Abstract
Importance
Composite grafting in nasal reconstruction involves transplanting auricular chondrocutaneous grafts, but the optimal design of these grafts is unknown.
Objectives
To investigate the ideal ratio of skin to cartilage as well as study the importance of the perichondrial attachment for graft survival.
Design, Setting, and Participants
A New England white rabbit model was used in this study, performed at the Laboratory for Animal Research at University of Kansas Medical Center from January 25 to March 18, 2016. Four varying designs of chondrocutaneous auricular grafts were transplanted to dorsal back defects, with a total of 10 grafts per treatment arm completed. The following 4 chondrocutaneous circular grafts were designed: group A, 1.5-cm diameter graft of equal skin to cartilage ratio; group B, 2.0-cm diameter skin and 1.5-cm diameter cartilage; group C, 1.5-cm diameter skin and 2.0-cm diameter cartilage; and group D, 1.5-cm diameter skin and cartilage separated and placed back together in a layered fashion. Grafts were observed until postoperative day 21, harvested, and evaluated with visual observation as well as histopathologic assessment.
Main Outcomes and Measures
Visually graded areas of survival were marked by 2 blinded academic facial plastic surgeons and calculated for approximate survival. Hematoxylin-eosin–stained, paraffin-embedded 5-μm slides were evaluated for overall survival rate, rate of cartilage necrosis, and mean vessel density per high-power field. In both cases, observers were blinded as to the study group.
Results
Visual assessments of the 5 female rabbits showed significant agreement between surgeons and consistency, with a Spearman coefficient of 0.84 and an intraclass correlation of 0.98. Group D (skin and cartilage separation) was visually graded to have significantly decreased mean survival (45.4%; 95% CI, 23.3%-67.4%) compared with group A (mean survival, 97.4%; 95% CI, 94.8%-99.9%; P < .001), group B (mean survival, 87.6%; 95% CI, 69.9%-100%; P = .004), and group C (mean survival, 82.1%; 95% CI, 66.0%-98.1%; P = .008). Histopathologic assessment revealed that group D again showed significantly inferior overall survival, increased cartilage necrosis, and decreased mean vessel density compared with group A. Group C additionally showed significantly decreased cartilage survival compared with group A (65% vs 0%; P < .001) and group B (65% vs 35%; P = .02).
Conclusions and Relevance
These results represent preliminary evidence that the attachment of skin to perichondrium in a composite graft plays an important role for graft survival. Clinicians performing nasal reconstruction with chondrocutaneous composite grafts should consider preserving attachments at this junction to improve graft survival.
Levels of Evidence
NA.
This animal model study investigates the ideal ratio of skin to cartilage and studies the importance of the perichondrial attachment for graft survival.
Key Points
Question
Does changing the skin to cartilage ratio or separating skin from cartilage have a negative association with composite graft survival?
Findings
In this animal model, changing the skin to cartilage ratio only had a negative association with survival when the size of the cartilage was larger than the skin. Separating skin and cartilage prior to transfer significantly decreased composite graft survival.
Meaning
These results suggest that composite grafts will tolerate some variation in the skin to cartilage ratio, but survival may decrease with increased cartilage size; when transferring composite grafts, maintaining an intact skin-to-cartilage interface may improve graft survival.
Introduction
Chondrocutaneous composite grafts were initially described by König1 in 1902 for nasal reconstruction. Initial success rates were reported at 53%, with later studies improving survival rate by limiting the graft size to less than 1.5 to 1.7 cm in diameter.1,2 This limitation restricts the applications of a composite graft, committing patients with larger defects to more complicated procedures to achieve optimal results.
Previous studies have investigated pharmacologic agents, cooling therapy, hyperbaric oxygen, platelet-rich plasma, and modifying recipient beds, with variable success rates in improving graft survival.3,4,5,6,7,8,9,10,11,12 Chandawarkar et al10 described using an extended dermal pedicle of skin attached to a composite graft to augment survival in humans by providing a larger area for vascular ingrowth. Prior studies in the rabbit model have shown decreased chondrocyte viability and increased fibrosis in implanted, crushed cartilage when perichondrium is absent.12 Recently, Belaldavar et al13 studied the histopathologic characteristics of crushed vs uncrushed autologous, auricular cartilage implantation in the rabbit model. They found that, with the perichondrium intact, crushing auricular cartilage did not decrease viability of the grafts.
