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Lipoinjection in the paralyzed feline vocal fold: Study of graft survival
Lipoinjection in the paralyzed feline vocal fold' Study of graft survival
m
PHILLIPW. SACCOGNA, MD, JOHN W. WERNING,MD, DMD,SEBOUHSETRAKIAN,MD, and MELVINSTRAUSS,MD, Cleveland, Ohio Six adult domestic strain cats were used to study the long-term histologic outcome of injected autologous fat for augmentation of the paralyzed vocal fold. Each animal had surgically induced left vocal cord paralysis via sectioning of the recurrent laryngeal nerve, followed by injection of 0. I to 0.2 ml of autologous fat into the paralyzed vocal fold. The animals were killed at 6 weeks, and at 4, 6, 8, and 12 months after the injection. Photographic and videolaryngoscopic data were obtained. Histologic studies of the larynges were performed. The results documented histologic viability and persistence of a portion of the injected adipose tissue graft at 8 months after the injection, but only minimal graft survival at 12 months. The outcome suggests that autologous lipoinjection has potential use for short-term (several months) augmentation of the paralyzed vocal cord. Further investigation is warranted before recommending this technique for such use or as an alternative to currently available long-term injectable laryngeal biomaterials. (©tolaryngol Head Neck Surg 1997; 1 ! 7:465-70,)
Teflon (Polytef; Mentor Co., Norwell, Md.) is well established as the injectable biomaterial of choice for augmentation of the vocal cord in symptomatic unilateral laryngeal paralysis. 1,2 Arnold 3 originally described the use of Teflon as a vocal cord injectable biomaterial. Dedo et al. 4 and others have reported extensive experience with this procedure, and have described histologic studies of the larynx after injection. 5-7 With. this experience have come reports of problems with Teflon as a vocal cord injectable biomaterial. The problems include overcorrection with airway obstruction, vocal cord stiffening and irregularity, migration of the implant, granuloma formation, and foreign body reaction. 8-~1 Other materials have been investigated, including col-
From the Departments of Otolaryngology-Headand Neck Surgery (Drs. Saccogna, Werning, and Strauss) and Pathology (Dr. Setrakian), Case Western Reserve University School of Medicine, Cleveland, Ohio. This research is supported by The Head and Neck Medicine and Surgery Foundation. Reprint requests: Melvin Strauss, MD, Department of Otolaryngology-Headand Neck Surgery, University Hospitals of Cleveland, 11100 Euclid Ave., Cleveland,OH 44106. Presented at the Annual Meeting of the American Academy of Otolaryngology-Headand Neck Surgery. New Orleans, La., Sept. 17-20, 1995. Copyright © 1997 by the American Academy of OtolaryngologyHead and Neck SurgeryFoundation, Inc. 0194-5998/97/$5.00 + 0 23/1/73746
lagen, 12-t4 but none has achieved the status of an ideal injectable biomaterial for vocal cord medialization. Most recently, autologous fat has reportedly been used as an injectable material in the treatment of human laryngeal paralysis. 15,16 Reported advantages offered by use of injectable autologous fat include ready availability, ease of injectability, and lack of foreign body or hypersensitivity reactions. Interest in using autologous fat has been generated by reports of its use in contouring of facial and other body areas. 17-19 The results with the use of injected autologous fat grafting in various body areas have been conflicting. Ersek 2° reported poor long-term clinical graft survival in humans, while other researchers have shown graft viability ranging from 20% to 50% using histologic and volume studies in animal models. 21-23 Brandenburg et al. I6 reported that good clinical results with fat graft augmentation were obtained up to 1 year after injection in cases of human laryngeal paralysis. The survival of injected autologous fat in the paralyzed vocal cord has been histologically demonstrated in the canine model up to 6 months after injection. 2425 The purpose of this study is to demonstrate the longterm histologic fate of injected autologous fat in the paralyzed cat vocal cord. The hypothesis is that if longterm survival of the graft can be demonstrated in an animal model, then clinical trials in human beings can be used with confidence that injectable grafts do indeed have the potential for tong-term vocal fold augmenta-
46G
466 SACCOGNA et al.
OtolaryngologyHead and Neck Surgery November 1997
Fig. 1. Hematoxylin and eosin section of the injected true vocal cord at 4 months (×40). A significant volume of grafted fat is present within the fibroconnective tissue and the skeletal muscle of the true vocal cord. The inset (x300) demonstrates a multinucleated giant cell surrounded by the grafted fat.
