Characterization of Injury Induced by Routine Surgical Manipulations of Nasal Septal Cartilage
William W. Thomas, MD1; Robert M. Brody, MD1; Abdullah D. Alotaibi, MD1,2; et al Emilie C. Rabut, BS2; Noam A. Cohen, MD, PhD1,3; Robert Lyman, MD2; Milos Kovacevic, MD4; Oren Friedman, MD1; George R. Dodge, PhD1,2,5
Author Affiliations Article Information
JAMA Facial Plast Surg. Published online May 30, 2019. doi:10.1001/jamafacial.2019.0169
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Characterization of Injury Induced by Routine Surgical Manipulations of Nasal Septal Cartilage
Key Points
Question  What is the optimal surgical manipulation for cartilage autografts in rhinoplasty?

Findings  In this in vitro investigation of 4 distinct surgical manipulations compared with a control group using cartilage from bovine snouts, the crushing manipulation was statistically significantly worse than all other manipulations with shaving, dicing, and scoring following, respectively, by various quantitative assays.

Meaning  When clinical needs dictate the use of cartilage grafts, surgeons should consider the least damaging, yet adequate cartilage manipulation to enhance clinical outcomes.

Abstract
Importance  This study characterizes and compares common surgical manipulations’ effects on septal cartilage to understand their implications for rhinoplasty outcomes based on cell viability and cartilage health.

Objective  To illustrate distinct differences in the impact of various surgical manipulations on septal cartilage in an in vitro septal cartilage model. A secondary objective is to better understand the chondrocyte’s response to injury as well as how alterations in the extracellular matrix correspond to chondrocyte viability.

Design, Setting, and Participants  In this bench-top in vitro porcine model using juvenile bovine septal cartilage from bovine snouts, easily obtainable septal cartilage was used to generate large numbers of homogenous cartilage specimens. Quantitative outcomes at early and late time points were cell viability, cell stress, matrix loss, and qualitative assessment through histologic examination. The study was performed at a single academic tertiary care research hospital.

Interventions  Four common surgical manipulations were contrasted with a control group: crushed cartilage, scored cartilage, diced cartilage, and shaved cartilage.

Main Outcomes and Measures  Following the manipulation of the cartilage, the quantitative outcomes were glycosaminoglycan release to the media, lactate dehydrogenase release to the media, and cell death analysis through apoptosis staining. The qualitative outcomes were histologic staining of the manipulated cartilage with safranin-O/fast green stain to identify proteoglycan loss.

Results  The crushing followed by shaving manipulations were the most damaging as indicated by increased levels of lactate dehydrogenase release, glycosaminoglycans loss, and cell death. Matrix loss did not increase until after 48 hours postinjury. Furthermore, chondrocyte death was seen early after injury and accelerated to the late time point, day 9, in all manipulations. Conversely, cell stress was found to be greater at 48 hours postinjury, which then declined to the late time point, day 9.

Conclusions and Relevance  The crushing manipulation followed by shaving and then dicing were the most destructive methods of cartilage manipulation relative to control specimens. Collectively, these outcomes demonstrate the range of injury which occurs with all septal cartilage manipulations and can inform rhinoplasty practice to use the least damaging effective surgical manipulation to obtain the desired outcome.

Level of Evidence  NA.

Introduction
The repair of nasal and facial deformities is most commonly attempted by using transplanted autologous cartilage. Autologous cartilage is an ideal graft material owing to lower rates of extrusion and infection compared with artificial grafting materials.1

The cause of various facial anatomical deformities may be traumatic, congenital, or oncologic, but the common technique for repair requires manipulating autologous cartilage into a workable form from its source in the septum, auricle, or rib. Common uses for autologous cartilage grafts include dorsal onlay grafts, shield grafts, columellar strut grafts, alar rim grafts, spreader grafts, batten grafts, and others. These grafts require differing levels of cartilage strength, plasticity, and physical shape.2 To customize the shape and flexibility of the cartilage, the surgeon manipulates it to achieve the desired plasticity. Altering the harvested autologous cartilage results in a more useful shape and structure at surgery, but also likely changes the resorption rate, straightness, and long-term position and survival in the nose. Alternate methods of cartilage manipulations for rhinoplasties are in their infancy, and this work is being pioneered by Wong et al3,4 using electromechanical intervention or enzymatic digestion.

