Eur Arch Otorhinolaryngol DOI 10.1007/s00405-014-3192-2
RHINOLOGY
The comparison of the viability of crushed, morselized and diced cartilage grafts: a confocal microscopic study Gurkan Kayabasoglu • Elvan Ozbek • Sevinc Yanar • Fikrettin Sahin • Osman Nuri Keles Mahmut Sinan Yilmaz • Mehmet Guven
•
Received: 26 April 2014 / Accepted: 1 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract To compare the cellular viability of diced, crushed, and morselized cartilage used in nasal surgeries. In this study, cartilage was extracted from the ears of seven New Zealand rabbits and was subsequently either diced, crushed or morselized to an amorphous state, or left unmodified. The four types of grafts were then implanted in the back regions of the rabbits. After 3 months, the cellular viability from four groups was compared to a control group using confocal microscopy. Analysis of the data obtained from the enumeration of live cells showed no statistically significant difference between the unmodified graft group and the control group. The diced, crushed, and morselized cartilage groups did show a statistically significant difference in terms of live cell count with the highest number of live cells in diced cartilage group. A statistically significant decrease in
This study was presented at 35th Turkish National Otolaryngology Meeting, 2–6 November 2013 Antalya, Turkey. It was awarded as second prize of scientific research in Rhinology/Facial Plastic section. G. Kayabasoglu (&) M. S. Yilmaz M. Guven Department of Otorhinolaryngology, Faculty of Medicine, Sakarya University, Adnan Menderes Cad. No 145, Adapazarı, Sakarya 54100, Turkey e-mail: [email protected] E. Ozbek S. Yanar Department of Histology and Embryology, Faculty of Medicine, Sakarya University, Adapazarı, Sakarya, Turkey F. Sahin Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul, Turkey O. N. Keles Department of Histology and Embryology, Faculty of Medicine, Ataturk University, Erzurum, Turkey
live cell count was detected in crushed cartilage group. Our study shows that the viability of cells in diced cartilage grafts is greater than those in crushed or morselized cartilage grafts. Keywords Confocal microscopy Cartilage Cell viability Experimental Rhinoplasty Morselization Graft Diced Crushing Nasal surgery
Introduction Aside from being one of the most frequently performed procedures by otolaryngologists and plastic surgeons, rhinoplasty is an increasingly popular research topic and the subject of a great number of medical conferences and meetings. One of the more popular topics in the field of rhinoplasty is the use of harvested cartilage for grafts, reconstruction of the nasal skeleton, concealment of the irregularities, and augmentation of the nasal dorsum. Cartilage grafts have a wide range of uses and advantages, but along with that utility comes a source for complications. One such possible problem is the visibility of the sharp edges of cartilaginous grafts through the subcutaneous tissue as the elevated skin is restored in the long term. Various methods have been described to prevent this problem including morselization, dicing and/or crushing of the cartilage prior to grafting. Numerous experimental and clinical studies have been performed to investigate the viability, reliability and the long-term outcomes of cartilage prepared by different methods. Our study highlights the first published comparison of the cellular viability rates of crushed, diced and morselized cartilage. Over the past 20 years, free cartilage grafts have been used extensively to provide shaping and structural support in nasal surgery [1, 2]. Cartilage can also be modified into a
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more pliable, or even amorphous, state with the use of specialized tools. In recent years, many clinical and experimental studies have been performed regarding the use and survival times of the amorphous cartilage. Our study focuses on the modification of cartilage in three distinct ways: crushed, morselized, and diced. The final analysis and determination was done using confocal microscopy, which is similar to fluorescent microscopy with the exception of the light source being monochromatic as opposed to panchromatic. It is, therefore, a sensitive and reliable, often used method to determine live and dead cell count [3].
Methods Preparation of cartilages Our study was performed in the Experimental Research Center Laboratories of Yeditepe University School of Medicine following the confirmation granted on 11/30/ 2012 (approval no: number 298.) In this study, seven New Zealand female rabbits were used, all between 8 and 9 months old, weighing 2,000–2,400 g. In the first stage of study, after general anesthesia was achieved in rabbits by intramuscular injection of 50 mg/kg Ketamine Hydrochloride and 5 mg/kg Xylokain Hydrochloride (Fig. 1a), cartilage tissue was obtained by elevating the dorsal and ventral perichondral planes in both ears of the rabbits (Fig. 1b) and the grafts were subsequently divided into approximately 1 9 1 cm pieces (Fig. 1c). From the collected cartilage, four different subsets were created: The first group was moderately crushed (crushed cartilage group) by crusher (Cottle cartilage crusher, model 523900; Karl Storz GmbH & Co, Tuttlingen, Germany), a second group was moderately softened (morselized cartilage group) by a morselizer (WrightRubin septum morselizer forceps straight, model N5345 Karl Storz GmbH & Co, Tuttlingen, Germany), the third group was diced (diced cartilage group) into pieces of 0.5–1 mm or smaller with a No. 11 blade scalpel, and the remaining group was left intact (unmodified cartilage group) (Fig. 1d). Following the preparation, multiple 1 cm incisions were made on the back of rabbits and a dissection performed between the panniculus carnosus and the deep fascia (Fig. 1e). The prepared grafts were then placed into the pouches opened and incisions were closed with 3/0 silk suture. The 2nd stage of the study commenced after a waiting period of 3 months. The rabbits were first killed by injecting a fatal dose of ketamine hydrochloride, and their backs were widely explored and the previously placed
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cartilage grafts removed (Fig. 1f). At this point, a piece of the remaining cartilage was taken from the rabbits’ ears for use as a control group (control cartilage group,) and all the grafts were examined by confocal microscopy. No rabbit had any sign of wound site infection, seroma or serious weight loss. No rabbits died during the study. The study was completed with the harvesting of the cartilage grafts from all seven rabbits after 3 months duration. Preparation of confocal microscopic sections for assessment of cell viability After killing the animals, 500 lm thick cross-sectional slices were cut lengthwise from the dissected fresh cartilage tissue samples and put in bacteriostatic PBS solution. Live/dead assay staining solution was prepared by adding 4 lM ethidium homodimer solution to 2 lM calcein AM solution (Molecular probes, Invitrogen Ltd., LIVE/DEADÒ Viability/Cytotoxicity Kit, for mammalian cells, Product no: L3224, Paisley, UK). The assay, distinguishing live cells from dead cells, is based on the characteristics of live cells that have intracellular esterase activity and an intact plasma membrane. The green-fluorescent calcein AM indicates intracellular esterase activity and therefore stains viable cells. The red-fluorescent ethidium homodimer indicates loss of plasma membrane integrity and therefore stains dead cells. Tissue sections were washed with PBS three times and incubated in Live/Dead assay staining solution for 30 min at 37 °C. After incubation, the samples were washed again with PBS three times. Stained tissue samples were put on microscope slides and examined using a confocal laser scanning microscope (Leica TCS SP2 SE, Wetzlar, Germany). Consecutive high-resolution digital images were taken from all areas of each section and converted to TIF format and merged using Adobe Photoshop CS3, version 10.0. Estimation of numerical density of live and dead cells using stereologic investigation method Stereology is a medical analytic method that uses random, systematic sampling to provide unbiased and quantitative data on tissue slides and images. It provides practical techniques for obtaining quantitative information about a three-dimensional material from measurements conducted with two-dimensional planar sections of the material. The unbiased counting frame-fractionator combination is a stereological method for counting cells in tissue sections [4, 5]. In our study, we used the unbiased counting framefractionator combination method to estimate numerical density of cells in cartilage tissue stained with fluorescent dyes. The systematic randomized sampling was made
