cytokines initially secreted by platelets and leukocytes and later amplified by macrophages, including a large number in endothelial cells and migrating fibroblasts. PRP has been proved to be effective in wound and fracture healing, accelerating the proliferative phase in tendon healing, tendon regeneration, and tension strength of the tendons.15-21 Moreover, Anitua et al22 and Blanton et al23 demonstrated that the growth factors in PRP most efficiently enhanced fibroblast chemotaxis, neovascularization, collagen synthesis, and vessel densities after 2 weeks of administration, in accordance with elevated VEGF secretion. PRP is an autologous, safe, easily prepared bioproduct that is more cost-effective than other recombinant technique products.14,19 The present experimental study aimed at extending the safe length of composite chondrocutaneous grafts by enhancement of angiogenesis and re-epithelialization through the myriad of factors in PRP. We also investigated
the changes that occurred after PRP administration to the graft. In addition, the influence of the recipient site of the graft was examined. Determining the effect of PRP on attenuating programmed cell death was also targeted using the terminal transferase fluorescein-dUTP nick end labeling (TUNEL) assay.
Materials and Methods The animal ethics committee provided approval before the study began, and the animals were cared for in accordance with the protocols approved by the institutional animal care and use committee (reference no. 21.02.2012/74). This experiment was conducted using 31 New Zealand white rabbits aged 14 to 18 weeks (mean 14.5 0.3) and weighing 1.8 to 2.2 kg (mean 2.0 0.1). All the rabbits were screened for common diseases and had not been subjected to any experiments before the present study. EXPERIMENTAL MODEL
The animal model used in the present study was adapted from an experimental study conducted by Karaca and Gursu.8 The rabbits were divided into 3 groups, with 10 rabbits in each group: 1.5-, 2.0-, and 2.5-cm size triangle-shaped composite chondrocutaneous grafts or defects were created. One rabbit was isolated to provide a normal pathologic control view of the rabbit ear model (Fig 1). In group A (graft pretreated with PRP, n = 10 rabbits), a ‘ prepared’’ graft 1.5, 2.0, and 2.5 cm in size was planned for transfer to fresh beds. A ‘ prepared’’ graft was a graft treated with PRP gel on all 3 sides of the 1.5-, 2.0-, and 2.5-cm equilateral triangle of the graft (0.5 mL on each side of the triangle). In group B (recipient bed pretreated with PRP, n = 10 rabbits), a ‘‘prepared’’ recipient bed 1.5, 2.0, and 2.5 cm in size was planned for reconstruction by the fresh grafts. A prepared recipient bed was one that had been treated with PRP gel on all 3 sides (0.5 mL on each side of the triangle), after forming an equilateral triangle-shaped defect. On the day of grafting, the epithelium, scar, or granulation tissue was excised from the sides. In group C (control group, n = 10 rabbits), fullthickness triangle-shaped defects, 1.5, 2.0, and 2.5 cm in size, were created on the right ears of the rabbits. Seven days later, matching size chondrocutaneous grafts were adapted to the defect areas without PRP. EXPERIMENTAL PROTOCOL
Ketamine hydrochloride (50 mg/kg; Ketalar, Pfizer, Istanbul, Turkey) and xylazine hydrochloride (3 mg/kg; Rompun, Bayer, Leverkusen, Germany) were administered intramuscularly for anesthesia. After shaving the rabbit ear with an electric shaver, the surgical field
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FIGURE 1. A, Application of platelet-rich plasma gel to the central part of the planned composite chondrocutaneous grafts in group A. B, Equilateral 1.5-, 2.0-, and 2.5-cm triangle defects carved on rabbit ears and platelet-rich plasma (PRP) solution injected to the sides 7 days before grafting in group B. C, Group C (control group) with 1.5-, 2.0-, 2.5-cm fresh defects and 7 days after grafting with grafts from the opposite ear. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
