The Laryngoscope C 2014 The American Laryngological, V
Rhinological and Otological Society, Inc.
Efficacy of Platelet-Rich Fibrin Matrix on Viability of Diced Cartilage Grafts in a Rabbit Model _ Ismail G€ uler, MD, PhD; Deniz Billur, MD, PhD; Sevim Aydin, MD, PhD; Sinan Kocat€ urk, MD, PhD Objectives/Hypothesis: The objective of this study was to compare the viability of cartilage grafts embedded in platelet-rich fibrin matrix (PRFM) wrapped with no material (bare diced cartilage grafts), oxidized methylcellulose (Surgicel), or acellular dermal tissue (AlloDerm). Study Design: Experimental study. Methods: In this study, six New Zealand rabbits were used. Cartilage grafts including perichondrium were excised from each ear and diced into 2-mm-by 2-mm pieces. There were four comparison groups: 1) group A, diced cartilage (not wrapped with any material); 2) group B, diced cartilage wrapped with AlloDerm; 3) group C, diced cartilage grafts wrapped with Surgicel; and 4) group D, diced cartilage wrapped with PRFM. Four cartilage grafts were implanted under the skin at the back of each rabbit. All rabbits were sacrificed at the end of 10 weeks. The cartilages were stained with hematoxylin-eosin, Masson’s Trichrome, and Orcein. After that, they were evaluated for the viability of chondrocytes, collagen content, fibrillar structure of matrix, and changes in peripheral tissues. Results: When the viability of chondrocytes, the content of fiber in matrix, and changes in peripheral tissues were compared, the cartilage embedded in the PRFM group was statistically significantly higher than in the other groups (P < 0.05). Conclusion: We concluded that PRFM has significant advantages in ensuring the chondrocyte viability of diced cartilage grafts. It is also biocompatible, with relatively lesser inflammation and fibrosis. Key Words: Platelet-rich fibrin matrix (PRFM), diced cartilage, rhinoplasty, AlloDerm, Surgicel. Level of Evidence: N/A. Laryngoscope, 00:000–000, 2014
INTRODUCTION Autologous cartilage grafting is a widely used method in rhinoplasty.1,2 However, cartilage grafting has several drawbacks, such as risks of resorption and necrosis.2–4 In order to increase the viability of cartilage tissue and prevent its resorption, many techniques have been described. Among these techniques, Erol’s “Turkish delight” method of using diced cartilage grafts wrapped with oxidized methylcellulose (Surgicel; Johnson and Johnson Medical Inc., Arlington, TX) recently has been popular.5 In the long term, however, recent clinical studies have reported that the diced cartilage wrapped with Surgicel was easily resorbable and created a limitation in their application.6–8 Some researchers have used temporal muscle fascia with a wrapping method in order to avoid foreign-body reactions in particular, among others. Although the resorption rate and frequency of related foreign-body reactions were much lower than those of Surgicel,9,10
From the Department of Otolaryngology, School of Medicine, Ufuk University (I.G., S.K.); and the Department of Histology and Embryology, School of Medicine, Ankara University (D.B., S.A.), Ankara, Turkey Editor’s Note: This Manuscript was accepted for publication November 20, 2014. The authors have no funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Deniz Billur, MD, Department of Histology and Embryology, School of Medicine, Ankara University, Ankara Turkey. E-mail: [email protected] DOI: 10.1002/lary.25097
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this method has several disadvantages, including donorsite scarring and the need for a second incision. To overcome these disadvantages, a preserved human dermis (AlloDerm; LifeCell Corporation, Branchburg, NJ) was used.11 Unfortunately, the risk of immune reactions, its high price, and difficulty in harvesting graft material have limited the usage. Many investigations have been performed to extend the survival of autogenous cartilages with a goal to make them ideal implant materials.12 Platelet-rich plasma (PRP) is a plasma fraction that is produced from autologous blood samples containing relatively higher concentration of platelets.13 PRP has several different growth factors and other cytokines that stimulate healing of bone.14,15 It has been used for different purposes in surgery for approximately 30 years and conceivably increases the survival of cartilage grafts used in rhinoplasty.16–18 Platelet-rich fibrin matrix (PRFM) was firstly introduced by Choukroun in 200119 and undeniably is easily obtained. In this study, we aimed to compare the viability of cartilage wrapped with PRFM with the cartilage that was left bare, the cartilage wrapped with Surgicel, and the cartilage wrapped with AlloDerm.
MATERIALS AND METHODS The experimental design was reviewed and approved (no. 2012-22-134) by the ethics committee in animal experiments at Ankara University, and the study was carried out in compliance with the guidelines for animal experimentation at the
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Fig. 1. Four types of grafts. (A) diced cartilage; (B) graft wrapped with AlloDerm; (C) graft wrapped with Surgicel; and (D) graft wrapped with PRFM. [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]
Department of Laboratory Animal Science at Ankara University Medical School (Ankara, Turkey). All animals used for the experiments received care according to the Principles of Laboratory Animal Care recommended by the National Society of Medical Research and the Guide for the Care and Use of Laboratory Animals, proposed and prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication No. 85-23, revised 1996).
Animal Care Animals were housed in a controlled environment at a temperature of 18 C 6 1 C and with a relative humidity of 50% to 70%. They were also exposed to 12-hour light–dark cycles and fed with pellets and water ad libitum. All animal care services and procedures were performed without inflicting pain. A total of six New Zealand rabbits, each weighing approximately 2 kg, were used for the study. Each rabbit was anesthetized with 35 mg/kg ketamine and 5 mg/kg xylazine hydrochloride, both given intramuscularly.
Material Used in the Study 1. Biomaterials. AlloDerm: Pieces of thin layers of acellular human epidermal tissue with a thickness of 0.89 mm to 1.65 mm and measuring 2 cm by 4 cm were used. PRFM: Using Choukroun’s method, the blood samples from rabbits were harvested and put in 10-cc venous blood tubes without anticoagulant.19 Tubes were centrifuged (N€ uve Corporation CN 090, Ankara, Turkey) for 10 minutes at 3,000 rpm (400 g). The absence of anticoagulant implies the activation within a few minutes of most platelets in the blood sample that are in contact with the tube walls, as well as the release of coagulation cascades. Before the circulating thrombin transforms fibrinogen into fibrin, fibrinogen is concentrated in the upper part of the tube. Afterward, a fibrin clot is obtained in the middle of the tube, just between the red blood cells at the bottom and the acellular plasma at the top.20 The samples of platelet-rich fibrin (PRF), which accumulated in the middle of the tube after centrifuge, were picked up from the tubes by the help of a pincette and separated from the red blood cells. PRF was exposed to pressure between the two glass slides. After this process, the PRFM, which had risen between the slides, were cut into 2- by 2-cm pieces using scissors, and then the diced cartilages were wrapped. 2. Nonbiomaterials: Oxidized cellulose polymers in 5.1- by 7.6cm pieces (Surgicel) were used.