tion. If the grafts do not prove to be viable, then their use in permanent vocal cord augmentation may be inappropriate. METHODS AND MATERIAL
This animal protocol was approved by the Institutional Animal Care and Use Committee of Case Western Reserve University School of Medicine and was in compliance with all codes of behavior for animal research. (Guide for the Care and Use of Laboratory Animals, NIH publication No. 80-23, revised 1978). Six adult random source domestic strain cats weighing 3 to 6 kg each comprised the experimental population. The animals were placed under general endotracheal inhalational anesthesia at the start of the procedure. All animals received intravenous dexamethasone 0.5 mg/kg preoperatively and ampicillin 20 mg/kg preoperatively and for 3 days postoperatively. The anterior neck and lower abdomen were shaved, prepared, and draped under sterile conditions. Incisional injections of 0.5% bupivacaine with 1:100,000 diluted epinephrine were administered. A vertical midline neck incision was used to expose the larynx and upper trachea. Dissection with the aid of an
operating microscope was carried out in the left tracheoesophageal groove. The recurrent nerve was identified and sectioned with excision of a 1 cm segment. Immediate direct laryngoscopy was performed with confirmation of left vocal cord paralysis. The fat was harvested using two methods: (1) from the lower abdomen via a suction technique with a stab incision and the Toomey Syringe (Byron Medical Products, Tucson, Ariz.), and (2) from a midline lower abdominal incision by sharply excising the subcutaneous fat. The excised fat specimen was minced into 1 to 2 mm 3 fragments, rinsed with Ringer's solution, and loaded atraumatically into a 10 ml syringe. Samples of both the suctioned and excised fat specimens were ejected through the injection apparatus into formalin and examined for cell integrity using light microscopy. In our study, the vocal cord injected fat was harvested and prepared via the direct excision and mincing method as described above. The excised fat was injected using the Disposo-A-Ject fat injection gun (Byron Medical Products) through a 19-gauge needle into the lateral portion of the paralyzed left thyroarytenoid muscle at the mid-true vocal cord level at a depth of 3 to 4 mm. The injection was carried out under direct laryngoscopic visualization of the temporarily extubated lar-
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Fig. 2. Hematoxylin and eosin section of the noninjected right true vocal cord control from the same cat illustrated in Fig. 1 (x 40).
ynx, Each animal had 0.1 to 0.2 ml of autologous fat injected into its paralyzed left vocal fold. The larynx was reintubated after fat injection. The wounds were closed in a standard two-layer fashion. The animals were awakened from anesthesia, extubated, and immediately placed in a humidified oxygen chamber until the animals were ambulatory and without any stridor. The animals were housed in the institutional Animal Resource Center. One animal each was killed at 6 weeks, and at 4 , 8 , and 12 months after the injection. Two animals were killed 6 months after the injection. At the time the animals were killed, videolaryngoscopic and photographic data of the larynges were obtained with the animal sedated and breathing spontaneously. The animals were killed with an intravenous overdosage of pentobarbital and a laryngectomy was performed. Each glottis was fixed in formalin, individually sectioned at right angles to the vocal cords, and e m b e d d e d in paraffin. Hematoxylin and eosin-stained slides were studied to evaluate for the survival of the grafted adipose tissue.
Fig. 3. Endoscopic appearance of the same larynx (4 months postinjection) just before the anima~ was killed demonstrates paramedian position of the left vocal fold with slight abduction of the right vocal fold,
RESULTS
All the animals survived until the planned dates that they were to be killed. Minor postoperative complications occurred in two animals. The first animal had a small dehiscence of the neck incision, which healed without additional intervention. The second animal
exhibited prolonged postoperative stridor, which required additional dexamethasone administration and close observation in the humidified oxygen chamber for 48 hours postoperatively. This animal had a technically
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468 SACCOGNA et al.