was cleaned with povidone-iodine solution and draped. The experiment consisted of 3 stages (Fig 2). In the first stage for groups A and B, the critical size defects were created on the rabbit’s ears, and PRP gel was administered to the graft or recipient bed, followed by a 7-day waiting period for activation of platelets, observing wound healing, and angiogenesis. Mupirocin-based ointment (Bactroban 2%, GlaxoSmithKline, Istanbul, Turkey) was used to cover the ears of the rabbits, and the dressings were changed daily. For group C, the defects were created, and the wounds were covered with mupirocin and the dressings changed daily. In the second stage of the experiment (7 days after the first stage), for group A, the ‘‘prepared’’ composite grafts were transferred to the fresh beds and sutured with 5-0 Vicryl (Ethicon, Novartis, Germany) from the dorsal and ventral sides. Inflammation, angiogenesis, and graft take were observed for another 7 days. For group B, the 1.5-, 2.0-, and 2.5-cm size fresh grafts were transferred to the ‘‘prepared’’ recipient beds. Before transfer, the prepared wound edges were
scraped gently with the back of a no. 15 blade to move some of the granulation tissue away such that the transferred graft could adequately attach to the recipient site. For group C, the defect edges were scraped gently to enhance graft take, and untreated grafts were harvested from the left ears of the rabbits and adapted to the defects. These grafts were observed for 7 more days to analyze graft viability. In the third stage of the experiment, graft viability was observed macroscopically in groups A and B. Next, with the rabbits under anesthesia, biopsy samples were taken, including a strip of normal tissue from the sides of the graft for immunohistologic assessment and TUNEL assay. The animals were sacrificed using an overdose of anesthetic. PREPARATION OF PRP
PRP was prepared from each rabbit separately. A total of 10 mL of blood was drawn from the arteria carotis communis of the rabbits in groups A and B and placed
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PRP WITH COMPOSITE CHONDROCUATANEOUOS GRAFT
FIGURE 2. Flow chart of the experimental stages. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
into citrate-phosphate-dextrose (CPD) solution in a 10 mL of blood to 2 mL of CPD ratio. Centrifugation of the blood to separate the plasma from the cellular products was performed for 20 minutes at 150g (1,200 rpm; Eppendorf AG 22331 centrifuge, Hamburg, Germany). The supernatant was aspirated and centrifuged for another 10 minutes at 450g (2,000 rpm). The formed bilayer of plasma, containing the upper platelet-poor plasma and a lower layer of PRP, was then separated using an aspirator. Only the lower layer was used for our experiment. Each vial of prepared PRP was sampled and sent to the laboratory for a platelet count to ensure that a high concentration of platelets was present in the prepared plasma. The number of platelets was 250,000 to 550,000 platelets/mL (mean 342,000 10,000) approximately, 4 times greater than that of the peripheral blood. The PRP obtained from the rabbits was strictly administered to the corresponding rabbit from which the peripheral blood had been obtained. Before injection into the defect site, calcium chloride and bovine thrombin were mixed with the PRP at a ratio of 1:6 for platelet activation. The calcium chloride and bovine thrombin were obtained from Tisseal, which also provided the kit (reference no. VNT 1 J008) for mixing the materials. For the procedure, a viscous material formed within 50 to 60 seconds. With the applicator syringe, the gel was injected onto the sides of the composite grafts.