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Method 1. Surgical technique. To prepare the recipient site, the back and the right ear of each rabbit was shaved, cleaned, and disinfected with povidone iodine (Betadine Hydrochloride. Purdue Pharma L.P, Stamford, USA.), and 7- to 8-cm incisions were made on the dorsal surface of the ear. A 3- by 3cm (2 cc) auricular cartilage was harvested with its perichondrium. Incisions were closed with 4/0 vicryl sutures. Four separate incisions were made on the back of the rabbits. Then, 3- by 4-cm pockets were made on the back of each rabbit. Afterward, four cartilage grafts were inserted into the four subcutaneous pockets on the same animal. The incisions sutured with 4/0 vicryl sutures. The grafts left at the recipient sites for 10 weeks postoperatively. 2. Graft preparation. In our study, approximately 2 cc of cartilage was harvested from each ear of the rabbits. The excised cartilage, with its perichondrium, was diced into 2-mm- by 2mm pieces. Four pieces (each 0.5 cc) of the diced cartilage was wrapped with rehydrated AlloDerm (group B), Surgicel (group C), 2- by 2-cm PRFM (group D), and in the control group it was left bare (group A) (Fig. 1). 3. Implantation of the diced cartilage grafts. Several incisions, each 3 cm to 4 cm in length, were made on the back of each rabbit and the diced cartilage was implanted under the skin. The incisions were marked as diced cartilage (control group A), AlloDerm (group B), Surgicel (group C), and PRFM (group D). The incisions were closed with 4/0 vicryl sutures. 4. Harvesting. After 10 weeks, the implantation sites of the rabbits were shaved again and the cartilages were harvested from the original incision sites. Specimens were placed into 10% formalin solution for fixation.
Histological Examination and Analysis After formalin fixation for 72 hours, samples were dehydrated, cleared, and embedded in paraffin. Tissue sections (4–5 lm) were stained with hematoxylin and eosin (H&E), Masson’s Trichrome, and Orcein. The H&E-stained sections were used to evaluate chondrocyte viability and the status of the chondroid tissue. Masson’s Trichrome demonstrates collagen content of the matrix by staining the collagen fibrils green. Orcein stains elastic fibers in the matrix. Each sample was evaluated under light microscope (Carl Zeiss Axio Scope-A1, G€ ottingen, Germany) by two histologists with no knowledge of examination groups. They were used a scoring system for evaluation of morphology and histological parameters that modified from Kim et al.21 Ten histological parameters were reviewed in three groups: 1) loss of chondrocyte nuclei, peripheral proliferation; 2) collagen and
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TABLE I. Histological Parameters of Groups.
Histological Parameters
Diced Cartilage
Diced Cartilage With AlloDerm
Diced Cartilage With Surgicel
Diced Cartilage With PFRM
n56
n56
n56
n56
2
11
21
31
41
2
11
21
31
41
2
11
21
31
41
2
11
21
31
41
0 0
1 3
1 1
2 1
2 1
0 0
1 4
1 1
3 1
1 0
0 1
0 3
1 2
3 0
2 0
4 0
1 1
1 2
0 2
0 1
0 0
4 2
2 4
0 0
0 0
0 0
2 0
2 5
2 1
0 0
2 0
3 3
0 2
1 1
0 0
0 0
2 0
1 2
2 4
1 0
1 1
3 3
0 2
2 0
0 0
0 0
2 1
2 2
2 2
0 1
0 0
3 1
1 2
1 2
1 1
2 5
3 1
1 0
0 0
0 0
Cartilage viability Loss of chondrocyte nucleus Peripheral proliferation Matrix Collagen Elastic fiber Changes of peripheral tissue Fibrosis Inflammation Vascularization
1
3
2
0
0
1
4
1
0
0
2
3
1
0
0
0
4
1
1
0
Metaplastic bone formation Fragmentation
6 4
0 1
0 1
0 0
0 0
4 2
1 2
1 2
0 0
0 0
5 0
1 1
0 2
0 3
0 0
5 5
1 0
0 0
0 1
0 0
Resorption
1
4
0
1
0
4
1
0
1
0
0
1
2
2
1
4
2
0
0
0
(2) 5 none; (11) 5 minimal; (21) 5 moderate; (31) 5 moderate to severe; (41) 5 severe. PFRM 5 platelet-rich fibrin matrix.
elastic fiber content of the matrix; and 3) fibrosis, inflammation, vascularization, metaplastic bone formation, fragmentation and chondroid tissue resorption. Quantitative analyses of all parameters were performed between groups. Parameters were recorded as a percentage of all analyzed material: 0% as none (2), 1% to 25% as minimal (11), 26% to 50% as moderate (21), 51% to 75% as moderate to severe (31), and 76% to 100% as severe (41).
Statistical Analysis Statistical analyses were performed using the SPSS software package (version 18.0; Chicago, IL). In intergroup comparisons, a nonparametric test (Kruskal- Wallis) and MannWhitney test were used. The average of each group was calculated. Results were evaluated at a significance level of P < 0.05.
RESULTS Macroscopic Observation All harvested grafts showed adhesions. In the control group, the grafts were more irregular than those of the other experimental groups. In all experimental groups, graft unity was observed. The grafts that were wrapped with Surgicel were smaller in volume than those used in the other experimental groups.
Histological Examination Histopathological parameters reviewed in four groups were demonstrated in Table I. Group A (Diced Cartilage). H&E-stained preparations demonstrated that the content of chondrocyte nuclei was the indicator of viability (Fig. 2 a,b). In the matrix, we observed that the collagen fibers in the matrix stained green with Masson Trichrome and elastic Laryngoscope 00: Month 2014
fibers stained brown with orcein (Fig. 2 c,d). In the cartilage matrix, both types of fibers were in minimal amount. We observed minimal fibrosis, inflammation, and vascularization in the tissue around the cartilage. There was no metaplastic bone formation. Minimal cartilage fragmentation and resorption were noted (Fig. 2a). Group B (Diced Cartilage Grafts Wrapped With AlloDerm). Whereas the loss of nuclei in the specimens of diced cartilage grafts wrapped with AlloDerm was variable (moderate to severe loss), peripheral proliferation was moderate (Fig. 3 a,b). The presence of collagen and fiber contents was moderate (Fig. 3 c,d). However in fibers around the cartilage tissue, vascularization was minimal, and the level of fibrosis and inflammation increased moderately. Metaplastic bone formation was observed in two rabbits. Cartilage fragmentation was observed in our specimens, whereas the resorption of cartilage grafts was minimal (Fig. 3 a). Group C (Diced Cartilage Grafts Wrapped With Surgicel). When the specimens of cartilage grafts were wrapped with Surgicel, there was severe loss of nuclei in chondrocytes stained with H&E and minimal peripheral proliferation was seen (Fig. 4 a,b). It was observed that collagen and the content of elastic fiber were minimal (Fig. 4 c,d). Vascularization was minimal, fibrosis was moderate, and the level of inflammation was between moderate and severe in the tissue that surrounds the cartilage. Metaplastic bone formation was observed in one rabbit. The degree of fragmentation and resorption of cartilage grafts was between moderate and severe. (Fig. 4 a,b). Group D (Diced Cartilage Grafts Wrapped With Plasma-Rich Fibrin Matrix). In the specimens of cartilage grafts wrapped with PRFM, minimal loss of chondrocyte nuclei was observed as compared with the €ler et al.: Augmentation Materials for Rhinoplasty Gu
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Fig. 2. Histological examination of diced cartilage grafts. (A) Peripheral chondrocyte proliferation in diced cartilage group (pcp), loss of chondrocytic nucleoli (*), viable chondrocyte (arrow), minimal fibrosis (F), and neovascularization (arrowhead) (hematoxylin and eosin [H&E], magnification 3200). (B) Pericondrium (P), peripheral chondrocyte proliferation (pcp), loss of chondrocytic nucleoli (*), isogenous groups (*), and territorial matrix (arrow) (H&E, magnification 3400). (C) Minimal fibrosis (F), minimal level of collagen fiber content in the green-stained area of cartilage matrix (Masson’s Trichrome stain; magnification 3200). (D) Minimal level of collagen fiber content in the brown-stained area of cartilage matrix (arrow) (Orcein stain; magnification 3400). [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]
other groups. Additionally, moderate to severe degrees of peripheral proliferation were noted. Analysis of the peripheral tissue demonstrated minimal degrees of fibrosis and vascularization without any evidence of inflammation. Additionally, metaplastic bone formation was seen in only one specimen, and bone fragmentation and resorption was minimal (Fig. 5 a,b). When the content of collagen and elastic fiber was analyzed, it was seen that both types of fibers were preserved moderate and severe degree (Fig. 5 c,d).
