- Email: [email protected]
Evaluation of the Grafted Fascia in the Vocal Fold of Dogs: A Histologic Study
ARTICLE IN PRESS Evaluation of the Grafted Fascia in the Vocal Fold of Dogs: A Histologic Study *Eduardo G.B. Carvalho, *Henrique F. Pauna, *Almiro J. Machado Junior, *Ester M.D. Nicola, †Albina M.A.M. Altemani, and *Agrício N. Crespo, *†Campinas, Brazil Summary: Introduction. There is no consensus on the ideal graft for medialization surgery of the vocal folds in the literature. One of the most favorable proposals is the use of autologous fascia, which seems limited by the lack of information regarding the integration of grafted tissue. Our study aims to evaluate the degree of fully engrafted fascia integration in the vocal fold lamina propria of dogs. Materials and Methods. Fourteen adult mongrel dogs that underwent intravenous general anesthesia were selected and kept under spontaneous ventilation. A fascia lata fragment of 4 cm2 was obtained from the right leg of each dog. The dogs underwent laryngoscopy; a 3 mm incision was made in the vocal process, next to the vestibular process, and the fascia was grafted into the right vocal fold. The left vocal fold was used as a control. The animals were divided into two groups: group A, evaluated after 2 months of the procedure, and group B, evaluated after 6 months of the procedure. Histologic analysis was made semiquantitatively regarding the presence of inflammatory reaction, fibrosis, and neovascularization. Results. Our final studied group comprised 12 dogs. Microscopic examination of the larynx revealed the absence of any detectable inflammation in the incision site. The lamina propria of the grafted vocal fold showed identifiable compact, thick, and eosinophilic collagen bands. The surrounding tissue showed thin collagen bands with some organization, similar to the contralateral vocal fold. Conclusion. The grafted fascia integrates into the vocal fold lamina propria and seems not to cause inflammatory reaction response. Key Words: Vocal folds–Vocal fold grafting–Fascia lata–Animal model–Inflammatory reaction.
INTRODUCTION The correction of extracellular matrix components distribution is essential to restore the structure of the vocal fold lamina propria. Indeed, it remains a major dilemma for phonosurgery today. Hirano et al1 demonstrated the changes in organization and distribution of components of the extracellular matrix within the vocal fold, such as increased deposition of type I collagen and fibronectin, and decreased elastin, decorin, and hyaluronic acid in vocal fold scars. Since the 1990s, many studies focused on the reestablishment of the vocal fold lamina propria among animal models, tried to reduce postoperative fibrosis and scarring, and tried to improve flexibility of the vocal folds.2–4 Initially, Brunnings, in 1911, described the paraffin implant, but this substance produced intense inflammatory reaction. Recently, several substances, from synthetic to biological materials, have been used to prevent this problem and improve glottal closure: Teflon, gelfoam, intralesional steroids, bovine collagen, homologous collagen, muscular fascia, and fat.5–8 Autologous fascia has been used for otologic and plastic surgery for decades, without complications and with reproducible results. Fascia graft has similar consistency to collagen and has a low rate of metabolic activity. Rihkanen,9 in 1998, proposed the use Accepted for publication December 29, 2016. From the *Department of Otorhinolaryngology, Head and Neck Surgery, University of Campinas, UNICAMP, Campinas, São Paulo, Brazil; and the †Department of Pathology, University of Campinas, UNICAMP, Campinas, São Paulo, Brazil. Address correspondence and reprint requests to Henrique F. Pauna, Department of Otorhinolaryngology, Head and Neck Surgery, University of Campinas, UNICAMP, PO Box 6111, Campinas, SP 13081-970, Brazil. E-mail: [email protected] Journal of Voice, Vol. ■■, No. ■■, pp. ■■-■■ 0892-1997 © 2017 The Voice Foundation. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jvoice.2016.12.022
of minced fascia in the surgery of volumetric augmentation of the vocal fold. The graft remained histologically identifiable after 1 year of follow-up (in most cases without early or late complications). Later, Tsunoda et al10–12 grafted fascia into the Reinke space for the correction of glottal incompetence in a human model, with promising results. Because there are only few studies available in the literature that histologically assess the degree of autologous fascia integration grafted into vocal folds, we performed this study—in a canine model—to evaluate the presence and degree of inflammatory reaction, fibrosis, and neovascularization.
MATERIALS AND METHODS Specimens Fourteen adult mongrel dogs of both genders were used, weighing between 10 and 15 kg. The number of male and female animals was the same. All the animals were kept with water and food ad libitum from the moment of the procedure to the moment of their sacrifice. Dogs were divided into two groups: group A, with animals maintained under observation for 2 months, and group B, observed for a period of 6 months after the surgical procedure. Animals were randomly distributed into two groups. Many studies have already evaluated different time points. It is well established that within 2 months, we could expect an acute inflammatory reaction to be observed in histologic evaluation, and that within 6 months, the process of chronic inflammatory reaction has already ended.5,6 We intended to evaluate vocal folds within both periods (2 and 6 months) to analyze the changes in inflammatory reaction after our surgical procedure.