MACROSCOPIC ASSESSMENT
On postoperative day 14, for all groups, each composite graft was examined macroscopically. Nonviable tissue was debrided. The amount of graft was traced on acetate paper and recorded using Image J software (National Institutes of Health, Bethesda, MD). The surviving graft area was then recorded as a percentage of the initial graft harvested. HISTOLOGIC ASSESSMENT
The harvested specimens were fixed in 10% buffered formalin solution and embedded in paraffin blocks, and 3-mm-thick sections were obtained. The mounted histologic sections were stained using hematoxylin and eosin (H&E). The histologic grade of the healed sites in terms of epithelial regeneration, inflammation, and fibrosis was scored for each specimen as none (), mild (+), moderate (++), and severe (+++), according to a scale previously described by Myers et al,24 in which the cellular elements of the wound were counted on the basis of the number per high power field to obtain a value from none () to severe (+++). Immunohistochemically, CD34 and smooth muscle actin (SMA) primer antibodies were used to determine neovascularization. For immunohistochemistry, paraffin blocks prepared from routinely processed specimens were cut into 5-mm-thick
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sections on the charged slides and deparaffinized. For antigen retrieval, the sections were microwaved and endogenous peroxidase activity was blocked by incubating the sections with hydrogen peroxide. CD34 antibody (ready to use, qbeND/10, Thermo Scientific, Cheshire, UK) and SMA (ready to use, 1A4, Thermo Scientific) were applied. After this process, biotinylated secondary antibody (HRP Sc – 2030, Lot no. D1504, Santa Cruz Biotechnology, Santa Cruz, CA), streptavidin peroxidase, and substrate-chromogen solution were applied. Nuclear staining was conducted with hematoxylin. The staining intensity was defined as a percentage and given a score ranging from 1 to 100%: 0 (), 10 to 30% (+), 30 to 60% (++), and more than 60% (+++). The examination was performed by a pathologist who was unaware of the study groups. Using the TUNEL assay, analysis of DNA-fragmented cells from the sections harvested from the rabbits was performed using a commercially available kit (11684795910, Roche USA, New York, NY). In these sections, DNA-fragmented cells were counted in a blinded manner in 4 randomly defined regions (each 62,500 mm2). Apoptotic cells were identified by the characteristics of nuclear condensation and margination. STATISTICAL ANALYSIS
The results were analyzed using nonparametric analysis methods (Kruskal-Wallis and Mann-Whitney U tests) because of the small sample size. The Statistical Package for Social Sciences, version 20.0 (SPSS, Chicago, IL), was used for the analyses. Significant differences were considered present for P < .05.
Results No rabbit deaths or complications were observed during the entire study period. One of the composite grafts, 1.5 cm in size, presented with wound dehiscence followed by graft failure. No signs of autophagy of the ears were observed among the rabbits because the wounds had been dressed with mupirocin and covered with sponges. The platelet counts derived from the whole blood samples were elevated from 157,000 43,000 platelets/mL to an average of 425,000 37,000 platelets/mL. Regarding the composite graft survival ratios, no significant difference was found for the 1.5-cm grafts. Thus, in group A (n = 6), with the grafts prepared with PRP, the survival rate of the 1.5-cm grafts was 82.8% 15.4%, and in group B (n = 6), with the recipient bed prepared with PRP, the graft survival rate was 79.8% 12.1%. In group C, with the composite grafts transferred without any PRP treatment to the recipient beds, the survival rate was 74.4% 10.3%.
For the 2-cm composite grafts, a significant increase was seen in the percentage of survival in group A (65.43% 15.7%). In group B, the graft survival rate was 78.12% 12.8%. In the control group, group C, however, the survival rate was a dismal 41.31% 37.4% (Figs 3, 4). These results have shown that preparing the grafts or recipient bed with PRP solution 7 days before elevating the grafts increased the survival length of the composite grafts 2 cm in size. However, no significant difference was found between groups A and B (Fig 5). For the composite 2.5-cm grafts, significant graft survival was observed in group B (fresh graft to PRP-prepared recipient bed) compared with groups A and C. In group B, 65.4% 11% of the composite grafts survived. However, in group A, the survival rate was 43% 10.3%, and in group C, it was only 10.5% 5.8%. HISTOLOGIC ASSESSMENT RESULTS