Statistical Analysis When the PRFM group (group D) was compared to the diced cartilage group (group A), there were significant differences in viability of the cartilage, fibrosis, inflammation, and resorption of the cartilage. When the PRFM group (group D) was compared to the AlloDerm group (group B), there were significant differences in the loss of chondrocytic nuclei, fibrosis, and inflammation. When PRFM group (group D) was compared to the Surgicel group (group C), there were significant differences in the loss of chondrocyte nuclei, peripheral proliferation, extent of elastic fiber, inflammation, fragmentation, and cartilage resorption (P < 0.05) (Table II). There was no significant difference among the groups regarding Laryngoscope 00: Month 2014
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metaplastic bone formation, vascularization, and collagen content of the fibers.
DISCUSSION Cartilage grafts have been used effectively in rhinoplasty surgery for the augmentation and correction of nasal dorsum asymmetry and irregularities.5,22 However, many surgeons have indicated a significant problem about the usage of cartilage harvested from septum, conchal cartilage, or costa as a block. The problem is that cartilage is disabled when used for dorsal irregularities because of the resorption and distortion of the graft material.23–25 In order to overcome these problems, diced cartilage was suggested for use instead of cartilage blocks. During reshaping, the diced cartilage method was preferred because of its many advantages.5 For this reason, we also prefer diced cartilage to block cartilage. In numerous experimental studies, the cartilage was wrapped with a material in order to minimize the problem of resorption and increase the viability of the implanted cartilage.8 The Turkish delight method of wrapping the cartilage with Surgicel, developed by Erol in 2000, was accepted and was a milestone. This method was very popular because the study included 2,365 €ler et al.: Augmentation Materials for Rhinoplasty Gu
Fig. 3. Histological examination of diced cartilage grafts wrapped with AlloDerm. (A) Pericondrium in the sample of cartilage tissue wrapped with AlloDerm (P), moderate fibrosis (F) and inflamation (star), neovascularization (*), and metaplastic bone formation (MB) (hematoxylin and eosin [H&E], magnification 3100). (B) Moderate peripheral chondrocyte proliferation (pcp), loss of chondrocytic nuclei (*), and viable chondrocyte (arrow) (H&E, magnification 3400). (C) The content of collagen in green-stained cartilage matrix (Masson’s Trichrome stain, magnification 3200). (D) Moderate elastic fiber content in the brown-stained area of cartilage matrix (arrow) (Orcein stain, magnification 3400). [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]
patients, and it had been used for 10 years with successful results.5 Many studies, which accepted or rejected this method, have been cited in the literature. Elahi also used cartilage wrapped with Surgicel and reported similar results in 2003.26 However, Yilmaz et al. reported that this surgical intervention prevented the oxygenation of cartilage tissue for at least 48 hours, and this relative hypoxia led to a loss of regeneration potential that is important for the survival of chondrocytes.6 Erdogmus et al. reported that the use of calcium hydroxylapatite in combination with diced cartilage grafts does not have any long-term negative effects on chondrocyte viability.27 A study reported by Cakmak showed that the diced cartilage wrapped with Surgicel inhibited the proliferation and viability of cartilage.7 Indeed, approximately 10% of the implanted cartilages maintained their viability.7 However, Daniel and Calvert observed that unexpected implant resorptions occurred a few months after a rhinoplasty operation, when diced cartilage wrapped with Surgicel was used.8 In our study, when cartilage graft wrapped with Surgicel was evaluated with respect to its histological characteristics, severe loss of chondrocyte nuclei, decreased peripheral proliferation, severe fibrosis, and inflammation were observed. Significant differences were found in the rate of cartilage resorption among the Laryngoscope 00: Month 2014
groups. Our study supported other studies for which the viability potential of cartilage graft wrapped with Surgicel was lower with higher resorption capability.6–8 When the higher level of resorption in cartilage grafts wrapped with Surgicel was considered, modification of the cartilage by wrapping it with biocompatible materials was suggested.10,28 Daniel and Calvert used diced cartilage wrapped with autologous temporal fascia in rhinoplasty and obtained consistent and significant results.8 Ozturk and Aydin used diced cartilage grafts wrapped with amniotic membrane.29 Bracaglia et al. used diced cartilage graft treated with human fibrin glue.30 Brenner et al. indicated that autologous fascia had no negative effect on durability of cartilage regeneration.28 Fascia is a durable material with a high resistance power that functions as a cellular support.31 Brenner et al. indicated that fascia functions as a pericondrium, increases viability of chondrocytes, prevents the resorption of grafts, and maintains the general regenerative potential of the cartilage.28 However, there are some limitations to our study, including using another donor site and incision scarring for autologous fascia. Also, we did not prefer the use of temporalis fascia wrapped with diced cartilage in our study because of the incision scar and also because Kim et al.21 demonsrated that AlloDerm was found to be significantly superior to temporalis fascia in terms of €ler et al.: Augmentation Materials for Rhinoplasty Gu