Fig. 4. Hematoxylin and eosin section of a larynx from a cat killed 6 months after autologous fat injection (x40). A small amount of grafted fat (arrow) is still present and a lipogranuloma with regenerative adipose cells can be seen (arrow head) (><40). Inset of lipogranuloma
(x300).
difficult injection and the added manipulation was considered to be responsible for the increased edema and temporary partial airway obstruction. A microscopic comparison of the free fat specimens harvested via the suction and excision techniques revealed histologic integrity of the adipocytes with minimal evidence of trauma or cellular disruption. The larynx studied 6 weeks after the injection revealed lipogranuloma formation with regenerative adipocytes and only a small volume of viable fat graft present within the thyroarytenoid muscle at the site of the injection. The larynx of the cat killed at 4 months demonstrated a significant volume of viable grafted fat within the vocal cord both medial to and in the muscle (Fig. 1). Occasional foreign body giant ceils were present within the fat (Fig. 1, inset). A comparison with a similar section from the noninjected right vocal cord (control) revealed no adipose tissue in the intramuscular vocal fold (Fig. 2). The photographic appearance of this larynx just before the cat was killed is shown in Fig. 3. The left vocal cord is in the paramedian position and the right vocal cord in abduction. A fullness of the left vocal fold is appreciated, although this is a reduction from the overcorrection at the time of injection. Two cats were killed 6 months after the fat injection. The first cat demonstrated a significant volume of
viable fat within the thyroarytenoid muscle slightly lateral and inferior to the true vocal fold. A chronic inflammatory infiltrate surrounded the margins of the viable fat. The second cat failed to demonstrate significant fat graft survival. Its larynx was histologically similar to the specimen obtained 6 weeks after the injection with lipogranuloma formation, regenerative adipocytes, and a small volume of viable fat graft (Fig. 4). The larynx studied at 8 months after the injection revealed a significant volume of viable graft identical in appearance to the 4-month and one of the @month specimens, again demonstrating adipose tissue with scant giant cell reaction within and medial to the muscular portion of the true vocal cord (Fig. 5). The noninjected right vocal cord was histologically normal. The larynx obtained 12 months after the injection demonstrated minimal survival of the fat graft within the thyroarytenoid muscle when compared with the 8month specimen. DISCUSSION
The results of this histologic study suggest that injectable autologous fat grafting in the paralyzed feline vocal fold allows short-term graft survival up to 8 months after the injection. Previous investigations have demonstrated variable results for the long-term viabili-
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Fig. 5. Hematoxylin and eosin section of a larynx from a cat killed 8 months after autologous fat injection (x40). Note the persistence of fat in this specimen with a paucity of inflammatory cells,
ty of implanted or injected fat grafts. Canine models reported by Wexler et al.26 and Jiang et al.27 demonstrated up to 3 months of histologic survival of the directly implanted fat grafts in the injured vocal fold mucosa. Injected fat has been shown to survive up to 6 months in a canine model. 24,a5 No histologic data, however, are available as to the survival of injected autologous fat for augmentation of vocal cord paralysis for a period longer than 6 months. Although prior reports 15,16 have mentioned promising long-term clinical results with lipoinjection in the paralyzed human vocal cord in a small number of patients, our animal model provides histologic support of those findings only for a period up to 8 months. Previous human and animal studies in nonlaryngeal fat grafting have not consistently supported the longterm viability of fat grafting. Bartynski et al. 22 studied lipoinjection in a rabbit ear model and found poor graft viability (20%) at 100 days after the injection. Others 2° have confirmed less than optimal clinical outcomes in body contouring lipoinjection. Chajchir and Benzaquen 19 performed histologic studies of punch biopsy specimens obtained up to 1 year after injection from grafts injected into facial defects. They showed no viable fat but only fibrosis at 1 year. Wetmore21 showed 9-month viability of injected fat in a rabbit back muscle
model. Our findings are consistent with these two studies in that we did find viable fat grafts at 8 months after injection, but failed to find significant survival of the grafts at 12 months. The results in the two animals killed at 6 weeks and 6 months after injection, which demonstrated only lipogranuloma and a small volume of viable fat grafts, are difficult to evaluate in light of the superior graft survival in the other specimens. A possible explanation for the findings includes extrusion of the graft through the injection site, which was noted to occur to some extent in all cases with reintubation of the larynx immediately after injection. In light of the small volume of fat injected, 0.1 to 0.2 ml, we believe that any significant extrusion could give poor results. The relatively small size of the cat airway limited our ability to inject larger volumes without airway obstruction. In addition, we used 19-gauge needles for the injections. This was the smallest needle size considered to allow atraumatic injection of adipocytes. % This needle was large relative to the size of the vocal cord injection site in the cat, making precise injection in all of the larynges technically difficult. The experience of the senior author with the use of injectable biomaterials in canines and humans suggests
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470 SACCOGNA et al.
that proper placement with lack of extrusion would be facilitated in larger larynges.