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Journal of Voice, Vol. ■■, No. ■■, 2017
Animals with congenital malformations of the larynx, or female dogs that were pregnant during the experiment, were excluded from this study. Surgical procedure The animals underwent intravenous general anesthesia using sodium thiopental (50 mg/kg/dose) and kept with spontaneous ventilation. A 2 × 2 cm fragment of fascia lata was obtained from the right posterior leg of each dog, extended over metallic surface, and exposed to ambient air for spontaneous dehydration. The dogs underwent direct laryngoscopy for exposure of the vocal folds (Figure 1). A 3 mm incision (in length) was made in front of the vocal process, in the superior surface, in both vocal folds, near the edge of coaptation. A 45° angled dissection instrument was introduced into the incision to make a submucosal tunnel parallel to the coaptation edge of the vocal fold, with approximately 7 mm extension, in both vocal folds. After dehydration, a fragment of 3 × 3 mm of the fascia lata was inserted into the submucosal pouch. The graft was first inserted anteriorly to ensure its complete coverage by the mucosa and prevent its extrusion during the movements of the vocal fold (Figures 2 and 3). This method seemed to be safe in keeping the graft inside the submucosal pouch, decreasing the chances of extrusion. Furthermore, this method seemed to be technically easier and faster to perform. The left vocal fold was not grafted, and remained as a control for histologic changes caused by isolated surgical trauma. In both vocal folds, we did not perform any suture to close the pouch, avoiding the surgical trauma that it can possible cause, and also avoiding another confusing factor for histologic analysis. Histologic analysis After the animals were sacrificed, their larynges were excised and kept in 10% formalin solution for 24 hours. The vocal folds were then resected and transversely sectioned, giving two fragments: the anterior half and the posterior half of the vocal fold. We felt that splitting the vocal fold into two halves could increase
FIGURE 1. Surgical procedure in a dog performed by direct laryngoscopy.
FIGURE 2. The figure illustrates how the graft was performed. The gray space represents the glottic opening, and the lines represent the area where the submucosal pouch was performed (bilaterally). The red square represents the area where the fascia lata graft was implanted for further histologic analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) the chances of locating the graft after transversally sectioning it. Additionally, because of our method of inserting the graft into the vocal fold, we could expect to locate it anteriorly. Fragments were histologically processed (4-μm thickness for each section) and embedded in paraffin. The slides were stained with hematoxylin and eosin (H&E) examined under light microscopy to assess the following parameters: (1) number and type of inflammatory cells (neutrophils, lymphocytes, and macrophages), (2) presence of fibrosis, (3) presence of neovascularization, (4) presence of foreign body reactions, and (5) persistence or absence of grafted tissue. Histologic analysis was first performed by three members of the research team (E.G.B.C., H.F.P., E.M.D.N.), and then the findings were confirmed by an experienced pathologist (A.M.A.M.A.), who was blind of which side (right or left) or half (anterior or posterior) of the vocal fold was being evaluated. Although the lamina propria is hypocellular and contains collagen elements, just as the implant is hypocellular and also contains collagen, the histologic appearance of the two is different. In the lamina propria, the collagen fibers are short and
FIGURE 3. The illustration represents how the transverse sectioning of the vocal folds was performed. This would increase the chances to locate the graft after 2 months (group A) and 6 months (group B).
ARTICLE IN PRESS Eduardo G B Carvalho, et al
3
Grafted Fascia into Dogs Vocal Folds
thin and contain corrugations, the Reinke space contains just few capillaries, and there is a lack of lymphatic structures. Also, it is poorly permeated by seromucinous glands. The graft, however, contains long, thick, more rectilinear collagen fibers. Inflammatory reaction was semiquantitatively assessed by comparing the small portion of the surrounding of the grafted fascia lata with the lamina propria beyond this area. It was then classified as: 0—absence of mature mononuclear cells; I—mild (1–10 mature mononuclear cells were observed); II—moderate (11–20 mature mononuclear cells were observed); and III—intense (more than 20 mature mononuclear cells were observed). Additionally, inflammatory reaction was classified as “acute” when there is presence of polymorphonuclear cells, and “chronic” when there is presence of a foreign body reaction with active macrophage predominance. Fibrosis was also semiquantitatively assessed by the following criteria: 0—absence of collagen fibers; I—mild (few foci of collagen fibers); II—moderate (continued collagen fibers around the grafted area); or III—severe (bigger area of continued collagen fibers around the grafted area, consisting a fibrous capsule).
The assessment of fibrosis can be performed by using H&E, Masson trichrome, picric acid, or picrosirius red staining. However, we decided to use H&E staining, which is widely accepted for this evaluation, and also, according to Pinna Bde et al,13 there is no financial advantage of using other methods because the final results are similar. Neovascularization was defined by the presence of thin-wall blood vessels within the grafted area, and lastly, we also assessed the presence or absence of fascia lata graft into the vocal fold lamina propria. The institutional review board of the University of Campinas approved this study (CEP 486-1). RESULTS Our final groups comprised six dogs in group A (2-month survival) and six dogs in group B (6-month survival). None of them presented any type of postoperative complications. Macroscopic examination of their larynges revealed no detectable inflammation and no identifiable location of the cordectomy. The comparison of the inflammatory reaction showed no difference between the two sides of vocal folds. It was mostly considered as a mild inflammatory response between groups A and B (Tables 1 and 2, Figures 4 and 5). Furthermore, the comparative analysis of cell predominance showed no significant differences between groups and between vocal folds. The main type of cells was represented by lymphohistiocytic cells. Yet none of the analyzed vocal folds (for both groups) met the criteria for chronic inflammatory reaction, with neither giant cell formation nor foreign body reaction.