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Fig. 4. Histological examination of diced cartilage grafts wrapped with Surgicel. (A) Minimal level of chondrocyte proliferation in the sample of cartilage tissue wrapped with Surgicel (pcp), variable level from moderate-to-severe inflammation (star), fibrosis (F), and fragmentation in cartilage tissue and metaplastic bone formation (MB) (hematoxylin and eosin [H&E], magnification 3100). (B) Severe loss of nucleus in chondrocyte (*) (H&E, magnification 3400). (C) Increasing fibrosis in peripheral tissue (F), minimal level of collagen fiber content in the green-stained area of cartilage matrix (Masson’s Trichrome stain, magnification 3200). (D) Minimal level of elastic fiber content in the brown-stained area of cartilage matrix (arrow) (Orcein stain, magnification 3400). [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]
histopathological criteria. To overcome donor-site problems, Achauer32 used AlloDerm on the nasal dorsum with a limited number of patients, and also Jacson et al. and Gryskiewicz et al. used Alloderm in dorsal nasal irregularities and the correction of nasal contour deformities.33–35 AlloDerm is an acellular human dermis matrix. After dermis is harvested from cadaveric skin, epidermis and all the cellular components are removed by scrubbing the epidermis layer with detergent and antiviral solutions. Afterward, the protein skeleton of the dermis is preserved in a frozen condition.36 Kim et al. compared cartilage grafts wrapped with AlloDerm to those wrapped with autologous fascia in their 2011 study.21 The observed results related to AlloDerm were histologically significant and showed the higher regeneration potential of the chondrocytes. Additionally, contents of matrix collagen were preserved and metaplastic bone formation did not occur.21 AlloDerm does not cause any immunological or foreign-body reactions, including rejection or reactions involving major histocompatibility class I and II antigens.37 For this reason, it tends to be treated as an autograft when used in the body. Many studies also have shown that AlloDerm does not cause inflammatory and allergic reactions. On the other hand, Bateman and Jones indiLaryngoscope 00: Month 2014
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cated the risks, including infection, tissue reaction, and rejection, caused by alloplastic materials in their study.12 In our study, when we compared the group of cartilage grafts wrapped with AlloDerm to other groups in terms of histological characteristics, we found that the loss of chondrocyte nuclei and peripheral proliferation in cartilage wrapped with AlloDerm were more significant than observed in cartilages wrapped with Surgicel. In both groups, fibrosis and inflammation were moderate. Metaplastic bone formation and undesirable results were observed in both groups. However, there were no statistically significant intergroup differences. We compared PRFM to AlloDerm in terms of inflammation and fibrosis and found that PRFM yielded significantly more successful outcomes. Originally, PRFM was identified as a secondgeneration platelet solution by Choukroun in 2001.19 It has been used in many fields of medicine such as dentistry, dermatology, and plastic surgery.20 PRFM in particular contains two important cytokines, interleukin (IL)24 and VEGF, in addition to other inflammatory cytokines (IL-1, IL-6, tumor necrosis factor [TNF] a, and TNF b), and is involved in the healing process of the tissues.38 Studies on healing bone have shown that PRFM caused fibroblastic proliferation and supported its €ler et al.: Augmentation Materials for Rhinoplasty Gu
Fig. 5. Histological examination of diced cartilage grafts wrapped with PRFM. (A) Severe peripheral chondrocyte proliferation in the samples of cartilage tissue wrapped with PRFM (pcp) (hematoxylin and eosin [H&E], magnification 3200). (B) Numerous viable chondrocytes (arrow) (H&E, magnification 3400). (C) Plenty of elastic fiber content in the green-stained area of cartilage matrix (Masson’s Trichrome stain, magnification 3200). (D) Plenty of elastic fiber content in the brown-stained area of cartilage matrix (arrow) (Orcein stain, magnification 3400). [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]
osteoblastic activity by triggering angiogenesis.39 In their study on rabbits, Kuo et al. created cartilage defects in rabbits and compared PRFM group to the con-
trol group.17 The authors reported significant healing in PRFM implanted rabbits based on the results of MR and histological observation.
TABLE II. The Results of Statistical Analysis for Histological Parameters Reviewed in Groups. Between Diced Cartilage and Diced Cartilage Wrapped With PRFM
Between Diced Cartilage Wrapped With AlloDerm and Diced Cartilage Wrapped With PRFM
Between Diced Cartilage Wrapped With Surgicel and Diced Cartilage Wrapped With PRFM Group
Group A Versus Group D
Group B Versus Group D
Group C Versus Group D
Loss of chondrocyte nucleus Peripheral proliferation
0.042* 0.407
0.026* 0.092
0.011* 0.038*
Collagen
0.12
0.616
0.065
Elastic fiber Fibrosis
0.014* 0.342
0.093 0.044*
0.041* 0.086
Inflammation
0.023*
0.004*
0.003*
Vascularization Metaplastic bone formation
0.589 0.317
0.293 0.4
0.179 0.317
Histopathologic Parameters
Fragmentation
0.674
0.209
0.022*
Resorption
0.045*
0.847
0.005*
*P value < 0.05 5 statistically significant. PRFM 5 platelet-rich fibrin matrix.
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CONCLUSION In our research, when we compared PRFM to other groups in terms of contents of chondrocyte nuclei and inflammation, we found that PRFM was superior among the other groups. Of note, it caused less fibrosis than AlloDerm grafts. In terms of the loss of chondrocyte nuclei, peripheral chondrocyte proliferation, elastic fiber content of matrix, fibrosis, inflammation, and resorption parameters, PRFM preserved the viability of cartilage grafts more successfully than Surgicel material. PRFM has significant advantages in that it ensures the chondrocyte viability of diced cartilage grafts and is also biocompatible, with relatively lesser inflammation and fibrosis.