The most common technique of composite grafting includes transplantation of cartilage and skin en bloc.14 However, some authors have advocated using separately harvested skin and cartilage layers to reconstruct a composite defect. This technique allows the surgeon flexibility to harvest skin and cartilage from different sites. Zopf et al15 describe using a supraclavicular, full-thickness skin graft overlying a separately harvested auricular cartilage graft for nasal alar reconstruction. Barlow16 similarly described this layered technique for composite reconstruction. He suggested that the keys to skin graft survival were maintaining the perichondrium as well as restricting the cartilage graft to less than 50% of the wound bed. To our knowledge, no prior studies have specifically investigated the significance of the skin to cartilage ratio in composite grafts. Our study aims to investigate how altering the skin to cartilage ratio and disrupting the skin-to-cartilage attachment is associated with composite graft survival.
Methods
Five adult female New Zealand white rabbits between 2 and 4 months of age were used in the study, performed from January 25 to March 18, 2016. Four composite graft treatment groups were designed (Figure 1). Group A consisted of a 1.5-cm diameter circular graft of equal skin to cartilage ratio. Group B consisted of a circular graft with 2.0-cm diameter skin and 1.5-cm diameter cartilage. Group C consisted of a circular graft with 1.5-cm diameter skin and 2.0-cm diameter cartilage. Group D consisted of the same-size grafts as in group A, with skin and cartilage separated and placed back together in a layered fashion. Care was taken to maintain the perichondrium on the cartilage side of all grafts, including in group D. Given the relatively tight adherence of perichondrium to auricular cartilage in these rabbits, this process was not a technically difficult maneuver. The Institutional Animal Care and Use Committee at the University of Kansas Medical Center approved this experimental protocol.
Figure 1. Schematic of Graft Treatment Groups.
A, Group A consisted of a 1.5-cm diameter circular graft of equal skin to cartilage ratio. B, Group B consisted of a circular graft with 2.0-cm diameter skin and 1.5-cm diameter cartilage. C, Group C consisted of a circular graft with 1.5-cm diameter skin and 2.0-cm diameter cartilage. D, Group D consisted of the same-size grafts as in group A, with skin and cartilage separated and placed back together in a layered fashion. White represents the cartilage layer and yellow represents the cutaneous layer.
In conjunction with a certified veterinarian, rabbits were administered intramuscular ketamine hydrochloride, 20 to 50 mg/kg, subcutaneous buprenorphine hydrochloride, 0.1 to 0.2 mg/kg, and intramuscular xylazine hydrochloride, 5 to 10 mg/kg, preoperatively for analgesia. Rabbits were intubated with an endotracheal tube. Isoflurane, 1% to 3%, was delivered throughout the procedure as needed to maintain a deep plane of general anesthesia. Maintenance intravenous fluids were given throughout the procedure and for the immediate postoperative period. Subcutaneous bupivacaine hydrochloride, 0.125%, was administered locally to the base of each ear and transplantation sites on the back. Total dosage of bupivacaine hydrochloride did not exceed 8 mg/kg to avoid toxic effects.
Circular defects were created along the paraspinous dorsal back of the animals in a randomized fashion with respect to treatment groups. Eight defect sites were created on the back of each animal, with spacing of at least 1.5 to 2.0 cm between neighboring defects (Figure 2). Grafts were harvested from randomized sites from the base of the ear using plastic templates printed for uniformity using a MakerBot Replicator 2X 3-dimensional printer (MakerBot Industries). Grafts in the 4 treatment groups were then transplanted to the defect sites, secured with 5-0 polydioxanone suture (Ethicon) in a running fashion with 12 throws each, and dressed with Telfa (Covidien). Bolsters were avoided to maintain animal comfort and to minimize anesthetic requirements for removal. The pressure applied from bolsters potentially hinders neovascularization, and bolsters are not routinely applied in the clinical setting.17
Figure 2. Schematic of Area of Graft Harvest From Base of Rabbit Auricle and Placement Along Dorsum.
Grafts were placed at least 1.5 to 2.0 cm apart, and both the graft harvest sites and placement along the dorsum were randomized.
The dressings were removed at 6 days after the procedure, and grafts were monitored for a total of 3 weeks. The animals were then killed and all grafts were photographed, then harvested and placed in formalin. Two academic facial plastic surgeons (J.D.K. and C.D.H.) were presented with blinded graft images at different locations and times to independently assess graft survival by marking the area of perceived necrosis. The graft images were then imported into Adobe Photoshop (Adobe Systems Inc). The lasso tool in Adobe Photoshop was used to trace the boundaries of the areas indicated. The pixels within the necrotic areas were compared with the total pixels of the graft boundaries and a survival rate was calculated and rounded to the nearest whole percentage (Table). Their results were compared for interrater reliability using the Spearman coefficient and consistency in the measurements with the intraclass correlation coefficient.18 Treatment groups were compared for survival outcomes with a Kruskal-Wallis test and Mann-Whitney test. All statistical analysis was performed with SPSS (IBM SPSS). All P values were from 2-sided tests, and results were deemed statistically significant at P < .05.
