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Survival characteristics of injected human cartilage slurry in a nude mouse model: a preliminary study.


We conducted a study to examine the viability, host response, and volume retention characteristics of drilled human septal cartilage slurry when injected into an athymic nude mouse model. We injected 0.2 ml of the slurry into the hind limbs of 6 mice. The mice were sequentially sacrificed over a period of 180 days. Histologic reviews of the hind limbs were performed to determine the viability of injected chondrocytes, host response, and volume retention. Specimens were obtained and histomorphologic analysis was performed at 1, 30, 90, and 180 days after injection. We identified viable cartilage throughout the study. Cartilage injection was well tolerated, and minimal inflammatory reaction occurred without significant adverse effects. The injected bolus of cartilage was found to have progressively dispersed throughout the muscle over time. Our findings warrant further investigation with a larger cohort of nude mice or possibly human subjects.


Unilateral vocal fold paralysis is a significant cause of morbidity. Nerve injury can occur in a variety of ways: as an iatrogenic recurrent laryngeal nerve injury during surgery, as a complication of endotracheal intubation, and as the result of blunt chest/neck trauma, a viral infection, or a tumor of the skull, neck and chest; some injuries are idiopathic. The resultant unilateral vocal fold paralysis can have a significant impact on a person's quality of life in terms of voice and swallowing, and it can cause aspiration.

The most common treatment of unilateral vocal fold paralysis is surgical medialization of the immobile vocal fold. Successful surgery allows for better apposition with the nonparalyzed vocal fold. One of the most popular medialization techniques involves the injection of a substance into the paralyzed fold. A number of synthetic and biologic injection materials are available, but no single ideal substance or technique has been identified, and the search for a safe, stable, and lasting injectable material continues. (1-6)

In 2008, Noordzij et al described a new technique for preparing human cartilage for injection. (7) This technique involves drilling the cartilage with a cutting bur (5 mm in diameter) and mixing it with saline, which produces an injectable slurry. The authors reported that cytologic examination of pieces of drilled nasal septal cartilage revealed good uniformity in the size of the pieces, with an average greatest dimension of 0.44 [+ or -] 0.33 mm. Moreover, 33% of the lacunae contained viable-appearing chondrocytes. The results of cytologic examination of drilled auricular cartilage were similar, except that only 10% of the lacunae were occupied by viable-appearing chondrocytes.

There are structural and anatomic differences between auricular and septal cartilage. The auricular type is composed of elastic cartilage that is flexible, while the septal type is composed of more rigid hyaline cartilage. For our experiment, we chose to use only septal cartilage to maximize the number of viable-appearing chondrocytes. Lee et al had used auricular rather than septal cartilage because it was easier to control the size of the pieces of cartilage with their method of harvesting, which involved the use of scalpels and scissors. (8,9)

Using the method described by Noordzij et al, (7) we conducted a study to examine the viability, host response, and volume retention characteristics of drilled human septal cartilage slurry injected into a nude mouse model. The long-term objective of this project is the development of a safe, stable, and lasting injectable material for vocal fold augmentation for the treatment of unilateral vocal fold paralysis in humans.

Materials and methods

Routine septoplasty often involves the removal of a portion of nasal septal cartilage to alleviate nasal obstruction caused by septal deviation. The removed cartilage is typically discarded. We obtained such cartilage from anonymous donors who had undergone septoplasty.

Our animal experiment was conducted in the Laboratory Animal Science Center at the Boston University Medical Center. Six athymic nude mice (NU/J Stock #002019; The Jackson Laboratory; Bar Harbor, Maine) served as the subjects for this experiment. Upon arrival at our site, the mice were housed in cages for 7 days to allow them to adapt to the environmental conditions prior to injection.

Within 24 hours of harvesting the cartilage from the human donors, the cartilage was washed with sterile saline and drilled down to flake-sized pieces with a 5-mm cutting bur. The flakes were collected in a bowl with a saline wash, and excess saline was aspirated away. The remaining cartilage slurry was aspirated into a 1-ml syringe through a 19-gauge needle.