4.
CONCLUSION The best method to harvest and prepare fat used for lipoinjection remains undetermined. 23,25,27 In our study, cellular integrity was maintained to a comparable degree when the fat was harvested by either a liposuction or excisional technique. We decided to use the excised fat for injection because cats have relatively small abdominal fat pads available for liposuction and empirical observations suggested that direct excision would be a less traumatic method of fat harvest. The technique of lipoinjection of autologous fat into the paralyzed vocal fold has been shown by photographic and histologic studies to offer short-term viability up to 8 months after injection in a feline model. Although the numbers of animals studied are too small to address the reasons for failure of the fat grafts to survive, these may well reflect difficulties resulting from the needle gauge required and the small size of cat larynges. Further long-term (12 months or longer) investigation combining histologic and photographic studies in a canine model would be ideal in documenting the value of this technique, while avoiding some of the technical difficulties encountered in our model. This study lends support, however, to the theory that autologous fat satisfies two of Arnold's 3 three criteria for the ideal laryngeal injectable material: ease of injectability and the ability to be well tolerated. The third criterion, long-term viability, is not applicable, as shown by the lack of significant graft survival in our 12-month specimen. We cannot recommend this technique as an alternative to Teflon injection or thyroplasty for permanent vocal fold augmentation until further studies demonstrate whether survival beyond 8 months can occur. If further study substantiates several months of fat survival in a significant number of cats, autologous fat may be useful in the paralyzed vocal cord to temporarily improve function in cases where compensation has not occurred and spontaneous recovery is still a possibility.
5. 6. 7.
8. 9. 10. 11. 12. 13. 14.
15. 16. 17. 18. 19. 20. 21. 22.
23.
24. 25. 26.
REFERENCES 1. Tucker HM. Vocal cord paralysis-1979: etiology and management. Laryngoscope 1980;90:585-90. 2. Ford CN. Laryngeal injection techniques. In: Ford CN, Bless DM, editors. Phonosurgery: assessment and surgical management of voice disorders. New York: Raven Press; 1991. 3. Arnold GE. Vocal rehabilitation of paralytic dysphonia. IX.
27. 28.
Technique of intracordal injection. Arch Otolaryngol 1962;79: 358-68. Dedo HH, Urrea RD, Lawson L. Intracordal injection of teflon in the treatment of 135 patients with dysphonia. Ann Otol Rhinol Laryngol 1973;82:661-7. Kirchner FR, Toledo PS, Svoboda DJ. Studies of the larynx after Teflon injection. Arch Otolaryngol 1966;83:350-4. Lewy RB. Responses of laryngeal tissue to granular Teflon in situ. Arch Otolaryngol 1966;83:355-9. Stone JW, Arnold GE, Stephens GB. Intracordal polytef (Teflon) injection: histologic study of three further cases. Arch Otolaryngol 1970;91:568-74. Rubin HJ. Pitfalls in treatment of dysphonias by intracordal inj ection of synthetics. Laryngoscope 1965 ;75:1381-97. Lewy RB. Teflon injection of the vocal cord: complications, errors, and precautions. Ann Otnl Rhinol Laryngol 1983;92: 473-4. Rubin HJ. Misadventures with injectable polytef (Teflon). Arch Otolaryngol 1975;101:1141-6. Tucker HM. Complications after surgical management of the paralyzed larynx. Laryngoscope 1983;93:295-8. Ford CN, Bless DM. A preliminary study of injectable collagen in human vocal fold augmentation. Otolaryngol Head Neck Surg • 1986;94:104-12. Ford CN, Martin DW, Warner TF. Injectable collagen in laryngeal rehabilitation. Laryngoscope 1984;94:513-8. Ford CN. Histologic studies of the fate of soluble collagen injected into canine vocal cords. Laryngoscope 1986;96:124857. Mikaelian DO, Lowry LD, Sataloff RT. Lipoinjection for unilateral vocal cord paralysis. Laryngoscope 1991; 101:465-8. Brandenburg JH, Kirkham W, Koschkee D. Vocal cord augmentation with autogenous fat. Laryngoscope 1992;102:495-500. Agris J. Autologous fat transplantation: a 3-year study. Am J Cosmetic Surgery 1987;4:95-102. Newman J, Ftaiha Z. The biographical history of fat transplant surgery. Am J Cosmetic Surgery 1987;4:85-7. Chajchir A, Benzaquen I. Fat-grafting