Ann 0101 Rhinal LaryngoII13:2004

INJECTION LARYNGOPLASTY WITH CALCIUM HYDROXYLAPATITEGEL IMPLANT IN AN IN VIVO CANINE MODEL

DINESH K. CHHETRI, MD

BABAK JAHAN-PARWAR, MD

SUNITA M. BHUTA, MD

Los ANGELES, CALIFORNIA

STEPHEN D. HART, MD

GERALD S. BERKE, MD

The ideal injectable agent for vocal fold medialization is biocompatible, durable, sized to prevent phagocytosis and migration,and formulated for easy injection and does not adversely affect the viscoelastic properties of the vocal fold. We tested a cohesiveimplant of calcium hydroxylapatite (CaHA) particles in a gel carrier in an in vivo canine model of phonation. Six dogs underwentunilateral recurrent laryngeal nerve section and injection laryngoplasty of the paralyzed vocal fold with a CaHA implant. The sixfollow-up examinations were performed at 1,2,3,6,9, and 12 months, and the larynx and bilateral neck lymphatic system wereharvested for histologic analysis. The CaHA implant adequately medialized the vocal fold to regain glottal closure. The mucosalwaves remained unaltered from baseline. The implant remained soft in the larynx and did not migrate to the neck lymphatic system.A localized foreign body giant cell reaction was present on histologic evaluation, but not acute or other chronic inflammation. A sizeanalysis revealed no resorption of the CaHA particles. A decrease in medialization was noted at all follow-up intervals related toresorption of the aqueous-based gel carrier. The CaHA implant appears to be relatively safe and suitable for injection laryngoplasty.

KEY WORDS - calcium hydroxylapatite, injection laryngoplasty, vocal fold paralysis.

INTRODUCTION

The vital laryngeal functions of airway protection,respiration, and phonation are often impaired by in­adequate glottal closure from vocal fold paralysis. Avariety of medialization laryngoplasty techniques in­volving either laryngeal framework surgery or injec­tion laryngoplasty have been proposed to improveclosure. The advantages of injection laryngoplastyover laryngeal framework surgery include avoidanceof an open surgical procedure, potential lower costand morbidity, and the possibility of performing theprocedure in an office setting, thus avoiding the needfor deep sedation or general anesthesia.

Since the tum of the previous century, a variety ofmaterials for injection laryngoplasty have been in­troduced. Brunings' introduced paraffin injection intothe vocal fold in 1911. Paraffin injection was aban­doned because of the frequent extrusion of the im­plant and development of paraffinomas. Materialsintroduced since then include autogenous and homo­geneous cartilage particles, bovine bone paste.? tan­talum oxide.t silicone.t Teflon," collagen," and autol­ogous fat and fascia.e? None of the materials havebeen ideal; rapid resorption and adverse immune re-

action have been the major difficulties encountered.Teflon was close to ideal and remained the work­horse medialization agent for almost 3 decades afterArnold'? introduced its use in 1962. Teflon has nowbeen abandoned because of widespread reports offoreign body reaction with formation of granuloma.Collagen is frequently used for vocal fold medial­ization and augmentation, but it is reabsorbed within3 to 6 months. I I Although the concern for immuno­reactivity is negligible with injection of autologousfat or fascia, their major drawback is also limiteddurability.

An ideal vocal fold implant is injectable, nonvola­tile, nondegradable, sized to prevent phagocytosisand migration, and formulated to reduce toxicity fromdirect cytotoxicity or immune mechanisms and doesnot adversely affect the viscoelastic properties of thevocal fold. A synthetic calcium hydroxylapatite(CaHA) implant has recently been introduced andapproved for use in the vocal fold. Calcium hydroxyl­apatite is a biocompatible material that is a principalcomponent of bone. In the head and neck, it has beenused to augment maxillofacial defects, obliteratefrontal sinus and mastoid cavities, fill periodontal

From the Division of Head and Neck Surgery (Chhetri, Jahan-Parwar, Berke) and the Department of Pathology and Laboratory Medicine (Hart,Bhuta), University of California, Los Angeles, School of Medicine, Los Angeles, California. This research was supported by Bioform Inc. Thisstudy was performed in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, the NIH Guide for the Care and UseofLaboratory Animals, and the Animal Welfare Act (7 U.S.C. et seq.): the animal use protocol was approved by the Institutional Animal Careand Use Committee (IACUC) of the University of California, Los Angeles.