TABLE 1. Histologic Aspects of Group A Infiltrated Compound Animal Number 1 2 3 4 5 6
Cellularity
Fibrosis
Chronic Reaction
Neovascularization
Grafted
Control
Grafted
Control
Grafted
Control
Grafted
Control
Grafted
Control
I II I I I I
I I I I I I
H LH L LH LH LH
LH L LH LH LH LH
I I 0 I 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
I I I I I I
0 0 0 0 0 0
H, histiocytic pattern; L, lymphocytic pattern; LH, lymphohistiocytic pattern; 0, absence; I, mild; II, moderate.
TABLE 2. Histologic Aspects of Group B Infiltrated Compound Animal Number 7 8 9 10 11 12
Chronic Reaction
Neovascularization
Grafted
Control
Grafted
Cellularity Control
Grafted
Fibrosis Control
Grafted
Control
Grafted
Control
I I II I I I
I I I 0 I I
LH LH LH LH LH L
L LH L LH LH LH
I I 0 I 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
I I I I I I
0 0 0 0 0 0
H, histiocytic pattern; L, lymphocytic pattern; LH, lymphohistiocytic pattern; 0, absence; I, mild; II, moderate.
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FIGURE 4. Histologic section of a right vocal fold of a dog from group A. The grafted fascia (within the area shown by the arrowheads) is constituted by a hypocellular fibrous tissue. Note the absence of inflammatory reaction or fibrosis around the graft (H&E, 40× magnification).
Journal of Voice, Vol. ■■, No. ■■, 2017
FIGURE 6. Histologic section of a right vocal fold of a dog from group B. The grafted fascia (within the area shown by the arrowhead) has a higher presence of cells within the lamina propria and its surroundings (arrows; H&E, 40× magnification).
FIGURE 7. Histologic section of a left vocal fold of a dog from group FIGURE 5. Histologic section showing a fragment of fascia lata. Observe how the collagen fibers are elongated (H&E, 40× magnification). The grafted vocal folds showed identifiable compact, thick, and eosinophilic collagen bands within the lamina propria. The surrounding tissue showed similar organization in both vocal folds (right and left), with thin collagen bands identified (Figures 6 and 7). Fifty percent of the grafted vocal folds met a minimum degree of focal fibrosis (mild) in groups A and B. The other 50% showed no difference when compared with the control side, in both groups. Ultimately, all grafted vocal folds showed the presence of neovascularization, in both analyzed groups. DISCUSSION Since the 1990s, several interventions to restore structural characteristics of vocal fold lamina propria have been tested on a trial basis, to reduce postoperative fibrosis and scarring, and to
A. Observe the similar features as in the aforementioned figures, with the grafted fascia. Note the absent inflammatory reaction or fibrosis within the lamina propria (H&E, 40× magnification).
improve the flexibility of the mucosa. In vitro studies with the use of fibroblast or hepatocyte growth factors—with or without stem cells associated—showed a reduction of type I collagen and an increased production of hyaluronic acid within Reinke space fibroblasts.2,3,14–20 Our study demonstrates, in a canine model, how the grafted fascia lata behaves when implanted into the vocal fold lamina propria, when one compares two different periods: 2 months and 6 months after surgical procedure. Carneiro et al7 studied 100 rabbits using fascia graft implanted into the vocal folds of one group, and fat graft implanted into the vocal folds of another group. Using polarized microscopy, they demonstrated that fat graft induced an increased collagen production or fibrosis, compared with fascia graft. Later, in 2006, the same authors conducted a literature review comparing
ARTICLE IN PRESS Eduardo G B Carvalho, et al
Grafted Fascia into Dogs Vocal Folds
the use of autologous fascia and fat to correct glottal incompetence. They concluded that both tissues play an important role to correct glottal incompetence because of their low rate of adverse reactions.21 Contradictorily, another study pointed out the rapid absorption of crushed autologous fascia injected after a 3-month period of observation. The authors of the study proposed that fascia is not suitable as a graft in the vocal fold. Interestingly, our study suggests that the fascia lata graft is well tolerated, hence, it did not trigger inflammatory reaction—acute or chronic— when implanted in the vocal fold lamina propria of a dog. The same result as ours was reported by Reijonen et al22 and Duke et al23 after histological assessment of shredded fascia lata injection into vocal muscles of a canine model. The evolution of a grafted tissue in a receptor site depends on a number of factors, such as nutritional support, immunogenic compatibility, and anatomical similarity between the graft and the host.24 Chemotactic stimulation, leading to a polymorphonuclear migration, resulting in phagocytosis and lysis of the graft, is seen with inflammatory reaction. However, it was not observed in our samples. The absence of activated macrophages may be related to the biocompatibility between the fascia lata and the vocal fold. These results are different from those described by other studies, where a fibrous capsule formation or a higher collagen deposition was identified around grafted fascia.22,25,26 We assume, based on our observations, that the foci of fibrosis, identified on half of the grafted vocal folds, may not be sufficient to affect the normal function of the vocal folds. One possible explanation for this difference is that the fascia is basically composed of the extracellular matrix of collagen fibers and fibroblasts, which is commonly found in the lamina propria.1,5,7 In our study, the fascia lata remained intact, with no evidence of breaking down of the collagen fibers. This fact can also be inferred by the absence of active macrophages in the grafted area. Apparently, the fascia not only remained inert, but also became well integrated into the vocal fold lamina propria (which is suggested by the presence of neovascularization at the periphery of the graft, and the absence of inflammatory foreign body reaction). Our results support the clinical observations by Tsunoda et al,10–12 because they had attributed the improvement of voice quality of patients with vocal sulcus to fascia transplantation in the vocal folds. The present study is, to the best of our knowledge, the first study that has histologically evaluated the effects caused by implanted fascia lata into the lamina propria of the vocal folds. CONCLUSION Our study supports the idea of fascia lata integration within the vocal fold lamina propria. Fascia lata integration did not induce either acute or chronic inflammatory reaction, it is slightly fibrogenic, and neovascularization can also be seen. We believe that our results can be useful to support the use of this material in humans. REFERENCES 1. Hirano M, Sato K, Nakashima T. Fine structure of the human newborn and infant vocal fold mucosae. Ann Otol Rhinol Laryngol. 2001;110(5 pt 1):417–424.
