Surgical approaches that directly
address pathology of the lamina propria (e.g., vocal fold scar and sulcus vocalis) are varied and have included excision, incision, scar manipulation and implantation of free fat grafts.3,4,17,18,30
These techniques can offer vocal improvement, but to date there has not been development of an autologous, vascularized augmentation material that is locally harvested and can be delivered into the lamina propria. The minithyrotomy technique as described by Gray et al.17
delineates how a direct approach to Reinke’s space can be achieved with a surgical skill set that incorporates elements of standard microlaryngoscopy and thyroplasty. Although the minithyrotomy offers attractive access, no appropriate implant has been designed to allow for durable augmentation of Reinke’s space once access is obtained. We sought to address this gap by designing two experimental flaps using adult human cadaveric larynges and whose performance parameters we explored in a canine model. We acknowledge that there are limitations of the canine model. Most importantly, the lamina propria does not have the same layered structure as the human adult vocal fold. However, as noted by Garrett et al.31
canines are suitable for microsuspension laryngoscopy and their vocal folds vibrated in a similar fashion to human vocal folds with mucosal waves and vertical phase differences under stroboscopic examination. Furthermore, the size of the dog was appropriate for the proposed surgical procedure; a larger animal would have been unwieldy and a smaller one would have prevented successful execution of the procedure.
As noted previously, it was necessary to compare the two procedures given that the fat contained in CTAP, but not in TAP, might offer potential advantages. We postulated that the fat component of CTAP would be less likely to induce additional tissue formation given that free perichondrial and periosteal grafts have been noted to produce cartilage and bone, respectively, when placed in nonnative surroundings.32
Fat, on the other hand, generally does not induce formation of associated tissue. One month was selected because in prior studies, cartilage was easily visible by 2 weeks, suggesting that the 1- month latency used in our study should be sufficient to demonstrate if neochondrogenesis or neoosteogenesis were to happen. Another potential advantage of CTAP relates to the rheologic properties of fat. As noted above, fat appears to have similar, although not identical viscoelastic properties as native lamina propria, which suggests that implanted native fat might perform similarly to a native vocal fold, as has been noted previously.18,21
Last, CTAPs are larger than TAPs making them good candidates for larger defects and allowing greater ability of the surgeon to tailor the implant to the defects. However, given that the fat component of the CTAP is at the distal end of the flap and might therefore be susceptible to vascular compromise, we also were interested in the viability of this flap versus the TAP.
Human Cadaveric Studies
After the example of Payr and others, we chose to perform our initial examinations in an ex vivo environment on adult human cadaveric larynges. This approach allowed us to make anatomic measurements not only to establish the fundamentals of surgical technique but also to verify that the proposed procedures might be feasible. We found that both TAP and CTAP were long enough to span the entire ipsilateral vocal fold and that their volume would be sufficient to reconstitute Reinke’s space in cases of physical defect. We considered that “extra” length might be important because the inset flap might be prone to migration due to laryngeal movement and because perichondrial flaps are known to contract somewhat over time. We considered that “extra” volume might be important not only because of tissue contraction but also because we anticipated some element of tissue loss of the flap as has been noted with previous reports.14
Another additional advantage of having extra volume is that of being able to tailor the flap to a defect. For example, a modest vocal fold scar might be best addressed by placement of a thinner TAP within the lamina propria, whereas a deep type II sulcus deformity might be better reconstructed with a larger CTAP. The authors considered technical adjustments to improve the chances of the flap staying in the recipient bed such as suturing the flap to the minithyrotomy site; we believed that this could compromise the blood supply, however, and chose to avoid this approach. We also had concerns that when the CTAP is rotated into the minithyrotomy tunnel, the fat is directed away from the surface potentially limiting its effect; we believe that fixing the ultimate orientation of the flap would be essentially unfeasible because of laryngeal motion and that ultimately any biologic effect in the lamina propria set into motion by the flap would take place regardless of flap orientation.