injection for soft-tissue augmentation. Plast Recorlstr Surg 1989;84:921-35. Ersek RA. Transplantation of purified autologous fat: a 3 year follow-up is disappointing. Plast Reconstr Surg 1991;87:219-28. Wetmore SJ. Injection of fat for soft tissue augmentation. Laryngoscope 1989;99:50-7. Bartynski J, Marion MS, Wang TD. Histopathologic evaluation of adipose autografts in a rabbit ear model. Otolaryngol Head Neck Surg 1990;102:314-21. Nguyen A, Pasyk KA, Bouvier TN, Hassett CA, Argenta LC. Comparative study of survival of autologous adipose tissue taken and transplanted by different techniques. Plast Reconstr Surg 1990;85:378-89. Zaretsky LS, Detar M, Shindo ML, Rice DH. Autologous fat injection for vocal fold paralysis: long term histologic evaluations. Ann Otol Rhinol Laryngol 1995;104:1-4. Mikus JL, Koufman JA, Kilpatrick SE. Fate of liposuctioned and purified autologous fat injection in the canine vocal fold. Laryngoscope 1995;105:17-22. Wexler DB, Gray SD, Jiang J, Titze IR. Phonosurgical studies: fat graft reconstruction of injured canine vocal cords. Ann Otot Rhinol Laryngol 1989;98:668-73. Jiang J, Wexler DB, Titze IR, Gray SD. Fundamental frequency and amplitude perturbation in reconstructed canine vocal folds. Ann Otol Rhinol Laryngol 1994;103:145-8. Campbell GH, Laudenslager N, Newman J. The effect of mechanical stress on adipocyte morphology and metabolism. Am J Cosmetic Surgery 1987;4:89-94.
m
PHILLIPW. SACCOGNA, MD, JOHN W. WERNING,MD, DMD,SEBOUHSETRAKIAN,MD, and MELVINSTRAUSS,MD, Cleveland, Ohio Six adult domestic strain cats were used to study the long-term histologic outcome of injected autologous fat for augmentation of the paralyzed vocal fold. Each animal had surgically induced left vocal cord paralysis via sectioning of the recurrent laryngeal nerve, followed by injection of 0. I to 0.2 ml of autologous fat into the paralyzed vocal fold. The animals were killed at 6 weeks, and at 4, 6, 8, and 12 months after the injection. Photographic and videolaryngoscopic data were obtained. Histologic studies of the larynges were performed. The results documented histologic viability and persistence of a portion of the injected adipose tissue graft at 8 months after the injection, but only minimal graft survival at 12 months. The outcome suggests that autologous lipoinjection has potential use for short-term (several months) augmentation of the paralyzed vocal cord. Further investigation is warranted before recommending this technique for such use or as an alternative to currently available long-term injectable laryngeal biomaterials. (©tolaryngol Head Neck Surg 1997; 1 ! 7:465-70,)
Teflon (Polytef; Mentor Co., Norwell, Md.) is well established as the injectable biomaterial of choice for augmentation of the vocal cord in symptomatic unilateral laryngeal paralysis. 1,2 Arnold 3 originally described the use of Teflon as a vocal cord injectable biomaterial. Dedo et al. 4 and others have reported extensive experience with this procedure, and have described histologic studies of the larynx after injection. 5-7 With. this experience have come reports of problems with Teflon as a vocal cord injectable biomaterial. The problems include overcorrection with airway obstruction, vocal cord stiffening and irregularity, migration of the implant, granuloma formation, and foreign body reaction. 8-~1 Other materials have been investigated, including col-
From the Departments of Otolaryngology-Headand Neck Surgery (Drs. Saccogna, Werning, and Strauss) and Pathology (Dr. Setrakian), Case Western Reserve University School of Medicine, Cleveland, Ohio. This research is supported by The Head and Neck Medicine and Surgery Foundation. Reprint requests: Melvin Strauss, MD, Department of Otolaryngology-Headand Neck Surgery, University Hospitals of Cleveland, 11100 Euclid Ave., Cleveland,OH 44106. Presented at the Annual Meeting of the American Academy of Otolaryngology-Headand Neck Surgery. New Orleans, La., Sept. 17-20, 1995. Copyright © 1997 by the American Academy of OtolaryngologyHead and Neck SurgeryFoundation, Inc. 0194-5998/97/$5.00 + 0 23/1/73746