The histologic assessment results revealed that groups A and B displayed a significantly increased rate of epithelial regeneration (P < .005) compared with the control group. However, when comparing groups A and B with each other, no significant difference was observed in epithelial regeneration (P > .05). The inflammation rates for the groups were also not significantly different (P > .05). In groups A and B, fibroblast proliferation was significantly different (P < .05). The inflammation, fibrosis, and epithelial regeneration rates for the 1.5-, 2.0- and 2.5-cm composite chondrocutaneous grafts are shown in Figure 6. In groups A and B, the 1.5-cm grafts histologically presented with regular wound healing and elastic cartilage formation and fibrosis. The grafts 2.0 cm in size displayed a thin epithelium with regenerative properties, and the 2.5-cm size grafts displayed partial necrotic debris
FIGURE 3. Immunohistochemical CD34 staining of the endothelial cells and precursors in group A. Original magnification 400. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
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FIGURE 4. Microscopic view of control group 2.0-cm composite grafts 7 days after grafting. Asterisk indicating ulcerated epithelium and granulation tissue and disintegrated cartilage tissue. Hematoxylin and eosin, original magnification x100. Microscopic view of group A 2.0-cm composite grafts 7 days after grafting. Asterisk shows mild inflammation in the dermis, fibrosis, and epithelial regeneration. Hematoxylin and eosin, original magnification x100. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
along the center part of the graft. In the control group, however, the 2.0- and 2.5-cm grafts presented with an ulcerated epithelium and granulation tissue, minimal fibrosis, and large amounts of debris (Figs 7, 8). To assess how inflammation contributes to wound healing, the H&E-stained slides were evaluated by the characteristics of the inflammatory cells in the grafts and the quality of the granulation tissue and fibrosis. A loss of integrity was seen from the epidermis to the
hypodermis, with inflammatory cellular infiltration and increased polymorphonuclear leukocytes in the control group specimens. In groups A and B, moderate histiocytic and neutrophilic infiltration, with notably increased amount of fibroblasts and macrophages, was seen. The density of the macrophage-type cells was significantly greater in groups A and B compared with that in the control group. Interstitial edema was seen more frequently in the control group slides.
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FIGURE 5. Microscopic view of group A 2.5-cm composite grafts, 7 days after grafting showing ulcerated epithelium and mounting debris with disintegrated cartilage tissue. Hematoxylin and eosin, original magnification x100. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
The control group 2.0- and 2.5-cm grafts displayed a viability percentage of 52% 8.1% and 22% 8.0%, respectively. IMMUNOHISTOCHEMICAL RESULTS
The CD34 and SMA-stained slides were scored in terms of angiogenesis, and the results confirmed that
the neovascularization scores in the PRP-prepared groups (groups A and B) displayed 30 to 60% (++) staining, which was a significant result compared with the control group (P < .001; Figs 6, 7). In the angiogenesis comparison of the 1.5-, 2.0-, and 2.5-cm composite grafts prepared with PRP, it was observed that in 2.0-cm composite graft, angiogenesis was
FIGURE 6. Immunohistochemical SMA presentation of neovascularization in groups A and B, 7 days after grafting. Original magnification, x400. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
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PRP WITH COMPOSITE CHONDROCUATANEOUOS GRAFT
FIGURE 7. Immunohistochemical CD 34 staining of the endothelial cells and precursors in group A. Original magnification 400. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
occurring (++) in 60% of the grafts. This result was also significant compared with the angiogenesis in the other sizes of composite grafts (P < .001). Microscopic slides of the 2.0-cm composite grafts (in groups A and B) displayed endothelial cell progenitors stained with CD34- and SMA-stained neovascularization. Also, pronounced granulation reaction was present around the vessels and was accompanied by fibroblastic proliferation, edema, and reparative processes in the dermis of groups A and B. TUNEL ASSAY RESULTS
Positive TUNEL-stained cells were detected and quantified in the composite chondrocutaneous grafts during wound healing in all groups. The TUNELpositive cell number was significantly decreased by PRP treatment in the 2.0-cm chondrocutaneous grafts from groups A and B compared with that in the control group (Table 1; P < .05). However, no significant
difference was noted between groups A and B (P > .05). TUNEL-positive cells were stained using the commercially used kit, revealed an increase in nuclear fluorescence intensity by changing their fluorescence from dark blue (normal cells) to a bright blue, white color, indicating chromatin condensation (Figs 9, 10).