Laser Doppler perfusion testing performed on all test animals before and immediately following flap harvest revealed a pattern similar to other harvested flaps of immediate perfusion reduction. For example, the pattern of immediate perfusion loss was consistent with a prior study on axial skin flaps in pigs.33
Comparison of data for perfusion changes between these and other flaps—whether random, axial, or free flaps—is difficult, however. Also, most measurements in the literature on other flaps are either not taken immediately after harvest or if they are, the raw data are not published, making easy comparisons impossible. Furthermore, most Doppler studies are performed on skin, which likely does not adhere to the same vascular principles as perichondrium. Notably, prior studies have shown subsequent increased perfusion to inset flaps and to the recipient bed after the initial perfusion drop.34,35
Finally, although the TAP is not novel in use, having been employed since the 1950s, we could find no other similar prior perfusion studies for comparison. There were no statistically significant differences between TAP and CTAP groups for perfusion loss immediately following flap harvest. However, the perfusion loss was 45.4% versus 30.3% for TAP and CTAP, respectively. This trend in the raw data suggests that CTAP may actually have better perfusion than that of the TAP. One might expect the opposite given that the additional tissue at the distal end of the CTAP could cause vascular compromise and would therefore increase the overall perfusion loss. This finding is pertinent because the additional tissue is largely fat, and fat may be highly useful in reconstituting the volume of the lamina propria in cases of injury. There is certainly the possibility of experimental error. For example, previous criticisms of laser Doppler imaging have cited tissue movement and single-site measurements as potential detractors from accurate readings. We attempted to account for this by assuring that the dogs and equipment were immobile and the Doppler unit used a scanning rather than a single-site measurement technique.
We performed an assessment of the biomechanical parameters of larynges implanted with TAP and CTAP to assess if there was a substantial alteration in aerodynamic and acoustic performance. Importantly, canine larynges have been noted to possess similar histologic and vibratory characteristics as human vocal folds, suggesting that the canine model is a reasonable model in this study.31
Therefore, we compared the vocal efficiency, laryngeal resistance, jitter, shimmer, and HNR to normal larynges. There were no statistically significant differences for laryngeal resistance and vocal efficiency, suggesting general similarity to the normal larynges. However, the standard errors were high relative to the mean values, demonstrating wide variability in animal to animal measures. These high error rates make valid interpretation of the data more difficult and may be explained by factors such as small numbers in each test group, differential healing between animals, and slightly different size and positioning of flaps within the lamina propria. This same pattern of statistically similar, but numerically different data is encountered when examining values for jitter, shimmer, and HNR. It is unclear if the appearance of high jitter and low shimmer values in the test larynges relative to normal vocal folds is accurate; if so it may indicate increased stiffness in the treated vocal folds, suggesting a treatment effect, albeit not a favorable one. For HNR the small number of animals and the wide variability in raw data may help to explain why the HNR values do not follow the same pattern as the jitter and shimmer values.
These performance measures appear to make sense because the implantation of a soft tissue flap into a normal vocal fold would almost certainly be expected to induce some alteration in oscillation due to asymmetry across the glottis (contour, tension, mass) and to healing effects. For example, if a flap were to have final placement on the subglottic edge rather than the free edge of the vocal fold, then substantial changes in acoustic performance might be anticipated.36
The fact that the measured changes are numerically different but not statistically different between test larynges and normals suggests that there has been a treatment effect in the test groups but that whatever effects have occurred at 1 month are not devastating to glottic performance. Lingering questions about glottic performance may in part be answered by using high-speed digital imaging in future studies.