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2. Hirano S, Bless DM, Heisey D, et al. Effect of growth factors on hyaluronan production by canine vocal fold fibroblasts. Ann Otol Rhinol Laryngol. 2003;112:617–624. 3. Hirano S, Bless DM, Rousseau B, et al. Fibronectin and adhesion molecules on canine scared vocal folds. Laryngoscope. 2003;113:966– 972. 4. Ohno T, Lesley C, Hirano S, et al. Effect of hepatocyte growth factor on gene expression of extracellular matrix during wound healing of the injured rat vocal fold. Ann Otol Rhinol Laryngol. 2008;117:696–702. 5. Ford CN. Advances and refinements in phonosurgery. Laryngoscope. 1999;109:1891–1899. 6. Akdogan O, Selcuk A, Ozcan I, et al. Activation of vocal fold healing with topical vitamin A in rabbits. Acta Otolaryngol. 2009;129:220–224. 7. Carneiro CG, Sennes LU, Saldiva PHN, et al. Assessment of collagen deposits after implant of fascia lata and fat in the vocal folds of rabbits: histomorphometric study. Braz J Otorhinolaryngol. 2005;71:798– 802. 8. Krishna P, Rosen CA, Branski RC, et al. Primed fibroblasts and exogenous decorin: potential treatments for subacute vocal fold scar. Otolaryngol Head Neck Surg. 2006;135:937–945. 9. Rihkanen H. Vocal fold augmentation by injection of autologous fascia. Laryngoscope. 1998;108:51–54. 10. Tsunoda K, Takanosawa M, Niimi S. Autologous transplantation of fascia into the vocal fold: a new phonosurgical technique for glottal incompetence. Laryngoscope. 1999;109:504–508. 11. Tsunoda K, Niimi S. Autologous transplantation of fascia into the vocal fold. Laryngoscope. 2000;110:680–682. 12. Tsunoda K, Baer T, Niimi S. Autologous transplantation of fascia into the vocal fold: long-term results of a new phonosurgical technique for glottal incompetence. Laryngoscope. 2001;111:453–457. 13. Pinna Bde R, Stavale JN, Lima Pontes PA, et al. Histological analysis of autologous fascia graft implantation into the rabbit voice muscle. Braz J Otorhinolaryngol. 2011;77:185–190. 14. Hirano M, Sato K, Nakashima T. Fibroblasts in human vocal fold mucosa. Acta Otolaryngol. 1999;119:271–276. 15. Hirano S, Bless DM, Massey RJ, et al. Morphological and functional changes of human vocal fold fibroblasts with hepatocyte growth factor. Ann Otol Rhinol Laryngol. 2003;112:1026–1033. 16. Kanemaru SI, Kojima H, Hirano S, et al. Regeneration of the vocal fold using autologous mesenchymal stem cells. Ann Otol Rhinol Laryngol. 2003;112:915–920. 17. Kanemaru S, Nakamura T, Yamashita M, et al. Destiny of autologous bone marrow-derived stromal cells implanted in the vocal fold. Ann Otol Rhinol Laryngol. 2005;114:907–912. 18. Thibeault SL, Rousseau B, Welham N, et al. Hyaluronan levels in acute vocal fold scar. Laryngoscope. 2004;114:760–764. 19. Luo Y, Kobler JB, Zeitels SM, et al. Effects of growth factors on extracellular matrix production by vocal fold fibroblasts in 3-dimensional culture. Tissue Eng. 2006;12:3365–3374. 20. Ohno T, Yoo MJ, Swanson ER, et al. Regenerative effects of basic fibroblasts growth factor on extracellular matrix production in aged rat vocal folds. Ann Otol Rhinol Laryngol. 2009;118:559–564. 21. Carneiro CG, Tsuji DH, Sennes LU, et al. Glottic insufficiency: the use of fat and fascia grafts. Braz J Otorhinolaryngol. 2006;72:140–144. 22. Reijonen P, Leivo I, Nevalainen T. Histology of injected autologous fascia in the paralyzed canine vocal fold. Laryngoscope. 2001;111:1068– 1074. 23. Duke SG, Salmon J, Blalock PD. Fascia augmentation of the vocal fold: graft yield in the canine and preliminary clinical experience. Laryngoscope. 2000;111:759–764. 24. Hom DB. The wound healing response to grafted tissues. Otolaryngol Clin North Am. 1994;27:13–24. 25. Tamura E, Kitahara S, Kohno N, et al. Use of freeze-dried autologous fascia to augment the vocal fold: an experimental study in dogs. Acta Otolaryngol. 2002;122:537–540. 26. Hachiya A, Imamura R, Parra ER, et al. Histologic study of perifascial areolar tissue implanted in rabbit vocal folds: an experimental study. Ann Otol Rhinol Laryngol. 2010;119:707–715.