lagen, 12-t4 but none has achieved the status of an ideal injectable biomaterial for vocal cord medialization. Most recently, autologous fat has reportedly been used as an injectable material in the treatment of human laryngeal paralysis. 15,16 Reported advantages offered by use of injectable autologous fat include ready availability, ease of injectability, and lack of foreign body or hypersensitivity reactions. Interest in using autologous fat has been generated by reports of its use in contouring of facial and other body areas. 17-19 The results with the use of injected autologous fat grafting in various body areas have been conflicting. Ersek 2° reported poor long-term clinical graft survival in humans, while other researchers have shown graft viability ranging from 20% to 50% using histologic and volume studies in animal models. 21-23 Brandenburg et al. I6 reported that good clinical results with fat graft augmentation were obtained up to 1 year after injection in cases of human laryngeal paralysis. The survival of injected autologous fat in the paralyzed vocal cord has been histologically demonstrated in the canine model up to 6 months after injection. 2425 The purpose of this study is to demonstrate the longterm histologic fate of injected autologous fat in the paralyzed cat vocal cord. The hypothesis is that if longterm survival of the graft can be demonstrated in an animal model, then clinical trials in human beings can be used with confidence that injectable grafts do indeed have the potential for tong-term vocal fold augmenta-
46G
466 SACCOGNA et al.
OtolaryngologyHead and Neck Surgery November 1997
Fig. 1. Hematoxylin and eosin section of the injected true vocal cord at 4 months (×40). A significant volume of grafted fat is present within the fibroconnective tissue and the skeletal muscle of the true vocal cord. The inset (x300) demonstrates a multinucleated giant cell surrounded by the grafted fat.
tion. If the grafts do not prove to be viable, then their use in permanent vocal cord augmentation may be inappropriate. METHODS AND MATERIAL
This animal protocol was approved by the Institutional Animal Care and Use Committee of Case Western Reserve University School of Medicine and was in compliance with all codes of behavior for animal research. (Guide for the Care and Use of Laboratory Animals, NIH publication No. 80-23, revised 1978). Six adult random source domestic strain cats weighing 3 to 6 kg each comprised the experimental population. The animals were placed under general endotracheal inhalational anesthesia at the start of the procedure. All animals received intravenous dexamethasone 0.5 mg/kg preoperatively and ampicillin 20 mg/kg preoperatively and for 3 days postoperatively. The anterior neck and lower abdomen were shaved, prepared, and draped under sterile conditions. Incisional injections of 0.5% bupivacaine with 1:100,000 diluted epinephrine were administered. A vertical midline neck incision was used to expose the larynx and upper trachea. Dissection with the aid of an
operating microscope was carried out in the left tracheoesophageal groove. The recurrent nerve was identified and sectioned with excision of a 1 cm segment. Immediate direct laryngoscopy was performed with confirmation of left vocal cord paralysis. The fat was harvested using two methods: (1) from the lower abdomen via a suction technique with a stab incision and the Toomey Syringe (Byron Medical Products, Tucson, Ariz.), and (2) from a midline lower abdominal incision by sharply excising the subcutaneous fat. The excised fat specimen was minced into 1 to 2 mm 3 fragments, rinsed with Ringer's solution, and loaded atraumatically into a 10 ml syringe. Samples of both the suctioned and excised fat specimens were ejected through the injection apparatus into formalin and examined for cell integrity using light microscopy. In our study, the vocal cord injected fat was harvested and prepared via the direct excision and mincing method as described above. The excised fat was injected using the Disposo-A-Ject fat injection gun (Byron Medical Products) through a 19-gauge needle into the lateral portion of the paralyzed left thyroarytenoid muscle at the mid-true vocal cord level at a depth of 3 to 4 mm. The injection was carried out under direct laryngoscopic visualization of the temporarily extubated lar-
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Fig. 2. Hematoxylin and eosin section of the noninjected right true vocal cord control from the same cat illustrated in Fig. 1 (x 40).