Table. Clinical Assessments of Graft Survival Rates by Surgeon.
| Graft Survival, % | |||||||
|---|---|---|---|---|---|---|---|
| Group Aa | Group Bb | Group Cc | Group Dd | ||||
| Surgeon 1 | Surgeon 2 | Surgeon 1 | Surgeon 2 | Surgeon 1 | Surgeon 2 | Surgeon 1 | Surgeon 2 |
| 85 | 100 | 36 | 29 | 52 | 33 | 3 | 0 |
| 87 | 92 | 48 | 53 | 54 | 63 | 6 | 0 |
| 91 | 100 | 98 | 99 | 68 | 55 | 22 | 27 |
| 95 | 100 | 93 | 100 | 63 | 66 | 27 | 42 |
| 97 | 100 | 98 | 100 | 98 | 100 | 40 | 31 |
| 98 | 100 | 98 | 100 | 97 | 100 | 62 | 66 |
| 100 | 100 | 98 | 100 | 97 | 100 | 61 | 64 |
| 100 | 100 | 100 | 100 | 100 | 99 | 69 | 42 |
| 100 | 100 | 100 | 100 | 100 | 100 | 71 | 82 |
| 100 | 100 | 100 | 100 | 100 | 100 | 91 | 100 |
Abbreviation: IQR, interquartile range.
Mean survival, 97.4% (95% CI, 94.8%-99.9%); median survival, 98.8% (IQR, 4.8%).
Mean survival, 87.6% (95% CI, 69.9%-100%); median survival, 99.0% (IQR, 15.0%).
Mean survival, 82.1% (95% CI, 66.0%-98.1%); median survival, 97.5% (IQR, 38.9%).
Mean survival, 45.4% (95% CI, 23.3%-67.4%); median survival, 45.8% (IQR, 48.0%).
Harvested grafts were paraffin embedded and cut in 5-μm representative sections, followed by hematoxylin-eosin staining. Microscopic evaluation was performed by an experienced clinical pathologist (O.T.). One slide per graft and 10 slides per treatment group were evaluated. The pathologist was blinded to the treatment groups. Slides were assessed for overall graft survival, cartilage necrosis, and mean vessel density per high-power field. Mean vessel density was calculated by averaging the number of vessels in a high-power field (×40) in 5 separate areas of each slide.
Results
Visual assessment of graft survival at 3 weeks showed significant agreement between the 2 surgeons, with a Spearman coefficient of 0.84 (P < .001) and consistency in the ratings, with an intraclass correlation of 0.98 (P < .001). Given the strong correlation of results, a mean survival rate was calculated for each graft. Group A had a mean survival of 97.4% (95% CI, 94.8%-99.9%) and median survival of 98.8% (interquartile range [IQR], 4.8%). Group B had a mean survival of 87.6% (95% CI, 69.9%-100%) and median survival of 99.0% (IQR, 15.0%). Group C had a mean survival of 82.1% (95% CI, 66.0%-98.1%) and median survival of 97.5% (IQR, 38.9%). Group D had a mean survival of 45.4% (95% CI, 23.3%-67.4%) and median survival of 45.8% (IQR, 48.0%). Group D (skin and cartilage separation) was visually graded to have significantly decreased survival when compared with group A (P < .001), group B (P = .004), and group C (P = .008). No significant difference in visual grading was found between groups A (mean, 97.4%), B (mean, 87.6%), and C (mean, 82.1%) (lowest P = .22).
Histopathologic assessment revealed that overall graft survival correlated with visual assessment results. Group D showed significant decrease in overall survival compared with group A, group B, and group C (Figure 3A). Group C additionally showed significantly decreased overall survival when compared with group A. Cartilage necrosis showed an inverse relationship with overall graft survival. Group A showed a significant decrease in cartilage necrosis compared with other groups. In addition, group C had a significant decrease in cartilage survival compared with group B (Figure 3B). Mean vessel density per high-power field showed that group A had significantly increased density compared with group C and group D but no difference compared with group B (Figure 3C).
Figure 3. Histopathologic Grading of Hematoxylin-Eosin–Stained Slides.