INTRODUCTION The correction of extracellular matrix components distribution is essential to restore the structure of the vocal fold lamina propria. Indeed, it remains a major dilemma for phonosurgery today. Hirano et al1 demonstrated the changes in organization and distribution of components of the extracellular matrix within the vocal fold, such as increased deposition of type I collagen and fibronectin, and decreased elastin, decorin, and hyaluronic acid in vocal fold scars. Since the 1990s, many studies focused on the reestablishment of the vocal fold lamina propria among animal models, tried to reduce postoperative fibrosis and scarring, and tried to improve flexibility of the vocal folds.2–4 Initially, Brunnings, in 1911, described the paraffin implant, but this substance produced intense inflammatory reaction. Recently, several substances, from synthetic to biological materials, have been used to prevent this problem and improve glottal closure: Teflon, gelfoam, intralesional steroids, bovine collagen, homologous collagen, muscular fascia, and fat.5–8 Autologous fascia has been used for otologic and plastic surgery for decades, without complications and with reproducible results. Fascia graft has similar consistency to collagen and has a low rate of metabolic activity. Rihkanen,9 in 1998, proposed the use Accepted for publication December 29, 2016. From the *Department of Otorhinolaryngology, Head and Neck Surgery, University of Campinas, UNICAMP, Campinas, São Paulo, Brazil; and the †Department of Pathology, University of Campinas, UNICAMP, Campinas, São Paulo, Brazil. Address correspondence and reprint requests to Henrique F. Pauna, Department of Otorhinolaryngology, Head and Neck Surgery, University of Campinas, UNICAMP, PO Box 6111, Campinas, SP 13081-970, Brazil. E-mail: [email protected] Journal of Voice, Vol. ■■, No. ■■, pp. ■■-■■ 0892-1997 © 2017 The Voice Foundation. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jvoice.2016.12.022
of minced fascia in the surgery of volumetric augmentation of the vocal fold. The graft remained histologically identifiable after 1 year of follow-up (in most cases without early or late complications). Later, Tsunoda et al10–12 grafted fascia into the Reinke space for the correction of glottal incompetence in a human model, with promising results. Because there are only few studies available in the literature that histologically assess the degree of autologous fascia integration grafted into vocal folds, we performed this study—in a canine model—to evaluate the presence and degree of inflammatory reaction, fibrosis, and neovascularization.
MATERIALS AND METHODS Specimens Fourteen adult mongrel dogs of both genders were used, weighing between 10 and 15 kg. The number of male and female animals was the same. All the animals were kept with water and food ad libitum from the moment of the procedure to the moment of their sacrifice. Dogs were divided into two groups: group A, with animals maintained under observation for 2 months, and group B, observed for a period of 6 months after the surgical procedure. Animals were randomly distributed into two groups. Many studies have already evaluated different time points. It is well established that within 2 months, we could expect an acute inflammatory reaction to be observed in histologic evaluation, and that within 6 months, the process of chronic inflammatory reaction has already ended.5,6 We intended to evaluate vocal folds within both periods (2 and 6 months) to analyze the changes in inflammatory reaction after our surgical procedure.
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Journal of Voice, Vol. ■■, No. ■■, 2017
Animals with congenital malformations of the larynx, or female dogs that were pregnant during the experiment, were excluded from this study. Surgical procedure The animals underwent intravenous general anesthesia using sodium thiopental (50 mg/kg/dose) and kept with spontaneous ventilation. A 2 × 2 cm fragment of fascia lata was obtained from the right posterior leg of each dog, extended over metallic surface, and exposed to ambient air for spontaneous dehydration. The dogs underwent direct laryngoscopy for exposure of the vocal folds (Figure 1). A 3 mm incision (in length) was made in front of the vocal process, in the superior surface, in both vocal folds, near the edge of coaptation. A 45° angled dissection instrument was introduced into the incision to make a submucosal tunnel parallel to the coaptation edge of the vocal fold, with approximately 7 mm extension, in both vocal folds. After dehydration, a fragment of 3 × 3 mm of the fascia lata was inserted into the submucosal pouch. The graft was first inserted anteriorly to ensure its complete coverage by the mucosa and prevent its extrusion during the movements of the vocal fold (Figures 2 and 3). This method seemed to be safe in keeping the graft inside the submucosal pouch, decreasing the chances of extrusion. Furthermore, this method seemed to be technically easier and faster to perform. The left vocal fold was not grafted, and remained as a control for histologic changes caused by isolated surgical trauma. In both vocal folds, we did not perform any suture to close the pouch, avoiding the surgical trauma that it can possible cause, and also avoiding another confusing factor for histologic analysis. Histologic analysis After the animals were sacrificed, their larynges were excised and kept in 10% formalin solution for 24 hours. The vocal folds were then resected and transversely sectioned, giving two fragments: the anterior half and the posterior half of the vocal fold. We felt that splitting the vocal fold into two halves could increase
FIGURE 1. Surgical procedure in a dog performed by direct laryngoscopy.
