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Development of A Tissue Engineered Vocal Fold Cover Replacement from Rabbit Adipose Derived Stem Cells

Travis Shiba, MD1; Jordan Hardy BS 2; Jennifer Long, MD, PhD1,2

1Department of Head and Neck Surgery, University of California Los Angeles, Los Angeles, CA 2Research Service, Greater Los Angeles Veterans Administration, Los Angeles California

Travis L. Shiba University of California Los Angeles Department of Head and Neck Surgery Email: tshiba@mednet.ucla.edu Phone: (310) 825-6301

Contact 1. Welham NV, Choi SH, Dailey SH, Ford CN, Jiang JJ, et al. Prospective multi-arm evaluation of surgical treatments for vocal fold scar and pathologic sulcus vocalis. Laryngoscope (2011); 121:1252–60. 2. Chhetri D, Berke G. Injection of cultured autologous fibroblasts for human vocal fold scars. (2011)a Laryngoscope 121:785-92 3. Thibeault SL, Klemuk S, Smith ME, Leugers C, Prestwich G, et al. In vivo comparison of biomimetic approaches for tissue regeneration of the scarred vocal fold (2009) Tissue Eng: Part A; 15:1481-7. 4. Johnson BQ, Fox R, Chen X, Thibeault S. Tissue regeneration of the vocal fold using bone marrow mesenchymal stem cells and synthetic extracellular matrix injection in rats (2010). Laryngoscope; 120:537-55. 5. Long J, Zuk P, Berke G, Chhetri D. Epithelial differentiation of adipose-derived stem cells for laryngeal tissue engineering (2010b). Laryngoscope; 120:125-31. 6. Ge P, French L, Ohno T, Zealear D, Rousseau B. Model of evoked rabbit phonation (2009). Ann Otol Rhinol Laryngol;118:51-5 7. Bless DM, Welham NV. Characterization of vocal fold scar formation, prophylaxis, and treatment using animal models (2010). Curr Opin Otolaryngol Head Neck Surg;18: 481-486 8. Maytag AL, Robitaille MJ, Rieves AL, Madsen J, Smith BL, et al. Use of the rabbit larynx in an excised larynx setup(2013). J Voice; 27:24-28 9. Kim YM, Oh SH, Choi JS, Lee S, Ra JC, et al. Adipose Derived Stem cell containing hyaluronic acid/alginate hydrogel improves vocal fold wound healing (2013). Laryngoscope; Mar;124(3):E64-72 10. Li H, Xu Y, Fu Q, Li C. Effects of multiple agents on epithelial differentiation of rabbit adipose-derived stem cells in 3D culture (2012). Tissue Eng Part A;18:1760-70Cedervall J, Ahrlund-Richter L, Svensson B, Fosgren K, Maurer FH, et al.

Injection of embryonic stem cells into scarred rabbit vocal folds enhances healing and improves viscoelasticity: short-term results (2007). Laryngoscope; 117:2075-2081 11. Hu R, Ling W, Xu W, Han D. Fibroblast-like cells differentiated from adipose-dreived mesenchymal stem cells for vocal fold wound healing (2014). PLoS One; 9(3):e92676. 12. Hiwatashi N, Hirano S, Mizuta M, Tateya I, Kanemaru SI, et al. Adipose-derived stem cells versus bone marrow derived stem cells for vocal fold regeneration (2014). Laryngoscope. doi: 10.1002/lary.24816. [Epub ahead of print] 13. Svensson B, Nagubothu SR, Cedervall J, Chan RW, Le Blanc K, et al. Injection of Human Mesenchymal Stem Cells Improves Healing of Vocal folds after Scar Excision – A Xenograft Analysis (2011). Laryngoscope. 121: 2185-90 14. Liang Q, Liu S, Han P, Li X, Li X, et al. Micronized acellular dermal matrix as an efficient expansion substrate and delivery vehicle of adipose-derived stem cells for vocal fold regeneration(2012). Laryngoscope. 122:1815-25

References

Tissue engineering of a vocal fold replacement is a promising potential treatment for severe vocal fold scarring. Development and testing of such a tissue construct is an ongoing project. We have previously demonstrated that human adipose derived stem cells (ASC) can produce a bilayered construct with suitable properties for implantation and phonation. This phase of the project developed a similar construct for pre-clinical animal studies, using rabbit cells. Rabbit adipose derived stem cells were isolated, cultured and embedded in fibrin gels under air-liquid interface conditions with epidermal growth factor. After culture periods of one to four weeks, constructs were harvested, sectioned and examined. Results: Rabbit cells attached and survived within rabbit fibrin gels which were capable of handling and suturing. rASC and human cryoprecipitate were not capable of handling. Osteogenic and adipogenic differentiation was induced in cultured ASC, confirming their multipotency. Differentiation to these unwanted lineages was not seen in vocal fold tissue constructs. Conclusions: Rabbit ASC are suitable for use in a tissue-engineered vocal fold replacement. This model will be used in future implantation trials in rabbits.