Presented at the meeting of the American Laryngological Association, Nashville, Tennessee, May 2-3, 2003.

CORRESPONDENCE - Gerald S. Berke, MD, 62-132 CHS, Head and Neck Surgery, Mail Code 162418, UCLA Medical Center, Los Angeles,CA 90095.

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260 Chhetri et al, Injection Laryngoplasty

pockets, promote bone formation in orthopedic anddental implant surfaces, and serve as an implant forcosmetic, otologic, and reconstructive purposes. Thebiocompatibility of hydroxylapatite has been dem­onstrated in both animal and human studies.

The objective of this study was to gain initial ex­perience with CaHA implants in animals before theiruse in humans. The specific aims were to 1) evalu­ate laryngeal tissue reaction to CaHA particles, 2)evaluate vocal fold mucosal pliability after CaHAinjection, 3) assess the durability of vocal fold me­dialization achieved by CaHA injection, and 4) evalu­ate the neck lymphatic system to assess potential lym­phatic migration of CaHA particles from the larynx.An in vivo canine model was used for the study.

MATERIALS AND METHODS

Six dogs underwent unilateral recurrent laryngealnerve (RLN) section followed by medialization ofthe paralyzed fold with an injectable CaHA gel im­plant. Each of the animals was observed for a differ­ent length oftime: 1,2,3,6,9, or 12 months. Video­stroboscopic assessment of phonation was performedat baseline, after unilateral paralysis, immediatelyafter medialization of the paralyzed vocal fold withthe CaHA gel implant, and at the end of the observa­tion period. The animals were humanely killed at theend of the observation periods for histologic analy­sis of the larynx and the neck lymphatic system andparticle size analysis of the CaHA explants.

CaHA Particles.The injectable hydroxylapatite gelimplant (Coaptite, BioForm Inc, Franksville, Wis­consin) consists of spherical particles of synthetic,high-density, nonporous CaHA. The samples pro­vided for this study had a particle size ranging from38 to 63 11m and a mean particle size of 44.9 11m.The particles come in prefilled, ready-to-use, I-mLsyringes in an aqueous-based gel suspension that al­lows delivery through a needle. The gel content inthe product used was 45% by mass and 70% by vol­ume. The gel carrier consists of water and glycerinwith approximately 3% sodium carboxymethylcellu­lose. The gel dissipates in vivo, and the particles re­main to provide long-term bulking.

In Vivo Canine Model of Phonation. An in vivocanine model of phonation, established in the Univer­sity of California, Los Angeles, Laryngeal Physiol­ogy Laboratory for almost 2 decades, was used tostudy laryngeal physiology in situ.'? Briefly, upperand lower tracheotomies are made in the animal aftergeneral anesthesia is induced. The lower tracheotomyis used to assist-ventilate the animal, and the uppertracheotomy is used to provide rostral airflow via acuffed endotracheal tube to drive phonation. Neck

exploration is performed to locate both recurrent andsuperior laryngeal nerves close to their entrance intothe larynx. Custom-designed monopolar electrodeswith silicone insulation are applied to the isolatednerves. The electrodes are attached to a constant-cur­rent nerve stimulator (model 2SLH, WR MedicalElectronics Co, St Paul, Minnesota). The nerves aretypically stimulated at 80 Hz with 0 to 3.0 rnA for aI.5-ms pulse duration to achieve complete adductionof the vocal folds. Once the vocal folds are adducted,airflow drives phonation and sound is generated.