Although surgical manipulations improve plasticity in cartilage autografts, they can also be considered varying degrees of injury. Common surgical cartilage manipulations used by facial plastic surgeons include dicing, shaving, scoring, and crushing of cartilage to achieve a range of plasticity. In addition, implanted cartilage may be wrapped in biologic or artificial materials to ensure conformity of shape. Specific to crushing of autologous cartilage, a standardized grading scale of slight, moderate, significant, or severe has been proposed. Furthermore, the viability of chondrocytes undergoing these manipulations has been partially characterized from traditional donor sites including septum, auricle, and rib.5,6 Other manipulations have been described, which could include covering with fascia, perichondrium, or Surgicel to prevent migration of diced cartilage fragments.

There is controversy surrounding the viability of cartilage autografts after the addition of a covering with either alloplastic material or autologous tissue. Wrapping cartilage in a cellulose polymer (Surgicel) was found to incite an inflammatory reaction and subsequent reabsorption of the cartilage relative to the autologous tissue.7 Clinically, however, this method of wrapping prior to placement along the dorsum of the nose has been found to produce long-lasting and consistent results.8 Other basic science evidence suggests that a perichondrium wrap allows for better nutrient delivery to diced cartilage because perichondrium was found to yield less resorption and increased viability relative to fascia in a rabbit model.9 After a review of the relevant literature related to single cartilage manipulations,5,10 there exists no comparison of cartilage manipulations alone against one another. This study provides insight on the impact of various surgical manipulations to autologous septal cartilage grafts in a uniform manner and lends evidence in the healing paradigm of rhinoplastic procedures.

Our hypothesis was that increasing plasticity from various manipulations is related to a loss of structural content in the form of sulfated glycosaminoglycans (GAG) and that this release of GAG is indicative of tissue injury, along with increased cell stress and death. Further understanding of the range of injury occurring with various manipulations can potentially offer practical information for rhinoplasty physicians to use the least damaging effective surgical manipulation to obtain the desired outcome.

Methods
Fresh juvenile bovine snouts were obtained from a commercial source (Research 87) and septal cartilage was sterilely dissected free of perichondrium. The harvested portion of the septum most devoid of blood vessels was partitioned into 10 equivalent sections. Each section immediately underwent a manipulation: no manipulation (control), scored in 1-mm increments to a partial thickness, moderately crushed11 with the Cottle Cartilage Crusher (Surgipro, Inc), diced into 1-mm3 pieces, or shaved into 1-mm thick translucent sheets. These cartilaginous samples were maintained in tissue culture for up to 9 days with tissue sampling at 2 and 9 days and media sampling and changing at day 2, 4, 6, 8, and 9. All cartilage was maintained in tissue culture in Dulbeccos Modified Eagles Medium with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL Fungizone, 1% MEM vitamin solution, 25 mM HEPES buffer, 50 μg/mL ascorbic acid). All tissue culture reagents used were from Life Technologies. On days 2 and 9, samples from each manipulation were harvested and divided into thirds. The first portion was fixed in 4% paraformaldehyde followed by processing into paraffin blocks and 7 micron sections were made. The second portion was for RNA analysis by snap-freezing the tissues in liquid nitrogen and storing in −80 °C freezer until further use. Finally, the third portion of the tissue was collected for biochemical analysis. Each sample was weighed for wet weight, and dry weight was obtained after a 4- to 6-hour drying period using an acid concentrator. Subsequently, the dried cartilage was digested with 0.5 mg/mL Proteinase-K (Roche Diagnostics) in 100 mM phosphate and 5 mM EDTA (PBE, pH 7.1) in a 60 °C waterbath for 16 hours. The digested solutions were then stored at −20 °C until use.

Media samples were analyzed on days 2 and 9 to assess proteoglycan content using a spectrophotometric assay for sulfated GAG using 1,9-dimethylmethylene blue (DMMB) dye assay.12 Briefly, the DMMB dye solution consisted of 1.6% (w/v) of DMMB (Polysciences, Inc) in sodium formate (Sigma-Aldrich) in MilliQ water, pH of 3.5. Triplicate 40-μL aliquots of media samples were combined with 250 uL of the DMMB dye solution. Absorbance at 540 nm and 595 nm was measured and compared with standards made from shark chondroitin sulfate type C (C-4384; Sigma-Aldrich).

Sample DNA content was determined using the PicoGreen Assay kit (Invitrogen). As per assay instructions, triplicate 10 μL (1/10 in assay buffer) of each sample was combined with 100 μL of PicoGreen reagent and incubated in the dark for 2 to 5 minutes. Fluorescence was measured at 485 nm/emission (em) 528 nm and compared with a standard curve made using the kit-provided 100 μg/mL λ DNA standard.