Many vocal fold implants have been shown to be rheologically dissimilar to native vocal fold mucosa.21,37
Given that the oscillation of vocal folds is sensitive to rheologic alterations of the lamina propria, we measured whether the insertion of TAP or CTAP would significantly change any of four rheologic properties—damping ratio, dynamic viscosity, elastic shear modulus, and viscous shear modulus—when compared to the untreated side within the same animal. The values we obtained for damping ratio, dynamic viscosity, elastic shear modulus, and viscous shear modulus were comparable to those found in earlier studies on human cadaveric vocal fold lamina propria, suggesting the validity of our results.38
When we found no statistically significant differences for any group, and for a comparison of TAP to CTAP groups, this was reassuring because substantial alterations in rheology might lead to poor voice outcomes. Importantly, dynamic viscosity has been noted to be strongly correlated to the ease of phonation; increased dynamic viscosity in the treated vocal folds might have been a “red flag” for increased phonatory energy requirements.39
Mere statistical examination, however, may not be sufficient for full data interpretation here. For example, there was substantial variability to the raw data for each of the parameters tested. This variability could be explained by differential healing of the flaps within their recipient beds or even the testing method itself; each sample was tested at 14 frequencies and undergoes tissue alteration over time. Order of magnitude differences in normal human cadaveric vocal folds in the measurement of dynamic viscosity and elastic shear modulus have been previously partially attributed to age differences between subjects as well as differences in the densities and distributions of different vocal fold matrix proteins among the subjects, which could also be the case for the test animals in our study.38
This investigation was limited by a small sample size (4), limiting the confidence that detrimental rheologic change is not likely with this procedure. However, for many parameters, values for treated vocal folds were often both above and below the untreated vocal folds, suggesting that testing a larger number of animals still might not yield a statistical difference due to the large variability.
Given the risk of disruption of the specific components and layered structure of the vocal fold lamina propria after flap placement, we assessed histologic changes in the lamina propria.40
We were particularly concerned that the perichondrially based flaps might induce either neochondrogenesis or neoosteogenesis, which could severely impede vocal fold oscillation. Neither TAP nor CTAP was noted to induce surrounding neochondrogenesis or neoosteogenesis. It may be that a different recipient environment could change the outcome, however. For example, when a clot of blood was applied to free perichondrial grafts placed into rabbit dermis, the grafts produced abundant cartilage, whereas they did not produce cartilage when the blood clot was omitted.32
Given that graft manufacture of new tissue has been noted previously by 2 weeks and that our examination time point was 1 month, it seems unlikely we would observe cartilage or bone production at a later time point.32
Another concern was that the inset of the flaps might incite abundant collagenesis and disruption of the amounts of normal lamina propria components such as glycosaminoglycans and elastin, leading to reduced glottic performance. There was no statistically significant difference between the treated and untreated vocal folds, suggesting no detectable changes. Again, perhaps because of the small number of test animals, sampling error in sectioning, variability in healing, and a different time point standard errors were high, making definitive conclusions challenging.
Despite no statistical significance between groups, there were interesting trends. For example, in vocal folds treated with CTAP, there was reduced collagen, increased HA and elastin, relative to untreated vocal folds within the same animal. TAP vocal folds followed the same trend except for a small reduction of HA rather than an increase. One might hypothesize that the interaction of the flaps with the local environment induced these changes because the mere presence of the flap would have been likely to increase collagen amounts, given the perichondrium’s collagen content. These findings are both surprising and critically important because an abundant deposition of collagen or reduction of glycosaminoglycans and elastin would create the characteristics of a scarred vocal fold, which is exactly what these experimental approaches are ultimately intended to treat by reducing or eliminating the deposition.
We did not uniformly find obvious visual evidence of the flaps within the lamina propria of the vocal folds into which they were inset. It remains unclear whether the flaps may have retracted out of the vocal fold due to motion of the larynx, whether inadequate vascular supply may have compromised flap survival, or whether the flaps actually began to change their characteristics based upon influence from the recipient bed. The fact that the flap was not consistently seen histologically at 1 month may not necessarily indicate the flap was no longer in place nor that it didn’t survive. Given that the dogs were young and healthy with robust healing it is possible that rapid integration of the donor tissue within the recipient bed may have occurred, making the flaps difficult to identify histologically. It is also possible that variable healing due to nonuniformity of the animals could lead to nonuniformity of histologic findings. Furthermore, the flaps are not easily distinguished histologically from the cellular components of the lamina propria because the major cell type in each case is the fibroblast. The CTAP flap might be viewed as an exception to this notion given that it contains fat and the native laminia propria does not; however, as noted elsewhere in the manuscript, the young healthy beagles do not have significant amounts of fat in the preepiglottic space, and it therefore may not be easily seen. Finally, it is unlikely that the flaps did not survive given that these were all class A young healthy dogs, none had wound complications and there was no histologic evidence of tissue necrosis such as giant cell or lymphocytic infiltration. We hope to address these issues in future studies by possibly staining the donor flap with India ink to better show its outlines, making eventual histologic identification easier. We also plan to better secure the test flap in a more stable fashion, helping to reduce confusion about the fate of the donor flap.