FIGURE 2. The figure illustrates how the graft was performed. The gray space represents the glottic opening, and the lines represent the area where the submucosal pouch was performed (bilaterally). The red square represents the area where the fascia lata graft was implanted for further histologic analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) the chances of locating the graft after transversally sectioning it. Additionally, because of our method of inserting the graft into the vocal fold, we could expect to locate it anteriorly. Fragments were histologically processed (4-μm thickness for each section) and embedded in paraffin. The slides were stained with hematoxylin and eosin (H&E) examined under light microscopy to assess the following parameters: (1) number and type of inflammatory cells (neutrophils, lymphocytes, and macrophages), (2) presence of fibrosis, (3) presence of neovascularization, (4) presence of foreign body reactions, and (5) persistence or absence of grafted tissue. Histologic analysis was first performed by three members of the research team (E.G.B.C., H.F.P., E.M.D.N.), and then the findings were confirmed by an experienced pathologist (A.M.A.M.A.), who was blind of which side (right or left) or half (anterior or posterior) of the vocal fold was being evaluated. Although the lamina propria is hypocellular and contains collagen elements, just as the implant is hypocellular and also contains collagen, the histologic appearance of the two is different. In the lamina propria, the collagen fibers are short and
FIGURE 3. The illustration represents how the transverse sectioning of the vocal folds was performed. This would increase the chances to locate the graft after 2 months (group A) and 6 months (group B).
ARTICLE IN PRESS Eduardo G B Carvalho, et al
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Grafted Fascia into Dogs Vocal Folds
thin and contain corrugations, the Reinke space contains just few capillaries, and there is a lack of lymphatic structures. Also, it is poorly permeated by seromucinous glands. The graft, however, contains long, thick, more rectilinear collagen fibers. Inflammatory reaction was semiquantitatively assessed by comparing the small portion of the surrounding of the grafted fascia lata with the lamina propria beyond this area. It was then classified as: 0—absence of mature mononuclear cells; I—mild (1–10 mature mononuclear cells were observed); II—moderate (11–20 mature mononuclear cells were observed); and III—intense (more than 20 mature mononuclear cells were observed). Additionally, inflammatory reaction was classified as “acute” when there is presence of polymorphonuclear cells, and “chronic” when there is presence of a foreign body reaction with active macrophage predominance. Fibrosis was also semiquantitatively assessed by the following criteria: 0—absence of collagen fibers; I—mild (few foci of collagen fibers); II—moderate (continued collagen fibers around the grafted area); or III—severe (bigger area of continued collagen fibers around the grafted area, consisting a fibrous capsule).
The assessment of fibrosis can be performed by using H&E, Masson trichrome, picric acid, or picrosirius red staining. However, we decided to use H&E staining, which is widely accepted for this evaluation, and also, according to Pinna Bde et al,13 there is no financial advantage of using other methods because the final results are similar. Neovascularization was defined by the presence of thin-wall blood vessels within the grafted area, and lastly, we also assessed the presence or absence of fascia lata graft into the vocal fold lamina propria. The institutional review board of the University of Campinas approved this study (CEP 486-1). RESULTS Our final groups comprised six dogs in group A (2-month survival) and six dogs in group B (6-month survival). None of them presented any type of postoperative complications. Macroscopic examination of their larynges revealed no detectable inflammation and no identifiable location of the cordectomy. The comparison of the inflammatory reaction showed no difference between the two sides of vocal folds. It was mostly considered as a mild inflammatory response between groups A and B (Tables 1 and 2, Figures 4 and 5). Furthermore, the comparative analysis of cell predominance showed no significant differences between groups and between vocal folds. The main type of cells was represented by lymphohistiocytic cells. Yet none of the analyzed vocal folds (for both groups) met the criteria for chronic inflammatory reaction, with neither giant cell formation nor foreign body reaction.
TABLE 1. Histologic Aspects of Group A Infiltrated Compound Animal Number 1 2 3 4 5 6
Cellularity
Fibrosis
Chronic Reaction
Neovascularization
Grafted
Control
Grafted
Control
Grafted
Control
Grafted
Control
Grafted
Control
I II I I I I
I I I I I I
H LH L LH LH LH
LH L LH LH LH LH
I I 0 I 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
I I I I I I
0 0 0 0 0 0
H, histiocytic pattern; L, lymphocytic pattern; LH, lymphohistiocytic pattern; 0, absence; I, mild; II, moderate.