Cartilage was histologically processed, embedded in paraffin, 7 μM sections made and stained with safranin O/fast green to identify changes in GAG representative of proteoglycan loss. To determine cell viability, lactate dehydrogenase (LDH) release was measured in the sample medias. Samples were tested in triplicate using a CytoTox-ONE assay kit (Promega), which measures the LDH release from cells with damaged membranes. A total of 25 μL of media samples were mixed with 25 μL of substrate mix/assay buffer reagent. Samples were incubated for 10 minutes at room temperature before adding 12.5 μL of stop solution. Fluorescence was measure at 530 nm/em 590 nm using a plate reader (gain setting of 35). Averaged fluorescence for samples were normalized to control samples. To assess cell death samples were section stained using a terminal deoxynucleotidyl transferase (TUNEL) kit (Roche). The TUNEL kit uses fluorescein-dUTP to label DNA strand breaks and detect and quantify apoptotic cell death at single-cell levels in cells and tissues. Samples were permeabilized using 10 μg/mL Proteinase K in 10 mM Tris-hydrochloride for 15 minutes before the TUNEL reaction. Slides were then mounted with ProLong Gold DAPI (Invitrogen) and fluorescently imaged at 488 nm with detection in 515 to 565 nm (green).

All statistical tests were performed using GraphPad Prism, (version 6.07, GraphPad). Error bars in all figures represent manipulation mean (SD) values. A Grubbs test was performed on all 209 data points and 7 outliers were removed and all the remaining data passed normality using a Kolmogorov-Smirnov test. Statistical significance for all 3 assays performed (GAG, cell death, LDH) used an unpaired, 1-tailed t test to compare each manipulation group against control group. The P values are reported for each comparison and significance determined using Bonferroni’s correction (P < .01). These assessments can be found as Panel D in Figure 1, Figure 2, and Figure 3, for each assay performed.

Results
To assess the health of the cartilage after various manipulations, we assessed 4 primary metrics (GAG, cell death, LDH, and histologic analysis). Measuring GAG is a good proxy of cartilage integrity and proteoglycan degradation.12,13 The first evaluation of cartilage manipulations was an assessment of GAG release to media at day 2 and day 9 following manipulation. Potential variations in the mass of the cartilage sample (grossly controlled for at harvest) were eliminated by standardizing the GAG loss to DNA content of the sample.

The results of the GAG assay were performed in triplicate with all interventions having at least 6 replicates (Table). The results of the GAG assays are presented in Figure 1A-C. The scored and diced manipulation did not show significant GAG loss to media at 48 hours postmanipulation compared with controls, though crushed and shaved showed significant GAG loss. On day 9, the crushed and shaved manipulations continued to have significantly greater GAG release than day 9 control, with crushed having the highest average (Figure 1D). In all manipulations, the day 9 GAG release was on average greater than the day 2 GAG release indicating a basal rate of increased GAG loss per day even with the slightest manipulation. However, day 2 control GAG loss compared with day 9 control GAG loss was not significantly elevated, illustrating that manipulation is required to initiate GAG loss (Figure 1C). The magnification of GAG loss over time in the crushed and shaved cohorts was likely attributed to increased cell death with time.

A cell death/apoptosis analysis was performed to determine which manipulation resulted in the death of cells, which would be critical to the survival of the graft. We assessed the number of dead cells as part of a total from multiple regions of interest at day 2 and day 9 (Figure 2). Cell death, compared with controls, was significantly elevated at both time points for the crushed, diced, and shaved manipulations as well as scored samples at the day 2 time point (Figure 2D). Cell death percentages increased in all manipulations on day 9 compared with day 2 indicating continued cell death numerous days after the initial manipulation. In comparison of the manipulations, crushed and shaved had the highest percentage of dead cells. The rate of cell death/apoptosis increased with time and the slope of this increase was faster than baseline (control day 2 vs control day 9), with crushed having the most profound effect on cell death.

As a third metric of the condition of the cartilage explant we measured LDH levels, an indicator of cell stress.14,15 The LDH assay results (Figure 3A-C) indicate all manipulations at both time points had statistically increased LDH release compared with controls (Figure 3D). The control cohort in this assay was used to generate a baseline. A trend of very high early release of LDH on day 2 with continued but lessened LDH release on day 9 was noted. This pattern was most clearly indicated by the crushed manipulation, followed to a lesser degree by the shaved and diced manipulations. Decreased release of LDH at day 9 relates to the increased number of dead cells on day 9 compared with controls, allowing fewer injured yet living cells to release LDH.