ynx, Each animal had 0.1 to 0.2 ml of autologous fat injected into its paralyzed left vocal fold. The larynx was reintubated after fat injection. The wounds were closed in a standard two-layer fashion. The animals were awakened from anesthesia, extubated, and immediately placed in a humidified oxygen chamber until the animals were ambulatory and without any stridor. The animals were housed in the institutional Animal Resource Center. One animal each was killed at 6 weeks, and at 4 , 8 , and 12 months after the injection. Two animals were killed 6 months after the injection. At the time the animals were killed, videolaryngoscopic and photographic data of the larynges were obtained with the animal sedated and breathing spontaneously. The animals were killed with an intravenous overdosage of pentobarbital and a laryngectomy was performed. Each glottis was fixed in formalin, individually sectioned at right angles to the vocal cords, and e m b e d d e d in paraffin. Hematoxylin and eosin-stained slides were studied to evaluate for the survival of the grafted adipose tissue.
Fig. 3. Endoscopic appearance of the same larynx (4 months postinjection) just before the anima~ was killed demonstrates paramedian position of the left vocal fold with slight abduction of the right vocal fold,
RESULTS
All the animals survived until the planned dates that they were to be killed. Minor postoperative complications occurred in two animals. The first animal had a small dehiscence of the neck incision, which healed without additional intervention. The second animal
exhibited prolonged postoperative stridor, which required additional dexamethasone administration and close observation in the humidified oxygen chamber for 48 hours postoperatively. This animal had a technically
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468 SACCOGNA et al.
Fig. 4. Hematoxylin and eosin section of a larynx from a cat killed 6 months after autologous fat injection (x40). A small amount of grafted fat (arrow) is still present and a lipogranuloma with regenerative adipose cells can be seen (arrow head) (><40). Inset of lipogranuloma
(x300).
difficult injection and the added manipulation was considered to be responsible for the increased edema and temporary partial airway obstruction. A microscopic comparison of the free fat specimens harvested via the suction and excision techniques revealed histologic integrity of the adipocytes with minimal evidence of trauma or cellular disruption. The larynx studied 6 weeks after the injection revealed lipogranuloma formation with regenerative adipocytes and only a small volume of viable fat graft present within the thyroarytenoid muscle at the site of the injection. The larynx of the cat killed at 4 months demonstrated a significant volume of viable grafted fat within the vocal cord both medial to and in the muscle (Fig. 1). Occasional foreign body giant ceils were present within the fat (Fig. 1, inset). A comparison with a similar section from the noninjected right vocal cord (control) revealed no adipose tissue in the intramuscular vocal fold (Fig. 2). The photographic appearance of this larynx just before the cat was killed is shown in Fig. 3. The left vocal cord is in the paramedian position and the right vocal cord in abduction. A fullness of the left vocal fold is appreciated, although this is a reduction from the overcorrection at the time of injection. Two cats were killed 6 months after the fat injection. The first cat demonstrated a significant volume of
viable fat within the thyroarytenoid muscle slightly lateral and inferior to the true vocal fold. A chronic inflammatory infiltrate surrounded the margins of the viable fat. The second cat failed to demonstrate significant fat graft survival. Its larynx was histologically similar to the specimen obtained 6 weeks after the injection with lipogranuloma formation, regenerative adipocytes, and a small volume of viable fat graft (Fig. 4). The larynx studied at 8 months after the injection revealed a significant volume of viable graft identical in appearance to the 4-month and one of the @month specimens, again demonstrating adipose tissue with scant giant cell reaction within and medial to the muscular portion of the true vocal cord (Fig. 5). The noninjected right vocal cord was histologically normal. The larynx obtained 12 months after the injection demonstrated minimal survival of the fat graft within the thyroarytenoid muscle when compared with the 8month specimen. DISCUSSION
The results of this histologic study suggest that injectable autologous fat grafting in the paralyzed feline vocal fold allows short-term graft survival up to 8 months after the injection. Previous investigations have demonstrated variable results for the long-term viabili-
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Fig. 5. Hematoxylin and eosin section of a larynx from a cat killed 8 months after autologous fat injection (x40). Note the persistence of fat in this specimen with a paucity of inflammatory cells,