There are fascinating questions that arise related to the interaction of the donor flap and recipient bed. For example, mechanical forces have been noted to change the gene expression in fibroblasts subjected to different conditions in a bioreactor, suggesting that the compartment is not static and is subject to alteration by physical forces.41
One might postulate that the forces of phonation could influence the tissue fate of the flap to appear and act more like native lamina propria. It may also be that the donor flap influences the recipient bed; for example, adipose-derived stem cells have been noted to exert a paracrine effect in the dermis, suggesting that the fat present in the CTAP could exert an influence on the lamina propria into which it is inset.42
We sought to evaluate the viability of TAP and CTAP 1 month after inset by staining the lamina propria for markers of vascular (vWF) and cellular proliferation (Ki-67). Flap viability would presumably be demonstrated by higher staining levels for these markers in treated versus untreated vocal folds within an animal. We did not find statistical significance for either TAP or CTAP groups, suggesting questionable viability for either procedure. Higher values for certain animals, however, and the visual evidence of viable flaps within the lamina propria () seem to point to variable healing rather than uniform flap loss. It may be that influences such as laryngeal movement, flap contraction, or rapid closure of the minithyrotomy around the flap—thereby choking off its vascular supply—could detract from uniform flap survival. One would anticipate thyroid ala perichondrial flaps to survive well, however, because they have been used successfully in relining the endolarynx following extirpation of laryngeal tumors and have been shown to be robust in animal models.25,43,44
Technical adaptations such as subepithelial fixation of TAP or CTAP to the arytenoid and a mechanism to reduce the risk of flap constriction at the minithyrotomy site may be useful in future studies.
Fig. 10 Histologic images of canine vocal folds in coronal section stained with EVG (4× magnification). (A) TAP-treated vocal fold. The black arrow points to a viable TAP within the lamina propria. (B) Untreated vocal fold. (C) CTAP-treated vocal fold. (more ...)
Technical Observations and Implications
Some observations have informed our understanding of this procedure with implications for the future. The combined endoscopic and open procedure for flap placement is strikingly similar in dogs and humans. Although not necessarily easy, the procedure involves familiar skill sets. Development of the tunnel within the lamina propria requires care not to violate the epithelium because this would compromise easy placement of the donor flap within Reinke’s space and would expose it to secretions from the endolarynx, potentially causing infection or even altering its fate. The authors found that development of the lamina propria tunnel was not uniform and that differentiating different layers of the lamina propria was not obvious. The use of instruments borrowed from otologic procedures was helpful, but we believe that more specialized instruments would enhance the precision of this critical element. The authors also found that although placement of the flap into the tunnel was straightforward, assuring that it would stay in place was not. Again, specialized instrumentation, perhaps designed to affix the flap to the arytenoid, would be beneficial.
It is also important to note that there were no canine mortalities in this study, helping to assure the safety of these procedures. Furthermore, no dog needed additional medication for airway compromise or altered diet for postoperative dysphagia. One might have expected dysphagia given the manipulation of the anterior neck structures. There was also concern that preepiglottic space dissection in the CTAP animals might induce significant dysphagia due to dissection near the epiglottis and near the internal branch of the superior laryngeal nerve, which provides sensation to the endolarynx. Dysphagia in the test animals was not observed. No local wound complications were noted.