To characterize physical damage and changes in cartilage architecture, we performed histologic assessments using cationic dye to see distribution of GAG (proteoglycan). Cartilage manipulations at day 2 and day 9 were stained with safranin O and fast green for histologic evaluation (Figure 4). These gross images indicate the continuum changes induced in the cartilage through manipulation. The crushing manipulation did not produce significant bending compared with the scoring manipulation, but at magnification ×10 there were areas of decreased safranin O staining, indicating diffuse loss of proteoglycan. The cut edges of the histologic images did not show proteoglycan loss. However, cell death imaging illustrates localization of the dead cells in the cutting manipulations (scoring, dicing, shaving) to the cut edges as would be expected.

Discussion
Many different surgical manipulations of cartilage autografts can be performed to ensure the appropriate size, plasticity, texture, and appearance. As time passes, reabsorption and degradation of transplanted cartilage can occur and turn an excellent surgical result into a long-term surgical disappointment. Various degrees of cartilage crushing and viability analyses have been performed in both human and animal models. Some degree of conflicting results have been presented, with work by Verwoerd-Verhoef et al16 showing extensive cell death following crushing manipulation and an inability to provide structural support, whereas work by Yilmaz et al17 and Guyuron and Friedman18 showing 80% to 90% crushed cartilage graft viability and consistent graft structural sustainability in an animal model and clinical experience even with cryogenic preservation methods, respectively. The differences in the literature, with this study in line with other in vitro studies, may be attributed to artificially worsened cell death in in vitro models owing to fewer cell rescue mechanisms compared with in vivo or, as stated by Yilmaz et al, “it is not always possible to determine in-vivo viability of the cartilage with high accuracy.”17(p1058) Furthermore, inconsistency surrounding the crushing manipulation in the literature was partially clarified by the work of Cakmak et al5 in 2005, showing that viability was strongly tied to severity of crushing manipulation.

This study compared a standardized crushing manipulation, moderately crushed, on a continuum of cutting manipulations, which are also regularly used in clinical practice. Moderately crushing bovine septal cartilage is shown to be a more severe manipulation than scoring, dicing, or shaving. However, all of these manipulations were significantly more damaging than the control manipulation. The 3 manipulations of scoring, dicing, and shaving induce additional cut surfaces into the cartilage specimen greater than the initial cuts to create the control specimen and these cut surfaces can induce considerable bending of cartilage. These cut surfaces also induce cell stress, as seen by LDH release, which leads to increased GAG release at day 9 and cell death at day 2 and day 9. The degree of bending of cartilage has been quantified by ten Koppel et al19 in a rabbit model, and our manipulations produced considerable bending. Bending of cartilage is generally unfavorable in rhinoplasty because it can distort the nasal shape over the long term, often leading to a crooked nose or other asymmetries, and multiple techniques are reported in the literature for preventing this bending.20-24 The cutting manipulations contrast crushing of cartilage because they induce specific fracture patterns into cartilage whereas the force of crushing is distributed diffusely across all chondrocytes and extracellular matrix. Histologic evaluation of crushed cartilage in Figure 4 exemplifies this diffuse distribution of proteoglycan loss.

There is a dichotomy between the negative results of cartilage manipulation seen in in vitro studies compared with the excellent long-term clinical results presented by many retrospective rhinoplasty series. This dichotomy may be owing to the greatly increased sensitivity of in vitro studies to determine cartilage injury compared with the multifactorial nature of a rhinoplasty; reliant only in part on the structural integrity and shape of the autologous cartilage graft, and surgeon experience may help guide anticipated cartilage graft changes and wound healing over time. For example, scar tissue may fill the dead space created by reabsorbed cartilage and may yield as an acceptable cosmetic result as may occur when crushed cartilage camouflage grafts are applied to the tip. Postrhinoplastic surgery tissue healing would involve the classic triad of events, inflammation, tissue biosynthesis, and remodeling. Healing in vivo would be impacted by the survival of the chondrocytes and presumably by diffusion growth factors such as TGF-β or PDGF from resident cells or blood supply on closure and resumption of circulation.25

The individual techniques a rhinoplasty surgeon chooses to use in each individual rhinoplasty is a multifactorial decision based on surgeon’s training and experience along with a multitude of patient factors. The dichotomy between in vitro studies such as ours and retrospective case series does present a challenges in direct translation of in vitro work to the bedside. This study presents new knowledge in the in vitro comparison between surgical techniques but unlike randomized control trials it can only suggest best practices and cannot warrant direct recommendation for in vivo applications. Because this study compares multiple techniques, rhinoplasty surgeons can begin to compare their own techniques and variation relative to the major techniques presented here and in doing so approximate their impact on the septal cartilage and in vivo outcomes.