ty of implanted or injected fat grafts. Canine models reported by Wexler et al.26 and Jiang et al.27 demonstrated up to 3 months of histologic survival of the directly implanted fat grafts in the injured vocal fold mucosa. Injected fat has been shown to survive up to 6 months in a canine model. 24,a5 No histologic data, however, are available as to the survival of injected autologous fat for augmentation of vocal cord paralysis for a period longer than 6 months. Although prior reports 15,16 have mentioned promising long-term clinical results with lipoinjection in the paralyzed human vocal cord in a small number of patients, our animal model provides histologic support of those findings only for a period up to 8 months. Previous human and animal studies in nonlaryngeal fat grafting have not consistently supported the longterm viability of fat grafting. Bartynski et al. 22 studied lipoinjection in a rabbit ear model and found poor graft viability (20%) at 100 days after the injection. Others 2° have confirmed less than optimal clinical outcomes in body contouring lipoinjection. Chajchir and Benzaquen 19 performed histologic studies of punch biopsy specimens obtained up to 1 year after injection from grafts injected into facial defects. They showed no viable fat but only fibrosis at 1 year. Wetmore21 showed 9-month viability of injected fat in a rabbit back muscle
model. Our findings are consistent with these two studies in that we did find viable fat grafts at 8 months after injection, but failed to find significant survival of the grafts at 12 months. The results in the two animals killed at 6 weeks and 6 months after injection, which demonstrated only lipogranuloma and a small volume of viable fat grafts, are difficult to evaluate in light of the superior graft survival in the other specimens. A possible explanation for the findings includes extrusion of the graft through the injection site, which was noted to occur to some extent in all cases with reintubation of the larynx immediately after injection. In light of the small volume of fat injected, 0.1 to 0.2 ml, we believe that any significant extrusion could give poor results. The relatively small size of the cat airway limited our ability to inject larger volumes without airway obstruction. In addition, we used 19-gauge needles for the injections. This was the smallest needle size considered to allow atraumatic injection of adipocytes. % This needle was large relative to the size of the vocal cord injection site in the cat, making precise injection in all of the larynges technically difficult. The experience of the senior author with the use of injectable biomaterials in canines and humans suggests
OtolaryngologyHead and Neck Surgery November 1997
470 SACCOGNA et al.
that proper placement with lack of extrusion would be facilitated in larger larynges.
4.
CONCLUSION The best method to harvest and prepare fat used for lipoinjection remains undetermined. 23,25,27 In our study, cellular integrity was maintained to a comparable degree when the fat was harvested by either a liposuction or excisional technique. We decided to use the excised fat for injection because cats have relatively small abdominal fat pads available for liposuction and empirical observations suggested that direct excision would be a less traumatic method of fat harvest. The technique of lipoinjection of autologous fat into the paralyzed vocal fold has been shown by photographic and histologic studies to offer short-term viability up to 8 months after injection in a feline model. Although the numbers of animals studied are too small to address the reasons for failure of the fat grafts to survive, these may well reflect difficulties resulting from the needle gauge required and the small size of cat larynges. Further long-term (12 months or longer) investigation combining histologic and photographic studies in a canine model would be ideal in documenting the value of this technique, while avoiding some of the technical difficulties encountered in our model. This study lends support, however, to the theory that autologous fat satisfies two of Arnold's 3 three criteria for the ideal laryngeal injectable material: ease of injectability and the ability to be well tolerated. The third criterion, long-term viability, is not applicable, as shown by the lack of significant graft survival in our 12-month specimen. We cannot recommend this technique as an alternative to Teflon injection or thyroplasty for permanent vocal fold augmentation until further studies demonstrate whether survival beyond 8 months can occur. If further study substantiates several months of fat survival in a significant number of cats, autologous fat may be useful in the paralyzed vocal cord to temporarily improve function in cases where compensation has not occurred and spontaneous recovery is still a possibility.
5. 6. 7.
8. 9. 10. 11. 12. 13. 14.
15. 16. 17. 18. 19. 20. 21. 22.
23.
24. 25. 26.
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