TABLE 2. Histologic Aspects of Group B Infiltrated Compound Animal Number 7 8 9 10 11 12
Chronic Reaction
Neovascularization
Grafted
Control
Grafted
Cellularity Control
Grafted
Fibrosis Control
Grafted
Control
Grafted
Control
I I II I I I
I I I 0 I I
LH LH LH LH LH L
L LH L LH LH LH
I I 0 I 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
I I I I I I
0 0 0 0 0 0
H, histiocytic pattern; L, lymphocytic pattern; LH, lymphohistiocytic pattern; 0, absence; I, mild; II, moderate.
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FIGURE 4. Histologic section of a right vocal fold of a dog from group A. The grafted fascia (within the area shown by the arrowheads) is constituted by a hypocellular fibrous tissue. Note the absence of inflammatory reaction or fibrosis around the graft (H&E, 40× magnification).
Journal of Voice, Vol. ■■, No. ■■, 2017
FIGURE 6. Histologic section of a right vocal fold of a dog from group B. The grafted fascia (within the area shown by the arrowhead) has a higher presence of cells within the lamina propria and its surroundings (arrows; H&E, 40× magnification).
FIGURE 7. Histologic section of a left vocal fold of a dog from group FIGURE 5. Histologic section showing a fragment of fascia lata. Observe how the collagen fibers are elongated (H&E, 40× magnification). The grafted vocal folds showed identifiable compact, thick, and eosinophilic collagen bands within the lamina propria. The surrounding tissue showed similar organization in both vocal folds (right and left), with thin collagen bands identified (Figures 6 and 7). Fifty percent of the grafted vocal folds met a minimum degree of focal fibrosis (mild) in groups A and B. The other 50% showed no difference when compared with the control side, in both groups. Ultimately, all grafted vocal folds showed the presence of neovascularization, in both analyzed groups. DISCUSSION Since the 1990s, several interventions to restore structural characteristics of vocal fold lamina propria have been tested on a trial basis, to reduce postoperative fibrosis and scarring, and to
A. Observe the similar features as in the aforementioned figures, with the grafted fascia. Note the absent inflammatory reaction or fibrosis within the lamina propria (H&E, 40× magnification).
improve the flexibility of the mucosa. In vitro studies with the use of fibroblast or hepatocyte growth factors—with or without stem cells associated—showed a reduction of type I collagen and an increased production of hyaluronic acid within Reinke space fibroblasts.2,3,14–20 Our study demonstrates, in a canine model, how the grafted fascia lata behaves when implanted into the vocal fold lamina propria, when one compares two different periods: 2 months and 6 months after surgical procedure. Carneiro et al7 studied 100 rabbits using fascia graft implanted into the vocal folds of one group, and fat graft implanted into the vocal folds of another group. Using polarized microscopy, they demonstrated that fat graft induced an increased collagen production or fibrosis, compared with fascia graft. Later, in 2006, the same authors conducted a literature review comparing
ARTICLE IN PRESS Eduardo G B Carvalho, et al
Grafted Fascia into Dogs Vocal Folds
the use of autologous fascia and fat to correct glottal incompetence. They concluded that both tissues play an important role to correct glottal incompetence because of their low rate of adverse reactions.21 Contradictorily, another study pointed out the rapid absorption of crushed autologous fascia injected after a 3-month period of observation. The authors of the study proposed that fascia is not suitable as a graft in the vocal fold. Interestingly, our study suggests that the fascia lata graft is well tolerated, hence, it did not trigger inflammatory reaction—acute or chronic— when implanted in the vocal fold lamina propria of a dog. The same result as ours was reported by Reijonen et al22 and Duke et al23 after histological assessment of shredded fascia lata injection into vocal muscles of a canine model. The evolution of a grafted tissue in a receptor site depends on a number of factors, such as nutritional support, immunogenic compatibility, and anatomical similarity between the graft and the host.24 Chemotactic stimulation, leading to a polymorphonuclear migration, resulting in phagocytosis and lysis of the graft, is seen with inflammatory reaction. However, it was not observed in our samples. The absence of activated macrophages may be related to the biocompatibility between the fascia lata and the vocal fold. These results are different from those described by other studies, where a fibrous capsule formation or a higher collagen deposition was identified around grafted fascia.22,25,26 We assume, based on our observations, that the foci of fibrosis, identified on half of the grafted vocal folds, may not be sufficient to affect the normal function of the vocal folds. One possible explanation for this difference is that the fascia is basically composed of the extracellular matrix of collagen fibers and fibroblasts, which is commonly found in the lamina propria.1,5,7 In our study, the fascia lata remained intact, with no evidence of breaking down of the collagen fibers. This fact can also be inferred by the absence of active macrophages in the grafted area. Apparently, the fascia not only remained inert, but also became well integrated into the vocal fold lamina propria (which is suggested by the presence of neovascularization at the periphery of the graft, and the absence of inflammatory foreign body reaction). Our results support the clinical observations by Tsunoda et al,10–12 because they had attributed the improvement of voice quality of patients with vocal sulcus to fascia transplantation in the vocal folds. The present study is, to the best of our knowledge, the first study that has histologically evaluated the effects caused by implanted fascia lata into the lamina propria of the vocal folds. CONCLUSION Our study supports the idea of fascia lata integration within the vocal fold lamina propria. Fascia lata integration did not induce either acute or chronic inflammatory reaction, it is slightly fibrogenic, and neovascularization can also be seen. We believe that our results can be useful to support the use of this material in humans. REFERENCES 1. Hirano M, Sato K, Nakashima T. Fine structure of the human newborn and infant vocal fold mucosae. Ann Otol Rhinol Laryngol. 2001;110(5 pt 1):417–424.