Eur Arch Otorhinolaryngol Fig. 1 Preparation steps of cartilage grafts. a Rabbits prepared under general anesthesia. b Cartilage tissue obtained by elevating the dorsal and ventral perichondral planes in both ears of the rabbits. c Cartilage grafts were divided into pieces measuring 1 9 1 cm. d Diced cartilage (left,) crushed cartilage (middle), and morselized cartilage (right). Unmodified cartilage not shown (refer to Fig. 1c). e Multiple 1 cm incisions were made rabbits’ back and dissection was performed between panniculus carnosus and deep fascia. f The rabbits were sacrificed and their backs were widely explored and the previously placed cartilage grafts were removed
according to an agreeable coefficient error for all cases in this study. A pilot application was performed to determine a suitable coefficient error (CE of 5 % or less) for a systematic randomized sampling [5–7]. In merged confocal microscopic images, the key area was counted via a stereology workstation using stereology software (Stereo Investigator version 8.0, Micro-brightfield, Colchester, VT). Unbiased counting frame was automatically and systematic-randomly distributed with equal intervals (x, y-step area) throughout the delineated area. Merged images of each section were sampled at low magnification using the fractionator principle of the stereology software. Cells were counted at high magnification,
which allowed accurate recognition. Cells were scored in unbiased counting frames on whole sections in each tissue sample (Fig. 2). After all these applications, the numerical density of live and dead cells was estimated using the following formula: ND ¼
TM CFA NSS
where ND is numerical density, TM is total markers, CFA is counting frame area (XY) (lm2), and NSS is number of sampling sites. The coefficient of error (CE) for calculations of numerical density was the last calculated value. Generally accepted highest limit of CE is 5 % [8, 9].
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Fig. 2 Viable cell counts by using stereoinvestigator in a specific area of confocal microscopy images. Orange arrows point at living cartilage cells
Statistical analysis Statistical analysis of obtained results was performed using one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test (DMRT) via SPSS software package, version 19.00 (SPSS Inc., Chicago, IL). p \ 0.05 was considered statistically significant. All results were expressed as mean standard error of the mean (SE) for seven rabbits in each group.
Comparing the groups in terms of the means of the dead cell amount, the diced cartilage group was the only group not statistically different from the control cartilage group. The numbers of dead cells found in the unmodified, crushed and morselized cartilage groups was significantly higher than both the control cartilage group and the diced cartilage group. The minimum number of dead cells was determined to be in the control cartilage group, followed by the diced cartilage group. There was no significant difference in the number of dead cells among unmodified, crushed and morselized cartilage groups (Table 1).
Results Examining the data obtained from viable cell counts, no statistical significance was defined between control cartilage group and unmodified cartilage group. However a statistically significant difference was present in the number of viable cells between the crushed, diced, and morselized cartilage groups and the control cartilage group. The numerical density of viable cells in the diced cartilage group and morselized cartilage group was found to be higher than the control cartilage group (Fig. 3). The maximum number of viable cells was determined to be in the in the diced cartilage group, and the minimum number of viable cells was found in crushed cartilage group (Fig. 4).
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Discussion For a successful rhinoplasty outcome, it is not only the careful reconstruction of osteocartilagenous skeleton but also the smooth appearance of the nasal contours that bears great significance. Irregularity of the nasal contours is often the sole impetus for revision rhinoplasty procedures. Various materials have been used to conceal the irregularities encountered during primary nasal operations, among these: cartilage grafts, dermal grafts, temporoparietal fascial grafts, alloplastic materials and even the replacement of resected dorsum tissue have been described [10–16].
Eur Arch Otorhinolaryngol
Fig. 3 Confocal laser screening microscopy images of tissue sections. To identify cellular viability, LIVE/DEADÒ Viability/Cytotoxicity Kit has been used. Viable cells have been stained with calcein AM (green) and dead cells have been stained with ethidium homodimer (red). Comparison of control group (c) with the composite (a) and diced (b) cartilage groups
Fig. 4 Confocal laser screening microscopy images of tissue sections. To identify cellular viability, LIVE/DEADÒ Viability/Cytotoxicity Kit has been used. Viable cells have been stained with calcein AM (green) and dead cells have been stained with ethidium homodimer (red). Comparison of control group (c) with the crushed (a) and morselized (b) cartilage groups
Cartilage grafts have become the most widely accepted material due to its long-term success and low rate of complications [17]. The fundamental difference between cartilage grafts used for structural grafts and correctional grafts is: Hardness, strength, shape specificity, and preservation of shape
are expected from structural grafts (or any grafts used for recreation or support of the nasal skeleton) whereas amorphous, soft, and conformation are expected from correctional grafts (or any grafts used contour concealing or dorsal augmentation.) In the long-term follow-up of correctional grafts, it is very important that there is no
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Eur Arch Otorhinolaryngol Table 1 Effects of different treatments on the numerical density of live and dead cells in cartilage tissue of rabbits Treatment
Number of rabbits
Numerical density of live cells (n/lm3)
Numerical density of dead cells (n/lm3)
Control
7
0.000095 ± 0.000001
0.00000 ± 0.000000
Unmodified
7
0.000098 ± 0.000005
0.00009 ± 0.000040x
Crushed
7
0.000030 ± 0.000012x
0.00008 ± 0.000031x
Diced
7
0.000137 ± 0.000006x
0.00001 ± 0.000003
Morselized
7
0.000111 ± 0.000008x
0.00012 ± 0.000040x
Results are mean ± standard deviation. Means in the same column by ‘‘x’’ superscript letter are statistically significantly different under the Duncan test (a = 0.05)