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18. Choukroun J, Diss A, Simonpieri A, et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part IV: clinical effects on tissue healing. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101: 56–60. 19. Choukroun J, Adda F, Schoeffler C, Vervelle A. Une opportunite en paroimplantologie: Le PRF. Implantodontie 2001;42:55–62. 20. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: Technological concepts and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Oral Endod 2006;101:37–44. 21. Kim HK, Chu LS, Kim JW, et al. The viability of diced cartilage grafts wrapped inautogenous fascia and AlloDerm in a rabbit model. J Plast Reconstr Aesthet Surg 2011;64:e193–e200. 22. Sheen JH. The ideal dorsal graft: a continuing quest. Plast Reconstr Surg 1998;102:2490–2493. 23. McKinney P, Loomis MG, Wiedrich TA. Reconstruction of the nasal cap with a thin septal graft. Plast Reconstr Surg 1993;92:346–351. 24. Guerrerosantos, J. Temporoparietal free fascia grafts in rhinoplasty. Plast Reconstr Surg 1984;74:465–475. 25. Bujia, J. Determination of the viability of crushed cartilage grafts: clinical implications for wound healing in nasal surgery. Ann Plast Surg 1994; 32:261–265. 26. Elahi MM, Jackson IT, Moreira-Gonzalez A, Yamini D. Nasal augmentation with Surgicel-wrapped diced cartilage: a review of 67 consecutive cases. Plast Reconstr Surg 2003;111:1309–1321. 27. Erdogmus N, Cingi C, Canaz F, et al. Survival of diced and block cartilage grafts in combination with injectable calcium hydroxylapatite. Laryngoscope 2013;123:E17–E22. 28. Brenner KA, McConnell MP, Evans GR, Calvert JW. Survival of diced cartilage grafts: an experimental study. Plast Reconstr Surg 2006;117:105– 115. 29. Ozturk M, Aydin O. Use of diced cartilage grafts wrapped with amniotic membrane in soft tissue augmentation: experimental study. Ann Otol Rhinol Laryngol 2013;122:66–70. 30. Bracaglia R, Tambasco D, D’Ettorre M, Gentileschi S. "Nougat graft": diced cartilage graft plus human fibrin glue for contouring and shaping of the nasal dorsum. Plast Reconstr Surg 2012;130:741e–743e. 31. Dubay DA, Wang X, Kirk S, Adamson B, Robson M, Franz MG. Fascial fibroblast kinetic activity is increased during abdominal wall repair compared to dermal fibroblasts. Wound Repair Regen 2004;12:539–545. 32. Achauer BM, VanderKam VM, Celikoz B, Jacobson DG. Augmentation of facial soft-tissue defects with Alloderm dermal graft. Ann Plast Surg 1998;41:503–507. 33. Jackson IT, Yavuzer R, Silverstein P. AlloDerm for dorsal nasal irregularities. Plast Reconstr Surg 2001;107:559–560. 34. Gryskiewicz JM, Rohrich RJ, Reagan BJ. The use of alloderm for the correction of nasal contour deformities. Plast Reconstr Surg 2001;107:561– 571. 35. Gryskiewicz JM. Waste not, want not: the use of AlloDerm in secondary rhinoplasty. Plast Reconstr Surg 2005;116:1999–2004. 36. Livesey SA, Herndon DN, Hollyoak MA, Atkinson YH, Nag A. Transplanted acellular allograft dermal matrix. Potential as a template for the reconstruction of viable dermis. Transplantation 1995;60:1–9. 37. Reagan BJ, Madden MR, Huo J, Mathwich M, Staiano-Coico L. Analysis of cellular and decellular allogeneic dermal grafts for the treatment of full-thickness wounds in a porcine model. J Trauma 1997;43:458–466. 38. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part III: leucocyte activation: a new feature for platelet concentrates? Oral Surg Oral Med Oral Pathol Oral Radiol Oral Endod 2006;101:e51–e55. 39. Choukroun J, Diss A, Simonpieri A, et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part IV: clinical effects on tissue healing. Oral Surg Oral Med Oral Pathol Oral Radiol Oral Endod 2006; 101:56–60.
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Rhinological and Otological Society, Inc.
Efficacy of Platelet-Rich Fibrin Matrix on Viability of Diced Cartilage Grafts in a Rabbit Model _ Ismail G€ uler, MD, PhD; Deniz Billur, MD, PhD; Sevim Aydin, MD, PhD; Sinan Kocat€ urk, MD, PhD Objectives/Hypothesis: The objective of this study was to compare the viability of cartilage grafts embedded in platelet-rich fibrin matrix (PRFM) wrapped with no material (bare diced cartilage grafts), oxidized methylcellulose (Surgicel), or acellular dermal tissue (AlloDerm). Study Design: Experimental study. Methods: In this study, six New Zealand rabbits were used. Cartilage grafts including perichondrium were excised from each ear and diced into 2-mm-by 2-mm pieces. There were four comparison groups: 1) group A, diced cartilage (not wrapped with any material); 2) group B, diced cartilage wrapped with AlloDerm; 3) group C, diced cartilage grafts wrapped with Surgicel; and 4) group D, diced cartilage wrapped with PRFM. Four cartilage grafts were implanted under the skin at the back of each rabbit. All rabbits were sacrificed at the end of 10 weeks. The cartilages were stained with hematoxylin-eosin, Masson’s Trichrome, and Orcein. After that, they were evaluated for the viability of chondrocytes, collagen content, fibrillar structure of matrix, and changes in peripheral tissues. Results: When the viability of chondrocytes, the content of fiber in matrix, and changes in peripheral tissues were compared, the cartilage embedded in the PRFM group was statistically significantly higher than in the other groups (P < 0.05). Conclusion: We concluded that PRFM has significant advantages in ensuring the chondrocyte viability of diced cartilage grafts. It is also biocompatible, with relatively lesser inflammation and fibrosis. Key Words: Platelet-rich fibrin matrix (PRFM), diced cartilage, rhinoplasty, AlloDerm, Surgicel. Level of Evidence: N/A. Laryngoscope, 00:000–000, 2014
INTRODUCTION Autologous cartilage grafting is a widely used method in rhinoplasty.1,2 However, cartilage grafting has several drawbacks, such as risks of resorption and necrosis.2–4 In order to increase the viability of cartilage tissue and prevent its resorption, many techniques have been described. Among these techniques, Erol’s “Turkish delight” method of using diced cartilage grafts wrapped with oxidized methylcellulose (Surgicel; Johnson and Johnson Medical Inc., Arlington, TX) recently has been popular.5 In the long term, however, recent clinical studies have reported that the diced cartilage wrapped with Surgicel was easily resorbable and created a limitation in their application.6–8 Some researchers have used temporal muscle fascia with a wrapping method in order to avoid foreign-body reactions in particular, among others. Although the resorption rate and frequency of related foreign-body reactions were much lower than those of Surgicel,9,10
From the Department of Otolaryngology, School of Medicine, Ufuk University (I.G., S.K.); and the Department of Histology and Embryology, School of Medicine, Ankara University (D.B., S.A.), Ankara, Turkey Editor’s Note: This Manuscript was accepted for publication November 20, 2014. The authors have no funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Deniz Billur, MD, Department of Histology and Embryology, School of Medicine, Ankara University, Ankara Turkey. E-mail: [email protected] DOI: 10.1002/lary.25097
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this method has several disadvantages, including donorsite scarring and the need for a second incision. To overcome these disadvantages, a preserved human dermis (AlloDerm; LifeCell Corporation, Branchburg, NJ) was used.11 Unfortunately, the risk of immune reactions, its high price, and difficulty in harvesting graft material have limited the usage. Many investigations have been performed to extend the survival of autogenous cartilages with a goal to make them ideal implant materials.12 Platelet-rich plasma (PRP) is a plasma fraction that is produced from autologous blood samples containing relatively higher concentration of platelets.13 PRP has several different growth factors and other cytokines that stimulate healing of bone.14,15 It has been used for different purposes in surgery for approximately 30 years and conceivably increases the survival of cartilage grafts used in rhinoplasty.16–18 Platelet-rich fibrin matrix (PRFM) was firstly introduced by Choukroun in 200119 and undeniably is easily obtained. In this study, we aimed to compare the viability of cartilage wrapped with PRFM with the cartilage that was left bare, the cartilage wrapped with Surgicel, and the cartilage wrapped with AlloDerm.
MATERIALS AND METHODS The experimental design was reviewed and approved (no. 2012-22-134) by the ethics committee in animal experiments at Ankara University, and the study was carried out in compliance with the guidelines for animal experimentation at the
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Fig. 1. Four types of grafts. (A) diced cartilage; (B) graft wrapped with AlloDerm; (C) graft wrapped with Surgicel; and (D) graft wrapped with PRFM. [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]
Department of Laboratory Animal Science at Ankara University Medical School (Ankara, Turkey). All animals used for the experiments received care according to the Principles of Laboratory Animal Care recommended by the National Society of Medical Research and the Guide for the Care and Use of Laboratory Animals, proposed and prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication No. 85-23, revised 1996).