The authors also noted that endoscopy performed 1 month after the procedure and immediately prior to humane sacrifice did not reveal endolaryngeal trauma such as web formation, hematoma, or superstructural alteration. No superstructural defect was noted in the thyroid ala once the larynx had been excised; one might postulate that removal of the thyroid ala perichondrium could predispose the cartilage to necrosis due to a reduced blood supply. This was not observed and would be consistent with experience in humans during thyroplasty and open partial laryngectomies. An important observation was made, however, at the minithyrotomy sites, which were all noted to have completely shut. Although one might expect closure of this dead space over the 1-month postoperative period, the authors are concerned that early closure of the minithyrotomy might compromise the vascular supply of either flap. Refinement of this element of the procedure may help to ensure that the vascular supply of the flaps is not choked off by early constriction at the minithyrotomy.
Flap Comparison and Potential
Throughout this study, the small number of test animals has limited our ability to tell a difference between groups treated with TAP and CTAP. However, some trends are notable and may offer insight into future applications. For example, the initial loss of perfusion was less with the CTAP group versus the TAP group, suggesting better perfusion for that flap. If the perfusion is indeed better it helps to alleviate concerns about distal flap perfusion; the distal flap contains the majority of the fat in the CTAP. Rheologically, the CTAP group had lower values (more favorable) than the TAP group for dynamic viscosity, which has been correlated to vocal effort, although the values for G″, which correlates to required energy for sustaining phonation were the inverse. Intergroup differences, were noted for laryngeal resistance and vocal efficiency but there was no consistent pattern, again making biomechanical conclusions difficult.
Histologically, both groups showed favorable trends overall with increased elastin and decreased collagen. The CTAP group revealed a slight increase in HA, a key component of the lamina propria. Last, although there appeared to be no difference between the groups when measuring vascular proliferation, the cellular proliferation difference between the CTAP group and TAP group approached statistical significance (0.08), suggesting that CTAP may ultimately be more viable.
The modest differences between groups do not prevent us from speculating about potential appropriate uses and pathways. Certainly due to size differences, TAP may be preferable for small defects and CTAP for larger ones. The amount of human preepiglottic space fat is much more abundant than in beagles, allowing for further options for contouring the size and shape of the inset flap. Furthermore, there is recent evidence supporting the presence of adipose-derived mesenchymal stem cells in peripheral fat.45
Mesenchymal stem cells have already been shown to have useful properties in the regeneration of damaged vocal folds.46
Although it is unknown if the preepiglottic space fat donated to CTAP contains a population of mesenchymal stem cells, certainly the notion of presenting a damaged vocal fold with a fresh population of stem cells is attractive.
Although not seen in this study, one might suspect improved rheologic performance with CTAP, given the rheologic similarities of fat to lamina propria. Also, due to biologic potential of paracrine effects from adipose tissue, one might be inclined to favor the use of CTAP.42
Variations of the CTAP suggest interesting possibilities for laryngeal reconstruction with wide-ranging applications. To wit, if the base of the flap were shifted to an inferior position instead of a lateral position, then simultaneous bilateral CTAPs could be harvested and applied in cases of bilateral pathology (). Furthermore, placement of either unilateral or bilateral flaps through a more laterally positioned minithyrotomy aperture would permit flap inset into the musculature of the paraglottic space. This approach might function well for correction of glottic volumetric defects that are not exclusive to the lamina propria such as paresis or age-related changes or indeed any cause creating a gap at the glottic level. It may also be that the musculature of the paraglottic space has a more robust vascular bed and might support donor flaps more generously, making this an attractive option for correction of glottic insufficiency in general.
Sketch of a human adult larynx from an anterior view. Bilateral inferiorly based CTAPs are mobilized. The left CTAP is held by forceps.
Last, the introduction of a vascularized bed may provide a healthy environment for introduction of stem cells. “Take rates” of injected stem cells are consistently low and, given the relative paucity of cells and vascularity in the lamina propria relative to the paraglottic space musculature, the introduction of an enhanced vascular bed appears attractive. Post hoc alteration of the flaps may also be possible with the introduction of growth factors that have recently made the transition from experimental to human use.47