atrophy, but instead protected viability, as to reduce the possibility of irregularities. The procedures to modify cartilage into an amorphous state themselves cause varying degrees of damage and have varying degrees of effect on the viability rates of grafts. There are studies in the literature reporting that the crushing degree of crushed cartilages can cause important differences on cellular viability rates [18]. It has been reported that viability rates of cartilages decrease with the increase in crushing degree. In a study published by Cakmak and Altintas [18], it was reported that crushed cartilage could be prepared at four varying degrees of thickness if a Cottle crusher is used. However the same does not apply for morselization; a morselizer can crush cartilage to either a mild or moderate degree. It has been reported that cartilage crushed to a moderate degree has the highest rate of cellular viability, and therefore that degree of morselization was used in this study to provide a standardization of procedure. Although there are publications about cartilage softened using a Cottle crusher, for example the study done by Garg et al., there are no studies on cartilage softened by using a morselizer [19, 20]. As far as we know, our study is the first in the literature evaluating the effects of cartilage morselization on cellular viability. Many surgeons use cartilage divided into very thin pieces (diced) by scalpel blade to correct depressions or irregularities in the nasal dorsum. The use of diced cartilage was first explained in 1943 by Peer et al. [21] however the number of studies on this subject has been increasing considerably over the last 10 years [22, 23]. Cartilage can be placed into the nose in various ways: wrapping it in Surgicel (Ethicon, Inc, Somerville, NJ), in AlloDerm (LifeCell Corp, Branchburg, New Jersey), wrapping it in the fascia of temporal muscle, or without wrapping, after achieving a high viscosity by mixing it with a few drops of blood. Erol et al. [24] reported high rates of success when used with Surgicel, and Daniel et al. have claimed that grafts wrapped with fascia have higher rates of viability. Kazikdas et al. and Brenner et al. [17, 25] support the results of Daniel et al. in their studies. Gordon et al. [22]
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have reported that the cartilages wrapped in AlloDerm were successful, did not cause donor site morbidity, and required a shorter operation time. However some conflicting studies do exist, reporting that long-term outcomes of AlloDerm are not reliable due to high rates of resorption [10, 22]. Rather than making a comparison of each application method of diced cartilage, we aimed to compare the advantages of diced cartilages with crushed and morselized cartilage in our study. Viability tests of tissue and cell cultures are used to determine the sensitivity of cells to various chemical, physical and biological factors. Many tests have been developed to define cellular viability in which some different criteria are evaluated such as the proliferative capacity of cells, amount of surviving cells and enzymatic activities or plasma membrane integrity. Tests evaluating the cellular metabolic activity such as ATP or MTT tests usually give fast and accurate results. Measurement of the amount of ATP by bioluminescence is very sensitive (as little as 20 cells/ml). MTT test is a colorimetric test based on the principle of tetrazolium dye reduction to a formazan blue product and measured with spectrophotometry. The MTT test is frequently used for viability evaluation in cell cultures. This test, which is easy to apply, reliable and highly reproducible has a disadvantage of having lower sensitivity [26]. In paint adhesion tests such as trypan blue, only the dead cells are stained due to disruption of plasma membrane. But it is not possible to distinguish healthy cells from the live cells that have lost their function using this colorimetric method. Thus, injury in tissues or cells cannot be detected exactly [27, 28]. Techniques with fluorescent dyes have many biological and clinical applications because they are more accurate and selective than the colorimetric methods. By a combination of different dyes, determination of both live and dead cells becomes possible with a single test. As in this study, staining the tissues or cells together with calcein AM and ethidium homodimer and examination by confocal laser screening microscopy presents quantitative data by ensuring determination of the amount of live and dead cells. Additionally, the optical sectioning capability of
Eur Arch Otorhinolaryngol
confocal microscopy allows the cells to be displayed even in thick tissue samples [26, 27]. Another advantage of this method is the determination of cellular viability by evaluating both the morphological and functional properties in a way that ethidium homodimer measures the membrane integrity and calcein AM measures the esterase activity [28]. The use of confocal microscopy to detect cellular viability has been established in literature. Lu et al. [29] showed that confocal microscopy with vital cell staining is an accurate and sensitive method of determining cell viability. In their study they also concluded that light microscopic techniques could be inadequate and insensitive in some cases where confocal microscopy exhibit successful and satisfying results. In another research Frenz et al. showed that confocal microscopy is a powerful tool for assessing changes in tissue structure [6]. Gantenbein-Ritter et al. clarified that using confocal microscopy to detect viability is advantageous and reasonable because it has the ability to produce optical sections through a 3D sample. Since optical sectioning during confocal microscopy is non-invasive, the 3D distribution and relative spatial relationship of stained living and dead cells could be observed with reasonable clarity [30]. To conclude, from studies conducted so far, confocal microscopy is a reliable, sensitive and sufficient method to detect cellular viability, which is why it was chosen as the method of determining and investigating the viability of cartilage samples in this study. One of the other methods for evaluation of cellular viability is the histological examination performed using different staining techniques. In the cartilage tissue stained by Hematoxylin and Eosin (H and E), chondrocyte viability is analyzed by evaluating the nucleated lacunae. Masson trichrome stain provides an evaluation of connective tissue by showing the collagen content of matrix. Safranin O and PAS stains the proteoglycan content, whereas van Gieson stains the elastic fibers. Evaluation of the viability of cartilage tissue at light microscopic level is performed as looking at the staining properties of chondroid matrix. Disappearance of matrix metachromasia and absence of nuclei in lacunae indicates a non-living chondroid tissue. Counting the nucleated lacunae with specialized software programs provide evaluation of chondrocyte viability and provides quantitative data [31, 32]. Presence of time-consuming steps in routine histological procedures such as tissue processing, sectioning, staining and microscopic examination makes this method more troublesome than others and is therefore a disadvantage. Aside from the advantage of confocal microscopic analysis showing live and dead cells together, it has a disadvantage due to its inability to distinguish cell types in tissue. As there is a possibility of the presence of fibroblasts
and inflammatory cells in addition to the chondrocytes among the cells identified as alive or dead by this technique, we believe that supporting the confocal microscopic data with the findings that achieved via conventional and immunohistochemical histological methods will provide valuable information (such as iNOS/eNOS, bcl/bax etc.). In a study such as this, it is natural to observe various inflammatory tissue responses resulting from varying levels of cellular damage based on the technique that was used to prepare grafts from the excised cartilage. Therefore we believe that some differences will also occur in the amounts of histopathological evaluation indices such as cellular viability, fibrosis, mitotic and chondrogenic activity in different graft groups. The confocal microscopic method used to evaluate cellular viability and cytotoxicity in this study is a novel method and has provided advanced technological data. In this study, we have found that the cellular viability in diced cartilage is greater than in morselized, crushed, unmodified, and control cartilage. Considering that the cartilage tissue is fed by diffusion from its environment in natural conditions, we can assert that the cellular proliferation could be stimulated even more as a result of the increased contact surface with the environment in diced graft group.