Animal Care Animals were housed in a controlled environment at a temperature of 18 C 6 1 C and with a relative humidity of 50% to 70%. They were also exposed to 12-hour light–dark cycles and fed with pellets and water ad libitum. All animal care services and procedures were performed without inflicting pain. A total of six New Zealand rabbits, each weighing approximately 2 kg, were used for the study. Each rabbit was anesthetized with 35 mg/kg ketamine and 5 mg/kg xylazine hydrochloride, both given intramuscularly.
Material Used in the Study 1. Biomaterials. AlloDerm: Pieces of thin layers of acellular human epidermal tissue with a thickness of 0.89 mm to 1.65 mm and measuring 2 cm by 4 cm were used. PRFM: Using Choukroun’s method, the blood samples from rabbits were harvested and put in 10-cc venous blood tubes without anticoagulant.19 Tubes were centrifuged (N€ uve Corporation CN 090, Ankara, Turkey) for 10 minutes at 3,000 rpm (400 g). The absence of anticoagulant implies the activation within a few minutes of most platelets in the blood sample that are in contact with the tube walls, as well as the release of coagulation cascades. Before the circulating thrombin transforms fibrinogen into fibrin, fibrinogen is concentrated in the upper part of the tube. Afterward, a fibrin clot is obtained in the middle of the tube, just between the red blood cells at the bottom and the acellular plasma at the top.20 The samples of platelet-rich fibrin (PRF), which accumulated in the middle of the tube after centrifuge, were picked up from the tubes by the help of a pincette and separated from the red blood cells. PRF was exposed to pressure between the two glass slides. After this process, the PRFM, which had risen between the slides, were cut into 2- by 2-cm pieces using scissors, and then the diced cartilages were wrapped. 2. Nonbiomaterials: Oxidized cellulose polymers in 5.1- by 7.6cm pieces (Surgicel) were used.
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Method 1. Surgical technique. To prepare the recipient site, the back and the right ear of each rabbit was shaved, cleaned, and disinfected with povidone iodine (Betadine Hydrochloride. Purdue Pharma L.P, Stamford, USA.), and 7- to 8-cm incisions were made on the dorsal surface of the ear. A 3- by 3cm (2 cc) auricular cartilage was harvested with its perichondrium. Incisions were closed with 4/0 vicryl sutures. Four separate incisions were made on the back of the rabbits. Then, 3- by 4-cm pockets were made on the back of each rabbit. Afterward, four cartilage grafts were inserted into the four subcutaneous pockets on the same animal. The incisions sutured with 4/0 vicryl sutures. The grafts left at the recipient sites for 10 weeks postoperatively. 2. Graft preparation. In our study, approximately 2 cc of cartilage was harvested from each ear of the rabbits. The excised cartilage, with its perichondrium, was diced into 2-mm- by 2mm pieces. Four pieces (each 0.5 cc) of the diced cartilage was wrapped with rehydrated AlloDerm (group B), Surgicel (group C), 2- by 2-cm PRFM (group D), and in the control group it was left bare (group A) (Fig. 1). 3. Implantation of the diced cartilage grafts. Several incisions, each 3 cm to 4 cm in length, were made on the back of each rabbit and the diced cartilage was implanted under the skin. The incisions were marked as diced cartilage (control group A), AlloDerm (group B), Surgicel (group C), and PRFM (group D). The incisions were closed with 4/0 vicryl sutures. 4. Harvesting. After 10 weeks, the implantation sites of the rabbits were shaved again and the cartilages were harvested from the original incision sites. Specimens were placed into 10% formalin solution for fixation.
Histological Examination and Analysis After formalin fixation for 72 hours, samples were dehydrated, cleared, and embedded in paraffin. Tissue sections (4–5 lm) were stained with hematoxylin and eosin (H&E), Masson’s Trichrome, and Orcein. The H&E-stained sections were used to evaluate chondrocyte viability and the status of the chondroid tissue. Masson’s Trichrome demonstrates collagen content of the matrix by staining the collagen fibrils green. Orcein stains elastic fibers in the matrix. Each sample was evaluated under light microscope (Carl Zeiss Axio Scope-A1, G€ ottingen, Germany) by two histologists with no knowledge of examination groups. They were used a scoring system for evaluation of morphology and histological parameters that modified from Kim et al.21 Ten histological parameters were reviewed in three groups: 1) loss of chondrocyte nuclei, peripheral proliferation; 2) collagen and
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TABLE I. Histological Parameters of Groups.
Histological Parameters
Diced Cartilage
Diced Cartilage With AlloDerm
Diced Cartilage With Surgicel
Diced Cartilage With PFRM
n56
n56
n56
n56
2
11
21
31
41
2
11
21
31
41
2
11
21
31
41
2
11
21
31
41
0 0
1 3
1 1
2 1
2 1
0 0
1 4
1 1
3 1
1 0
0 1
0 3
1 2
3 0
2 0
4 0
1 1
1 2
0 2
0 1
0 0
4 2
2 4
0 0
0 0
0 0
2 0
2 5
2 1
0 0
2 0
3 3
0 2
1 1
0 0
0 0
2 0
1 2
2 4
1 0
1 1
3 3
0 2
2 0
0 0
0 0
2 1
2 2
2 2
0 1
0 0
3 1
1 2
1 2
1 1
2 5
3 1
1 0
0 0
0 0
Cartilage viability Loss of chondrocyte nucleus Peripheral proliferation Matrix Collagen Elastic fiber Changes of peripheral tissue Fibrosis Inflammation Vascularization
1
3
2
0
0
1
4
1
0
0
2
3
1
0
0
0
4
1
1
0
Metaplastic bone formation Fragmentation
6 4
0 1
0 1
0 0
0 0
4 2
1 2
1 2
0 0
0 0
5 0
1 1
0 2
0 3
0 0
5 5
1 0
0 0
0 1
0 0
Resorption
1
4
0
1
0
4
1
0
1
0
0
1
2
2
1
4
2
0
0
0
(2) 5 none; (11) 5 minimal; (21) 5 moderate; (31) 5 moderate to severe; (41) 5 severe. PFRM 5 platelet-rich fibrin matrix.
elastic fiber content of the matrix; and 3) fibrosis, inflammation, vascularization, metaplastic bone formation, fragmentation and chondroid tissue resorption. Quantitative analyses of all parameters were performed between groups. Parameters were recorded as a percentage of all analyzed material: 0% as none (2), 1% to 25% as minimal (11), 26% to 50% as moderate (21), 51% to 75% as moderate to severe (31), and 76% to 100% as severe (41).
Statistical Analysis Statistical analyses were performed using the SPSS software package (version 18.0; Chicago, IL). In intergroup comparisons, a nonparametric test (Kruskal- Wallis) and MannWhitney test were used. The average of each group was calculated. Results were evaluated at a significance level of P < 0.05.
RESULTS Macroscopic Observation All harvested grafts showed adhesions. In the control group, the grafts were more irregular than those of the other experimental groups. In all experimental groups, graft unity was observed. The grafts that were wrapped with Surgicel were smaller in volume than those used in the other experimental groups.