Upon completion of the preparation of the slurry, anesthesia was administered to a particular mouse. (The same protocol was followed for all mice.) When a sufficient state of anesthesia was obtained, the mouse's hind limbs were sterilized, and 5- to 10-mm skin incisions were made to expose the quadriceps muscles. Then the 19-gauge needle was used to inject the slurry into the muscle on both the right and left sides.

After injection, the incisions were closed with absorbable suture. The injection sites were marked with a permanent tattoo to facilitate localization of the cartilage slurry. Two days before and 7 days after injection, each mouse was administered trimethoprim/sulfamethoxazole suspension to prevent infection at the surgical site.

Euthanization was performed in a C[O.sub.2] chamber and by cervical dislocation. Two mice were sacrificed 1 day after injection, and 1 mouse each after 30, 90, and 180 days. The sixth mouse had been scheduled for sacrifice on day 360, but it died before then.

Following sacrifice, the hind limbs were removed and placed in 10% neutral buffered formalin for 7 days. The average size of the hind limbs was 2 x 1 x 1 cm. Both the right and left quadriceps muscles were removed from each limb, inspected for graft material, and placed into a cassette embedded in paraffin and sectioned. Starting at the top of each specimen, we obtained 10 sections located 1,000 [micro]m apart and stained them with hematoxylin and eosin. (10)

With the aid of our pathologist, we evaluated the viability, host response, and volume retention characteristics of the injected slurry. The area of cartilage present was determined by morphometric analysis with image analysis software (Optimas v. 6.51; Media Cybernetics; Bethesda, Md.). Viability was indicated by the presence of intact nucleoli within the cartilage lacunae. Host response was indicated by the presence of inflammatory cells, granuloma formation, and the mice's tolerance to the procedures. Volume approximation was performed with the 10 sections by multiplying the area of each section by the distance of 1,000 pm between each side. Stained specimen slides were viewed through a Leitz microscope at x40, x100, x200, and x400 magnification.

The donor cartilage was obtained with the approval of the Boston University Institutional Review Board (IRB) for human subjects, and all procedures were approved and monitored by the university's Institutional Animal Care and Use Committee as well as the IRB for human subjects.


All animals tolerated the intervention without infection, rejection, or difficulty with limb movement. As mentioned, 5 survived until the day they were to be sacrificed, and 1 died prematurely at 4 months. We presume that this mouse died of natural causes unrelated to the experiment.

Histomorphologic findings. Specimens were analyzed at 1, 30, 90, and 180 days:

Day 1. The injected cartilage remained compact, easily identifiable, and localized within the muscle (figure 1, A and B). An acute inflammatory reaction was seen surrounding the cartilage, and degenerative changes were seen in the peripheral areas of the cartilage (figure 1, C). No granuloma, fibrosis, or foreign-body reaction was noted.

Both viable and nonviable chondrocytes were seen. The viable chondrocytes were identified by their intact nucleoli. The nonviable cells exhibited degenerative changes in the cartilaginous matrix and empty cartilage lacunae from which the cartilage cells were lost.

Day 30. No inflammatory cells or degenerative changes were seen within the cartilage or at the periphery, and the viable chondrocytes appeared to be dividing (figure 2, A). Again, no granuloma, fibrosis, or foreign-body reaction was seen surrounding the cartilage. In the nonviable chondrocytes, the outline of grafted cartilage was still preserved (figure 2, B).

Day 90. Viable dividing chondrocytes were seen intermixed with skeletal muscle. The outlines of the injected bolus of cartilage had become less identifiable, and the cartilage appeared to have dispersed throughout the muscle.

Day 180. Viable dividing chondrocytes were still seen intermixed with skeletal muscle (figure 3). The acute inflammatory reaction and degenerative changes had disappeared from the interstitial tissues around the cartilage. Granuloma, fibrosis, or a foreign-body reaction was still not observed. Although some chondrocytes appeared to have lost their viability (i.e., nucleoli were absent), the cartilage matrix was preserved. Again, the overall outlines of the injected cartilage bolus became less identifiable as they appeared to have diffusely dispersed throughout the muscle.