Conclusion In this study, we aimed to evaluate the cellular viabilities of crushed, morselized and diced cartilage grafts by confocal microscopic examination and to compare them with the unmodified cartilage grafts. It was found that the viability of diced cartilage grafts is greater than the crushed, morselized, unmodified, and control cartilage. It is important to keep in mind that this is an experimentally induced model in rabbits and this model may yield different results in humans. Thus further studies are needed to evaluate long-term effects and clinical applications of these methods in rhinoplasty procedures. Acknowledgments Authors wish to thank Leyla Tarhan and Serap Kaya for their technical assistance in using confocal microscope. All financial and material support for this research and work were provided by the scientific research fund of Sakarya University. Our study is a multi-centered and multidisciplinary, single-blind randomized controlled animal trial supported by the scientific research fund of our university. Conflict of interest Authors declare that there is no conflict of interest or ethical adherence in this scientific study. Ethical standard The experimental study design was reviewed and approved (no. 298, 30/11/12) by the Committee for Ethics in Animal Experiments at Yeditepe University School of Medicine, and this study was carried out in compliance with the guidelines for animal
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Eur Arch Otorhinolaryngol experimentation at the Department of Laboratory Animal Science at Yeditepe University (Istanbul, Turkey). All animals used for these experiments received care according to the ‘‘Principles of Laboratory Animal Care’’ from the National Society of Medical Research and the ‘‘Guide for the Care and Use of Laboratory Animals’’ prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication No 85-23, revised 1996).
References 1. Ansari K, Asaria J, Hilger P, Adamson PA (2008) Grafts and implants in rhinoplasty—Techniques and long-term results. Op Tech Otolaryngol 19:42–58 2. Hamra ST (1993) Crushed Cartilage Grafts over Alar Dome Reduction in Open Rhinoplasty. Plast Reconstr Surg 92:352 3. Anon (2007v) Theory of confocal microscopy. Available at: www.olympusconfocal.com. Accessed 20 Nov 2007 4. Lu Y, Meyer ML, Bogdanske JJ, Markel MD (2007) The effects of radiofrequency energy probe speed and application force on chondrocyte viability. Vet Comp Orthop Traumatol. 20:34–37 5. Sterio DC (1984) The Unbiased estimation of number and size of arbitrary particles using the dissector. J Microsc 134:127–136 6. Mainil-Varlet P, Monin D, Weiler C, Grogan S, Schaffner T, Zu¨ger B, Frenz M (2001) Quantification of laser-induced cartilage injury by confocal microscopy in an ex vivo model. J Bone Joint Surg Am 83:566–571 7. Gundersen HJ, Jensen EB (1987) The efficiency of systematic sampling in stereology and its prediction. J Microsc 147:229–263 8. Wong DM, Franz AK (2013) A comparison of lipid storage in Phaeodactylum tricornutum and Tetraselmis suecica using laser scanning confocal microscopy. J Microbiol Methods 95:122–128 9. Howard V, Reid S, Baddeley A (1985) Unbiased estimation of particle density in the tandem scanning reflected light microscope. J Microsc 138:203–212 10. Tarhan E, Cakmak O (2008) Ozdemir BH, Akdogan V, Suren D, MD. Comparison of AlloDerm, fat, fascia, cartilage and dermal grafts in rabbits. Arch Facial Plast Surg 10:187–193 11. McKinney P, Loomis MG, Wiedrich TA (1993) Reconstruction of the nasal cap with a thin septal graft. Plast Reconstr Surg 92:346–351 12. Skoog T (1966) A method of hump reduction in rhinoplasty: a technique for preservation of the nasal roof. Arch Otolaryngol 83:283–287 13. Reich J (1983) The application of dermis grafts in deformities of the nose. Plast Reconstr Surg 71:772–782 14. Baker TM, Courtiss EH (1994) Temporalis fascia grafts in open secondary rhinoplasty. Plast Reconstr Surg 93:802–810 15. Gilmore J (1989) Use of Vicryl mesh in prevention of post rhinoplasty dorsal irregularities. Ann Plast Surg 22:105–107
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16. Stoll W (1991) Use of polytetrafluoroethylene for particular augmentation of the nasal dorsum. Aesthetic Plast Surg 15:233–236 17. Kazikdas KC, Ergur B, Tugyan K, Guneli E, Kaya D, Sahan M (2007) Viability of crushed and diced cartilage grafts wrapped in oxidized regenerated cellulose and esterified hyaluronic acid: an experimental study. Laryngoscope 117:1728–1734 18. Cakmak O, Altintas H (2010) A Classification for degree of crushed cartilage. Arch Facial Plast Surg 12:435–436 19. Cakmak O, Buyuklu F (2007) Crushed cartilage grafts for concealing irregularities in rhinoplasty. Arch Facial Plast Surg 9:352–357 20. Garg R, Shaikh M, Foulad A, Wong B (2010) Chondrocyte viability in human nasal septum after morselisation. Arch Facial Plast Surg 12:204–206 21. Peer LA (1943) Diced cartilage grafts. Arch Otolaryngol 38:156–165 22. Gordon CR, Alghoul M, Goldberg JS, Habal MB, Papay F (2001) Diced cartilage grafts wrapped in Alloderm for dorsal nasal augmentation. J Craniofac Surg 22:1196–1199 23. Daniel RK (2008) Diced cartilage grafts in rhinoplasty surgery: current techniques and applications. Plast Reconstr Surg 122:1883 24. Erol OO (2000) The Turkish delight: apliable graft for rhinoplasty. Plast Reconstr Surg 105:229–243 25. Brenner KA, McConnel MP, Evans GR, Calvert JW (2006) Survival of diced cartilage grafts: an experimental study. Plast Reconstr Surg 117:105–115 26. O’Riordan TC, Buckley D, Ogurtsov V, O’Connor R, Papkovsky DB (2000) A cell viability assay based on monitoring respiration by optical oxygen sensing. Anal Biochem 278:221–227 27. Monette R, Small DL, Mealing G, Morley P (1998) A fluorescence confocal assay to assess neuronal viability in brain slices. Brain Res Proc 2:99–108 28. Jimenez-Hernandez ME, Orellana G, Montero F, Portoles MT (2000) A ruthenium probe for cell viability measurement using flow cytometry. Photochem Photobiol 72:28–34 29. Yan Lu, Edwards RB III, Kalscheur VL et al (2001) Effect of bipolar radiofrequency energy on human articular cartilage: comparison of confocal laser microscopy and light microscopy. Arthroscopy 17:117–123 30. Benjamin Gantenbein-Ritter B, Sprecher CM, Chan S et al (2011) Confocal imaging protocols for live/dead staining in threedimensional carriers. Methods Mol Biol 740:127–140 31. Imbert D, Cullander C (1999) Buccal mucosa in vitro experiments. Confocal imaging of vital staining and MTT assays for the determination of tissue viability. J Control Release 58:39–50 32. Orbay H, Tobita M, Hyakusoku H, Mizuno H (2012) Effects of adipose-derved stem cells on improving the viability of diced cartilage grafts. Plast Reconstr Surg 129:369–377
RHINOLOGY
The comparison of the viability of crushed, morselized and diced cartilage grafts: a confocal microscopic study Gurkan Kayabasoglu • Elvan Ozbek • Sevinc Yanar • Fikrettin Sahin • Osman Nuri Keles Mahmut Sinan Yilmaz • Mehmet Guven
•
Received: 26 April 2014 / Accepted: 1 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract To compare the cellular viability of diced, crushed, and morselized cartilage used in nasal surgeries. In this study, cartilage was extracted from the ears of seven New Zealand rabbits and was subsequently either diced, crushed or morselized to an amorphous state, or left unmodified. The four types of grafts were then implanted in the back regions of the rabbits. After 3 months, the cellular viability from four groups was compared to a control group using confocal microscopy. Analysis of the data obtained from the enumeration of live cells showed no statistically significant difference between the unmodified graft group and the control group. The diced, crushed, and morselized cartilage groups did show a statistically significant difference in terms of live cell count with the highest number of live cells in diced cartilage group. A statistically significant decrease in