Abstract

rASC differentiate into mesenchymal phenotypes. Light microscopy demonstrated that cultures induced to differentiate into adipogenic (left panel) and osteogenic cell (center panel) types showed morphological changes after 2 weeks in culture (control media only, right):

No adipogenic or osteogenic changes occurred in the untreated ASC nor in the tissue constructs Rabbit ASC constructs require species-specific fibrin scaffold. Rabbit ASC embedded within fibrin gels attached and survived. Constructs formed with rabbit fibrinogen could withstand handling, manipulation, and placement of 5-0 sutures, irrespective of treatment group or culture period. Microscopy showed a fibrin lattice (red stain) with similar gross morphology to the vocal fold lamina propria and epithelium. Cell nuclei were identified throughout the construct (blue stain) and spindle shape morphology at the air interface was noted:

Constructs formed with human cryoprecipitate degenerated rapidly, could not withstand manipulation past day four, and were not pursued further.

(Cont.)

rASC collection: rASC were isolated from inguinal fat pads, treated with collagenase and centrifuged. The stromovascular fraction was collected and cultured. rASC Differentiation: osteogenic differentiation was induced with 1,25 dihydroxy vitamin D, ascorbate 3 phosphate and B glycerophosphate. Adipogenic differentiation was induced by dexamethasone, insulin and indomethacin. Fibrin-ASC constructs: human cryoprecipitate and rabbit fibrinogen were used as fibrin sources and rASC were seeded onto both in a trans well 3D air liquid interface with and without EGF (Fig 2 & 3)

Methods and Materials

The construct developed here is suitable for implantation as a vocal fold mucosa graft in rabbits. The in vivo microenvironment may direct differentiation of the superficial layer of cells to a more mature epithelial phenotype; this will be tested further in implant studies. ASC or bone marrow derived stem cell injections into animal vocal folds in vivo decrease scarring and dense collagen deposition; improve viscoelasticity and mobility; and increase elastin production, vocal fold smoothness and collagen organization. Furthermore, adding a micronized acellular dermal matrix or hydrogel scaffold to the injection decreased scarring9, 11-14. The wound healing properties of ASC may be adequate to prevent scar formation after implantation, while the fibrin scaffold may further support ASC survival and differentiation. These aspects will be tested in a rabbit implantation trial.

Discussion

Rabbit adipose-derived stem cells were used to create a three-dimensional tissue-engineered construct suitable for vocal fold cover replacement. The three dimensional construct was bilayered and resembled the vocal fold lamina propria and mucosa in microstructure although not in cell phenotype. The construct was able to withstand handling and suturing similar to a native rabbit vocal fold cover layer. This construct will be used for future in vivo implantation studies in rabbits.

Conclusions

Results

Figure 1. Patient with vocal fold scar before and after scar excision without good options for preventing re-scarring

Introduction The specialized extracellular matrix of the vocal fold lamina propria and its attached epithelium are, thus far, irreplaceable after severe scarring 1-

4. Tissue engineering of the vibratory vocal fold has been proposed as a treatment for severe vocal fold scarring. This laboratory previously developed a human ASC-populated three-dimensional tissue-engineered construct intended for vocal fold cover layer replacement5. Here we attempt to take the next step toward human application by creating an animal model. We set out to develop a tissue-engineered construct suitable for implantation in rabbit vocal folds. Rabbits are a favorable animal model for study of the larynx due to their histologic and size similarities to human larynges, easy animal husbandry, ability to phonate in an excised setting and inherently silent behavior6-9. Rabbit ASC have been shown to differentiate into an epithelial phenotype when exposed to an air liquid interface and multiple growth factors10. Here we report the production of a tissue-engineered mucosa replacement from rabbit ASC that will allow for preclinical construct implantation studies. Key differences in the in vitro development

between human and rabbit cells are highlighted.

Figure 2. Concept behind fibrin-ASC construct

+

Fibrinogen

Adipose-derived stem cells (ASC)

Homogeneous fibrin-ASC construct Ideal mature construct with

stratified cell differentiation for cover layer replacement

Figure 3. Air Liquid Interface

Semipermeable

membrane Fibrin gel with cells

Culture fluid with growth factors

bathes gel from bottom surface

(cont.) Epithelial differentiation signals differ for rabbit and human ASC. Previous work with human ASC embedded in cryoprecipitate demonstrated epithelial differentiation of cells cultured in fibrin at an air-liquid interface and treated with epidermal growth factor5. Those conditions were replicated here but did not produce consistent expression of the epithelial marker protein CK 19, nor did EGF create a significant difference. Immunohistochemistry demonstrated that vimentin, a general mesenchymal cell marker, was preserved in all cells in both conditions. Cell nuclei are blue and vimentin is red.

Acknowledgements This material is based upon work supported by the Department of Veteran Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development as well as the American Academy of Otolaryngology AAO-HNSF Resident Research Award. A manuscript based on the content of this work was submitted for review 8/31/14

Figure 6. Tissue Engineered Construct

Adipogenic Media Osteogenic Media Control Media

Figure 4

Figure 7. Rabbit vocal cord (A); construct (B). rASC have retained vimentin

Figure 5

Rabbit Vocal fold rASC+ fibrin Construct