Discussion In the case of a severe tissue deficiency with a secondary cleft lip nasal deformity, it has not been easy to correct the deficiency completely using only nasal tissue. Stenosis of the nasal vestibule and septal deviation intruding in the contralateral airway will result in severe nasal obstruction. Asymmetry of the nasal base has been the most striking esthetic problem, involving the alar rim and anterior nares region. The nostril sill will often be absent in these patients. Composite
FIGURE 8. Microscopic view of group A 2.5-cm composite grafts, 7 days after grafting showing ulcerated epithelium and mounting debris with disintegrated cartilage tissue. Hematoxylin and eosin, original magnification 100. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
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Table 1. APOPTOTIC CELLS IN 2.0-CM CHONDROCUTANEOUS GRAFTS, QUANTIFIED BY TUNEL ASSAY (CELLS/62.500 2 mM )
Group Group A (PRP prepared graft; n = 10) Group B (PRP prepared recipient site; n = 10) Group C (control; n = 10)
Region 1
Region 2
Region 3
Region 4
10 3 51 16 2
32 5 18 3 45 1
45 6 50 1 125 5
60 1 37 5 65 1
P < .05, groups A and B (PRP pretreatment applied) were compared with the control group. P > .05, groups A and B were compared with each other. Abbreviations: PRP, platelet-rich plasma; TUNEL, terminal transferase fluorescein-dUTP nick end labeling. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
chondrocutaneous grafts might be placed to reconstruct all these areas. Additionally, it is well known that if a more than 5-mm disparity of columellar length is present compared with the normal side, composite grafting could be useful.25 However, patients with cleft lip nasal deformity undergo numerous operation, and the scars on the vestibular floors and alar base from the previous operations disturb the normal anatomy, making composite graft take more challenging.26 The aim of our study was to describe a surgical enhancement technique that increases the size of the available composite chondrocutaneous grafts by either preparing the grafts or the nonideal recipient beds to enhance angiogenesis and fibrosis using a PRP solution. Composite grafts, by definition, are composed of 2 or more germ layers of tissue or 2 separate tissues (ie, skin + cartilage, mucosa + cartilage, skin + fat, skin + mucosa + nail, skin + fat + periosteum). The principles of reconstruction with composite grafts were defined by Konig in 1902.27 Interest in composite grafts was revitalized in 1946 by Dupertuis,28 when 15 nasal reconstructions were successfully performed with composite grafts prepared from the ear lobule. Composite grafts can be harvested from the ear lobule, helical rim, root of helix, nipple areola, or nail incorporated into skin and mucosa.29-31 Composite tissue grafts have been used as a convenient and esthetic method of head and neck reconstruction, nipple areola reconstruction, anophthalmic socket reconstruction, and finger pulp reconstruction for more than 1 century.4,7 Anastomosis of the recipient and donor vascular tissue on the subdermal plexus level is an essential aspect of the success of composite grafts.32,33 McLaughlin34 defined the life cycle of a composite graft and stated that they initially present with a dead white color when harvested, followed by a pale pink color after 6 hours, cyanotic color until 5 days after transplant, and healthy pink within approximately 10 days. The disadvantages of this useful, relatively easy, reconstructive option is that the available size of composite grafts has been limited to 1 cm, color discrepancies occur, composite grafts tend to shrink 10 to 20%, and it is challenging to the inexperienced sur-