Histological Examination Histopathological parameters reviewed in four groups were demonstrated in Table I. Group A (Diced Cartilage). H&E-stained preparations demonstrated that the content of chondrocyte nuclei was the indicator of viability (Fig. 2 a,b). In the matrix, we observed that the collagen fibers in the matrix stained green with Masson Trichrome and elastic Laryngoscope 00: Month 2014
fibers stained brown with orcein (Fig. 2 c,d). In the cartilage matrix, both types of fibers were in minimal amount. We observed minimal fibrosis, inflammation, and vascularization in the tissue around the cartilage. There was no metaplastic bone formation. Minimal cartilage fragmentation and resorption were noted (Fig. 2a). Group B (Diced Cartilage Grafts Wrapped With AlloDerm). Whereas the loss of nuclei in the specimens of diced cartilage grafts wrapped with AlloDerm was variable (moderate to severe loss), peripheral proliferation was moderate (Fig. 3 a,b). The presence of collagen and fiber contents was moderate (Fig. 3 c,d). However in fibers around the cartilage tissue, vascularization was minimal, and the level of fibrosis and inflammation increased moderately. Metaplastic bone formation was observed in two rabbits. Cartilage fragmentation was observed in our specimens, whereas the resorption of cartilage grafts was minimal (Fig. 3 a). Group C (Diced Cartilage Grafts Wrapped With Surgicel). When the specimens of cartilage grafts were wrapped with Surgicel, there was severe loss of nuclei in chondrocytes stained with H&E and minimal peripheral proliferation was seen (Fig. 4 a,b). It was observed that collagen and the content of elastic fiber were minimal (Fig. 4 c,d). Vascularization was minimal, fibrosis was moderate, and the level of inflammation was between moderate and severe in the tissue that surrounds the cartilage. Metaplastic bone formation was observed in one rabbit. The degree of fragmentation and resorption of cartilage grafts was between moderate and severe. (Fig. 4 a,b). Group D (Diced Cartilage Grafts Wrapped With Plasma-Rich Fibrin Matrix). In the specimens of cartilage grafts wrapped with PRFM, minimal loss of chondrocyte nuclei was observed as compared with the €ler et al.: Augmentation Materials for Rhinoplasty Gu
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Fig. 2. Histological examination of diced cartilage grafts. (A) Peripheral chondrocyte proliferation in diced cartilage group (pcp), loss of chondrocytic nucleoli (*), viable chondrocyte (arrow), minimal fibrosis (F), and neovascularization (arrowhead) (hematoxylin and eosin [H&E], magnification 3200). (B) Pericondrium (P), peripheral chondrocyte proliferation (pcp), loss of chondrocytic nucleoli (*), isogenous groups (*), and territorial matrix (arrow) (H&E, magnification 3400). (C) Minimal fibrosis (F), minimal level of collagen fiber content in the green-stained area of cartilage matrix (Masson’s Trichrome stain; magnification 3200). (D) Minimal level of collagen fiber content in the brown-stained area of cartilage matrix (arrow) (Orcein stain; magnification 3400). [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]
other groups. Additionally, moderate to severe degrees of peripheral proliferation were noted. Analysis of the peripheral tissue demonstrated minimal degrees of fibrosis and vascularization without any evidence of inflammation. Additionally, metaplastic bone formation was seen in only one specimen, and bone fragmentation and resorption was minimal (Fig. 5 a,b). When the content of collagen and elastic fiber was analyzed, it was seen that both types of fibers were preserved moderate and severe degree (Fig. 5 c,d).
Statistical Analysis When the PRFM group (group D) was compared to the diced cartilage group (group A), there were significant differences in viability of the cartilage, fibrosis, inflammation, and resorption of the cartilage. When the PRFM group (group D) was compared to the AlloDerm group (group B), there were significant differences in the loss of chondrocytic nuclei, fibrosis, and inflammation. When PRFM group (group D) was compared to the Surgicel group (group C), there were significant differences in the loss of chondrocyte nuclei, peripheral proliferation, extent of elastic fiber, inflammation, fragmentation, and cartilage resorption (P < 0.05) (Table II). There was no significant difference among the groups regarding Laryngoscope 00: Month 2014
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metaplastic bone formation, vascularization, and collagen content of the fibers.
DISCUSSION Cartilage grafts have been used effectively in rhinoplasty surgery for the augmentation and correction of nasal dorsum asymmetry and irregularities.5,22 However, many surgeons have indicated a significant problem about the usage of cartilage harvested from septum, conchal cartilage, or costa as a block. The problem is that cartilage is disabled when used for dorsal irregularities because of the resorption and distortion of the graft material.23–25 In order to overcome these problems, diced cartilage was suggested for use instead of cartilage blocks. During reshaping, the diced cartilage method was preferred because of its many advantages.5 For this reason, we also prefer diced cartilage to block cartilage. In numerous experimental studies, the cartilage was wrapped with a material in order to minimize the problem of resorption and increase the viability of the implanted cartilage.8 The Turkish delight method of wrapping the cartilage with Surgicel, developed by Erol in 2000, was accepted and was a milestone. This method was very popular because the study included 2,365 €ler et al.: Augmentation Materials for Rhinoplasty Gu
Fig. 3. Histological examination of diced cartilage grafts wrapped with AlloDerm. (A) Pericondrium in the sample of cartilage tissue wrapped with AlloDerm (P), moderate fibrosis (F) and inflamation (star), neovascularization (*), and metaplastic bone formation (MB) (hematoxylin and eosin [H&E], magnification 3100). (B) Moderate peripheral chondrocyte proliferation (pcp), loss of chondrocytic nuclei (*), and viable chondrocyte (arrow) (H&E, magnification 3400). (C) The content of collagen in green-stained cartilage matrix (Masson’s Trichrome stain, magnification 3200). (D) Moderate elastic fiber content in the brown-stained area of cartilage matrix (arrow) (Orcein stain, magnification 3400). [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]
patients, and it had been used for 10 years with successful results.5 Many studies, which accepted or rejected this method, have been cited in the literature. Elahi also used cartilage wrapped with Surgicel and reported similar results in 2003.26 However, Yilmaz et al. reported that this surgical intervention prevented the oxygenation of cartilage tissue for at least 48 hours, and this relative hypoxia led to a loss of regeneration potential that is important for the survival of chondrocytes.6 Erdogmus et al. reported that the use of calcium hydroxylapatite in combination with diced cartilage grafts does not have any long-term negative effects on chondrocyte viability.27 A study reported by Cakmak showed that the diced cartilage wrapped with Surgicel inhibited the proliferation and viability of cartilage.7 Indeed, approximately 10% of the implanted cartilages maintained their viability.7 However, Daniel and Calvert observed that unexpected implant resorptions occurred a few months after a rhinoplasty operation, when diced cartilage wrapped with Surgicel was used.8 In our study, when cartilage graft wrapped with Surgicel was evaluated with respect to its histological characteristics, severe loss of chondrocyte nuclei, decreased peripheral proliferation, severe fibrosis, and inflammation were observed. Significant differences were found in the rate of cartilage resorption among the Laryngoscope 00: Month 2014