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2. Hirano S, Bless DM, Heisey D, et al. Effect of growth factors on hyaluronan production by canine vocal fold fibroblasts. Ann Otol Rhinol Laryngol. 2003;112:617–624. 3. Hirano S, Bless DM, Rousseau B, et al. Fibronectin and adhesion molecules on canine scared vocal folds. Laryngoscope. 2003;113:966– 972. 4. Ohno T, Lesley C, Hirano S, et al. Effect of hepatocyte growth factor on gene expression of extracellular matrix during wound healing of the injured rat vocal fold. Ann Otol Rhinol Laryngol. 2008;117:696–702. 5. Ford CN. Advances and refinements in phonosurgery. Laryngoscope. 1999;109:1891–1899. 6. Akdogan O, Selcuk A, Ozcan I, et al. Activation of vocal fold healing with topical vitamin A in rabbits. Acta Otolaryngol. 2009;129:220–224. 7. Carneiro CG, Sennes LU, Saldiva PHN, et al. Assessment of collagen deposits after implant of fascia lata and fat in the vocal folds of rabbits: histomorphometric study. Braz J Otorhinolaryngol. 2005;71:798– 802. 8. Krishna P, Rosen CA, Branski RC, et al. Primed fibroblasts and exogenous decorin: potential treatments for subacute vocal fold scar. Otolaryngol Head Neck Surg. 2006;135:937–945. 9. Rihkanen H. Vocal fold augmentation by injection of autologous fascia. Laryngoscope. 1998;108:51–54. 10. Tsunoda K, Takanosawa M, Niimi S. Autologous transplantation of fascia into the vocal fold: a new phonosurgical technique for glottal incompetence. Laryngoscope. 1999;109:504–508. 11. Tsunoda K, Niimi S. Autologous transplantation of fascia into the vocal fold. Laryngoscope. 2000;110:680–682. 12. Tsunoda K, Baer T, Niimi S. Autologous transplantation of fascia into the vocal fold: long-term results of a new phonosurgical technique for glottal incompetence. Laryngoscope. 2001;111:453–457. 13. Pinna Bde R, Stavale JN, Lima Pontes PA, et al. Histological analysis of autologous fascia graft implantation into the rabbit voice muscle. Braz J Otorhinolaryngol. 2011;77:185–190. 14. Hirano M, Sato K, Nakashima T. Fibroblasts in human vocal fold mucosa. Acta Otolaryngol. 1999;119:271–276. 15. Hirano S, Bless DM, Massey RJ, et al. Morphological and functional changes of human vocal fold fibroblasts with hepatocyte growth factor. Ann Otol Rhinol Laryngol. 2003;112:1026–1033. 16. Kanemaru SI, Kojima H, Hirano S, et al. Regeneration of the vocal fold using autologous mesenchymal stem cells. Ann Otol Rhinol Laryngol. 2003;112:915–920. 17. Kanemaru S, Nakamura T, Yamashita M, et al. Destiny of autologous bone marrow-derived stromal cells implanted in the vocal fold. Ann Otol Rhinol Laryngol. 2005;114:907–912. 18. Thibeault SL, Rousseau B, Welham N, et al. Hyaluronan levels in acute vocal fold scar. Laryngoscope. 2004;114:760–764. 19. Luo Y, Kobler JB, Zeitels SM, et al. Effects of growth factors on extracellular matrix production by vocal fold fibroblasts in 3-dimensional culture. Tissue Eng. 2006;12:3365–3374. 20. Ohno T, Yoo MJ, Swanson ER, et al. Regenerative effects of basic fibroblasts growth factor on extracellular matrix production in aged rat vocal folds. Ann Otol Rhinol Laryngol. 2009;118:559–564. 21. Carneiro CG, Tsuji DH, Sennes LU, et al. Glottic insufficiency: the use of fat and fascia grafts. Braz J Otorhinolaryngol. 2006;72:140–144. 22. Reijonen P, Leivo I, Nevalainen T. Histology of injected autologous fascia in the paralyzed canine vocal fold. Laryngoscope. 2001;111:1068– 1074. 23. Duke SG, Salmon J, Blalock PD. Fascia augmentation of the vocal fold: graft yield in the canine and preliminary clinical experience. Laryngoscope. 2000;111:759–764. 24. Hom DB. The wound healing response to grafted tissues. Otolaryngol Clin North Am. 1994;27:13–24. 25. Tamura E, Kitahara S, Kohno N, et al. Use of freeze-dried autologous fascia to augment the vocal fold: an experimental study in dogs. Acta Otolaryngol. 2002;122:537–540. 26. Hachiya A, Imamura R, Parra ER, et al. Histologic study of perifascial areolar tissue implanted in rabbit vocal folds: an experimental study. Ann Otol Rhinol Laryngol. 2010;119:707–715.