Morphometric analysis of volume. At day 1, the injected cartilage remained compact, and clear borders were seen between the muscle and the cartilage interface. The area and volume were easily measured. The approximation of the remaining volume closely matched that of the injected volume of 0.2 ml; the average volume was 0.188 ml.

At days 30, 90, and 180, it appeared that the cartilage graft had become progressively dispersed throughout the muscle. In general, it was more difficult to measure the exact area of the injected cartilage over time, since it had become less compact and intermixed with muscle and fat; clear borders for measurement were not found. As a result of the intermixing and the lack of clear borders, cartilage volume could not be accurately quantified.


Many different synthetic and biologic materials--paraffin, bone paste, polytetrafluoroethylene (Teflon) and, more commonly, collagen, fat, and calcium hydroxyapatite--have been used for long-term augmentation, but they all have their drawbacks, including foreign-body reactions, resorption within a period of 2 to 6 months, immunologic reactions, and a lack of long-term success. (11,12) For example, polytetrafluoroethylene injection has fallen out of favor because of the unpredictable nature of the foreign-body reaction it causes, which often leads to the formation of endolaryngeal granulomas. (5,6)

Karpenko et al described an ideal injectable material as one that is characterized by:

* little or no resorption;

* high tissue biocompatibility to maintain the fluency of vibration of the vocal fold mucosa;

* no tissue reactivity at the injection site;

* reversibility or the potential for later modification;

* ease of handling and administration in either the operating room or office setting; and

* no migration of the injected material. (13)

Calcium hydroxyapatite has been reported to last for 6 to 12 months. (1) Longer-term follow-up has not been reported with calcium hydroxyapatite, and the biomechanical effects of injecting bone cement into the pliable vocal fold is of concern. (1)

Lee et al described the use of autologous minced cartilage as an injectable implant for vocal fold medialization in dogs. (8,9) Their histomorphologic experiments showed that injected autologous cartilage grafts were well tolerated. At 12 months, the outlines of the cartilage were preserved even after the chondrocytes had died out and fibrotic changes had occurred in the surrounding cartilage. Lee et al noted that human cartilage has many excellent autograft properties for use in vocal fold injection, including a low extrusion rate and a low blood supply requirement.

Other biologic compounds include xenograft materials (i.e., from different species) such as Gelfoam, bovine collagen, and hyaluronic acid; homograft materials (i.e., from the same species) such as skin (Cymetra); and autograft materials such as fat, collagen, fascia, and cartilage. (2-4,8,9,11,13)

Autologous fat can be easily extracted, it causes no hypersensitivity or inflammatory reaction, and it does not interfere with vocal fold oscillation. The major disadvantage of autologous fat is that 20 to 90% of the graft volume is absorbed, and therefore volume retention beyond 3 to 6 months has been inconsistent. (4,11,12)

Courey evaluated the histologic characteristics of homologous collagen compounds injected into the quadriceps muscles of nude mice. (2) The collagen was easily identifiable at 30 days, but at 2 and 6 months, significant resorption had occurred. Identification of the injection material was difficult because it had dispersed throughout the muscle.

Autologous cartilage is associated with a high survival rate and a low absorption rate, and therefore it is one of the more desirable injectable materials. (8,9,14) Salinger reported that grafted cartilage existed permanently and that the matrix continued to exist even several years after the cells of the grafted cartilage had lost viability. (14)

For our study, we chose to use an athymic nude mouse model to preclude the possibility of an immunologic rejection of the human septal cartilage. Our histomorphologic results show that this graft was well tolerated. At 1 day postinjection, the cartilage slurry remained intact, and at 30 days the acute inflammation and degenerative changes had disappeared. No granuloma formation, fibrosis, or foreign-body reaction was encountered at any time. At 180 days, the cartilage cells were identifiable, and viable chondrocytes were seen throughout the study period. The slurry became dispersed throughout the quadriceps muscles, and it became increasingly difficult to calculate the volume over time.

In conclusion, this preliminary study examined the viability, host response, and volume retention characteristics of drilled human cartilage slurry injected into a nude mouse model over a 6-month period. All animals tolerated the surgical intervention without infection or rejection. Human cartilage appears to be well tolerated when injected into the quadriceps of nude mice. Viable chondrocytes were seen throughout the study. Our findings warrant further investigation with a larger cohort of nude mice and possibly human subjects.