geon to properly evaluate the life cycle of the graft. Within the published data, multiple surgical treatment modalities have been proposed to increase the viability of composite grafts. In 1977, MacCollum and Grabb5 applied the concept of the advanced preparation of the recipient bed to composite grafts with Rhesus monkeys. They demonstrated 88% survival in auricular composite transfers when the recipient bed was ‘‘prepared’’ 10 days in advance versus 22% in fresh composite transfers. In our experiment, the preparation period was 7 days, and this period was shorter than the 10- to 11-day delay period in the study by MacCollum and Grabb.5 Park et al35 in 1996 studied the survival rate of composite grafts relative to the graft size in a rabbit ear model. They reported that from 5 to 15 mm, the percentage of survival area did not display any decrease relative to the increased graft size, although it did decrease with a graft size of 17 mm.35 Our study challenged the results of that study because we compared the graft survival of 1.5-, 2.0-, and 2.5-cm size composite grafts with PRP solution enhancement. Chandawarkar et al36 advocated harvesting a larger skin island relative to cartilage, in the belief that more perichondrium of the cartilage extension will be in contact with the overlying undermined dermis. Keck et al12 and Pilanci et al11 suggested enhancing the healing of composite grafts by locoregional skin flaps or dermal turnover flaps. Eley et al37 and Rashid et al38 sculptured skin tags to restore a nasal deformity using local flaps or transposition flaps. Tiengo et al39 proposed the use of dermal substitutes such as Integra (Integra Lifesciences, Plainsboro, NJ) for the reconstruction of nose tip avulsions. All these techniques, however, require a morbidity of donor site plus excessive scarring. The technique we investigated in our study is superior to these techniques, because it is less invasive and does not involve the use of neighboring tissue or expensive biomaterials, such as dermal substitutes. In the design of the experiment, realizing that the ear’s vascularity might differ from the proximal to distal ends, the 1.5-cm size defects were placed closer
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PRP WITH COMPOSITE CHONDROCUATANEOUOS GRAFT
FIGURE 9. A, Terminal transferase fluorescein-dUTP nick end labeling (TUNEL) assay micrograph of group A grafts. Arrow shows the nuclear decomposition and chromatin condensation. B, TUNEL assay micrograph of group B grafts. Arrow shows the nuclear decomposition and chromatin condensation. (Fig 9 continued on next page.) Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
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FIGURE 9 (cont’d). C, TUNEL assay micrograph of group C grafts. Arrow shows the increased amount of nuclear decomposition and chromatin condensation (P < .05). Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
to the helical root. The largest size defect, 2.5 cm, was placed in the middle, and 2.0-cm size defect was placed at the distal end. The order and placement of the created defects were the same for all rabbits and all composite grafts among the groups.
FIGURE 10. Quantitative analysis of terminal transferase fluorescein-dUTP nick end labeling (TUNEL)-positive cells per mm2 among all groups. Sevim et al. PRP With Composite Chondrocuataneouos Graft. J Oral Maxillofac Surg 2014.
Three main factors contribute to angiogenesis: formation of new blood vessel sprouts owing to inflammatory wound healing responses, endogenous release of angiogenic growth factors, and exogenous release of angiogenic growth factors, the preferred method of angiogenesis targeted with PRP therapy.40 It has been established that PRP releases growth factors and contains a high concentration of PDGF-a and PDGF-b. TGF-b, IGF, and fibrillar 3-dimensional scaffolds formed by fibrinogen from activated PRP that easily biodegrades and is a safe autologous biomaterial. The platelet lifespan is approximately 7 to 9 days. Therefore, in our experiment, we waited 7 days after the PRP had been administered. Many discrepancies are present in terms of PRP preparation, and standardization of the techniques has been lacking throughout published studies. Lower platelet concentrations can lead to suboptimal effects, and greater concentrations might have an inhibitory affect by secreting thrombospondin-1, which interferes with mitogenic effects of PRP.39 Platelets modulate inflammation by their ability to secrete a variety of chemokines. These chemokines control the amount of accumulation of leukocytes and monocytes in the injured tissue.18 Remembering this, one can deduce that the
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PRP WITH COMPOSITE CHONDROCUATANEOUOS GRAFT