This study was presented at 35th Turkish National Otolaryngology Meeting, 2–6 November 2013 Antalya, Turkey. It was awarded as second prize of scientific research in Rhinology/Facial Plastic section. G. Kayabasoglu (&) M. S. Yilmaz M. Guven Department of Otorhinolaryngology, Faculty of Medicine, Sakarya University, Adnan Menderes Cad. No 145, Adapazarı, Sakarya 54100, Turkey e-mail: [email protected] E. Ozbek S. Yanar Department of Histology and Embryology, Faculty of Medicine, Sakarya University, Adapazarı, Sakarya, Turkey F. Sahin Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul, Turkey O. N. Keles Department of Histology and Embryology, Faculty of Medicine, Ataturk University, Erzurum, Turkey
live cell count was detected in crushed cartilage group. Our study shows that the viability of cells in diced cartilage grafts is greater than those in crushed or morselized cartilage grafts. Keywords Confocal microscopy Cartilage Cell viability Experimental Rhinoplasty Morselization Graft Diced Crushing Nasal surgery
Introduction Aside from being one of the most frequently performed procedures by otolaryngologists and plastic surgeons, rhinoplasty is an increasingly popular research topic and the subject of a great number of medical conferences and meetings. One of the more popular topics in the field of rhinoplasty is the use of harvested cartilage for grafts, reconstruction of the nasal skeleton, concealment of the irregularities, and augmentation of the nasal dorsum. Cartilage grafts have a wide range of uses and advantages, but along with that utility comes a source for complications. One such possible problem is the visibility of the sharp edges of cartilaginous grafts through the subcutaneous tissue as the elevated skin is restored in the long term. Various methods have been described to prevent this problem including morselization, dicing and/or crushing of the cartilage prior to grafting. Numerous experimental and clinical studies have been performed to investigate the viability, reliability and the long-term outcomes of cartilage prepared by different methods. Our study highlights the first published comparison of the cellular viability rates of crushed, diced and morselized cartilage. Over the past 20 years, free cartilage grafts have been used extensively to provide shaping and structural support in nasal surgery [1, 2]. Cartilage can also be modified into a
123
Eur Arch Otorhinolaryngol
more pliable, or even amorphous, state with the use of specialized tools. In recent years, many clinical and experimental studies have been performed regarding the use and survival times of the amorphous cartilage. Our study focuses on the modification of cartilage in three distinct ways: crushed, morselized, and diced. The final analysis and determination was done using confocal microscopy, which is similar to fluorescent microscopy with the exception of the light source being monochromatic as opposed to panchromatic. It is, therefore, a sensitive and reliable, often used method to determine live and dead cell count [3].
Methods Preparation of cartilages Our study was performed in the Experimental Research Center Laboratories of Yeditepe University School of Medicine following the confirmation granted on 11/30/ 2012 (approval no: number 298.) In this study, seven New Zealand female rabbits were used, all between 8 and 9 months old, weighing 2,000–2,400 g. In the first stage of study, after general anesthesia was achieved in rabbits by intramuscular injection of 50 mg/kg Ketamine Hydrochloride and 5 mg/kg Xylokain Hydrochloride (Fig. 1a), cartilage tissue was obtained by elevating the dorsal and ventral perichondral planes in both ears of the rabbits (Fig. 1b) and the grafts were subsequently divided into approximately 1 9 1 cm pieces (Fig. 1c). From the collected cartilage, four different subsets were created: The first group was moderately crushed (crushed cartilage group) by crusher (Cottle cartilage crusher, model 523900; Karl Storz GmbH & Co, Tuttlingen, Germany), a second group was moderately softened (morselized cartilage group) by a morselizer (WrightRubin septum morselizer forceps straight, model N5345 Karl Storz GmbH & Co, Tuttlingen, Germany), the third group was diced (diced cartilage group) into pieces of 0.5–1 mm or smaller with a No. 11 blade scalpel, and the remaining group was left intact (unmodified cartilage group) (Fig. 1d). Following the preparation, multiple 1 cm incisions were made on the back of rabbits and a dissection performed between the panniculus carnosus and the deep fascia (Fig. 1e). The prepared grafts were then placed into the pouches opened and incisions were closed with 3/0 silk suture. The 2nd stage of the study commenced after a waiting period of 3 months. The rabbits were first killed by injecting a fatal dose of ketamine hydrochloride, and their backs were widely explored and the previously placed
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cartilage grafts removed (Fig. 1f). At this point, a piece of the remaining cartilage was taken from the rabbits’ ears for use as a control group (control cartilage group,) and all the grafts were examined by confocal microscopy. No rabbit had any sign of wound site infection, seroma or serious weight loss. No rabbits died during the study. The study was completed with the harvesting of the cartilage grafts from all seven rabbits after 3 months duration. Preparation of confocal microscopic sections for assessment of cell viability After killing the animals, 500 lm thick cross-sectional slices were cut lengthwise from the dissected fresh cartilage tissue samples and put in bacteriostatic PBS solution. Live/dead assay staining solution was prepared by adding 4 lM ethidium homodimer solution to 2 lM calcein AM solution (Molecular probes, Invitrogen Ltd., LIVE/DEADÒ Viability/Cytotoxicity Kit, for mammalian cells, Product no: L3224, Paisley, UK). The assay, distinguishing live cells from dead cells, is based on the characteristics of live cells that have intracellular esterase activity and an intact plasma membrane. The green-fluorescent calcein AM indicates intracellular esterase activity and therefore stains viable cells. The red-fluorescent ethidium homodimer indicates loss of plasma membrane integrity and therefore stains dead cells. Tissue sections were washed with PBS three times and incubated in Live/Dead assay staining solution for 30 min at 37 °C. After incubation, the samples were washed again with PBS three times. Stained tissue samples were put on microscope slides and examined using a confocal laser scanning microscope (Leica TCS SP2 SE, Wetzlar, Germany). Consecutive high-resolution digital images were taken from all areas of each section and converted to TIF format and merged using Adobe Photoshop CS3, version 10.0. Estimation of numerical density of live and dead cells using stereologic investigation method Stereology is a medical analytic method that uses random, systematic sampling to provide unbiased and quantitative data on tissue slides and images. It provides practical techniques for obtaining quantitative information about a three-dimensional material from measurements conducted with two-dimensional planar sections of the material. The unbiased counting frame-fractionator combination is a stereological method for counting cells in tissue sections [4, 5]. In our study, we used the unbiased counting framefractionator combination method to estimate numerical density of cells in cartilage tissue stained with fluorescent dyes. The systematic randomized sampling was made