After baseline videostroboscopy, the left RLN wascut to induce unilateral paralysis of the vocal fold. A3- to 4-cm segment of the nerve was also removed,and both ends of the cut nerve were ligated with silksutures to prevent reinnervation. The CaHA gel wasthen injected into the paralyzed left vocal fold. Theanimals were then awakened from anesthesia, decan­nulated, and returned to recovery kennels. Videostro­boscopy was again performed at the end of the des­ignated observation period for each dog.

Videostroboscopic Analysis. Videostroboscopywas performed with a rigid 0° endoscope attached toa charge-coupled device camera (Telecam 20 210120, Karl Storz, Culver City, California) with illumi­nation from a stroboscopic light source (model RLS9100, Kay Elemetrics, Lincoln Park, New Jersey).Recordings were performed on a 3/4-inch videocas­sette recorder (model V09850, Sony, Park Ridge, NewJersey). The use of videostroboscopy allowed slow­motion examination of the vibrating vocal folds andbetter delineation of the mucosal waves on the vocalfold surface. Mucosal waves were rated on a scaleof 1 to 5: 1 = absent, 2 = limited to the most medialedge, 3 =present laterally up to one quarter of thewidth of the vocal folds, 4 = between one quarterand one half the width of the vocal folds, and 5 =present more than half the width of the vocal folds(normal).

Vocal Fold Injection. The larynx was visualizedunder direct laryngoscopy. After vocal fold paraly­sis was induced by resection of the RLN, CaHA gelinjection was delivered percutaneously, lateral to thethyroarytenoid muscle, at the level of the vocal pro­cess with a 23-gauge needle. The goal of injectionwas to bring the paralyzed vocal fold toward a medi­alized position and to slightly overinject to compen­sate for expected resorption of the aqueous portionof the implant.

Histologic Analysis. The animals were broughtback into the operating room at the end of the desig­nated observation periods. After phonation parame­ters were recorded, the animals were humanely killedaccording to established protocols. Laryngectomy

Chhetri et al, Injection Laryngoplasty

Fig 1. Typical laryngoscopic views of study animals A) atbaseline, B) immediately after injection medialization laryn­goplasty with calcium hydroxylapatite gel implant, and C)at follow-up period after resorption of aqueous-based gelcarrier.

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and bilateral neck dissections were performed. Thelarynges were fixed in formaldehyde, decalcifiedwith ethylenediaminetetraacetic acid, and coronallycut into 0.5-cm slices from the anterior commissureto the vocal process of the arytenoid cartilage andembedded in paraffin blocks. Microscopic sections3 urn thick were then cut from the middle and poste­rior vocal fold areas and stained for histologic analy­sis with hematoxylin and eosin. The neck lymphaticsystem underwent routine histologic analysis.

Particle Size Measurement. Dissection was per­formed at the pyriform sinus of the injected side inthe harvested larynx until the injected CaHA boluswas encountered in its lateral aspect. A small pieceof the CaHA bolus was removed with fine forcepsand sent to the manufacturer (BioForm Inc) for par­ticle size analysis by validated techniques. Briefly,the tissue sample is first placed in a high-tempera­ture furnace to bum the organic components. TheCaHA particles are then washed to remove organicdebris, and a microscopic computerized image analy­sis is used to measure the size of the remaining CaHAparticles. More than 100 randomly chosen particleswere measured from each sample.

RESULTS

Laryngoscopy and Videostroboscopy. Unilateralvocal fold paralysis resulted in inadequate closurein all of the animals. Closure was reestablished afterinjection laryngoplasty with CaHA gel. However, aposterior glottal chink that is normally present in ca­nine larynges was often accentuated by the medialbulging of the membranous vocal fold after the in­jection, because the arytenoid cartilage could not berotated as well as in a human larynx. The vocal foldswere overinjected (Fig IB) with the expectation thatthe convexity would resolve with resorption of theaqueous-based carrier. This convexity had resolvedin all of the animals at the time of their follow-upexaminations (Fig 1C). Quantification of the decreasein vocal fold bulkiness after resorption of the aque­ous portion of the implant (decrease in medialization)was not performed at the various follow-up periods,but appeared to be of a similar degree in all animals(Fig 1).