This study illustrates the trade-offs of cartilage manipulation in viability, plasticity, and lack of bending with the crushing manipulation compared with the less traumatic but bend-inducing manipulations of scoring and shaving. Dicing represents an in-between manipulation with regard to plasticity and survival. However, in individuals with thin-skin who undergo dorsal augmentation with diced cartilage, the individual cartilage pieces may present as irregularities or may shift positions. Initially, in clinical settings, free diced cartilage was implanted into patients, and later, finely diced cartilage was injected into patients during surgery. Both methods were abandoned owing to the dispersion of cartilage in the long term after surgery. To mitigate this potential issue, different techniques of retaining diced cartilage have been used including addition of fibrin glue or temporalis fascia. One study by Kreutzer et al26 generated a cartilage paste by dicing cartilage to at most a 0.2-mm diameter. Their surgical technique included avoidance of squeezing the cartilage paste to maintain viability, but no further mechanistic detail is provided in this regard. In addition, this study found that the addition of autologous or allogenic fascia increased the probability of revision rhinoplasty for dorsal irregularities.26 Findings in this study could also inform developing techniques that use cartilage graft material and other factors such as combinations with tissue engineered supporting scaffolds, progenitor cells, and growth factors. As our understanding of facial and nasal cartilage characteristics improve, we anticipate a more informed approach to the use of various graft techniques as we aim for more predictable long-term surgical rhinoplasty outcomes.

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Article Information
Corresponding author: George R. Dodge, PhD, Department of Otorhinolaryngology–Head and Neck Surgery, Department of Orthopaedic Surgery, University of Pennsylvania, 36th and Hamilton Walk, 110A Stemmler Hall, Philadelphia, PA 19104 (gdodge@upenn.edu).

Accepted for Publication: March 1, 2019.

Published Online: May 30, 2019. doi:10.1001/jamafacial.2019.0169

Author Contributions: Dr Dodge had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Thomas, Brody, Cohen, Lyman, Kovacevic, Friedman, Dodge.

Acquisition, analysis, or interpretation of data: Thomas, Brody, Alotaibi, Rabut, Friedman.

Drafting of the manuscript: Thomas, Brody, Alotaibi, Rabut, Cohen, Lyman, Friedman, Dodge.

Critical revision of the manuscript for important intellectual content: Thomas, Brody, Rabut, Kovacevic, Friedman, Dodge.

Statistical analysis: Thomas, Brody, Alotaibi, Rabut, Lyman.

Obtained funding: Brody, Alotaibi, Cohen.

Administrative, technical, or material support: Brody, Rabut, Lyman, Friedman.

Study supervision: Brody, Cohen, Kovacevic, Friedman, Dodge.

Conflict of Interest Disclosures: None reported.

Funding/Support: The study was supported in part by an American Academy of Otolaryngology–Head and Neck Surgery, Resident Research Grant sponsored by Cook Medical (No. 418777, Thomas, Dodge), the Leslie Bernstein Resident Research Grant from the Educational and Research Foundation for the American Academy of Facial Plastic and Reconstructive Surgery Foundation (No. 352546, Brody, Dodge), and a grant from the Saudi Arabian Cultural Mission, Department of the Medical and Health Science Programs (Dodge, Friedman). Additional support was provided by grants from the National Institutes of Health, the Department of Veterans Affairs (R01 EB008722 and I01 RX001213), NIH T32 training grant (core facilities) (T32 AR007132).

Role of the Funder/Sponsor: The American Academy of Otolaryngology–Head and Neck Surgery, the American Academy of Facial Plastic and Reconstructive Surgery Foundation, the Saudi Arabian Cultural Mission, the National Institutes of Health, and the Department of Veterans Affairs had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: We thank Ryan Smalley, BS, Greg Meloni, MS, and Bhavana Mohanraj, PhD, at University of Pennsylvania, for input and excellent technical assistance. We acknowledge Andrew Cucchiara, PhD, and Carla Scanzello, MD, PhD, University of Pennsylvania, for their expert advice and statistical analysis.

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