Eur Arch Otorhinolaryngol Fig. 1 Preparation steps of cartilage grafts. a Rabbits prepared under general anesthesia. b Cartilage tissue obtained by elevating the dorsal and ventral perichondral planes in both ears of the rabbits. c Cartilage grafts were divided into pieces measuring 1 9 1 cm. d Diced cartilage (left,) crushed cartilage (middle), and morselized cartilage (right). Unmodified cartilage not shown (refer to Fig. 1c). e Multiple 1 cm incisions were made rabbits’ back and dissection was performed between panniculus carnosus and deep fascia. f The rabbits were sacrificed and their backs were widely explored and the previously placed cartilage grafts were removed
according to an agreeable coefficient error for all cases in this study. A pilot application was performed to determine a suitable coefficient error (CE of 5 % or less) for a systematic randomized sampling [5–7]. In merged confocal microscopic images, the key area was counted via a stereology workstation using stereology software (Stereo Investigator version 8.0, Micro-brightfield, Colchester, VT). Unbiased counting frame was automatically and systematic-randomly distributed with equal intervals (x, y-step area) throughout the delineated area. Merged images of each section were sampled at low magnification using the fractionator principle of the stereology software. Cells were counted at high magnification,
which allowed accurate recognition. Cells were scored in unbiased counting frames on whole sections in each tissue sample (Fig. 2). After all these applications, the numerical density of live and dead cells was estimated using the following formula: ND ¼
TM CFA NSS
where ND is numerical density, TM is total markers, CFA is counting frame area (XY) (lm2), and NSS is number of sampling sites. The coefficient of error (CE) for calculations of numerical density was the last calculated value. Generally accepted highest limit of CE is 5 % [8, 9].
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Fig. 2 Viable cell counts by using stereoinvestigator in a specific area of confocal microscopy images. Orange arrows point at living cartilage cells
Statistical analysis Statistical analysis of obtained results was performed using one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test (DMRT) via SPSS software package, version 19.00 (SPSS Inc., Chicago, IL). p \ 0.05 was considered statistically significant. All results were expressed as mean standard error of the mean (SE) for seven rabbits in each group.
Comparing the groups in terms of the means of the dead cell amount, the diced cartilage group was the only group not statistically different from the control cartilage group. The numbers of dead cells found in the unmodified, crushed and morselized cartilage groups was significantly higher than both the control cartilage group and the diced cartilage group. The minimum number of dead cells was determined to be in the control cartilage group, followed by the diced cartilage group. There was no significant difference in the number of dead cells among unmodified, crushed and morselized cartilage groups (Table 1).
Results Examining the data obtained from viable cell counts, no statistical significance was defined between control cartilage group and unmodified cartilage group. However a statistically significant difference was present in the number of viable cells between the crushed, diced, and morselized cartilage groups and the control cartilage group. The numerical density of viable cells in the diced cartilage group and morselized cartilage group was found to be higher than the control cartilage group (Fig. 3). The maximum number of viable cells was determined to be in the in the diced cartilage group, and the minimum number of viable cells was found in crushed cartilage group (Fig. 4).
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Discussion For a successful rhinoplasty outcome, it is not only the careful reconstruction of osteocartilagenous skeleton but also the smooth appearance of the nasal contours that bears great significance. Irregularity of the nasal contours is often the sole impetus for revision rhinoplasty procedures. Various materials have been used to conceal the irregularities encountered during primary nasal operations, among these: cartilage grafts, dermal grafts, temporoparietal fascial grafts, alloplastic materials and even the replacement of resected dorsum tissue have been described [10–16].
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Fig. 3 Confocal laser screening microscopy images of tissue sections. To identify cellular viability, LIVE/DEADÒ Viability/Cytotoxicity Kit has been used. Viable cells have been stained with calcein AM (green) and dead cells have been stained with ethidium homodimer (red). Comparison of control group (c) with the composite (a) and diced (b) cartilage groups
Fig. 4 Confocal laser screening microscopy images of tissue sections. To identify cellular viability, LIVE/DEADÒ Viability/Cytotoxicity Kit has been used. Viable cells have been stained with calcein AM (green) and dead cells have been stained with ethidium homodimer (red). Comparison of control group (c) with the crushed (a) and morselized (b) cartilage groups
Cartilage grafts have become the most widely accepted material due to its long-term success and low rate of complications [17]. The fundamental difference between cartilage grafts used for structural grafts and correctional grafts is: Hardness, strength, shape specificity, and preservation of shape
are expected from structural grafts (or any grafts used for recreation or support of the nasal skeleton) whereas amorphous, soft, and conformation are expected from correctional grafts (or any grafts used contour concealing or dorsal augmentation.) In the long-term follow-up of correctional grafts, it is very important that there is no
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Eur Arch Otorhinolaryngol Table 1 Effects of different treatments on the numerical density of live and dead cells in cartilage tissue of rabbits Treatment
Number of rabbits
Numerical density of live cells (n/lm3)
Numerical density of dead cells (n/lm3)
Control
7
0.000095 ± 0.000001
0.00000 ± 0.000000
Unmodified
7
0.000098 ± 0.000005
0.00009 ± 0.000040x
Crushed
7
0.000030 ± 0.000012x
0.00008 ± 0.000031x
Diced
7
0.000137 ± 0.000006x
0.00001 ± 0.000003
Morselized
7
0.000111 ± 0.000008x
0.00012 ± 0.000040x
Results are mean ± standard deviation. Means in the same column by ‘‘x’’ superscript letter are statistically significantly different under the Duncan test (a = 0.05)
atrophy, but instead protected viability, as to reduce the possibility of irregularities. The procedures to modify cartilage into an amorphous state themselves cause varying degrees of damage and have varying degrees of effect on the viability rates of grafts. There are studies in the literature reporting that the crushing degree of crushed cartilages can cause important differences on cellular viability rates [18]. It has been reported that viability rates of cartilages decrease with the increase in crushing degree. In a study published by Cakmak and Altintas [18], it was reported that crushed cartilage could be prepared at four varying degrees of thickness if a Cottle crusher is used. However the same does not apply for morselization; a morselizer can crush cartilage to either a mild or moderate degree. It has been reported that cartilage crushed to a moderate degree has the highest rate of cellular viability, and therefore that degree of morselization was used in this study to provide a standardization of procedure. Although there are publications about cartilage softened using a Cottle crusher, for example the study done by Garg et al., there are no studies on cartilage softened by using a morselizer [19, 20]. As far as we know, our study is the first in the literature evaluating the effects of cartilage morselization on cellular viability. Many surgeons use cartilage divided into very thin pieces (diced) by scalpel blade to correct depressions or irregularities in the nasal dorsum. The use of diced cartilage was first explained in 1943 by Peer et al. [21] however the number of studies on this subject has been increasing considerably over the last 10 years [22, 23]. Cartilage can be placed into the nose in various ways: wrapping it in Surgicel (Ethicon, Inc, Somerville, NJ), in AlloDerm (LifeCell Corp, Branchburg, New Jersey), wrapping it in the fascia of temporal muscle, or without wrapping, after achieving a high viscosity by mixing it with a few drops of blood. Erol et al. [24] reported high rates of success when used with Surgicel, and Daniel et al. have claimed that grafts wrapped with fascia have higher rates of viability. Kazikdas et al. and Brenner et al. [17, 25] support the results of Daniel et al. in their studies. Gordon et al. [22]