groups. Our study supported other studies for which the viability potential of cartilage graft wrapped with Surgicel was lower with higher resorption capability.6–8 When the higher level of resorption in cartilage grafts wrapped with Surgicel was considered, modification of the cartilage by wrapping it with biocompatible materials was suggested.10,28 Daniel and Calvert used diced cartilage wrapped with autologous temporal fascia in rhinoplasty and obtained consistent and significant results.8 Ozturk and Aydin used diced cartilage grafts wrapped with amniotic membrane.29 Bracaglia et al. used diced cartilage graft treated with human fibrin glue.30 Brenner et al. indicated that autologous fascia had no negative effect on durability of cartilage regeneration.28 Fascia is a durable material with a high resistance power that functions as a cellular support.31 Brenner et al. indicated that fascia functions as a pericondrium, increases viability of chondrocytes, prevents the resorption of grafts, and maintains the general regenerative potential of the cartilage.28 However, there are some limitations to our study, including using another donor site and incision scarring for autologous fascia. Also, we did not prefer the use of temporalis fascia wrapped with diced cartilage in our study because of the incision scar and also because Kim et al.21 demonsrated that AlloDerm was found to be significantly superior to temporalis fascia in terms of €ler et al.: Augmentation Materials for Rhinoplasty Gu
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Fig. 4. Histological examination of diced cartilage grafts wrapped with Surgicel. (A) Minimal level of chondrocyte proliferation in the sample of cartilage tissue wrapped with Surgicel (pcp), variable level from moderate-to-severe inflammation (star), fibrosis (F), and fragmentation in cartilage tissue and metaplastic bone formation (MB) (hematoxylin and eosin [H&E], magnification 3100). (B) Severe loss of nucleus in chondrocyte (*) (H&E, magnification 3400). (C) Increasing fibrosis in peripheral tissue (F), minimal level of collagen fiber content in the green-stained area of cartilage matrix (Masson’s Trichrome stain, magnification 3200). (D) Minimal level of elastic fiber content in the brown-stained area of cartilage matrix (arrow) (Orcein stain, magnification 3400). [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]
histopathological criteria. To overcome donor-site problems, Achauer32 used AlloDerm on the nasal dorsum with a limited number of patients, and also Jacson et al. and Gryskiewicz et al. used Alloderm in dorsal nasal irregularities and the correction of nasal contour deformities.33–35 AlloDerm is an acellular human dermis matrix. After dermis is harvested from cadaveric skin, epidermis and all the cellular components are removed by scrubbing the epidermis layer with detergent and antiviral solutions. Afterward, the protein skeleton of the dermis is preserved in a frozen condition.36 Kim et al. compared cartilage grafts wrapped with AlloDerm to those wrapped with autologous fascia in their 2011 study.21 The observed results related to AlloDerm were histologically significant and showed the higher regeneration potential of the chondrocytes. Additionally, contents of matrix collagen were preserved and metaplastic bone formation did not occur.21 AlloDerm does not cause any immunological or foreign-body reactions, including rejection or reactions involving major histocompatibility class I and II antigens.37 For this reason, it tends to be treated as an autograft when used in the body. Many studies also have shown that AlloDerm does not cause inflammatory and allergic reactions. On the other hand, Bateman and Jones indiLaryngoscope 00: Month 2014
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cated the risks, including infection, tissue reaction, and rejection, caused by alloplastic materials in their study.12 In our study, when we compared the group of cartilage grafts wrapped with AlloDerm to other groups in terms of histological characteristics, we found that the loss of chondrocyte nuclei and peripheral proliferation in cartilage wrapped with AlloDerm were more significant than observed in cartilages wrapped with Surgicel. In both groups, fibrosis and inflammation were moderate. Metaplastic bone formation and undesirable results were observed in both groups. However, there were no statistically significant intergroup differences. We compared PRFM to AlloDerm in terms of inflammation and fibrosis and found that PRFM yielded significantly more successful outcomes. Originally, PRFM was identified as a secondgeneration platelet solution by Choukroun in 2001.19 It has been used in many fields of medicine such as dentistry, dermatology, and plastic surgery.20 PRFM in particular contains two important cytokines, interleukin (IL)24 and VEGF, in addition to other inflammatory cytokines (IL-1, IL-6, tumor necrosis factor [TNF] a, and TNF b), and is involved in the healing process of the tissues.38 Studies on healing bone have shown that PRFM caused fibroblastic proliferation and supported its €ler et al.: Augmentation Materials for Rhinoplasty Gu
Fig. 5. Histological examination of diced cartilage grafts wrapped with PRFM. (A) Severe peripheral chondrocyte proliferation in the samples of cartilage tissue wrapped with PRFM (pcp) (hematoxylin and eosin [H&E], magnification 3200). (B) Numerous viable chondrocytes (arrow) (H&E, magnification 3400). (C) Plenty of elastic fiber content in the green-stained area of cartilage matrix (Masson’s Trichrome stain, magnification 3200). (D) Plenty of elastic fiber content in the brown-stained area of cartilage matrix (arrow) (Orcein stain, magnification 3400). [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]
osteoblastic activity by triggering angiogenesis.39 In their study on rabbits, Kuo et al. created cartilage defects in rabbits and compared PRFM group to the con-
trol group.17 The authors reported significant healing in PRFM implanted rabbits based on the results of MR and histological observation.
TABLE II. The Results of Statistical Analysis for Histological Parameters Reviewed in Groups. Between Diced Cartilage and Diced Cartilage Wrapped With PRFM
Between Diced Cartilage Wrapped With AlloDerm and Diced Cartilage Wrapped With PRFM
Between Diced Cartilage Wrapped With Surgicel and Diced Cartilage Wrapped With PRFM Group
Group A Versus Group D
Group B Versus Group D
Group C Versus Group D
Loss of chondrocyte nucleus Peripheral proliferation
0.042* 0.407
0.026* 0.092
0.011* 0.038*
Collagen
0.12
0.616
0.065
Elastic fiber Fibrosis
0.014* 0.342
0.093 0.044*
0.041* 0.086
Inflammation
0.023*
0.004*
0.003*
Vascularization Metaplastic bone formation
0.589 0.317
0.293 0.4
0.179 0.317
Histopathologic Parameters
Fragmentation
0.674
0.209
0.022*
Resorption
0.045*
0.847
0.005*
*P value < 0.05 5 statistically significant. PRFM 5 platelet-rich fibrin matrix.
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CONCLUSION In our research, when we compared PRFM to other groups in terms of contents of chondrocyte nuclei and inflammation, we found that PRFM was superior among the other groups. Of note, it caused less fibrosis than AlloDerm grafts. In terms of the loss of chondrocyte nuclei, peripheral chondrocyte proliferation, elastic fiber content of matrix, fibrosis, inflammation, and resorption parameters, PRFM preserved the viability of cartilage grafts more successfully than Surgicel material. PRFM has significant advantages in that it ensures the chondrocyte viability of diced cartilage grafts and is also biocompatible, with relatively lesser inflammation and fibrosis.
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