Some animals had reduced mucosal waves evenat baseline (Table 1). These animals likely had re­duced mucosal waves because of larger posterior glot­tal chinks and limitations in maintaining sustained

262 Chhetri et al, Injection Laryngoplasty

TABLE 1. MUCOSAL WAVE RATINGS AT BASELINE TABLE 2. PARTICLE SIZE ANALYSIS AT BASELINEAND AT VARIOUS FOLLOW-UP INTERVALS AFTER AND AT FOLLOW-UP PERIODS

UNILATERAL INJECTION OF CAHA PARTICLES No. ofFollow-Up Particles

Animal Interval Mucosal Wave Rating* CaHA Specimen Measured Diameter* (um)No. (rna) Baseline Follow-Up

CaHA implant (baseline) 302 44.9±4.91 I 3 3 l-rno explant 358 43.4 ± 4.32 2 3 3 3-rno explant 201 43.3 ±4.23 3 3 3 6-rno explant 135 46.8 ± 5.24 6 5 5 9-rno explant 123 48.2 ± 8.45 9 3 5 12-rno explant 187 47.0± 7.86 12 5 3 *Mean + SD.

CaHA - calcium hydroxylapatite.*I =absent, 2 =limited to most medial edge, 3 =present laterally upto one quarter of width of vocal fold, 4 =between one quarter andone half width of vocal fold, 5 =present more than half width ofvocal fold (normal).

phonation during the experiment. Mucosal wavescould not be generated after injection, because theconvex injected vocal fold prevented linear approxi­mation of the glottis. At all of the follow-up periods(all animals), the injected vocal fold edge was straight,glottal closure could be obtained, and mucosal pli­ability was generated (Table 1). In4 animals, the mu­cosal waves were unchanged from baseline. In 1ani­mal (No.5), the mucosal wave improved from re­duced to normal, and in another (No.6), it worsenedfrom normal to reduced. Careful examination of thevocal folds revealed no abnormal areas such as scar­ring or tethering of the epithelial surface to accountfor reduced mucosal waves in any of the animals.

Particle Size Analysis. A small amount of the in­jected CaHA gel implant was removed and used forparticle size analysis. The injection bolus was softand appeared well localized to the injected site, andit was relatively easy to dissect around the materialfor its removal. The particle size analysis was per­formed on 1-,3-,6-,9-, and 12-month explants. TheCaHA particle size distribution in the explants wasnot significantly different from the size distributionbefore injection (Table 2). The mean diameters ofall of the explanted particles were within 1 SD of themean of the original particles.

Histologic Analysis. The spaces occupied by hy­droxylapatite particles appeared as spherical emptyspaces after the decalcification process (Fig 2). A cel­lular infiltrate was present within the injected CaHAparticles. Adjacent tissue, including muscle and car­tilage, was unaffected and appeared normal (Fig 2A).Higher-power microscopic examination of the cel­lular infiltrate revealed a foreign body reaction char­acterized by mostly multinucleated giant cells, somelymphocytes, and moderate amounts of histiocytes(Fig 2B). The foreign body reaction was limited tothe CaHA particles only. There was no evidence of

acute inflammation such as polymorphonuclear leu­kocytes, plasma cells, or necrosis. Complete histolog­ic evaluation of the neck dissection specimens re­vealed no evidence of CaHA particle migration fromthe larynx to the neck lymphatic system.

DISCUSSION

The ideal injectable vocal fold medialization agentis biocompatible, durable, sized to prevent phagocy­tosis and migration, and formulated for easy injec­tion and does not adversely affect the viscoelasticproperties of the vocal fold. The CaHA gel implantappears to fulfill many of these criteria. All of theanimals tolerated the injection well. Medializationwas achieved in all of the animals. Particle analysisshowed no resorption of the implants up to a 12­month observation period. The implant particles didnot migrate from the larynx to the neck lymphaticsystem.