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have reported that the cartilages wrapped in AlloDerm were successful, did not cause donor site morbidity, and required a shorter operation time. However some conflicting studies do exist, reporting that long-term outcomes of AlloDerm are not reliable due to high rates of resorption [10, 22]. Rather than making a comparison of each application method of diced cartilage, we aimed to compare the advantages of diced cartilages with crushed and morselized cartilage in our study. Viability tests of tissue and cell cultures are used to determine the sensitivity of cells to various chemical, physical and biological factors. Many tests have been developed to define cellular viability in which some different criteria are evaluated such as the proliferative capacity of cells, amount of surviving cells and enzymatic activities or plasma membrane integrity. Tests evaluating the cellular metabolic activity such as ATP or MTT tests usually give fast and accurate results. Measurement of the amount of ATP by bioluminescence is very sensitive (as little as 20 cells/ml). MTT test is a colorimetric test based on the principle of tetrazolium dye reduction to a formazan blue product and measured with spectrophotometry. The MTT test is frequently used for viability evaluation in cell cultures. This test, which is easy to apply, reliable and highly reproducible has a disadvantage of having lower sensitivity [26]. In paint adhesion tests such as trypan blue, only the dead cells are stained due to disruption of plasma membrane. But it is not possible to distinguish healthy cells from the live cells that have lost their function using this colorimetric method. Thus, injury in tissues or cells cannot be detected exactly [27, 28]. Techniques with fluorescent dyes have many biological and clinical applications because they are more accurate and selective than the colorimetric methods. By a combination of different dyes, determination of both live and dead cells becomes possible with a single test. As in this study, staining the tissues or cells together with calcein AM and ethidium homodimer and examination by confocal laser screening microscopy presents quantitative data by ensuring determination of the amount of live and dead cells. Additionally, the optical sectioning capability of
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confocal microscopy allows the cells to be displayed even in thick tissue samples [26, 27]. Another advantage of this method is the determination of cellular viability by evaluating both the morphological and functional properties in a way that ethidium homodimer measures the membrane integrity and calcein AM measures the esterase activity [28]. The use of confocal microscopy to detect cellular viability has been established in literature. Lu et al. [29] showed that confocal microscopy with vital cell staining is an accurate and sensitive method of determining cell viability. In their study they also concluded that light microscopic techniques could be inadequate and insensitive in some cases where confocal microscopy exhibit successful and satisfying results. In another research Frenz et al. showed that confocal microscopy is a powerful tool for assessing changes in tissue structure [6]. Gantenbein-Ritter et al. clarified that using confocal microscopy to detect viability is advantageous and reasonable because it has the ability to produce optical sections through a 3D sample. Since optical sectioning during confocal microscopy is non-invasive, the 3D distribution and relative spatial relationship of stained living and dead cells could be observed with reasonable clarity [30]. To conclude, from studies conducted so far, confocal microscopy is a reliable, sensitive and sufficient method to detect cellular viability, which is why it was chosen as the method of determining and investigating the viability of cartilage samples in this study. One of the other methods for evaluation of cellular viability is the histological examination performed using different staining techniques. In the cartilage tissue stained by Hematoxylin and Eosin (H and E), chondrocyte viability is analyzed by evaluating the nucleated lacunae. Masson trichrome stain provides an evaluation of connective tissue by showing the collagen content of matrix. Safranin O and PAS stains the proteoglycan content, whereas van Gieson stains the elastic fibers. Evaluation of the viability of cartilage tissue at light microscopic level is performed as looking at the staining properties of chondroid matrix. Disappearance of matrix metachromasia and absence of nuclei in lacunae indicates a non-living chondroid tissue. Counting the nucleated lacunae with specialized software programs provide evaluation of chondrocyte viability and provides quantitative data [31, 32]. Presence of time-consuming steps in routine histological procedures such as tissue processing, sectioning, staining and microscopic examination makes this method more troublesome than others and is therefore a disadvantage. Aside from the advantage of confocal microscopic analysis showing live and dead cells together, it has a disadvantage due to its inability to distinguish cell types in tissue. As there is a possibility of the presence of fibroblasts
and inflammatory cells in addition to the chondrocytes among the cells identified as alive or dead by this technique, we believe that supporting the confocal microscopic data with the findings that achieved via conventional and immunohistochemical histological methods will provide valuable information (such as iNOS/eNOS, bcl/bax etc.). In a study such as this, it is natural to observe various inflammatory tissue responses resulting from varying levels of cellular damage based on the technique that was used to prepare grafts from the excised cartilage. Therefore we believe that some differences will also occur in the amounts of histopathological evaluation indices such as cellular viability, fibrosis, mitotic and chondrogenic activity in different graft groups. The confocal microscopic method used to evaluate cellular viability and cytotoxicity in this study is a novel method and has provided advanced technological data. In this study, we have found that the cellular viability in diced cartilage is greater than in morselized, crushed, unmodified, and control cartilage. Considering that the cartilage tissue is fed by diffusion from its environment in natural conditions, we can assert that the cellular proliferation could be stimulated even more as a result of the increased contact surface with the environment in diced graft group.