chemokines secreted from PRP in addition to the myriad of factors released from the prepared recipient site should result in an increased graft take percentage. In our study, the group B composite grafts had the greatest survival rate, although, statistically, nonsignificant results were obtained. The quantification of the concentration of some chemokines that platelets augment such as CXCL-5, monocyte chemoattractant protein 1, and interleukin-8 could be investigated in an additional study using this chondrocutaneous graft model.41 The results obtained from the present study revealed that 2.0-cm equilateral triangle chondrocutaneous grafts prepared with bovine thrombin and calcium chloride-enhanced PRP gel survived at a rate of 65.43% 15.7% in the groups in which PRP was administered to the graft and 78.12% 12.8% in the groups in which it was administered to the recipient bed. Both were significantly greater than that of the control group, for which the graft survival rate decreased to 41.3% 37.4% (P = .0364). Therefore, we deduced that the location of PRP administration (either the graft or recipient bed) is not crucially important, although it was minimally more effective in group B, as long as adequate angiogenesis can be achieved. Immunohistochemical analysis of CD34 and SMA staining revealed that in the PRP-prepared groups (groups A and B), a moderate rate of neovascularization (60%) in the graft specimens was observed and was significantly different from that of the control group (P > .005). In the present study, we demonstrated that the safe length of composite chondrocutaneous grafts can be extended to 2.0 cm with the application of autologous-prepared PRP 7 days before transfer. Furthermore, the TUNEL assay results obtained from our study have clearly shown that PRP pretreatment, to either the recipient site or the graftameliorated apoptosis, such that the available graft size can be increased to 2.0 cm using PRP. PRP contains a high concentration of growth factors and cytokines initially secreted by platelets and leukocytes and later amplified by macrophages, including a rapid increase in endothelial cells and migrating fibroblasts. The evident hypoxic environment within the composite graft will also increase angiogenesis. Secreted growth factors, including TGF-b, PDGF-b, and angiopoietin-1, are important for vessel stabilization.22 A comparison of angiogenesis among the 1.5-, 2.0-, and 2.5-cm composite grafts revealed that in the 2.0-cm composite grafts, angiogenesis was occurring (++) in 60% of the grafts. This result was significantly greater than the angiogenesis rate of the other composite graft sizes (P < .001). The initial in vivo experiments involving PRP were in the field of oral and maxillofacial surgery and in dentistry, in particular, periodontal therapy. As Marx et al42 reported, PRP has been used to allow denser bone regeneration and improving the contact surface of the
implants. Relating this to our study, we can conclude that larger size composite grafts prepared with PRP can be used with our method, to prepare dental implant sites, mandibular defects forming after removal of jaw cysts, anophthalmic sockets, or augmentation of maxillary sinus floors in elderly patients owing to its fibrinforming capacity and enhancing angiogenesis. In a study by Wei et al,43 in 2007, chondrocytes harvested from the auricular root of New Zealand rabbits were cultured, harvested, and prepared with PRP as autologous injectable scaffolds to cartilage defects. However, our technique is more feasible and easily obtained than that method. In conclusion, the results obtained from our study support the report by MacCollum and Grabb5 and indicate an advantage from advanced preparation of the recipient bed and not the graft itself. The repeated application of PRP or any drug delivery system might be practical for clinical application from the perspective of safety and invasiveness. A 0.5-cm increase in the possible graft size, and a possible 1.5-cm increase, is quite significant, because these grafts are mainly used in alar reconstruction or alar base reconstruction. The present study reports a reliable and easy-to-use rabbit model for studying composite graft survival for various sizes of grafts. Our aim was to initiate the clinical use of PRP in patients with cleft lip nasal deformity for whom extended-size composite grafts are in demand.