Reductions in medialization were noted at the vari­ous follow-up periods and were similar in degree inall of the animals. This finding is consistent with re­sorption of the aqueous-based gel carrier that is 70%in volume. Therefore, slight overcorrection of theparalyzed vocal fold should be attempted at the ini­tial injection. Repeated injections may be necessary.However, unlike the experience with collagen, onlya limited number of repeat injections should be nec­essary, because the body does not resorb CaHA par­ticles. Animal experiments are ill-suited to assesswhether the residual medialization is adequate to ef­fect an improved and long-lasting vocal quality.Therefore, in clinical practice each surgeon needs todevelop his or her own experience and criteria forinjection laryngoplasty with the CaHA implant. Inour limited clinical practice with several patients whoreceived CaHA implants, we have noted that only asingle injection resulted in long-term subjective im­provement of dysphonia.

The tissue reaction to CaHA particles is limited toa foreign body giant cell reaction. This was similarin all of the animals. There was no evidence of an

Chhetri et al, Injection Laryngoplasty 263

Fig 2. Laryngeal histologic specimen afterinjection medialization laryngoplasty withcalcium hydroxylapatite (CaHA) gel im­plant. A) CaHA particles appear as clearspheres (asterisks) due to decalcification.Superiorly to right, normal thyroarytenoid(TA) muscle lies adjacent to CaHA bolus(original xlOO). B) At higher magnification(original x200), cellular reaction to implantis foreign body reaction consisting mostlyof multinucleated giant cells (asterisks),some lymphocytes, and moderate amountof histiocytes.

acute inflammatory process or granuloma formation.Thus, the tissue reaction appears to remain stable overtime. Although the longer-term effects of CaHA inthe larynx are unknown, we have no reason to be­lieve that an adverse tissue reaction would developover a longer period of time. The mucosal wave re­mained unaltered by the presence of the particles inthe vocal fold, and the particles remained within theinjected site as a bolus that was soft in consistency.

CONCLUSIONSWe tested CaHA particles as an injection laryngo­

plasty agent for vocal fold paralysis in a canine mod­el. The implant appears to be biocompatible, durable,relatively safe, and suitable for injection laryngo­plasty. The vocal fold should be overcorrected at theinitial injection to account for the resorption of theaqueous-based gel carrier. Clinical experience isneeded with this implant.

REFERENCES

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2. Goff WE Laryngeal adductor paralysis treated by vocal laryngol 1961;73:290-4.cord injection of bone paste; a preliminary investigation. Trans 4. Rubin HJ. Pitfalls in treatment of dysphonias by intracor-Pac Coast Otoophthalmol Soc Annu Meet 1960;41:77-88. dal injection of synthetics. Laryngoscope 1965;75:1381-97.

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5. Arnold GE. Vocal rehabilitation of paralytic dysphonia.VIII. Phoniatric methods of vocal compensation. Arch Otolaryn­gol 1962;76:76-83.

6. Ford CN, Bless OM, Loftus JM. Role of injectable col­lagen in the treatment of glottic insufficiency: a study of 119patients. Ann Otol Rhinol LaryngoI1992;101:237-47.

7. Brandenburg JH, Kirkham W, Koschkee O. Vocal cordaugmentation with autogenous fat. Laryngoscope 1992;102:495­500.

8. Shaw GY, Szewczyk MA, Searle J, Woodroof J. Autolo­gous fat injection into the vocal folds: technical considerationsand long-term follow-up. Laryngoscope 1997;107: 177-86.

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9. Rihkanen H. Vocal fold augmentation by injection of au­tologous fascia. Laryngoscope 1998;108:51-4.

10. Arnold GE. Vocal rehabilitation of paralytic dysphonia.IX: Technique of intracordal injection. Arch Otolaryngol 1962;76:358-68.

II. Ford CN. Histologic studies on the fate of soluble colla­gen injected into canine vocal folds. Laryngoscope 1986;96:1248-57.

12. Berke GS, Moore OM, Hantke DR, Hanson OG, GerrattBR, Burstein F. Laryngeal modeling: theoretical, in vitro, invivo. Laryngoscope 1987;97:871-81.

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