Conclusion In this study, we aimed to evaluate the cellular viabilities of crushed, morselized and diced cartilage grafts by confocal microscopic examination and to compare them with the unmodified cartilage grafts. It was found that the viability of diced cartilage grafts is greater than the crushed, morselized, unmodified, and control cartilage. It is important to keep in mind that this is an experimentally induced model in rabbits and this model may yield different results in humans. Thus further studies are needed to evaluate long-term effects and clinical applications of these methods in rhinoplasty procedures. Acknowledgments Authors wish to thank Leyla Tarhan and Serap Kaya for their technical assistance in using confocal microscope. All financial and material support for this research and work were provided by the scientific research fund of Sakarya University. Our study is a multi-centered and multidisciplinary, single-blind randomized controlled animal trial supported by the scientific research fund of our university. Conflict of interest Authors declare that there is no conflict of interest or ethical adherence in this scientific study. Ethical standard The experimental study design was reviewed and approved (no. 298, 30/11/12) by the Committee for Ethics in Animal Experiments at Yeditepe University School of Medicine, and this study was carried out in compliance with the guidelines for animal
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Eur Arch Otorhinolaryngol experimentation at the Department of Laboratory Animal Science at Yeditepe University (Istanbul, Turkey). All animals used for these experiments received care according to the ‘‘Principles of Laboratory Animal Care’’ from the National Society of Medical Research and the ‘‘Guide for the Care and Use of Laboratory Animals’’ prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication No 85-23, revised 1996).
References 1. Ansari K, Asaria J, Hilger P, Adamson PA (2008) Grafts and implants in rhinoplasty—Techniques and long-term results. Op Tech Otolaryngol 19:42–58 2. Hamra ST (1993) Crushed Cartilage Grafts over Alar Dome Reduction in Open Rhinoplasty. Plast Reconstr Surg 92:352 3. Anon (2007v) Theory of confocal microscopy. Available at: www.olympusconfocal.com. Accessed 20 Nov 2007 4. Lu Y, Meyer ML, Bogdanske JJ, Markel MD (2007) The effects of radiofrequency energy probe speed and application force on chondrocyte viability. Vet Comp Orthop Traumatol. 20:34–37 5. Sterio DC (1984) The Unbiased estimation of number and size of arbitrary particles using the dissector. J Microsc 134:127–136 6. Mainil-Varlet P, Monin D, Weiler C, Grogan S, Schaffner T, Zu¨ger B, Frenz M (2001) Quantification of laser-induced cartilage injury by confocal microscopy in an ex vivo model. J Bone Joint Surg Am 83:566–571 7. Gundersen HJ, Jensen EB (1987) The efficiency of systematic sampling in stereology and its prediction. J Microsc 147:229–263 8. Wong DM, Franz AK (2013) A comparison of lipid storage in Phaeodactylum tricornutum and Tetraselmis suecica using laser scanning confocal microscopy. J Microbiol Methods 95:122–128 9. Howard V, Reid S, Baddeley A (1985) Unbiased estimation of particle density in the tandem scanning reflected light microscope. J Microsc 138:203–212 10. Tarhan E, Cakmak O (2008) Ozdemir BH, Akdogan V, Suren D, MD. Comparison of AlloDerm, fat, fascia, cartilage and dermal grafts in rabbits. Arch Facial Plast Surg 10:187–193 11. McKinney P, Loomis MG, Wiedrich TA (1993) Reconstruction of the nasal cap with a thin septal graft. Plast Reconstr Surg 92:346–351 12. Skoog T (1966) A method of hump reduction in rhinoplasty: a technique for preservation of the nasal roof. Arch Otolaryngol 83:283–287 13. Reich J (1983) The application of dermis grafts in deformities of the nose. Plast Reconstr Surg 71:772–782 14. Baker TM, Courtiss EH (1994) Temporalis fascia grafts in open secondary rhinoplasty. Plast Reconstr Surg 93:802–810 15. Gilmore J (1989) Use of Vicryl mesh in prevention of post rhinoplasty dorsal irregularities. Ann Plast Surg 22:105–107
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16. Stoll W (1991) Use of polytetrafluoroethylene for particular augmentation of the nasal dorsum. Aesthetic Plast Surg 15:233–236 17. Kazikdas KC, Ergur B, Tugyan K, Guneli E, Kaya D, Sahan M (2007) Viability of crushed and diced cartilage grafts wrapped in oxidized regenerated cellulose and esterified hyaluronic acid: an experimental study. Laryngoscope 117:1728–1734 18. Cakmak O, Altintas H (2010) A Classification for degree of crushed cartilage. Arch Facial Plast Surg 12:435–436 19. Cakmak O, Buyuklu F (2007) Crushed cartilage grafts for concealing irregularities in rhinoplasty. Arch Facial Plast Surg 9:352–357 20. Garg R, Shaikh M, Foulad A, Wong B (2010) Chondrocyte viability in human nasal septum after morselisation. Arch Facial Plast Surg 12:204–206 21. Peer LA (1943) Diced cartilage grafts. Arch Otolaryngol 38:156–165 22. Gordon CR, Alghoul M, Goldberg JS, Habal MB, Papay F (2001) Diced cartilage grafts wrapped in Alloderm for dorsal nasal augmentation. J Craniofac Surg 22:1196–1199 23. Daniel RK (2008) Diced cartilage grafts in rhinoplasty surgery: current techniques and applications. Plast Reconstr Surg 122:1883 24. Erol OO (2000) The Turkish delight: apliable graft for rhinoplasty. Plast Reconstr Surg 105:229–243 25. Brenner KA, McConnel MP, Evans GR, Calvert JW (2006) Survival of diced cartilage grafts: an experimental study. Plast Reconstr Surg 117:105–115 26. O’Riordan TC, Buckley D, Ogurtsov V, O’Connor R, Papkovsky DB (2000) A cell viability assay based on monitoring respiration by optical oxygen sensing. Anal Biochem 278:221–227 27. Monette R, Small DL, Mealing G, Morley P (1998) A fluorescence confocal assay to assess neuronal viability in brain slices. Brain Res Proc 2:99–108 28. Jimenez-Hernandez ME, Orellana G, Montero F, Portoles MT (2000) A ruthenium probe for cell viability measurement using flow cytometry. Photochem Photobiol 72:28–34 29. Yan Lu, Edwards RB III, Kalscheur VL et al (2001) Effect of bipolar radiofrequency energy on human articular cartilage: comparison of confocal laser microscopy and light microscopy. Arthroscopy 17:117–123 30. Benjamin Gantenbein-Ritter B, Sprecher CM, Chan S et al (2011) Confocal imaging protocols for live/dead staining in threedimensional carriers. Methods Mol Biol 740:127–140 31. Imbert D, Cullander C (1999) Buccal mucosa in vitro experiments. Confocal imaging of vital staining and MTT assays for the determination of tissue viability. J Control Release 58:39–50 32. Orbay H, Tobita M, Hyakusoku H, Mizuno H (2012) Effects of adipose-derved stem cells on improving the viability of diced cartilage grafts. Plast Reconstr Surg 129:369–377