(1.) Rosen CA, Gartner-Schmidt J, Casiano R, et al. Vocal fold augmentation with calcium hydroxylapatite: Twelve-month report. Laryngoscope 2009;119(5):1033-41.

(2.) Courey MS. Homologous collagen substances for vocal fold augmentation. Laryngoscope 2001;111(5):747-58.

(3.) McCulloch TM, Andrews BT, Hoffman HT, et al. Long-term follow-up of fat injection laryngoplasty for unilateral vocal cord paralysis. Laryngoscope 2002;112(7 Pt 1):1235-8.

(4.) Hill DP, Meyers AD, Harris J. Autologous fat injection for vocal cord medialization in the canine larynx. Laryngoscope 1991; 101(4 Pt 1):344-8.

(5.) Lewy RB. Teflon injection of the vocal cord: Complications, errors, and precautions. Ann Otol Rhinol Laryngol 1983;92(5 Pt 1):473-4.

(6.) Nakayama M, Ford CN, Bless DM. Teflon vocal fold augmentation: Failures and management in 28 cases. Otolaryngol Head Neck Surg 1993;109(3 Pt l):493-8.

(7.) Noordzij JP, Cates JM, Cohen SM, et al. Preparation techniques for the injection of human autologous cartilage: An ex vivo feasibility study. Laryngoscope 2008;118(1):185-8.

(8.) Lee BJ, Wang SG, Goh EK, et al. Intracordal injection of autologous auricular cartilage in the paralyzed canine vocal fold. Otolaryngol Head Neck Surg 2004;131(1):34-43.

(9.) Lee B J, Wang SG, Goh EK, et al. Histologic evaluation of intracordal autologous cartilage injection in the paralyzed canine vocal fold at two and three years. Otolaryngol Head Neck Surg 2006;134(4):627-30.

(10.) Rosen GD, Harry JD. Brain volume estimation from serial section measurements: A comparison ofmethodologies. J Neurosci Methods 1990;35(2):115-24.

(11.) Shindo ML, Zaretsky LS, Rice DH. Autologous fat injection for unilateral vocal fold paralysis. Ann Otol Rhinol Laryngol 1996;105(8):602-6.

(12.) Shaw GY, Szewczyk MA, Searle J, Woodroof J. Autologous fat injection into the vocal folds: Technical considerations and long-term follow-up. Laryngoscope 1997;107(2):177-86.

(13.) Karpenko AN, Dworkin JP, Meleca RJ, Stachler RJ. Cymetra injection for unilateral vocal fold paralysis. Ann Otol Rhinol Laryngol 2003;112(11):927-34.

(14.) Salinger S. Cartilage homograffs in rhinoplasty: A critical evaluation. Ann Otol Rhinol Laryngol 1952;61(2):533-41.

Bounmany Kyle Keojampa, MD; Jacob Pieter Noordzij, MD; Bohdana Burke, MD; Joseph Alroy, DVM; Vartan Mardirossian, MD; Alphi Elackttu, MD; Zhi Wang, MD

From the Department of Otolaryngology-Head and Neck Surgery (Dr. Keojampa, Dr. Noordzij, Dr. Mardirossian, Dr. Elackttu, and Dr. Wang) and the Department of Pathology (Dr. Burke), Boston University School of Medicine; and the Department of Pathology, Tufts Medical Center, Boston (Dr. Alroy). This experiment was conducted at the Laboratory Animal Science Center, Boston University Medical Center.

Corresponding author: Bounmany Kyle Keojampa, MD, Department of Otolaryngology-Head and Neck Surgery, Boston University School of Medicine, 820 Harrison Ave., FGH Bldg., 4th Floor, Boston, MA 02118. Email:
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Author:Keojampa, Bounmany Kyle; Noordzij, Jacob Pieter; Burke, Bohdana; Alroy, Joseph; Mardirossian, Vartan
Publication:Ear, Nose and Throat Journal
Article Type:Report
Date:Jul 1, 2014
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