A, Overall survival of grafts. Comparison between groups: A vs B, P = .20; A vs C, P = .01; A vs D, P < .001; B vs C, P = .71; B vs D, P = .03; and C vs D, P = .03. B, Rate of cartilage necrosis. Comparison between groups: A vs B, P < .001; A vs C, P < .001; A vs D, P < .001; B vs C, P = .02; B vs D, P = .17; and C vs D, P = .46. C, Mean vessel density per high-power field (×40). Comparison between groups: A vs B, P = .08; A vs C, P = .001; A vs D, P = .008; B vs C, P = .08; B vs D, P = .27; and C vs D, P = .85. For all panels, the horizontal lines in the boxes represent median values, while the top and bottom borders of the boxes represent the 75th and 25th percentiles, respectively (ie, the interquartile range). The whiskers above and below the box represent the 90th and 10th percentiles. The points beyond the whiskers are outliers.
Discussion
The physiologic characteristics of composite graft healing and survival are not well described. Controversy exists regarding the importance of the perichondrium for composite graft survival. Skin graft healing is well understood as a process of imbibition, inosculation, and neovascularization. This physiologic paradigm is often applied to composite graft healing, but composite graft healing is almost certainly more complex. The addition of a cartilage component in a composite graft would dictate that neovascularization occurs primarily at the edges of the graft. If one applies the skin graft model to composite graft healing, separating the skin from the cartilage should not be significantly associated with graft survival. However, our study suggests that disruption of the skin to the perichondrial junction leads to decreased graft survival, as judged by visual inspection as well as histopathologic analysis. We found poorer overall survival of the grafts as well as increased cartilage necrosis. Although Zopf et al15 reported success in combining separate cartilage and skin grafts in a layered fashion to repair nasal defects, their study differs from ours in that the skin and cartilage do not overlap exactly, as in our study. Our results indicate that maintaining the chondrocutaneous junction plays a significant role in composite graft survival. Layering separate cartilage and skin grafts to repair a composite defect might be successful if the layers are partially staggered; Barlow16 advocates that the cartilage graft should ideally occupy no more than 50% of the surface area of the wound bed when a full-thickness skin graft is applied over it.
When we varied the skin to cartilage ratio in an inverse fashion, grafts in both groups B and C demonstrated similar survival visually as well as histopathologically. In addition, the mean vessel density in both groups was identical. Despite having a larger skin diameter, grafts in group B did not demonstrate poorer outcomes compared with group C. However, when the cartilage component was larger than the skin component, cartilage necrosis was significantly increased.
Compared with group A, group B had the same size cartilage graft but a larger skin component. Despite a higher metabolic requirement of group B, the skin does not show poorer overall survival visually or by histopathologic examination. Despite having an equal-size cartilage component, grafts in group B did have more cartilage necrosis compared with group A. This difference in cartilage necrosis may be a function of increased manipulation during graft harvest that is technically more challenging owing to the different sizes of the skin and cartilage components in group B. This increased manipulation could result in graft trauma and shearing at the chondrocutaneous junction. Similar to the significant difference from group A in visual grading, group C had poorer survival compared with group A in all categories of histopathologic analysis, including overall survival, cartilage necrosis, and mean vessel density. As with group B, harvesting grafts in group C was more technically challenging. In addition, there was circumferential undermining required at the recipient site to implant the entire cartilage component, which required more disruption of the wound bed. In designing auricular chondrocutaneous composite grafts, surgeons should consider limiting the shearing between the skin and cartilage to optimize graft survival.
Limitations
The study has several limitations. First, the results were derived from an animal model and cannot be directly extrapolated to humans. The grafts were transferred to the animals’ dorsum owing to limited nasal soft tissue in this rabbit model. Although this model does not simulate a nasal defect, it does present a well-vascularized wound bed for each graft and eliminates this consideration as a variable. Both our sample size and inability to test a wider range of skin to cartilage ratios prohibited us from making definitive conclusions about the association with survival of changing skin to cartilage ratios in the grafts.
Additional studies are needed to better understand composite graft physiology. The skin-to-perichondrium attachment appears to play a role in survival. Therefore, applying the model of imbibition, inosculation, and neovascularization as it applies to skin grafts does not fully explain healing of composite grafts. Understanding the exact physiology behind healing and survival of composite grafts would allow us to design more ideal grafts.
Conclusions
Our results provide evidence that the skin-to-perichondrium junction in a chondrocutaneous composite graft has a significant association with graft survival in a rabbit model. Reconstructive surgeons should preserve the chondrocutaneous junction when using composite grafts to potentially increase graft survival. Future studies that improve our understanding of healing and physiology of composite grafts will allow us to further refine indications and optimal surgical technique for using these unique grafts.
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