References 1. Cho BC, Park JW, Baik BS: Correction of severe secondary cleft lip nasal deformity using a composite graft: Current approach and review. Ann Plast Surg 48:131, 2002 2. Douglas B: Successful replacement of completely avulsed portions of fingers as composite grafts. Plast Reconstr Surg 23: 213, 1959 3. Adams WM: Free transplantation of the nipples and areola. Surgery 34:140, 1944 4. Zampar AG, Salomons RL, Dornelles RF, et al: Chondrocutaneous auricular graft for reconstruction of ocular socket in anophthalmic cavities. J Craniofac Surg 22:602, 2011 5. MacCollum M, Grabb W: Increasing the incidence and the size of successful experimental composite ear grafts by advance preparation of the recipient bed. Plast Reconstr Surg 60:759, 1977 6. Musgrave RH, Lehman JA: Composite grafts, in Georgiade GS, Rieffkohl R, Levin LS, (eds). Georgiade Textbook of Plastic, Maxillofacial and Reconstructive Surgery (ed 2). Baltimore, Williams & Wilkins, 1992, pp 37–38 7. Davenport G, Bernard FD: Improving the take of composite grafts. Plast Reconstr Surg 24:175, 1959 8. Karaca C, Gursu G: The effects of delaying composite cartilage grafts and their recipient bed on the graft survival. Eur J Plast Surg 17:247, 1994 9. Fann PC, Hartman DF, Goode RL: Pharmacologic and surgical enhancement of composite graft survival. Arch Otolaryngol Head Neck Surg 119:313, 1993 10. Conley JJ, Von Fraenkel PH: The principle of cooling as applied to the composite graft in the nose. Plast Reconstr Surg 17:444, 1956 € Sac¸ak B, Kuvat SV, et al: Use of outsized composite 11. Pilanci O, chondrocutaneous grafts in conjunction with dermal turnover
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13 27. Koenig F: Zur Deckung von Defekten der Nasenflugel. Berl Klin Wochenschr 39:137, 1902 28. Dupertuis SM: Free ear lobe grafts of skin and fat. Plast Reconstr Surg 1:135, 1946 29. Meade RJ: Composite ear grafts for construction of columella. Plast Reconstr Surg 23:134, 1959 30. Ozek C, Gurler T, Uckan A, Bilkay U: Reconstruction of the distal third of the nose with composite ear-helix free flap. Ann Plast Surg 58:74, 2007 € € Kayalı MU, et al: Preliminary study on 31. Karsidag S, Ozkaya O, durability of nail as xenograft. J Craniofac Surg 21:1843, 2010 32. Ozaki W, Chaffoo RA, Vu KC, Markowitz BL: Comprehensive rhinoplasty technique to correct the bilateral cleft lip nasal deformity using conchal composite grafts. J Craniomaxillofac Surg 34:150, 2006 33. Musgrave RH: Surgery of nasal deformities associated with cleft lip. Plast Reconstr Surg 28:261, 1961 34. McLaughlin CR: Composite ear grafts and their blood supply. Br J Plast Surg 7:274, 1954 35. Park JB, Jang HJ, Hong IP, et al: Survival rate of composite graft according to graft size and microscopic examination: Experimental study on ear of rabbit. J Korean Soc Plast Reconstr Surg 23:1204, 1996 36. Chandawarkar RY, Cervino AL, Wells MD: Reconstruction of nasal deformities using modified composite grafts. Br J Plast Surg 56:26, 2003 37. Eley KA, Pleat JM, Wall SA: Reconstruction of a congenital nasal deformity using skin tags as a chondrocutaneous composite graft. J Craniofac Surg 20:573, 2009 38. Rashid M, Islam MZ, Tamimy MS, et al: Rotation-transposition correction of nasal deformity in Tessier number 1 and 2 clefts. Cleft Palate Craniofac J 46:674, 2009 39. Tiengo C, Amabile A, Azzena B: The contribution of a dermal substitute in the three-layer reconstruction of a nose tip avulsion. J Plast Rec Aesth Surg 65:573, 2012 40. Dong Z, Li B, Liu B, et al: Platelet-rich plasma promotes angiogenesis of prefabricated vascularized bone graft. J Oral Maxillofac Surg 70:2191, 2012 41. Flad H-D, Brandt E: Platelet-derived chemokines: Pathophysiology and therapeutic aspects. Cell Mol Life Sci 67:2363, 2010 42. Marx RE, Carlson ER, Eichstaedt RM, et al: Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85:638, 1998 43. Wu W, Fulin C, Yanpu L, et al: Autologous injectable tissueengineered cartilage by using platelet-rich plasma: Experimental study in a rabbit model. J Oral Maxillofac Surg 65: 1951, 2007
