No conflict of interests. 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 (Jennifer Long and Jordan Hardy), VA Career Development Award IK2BX001944 (Jennifer Long), and the American Academy of Otolaryngology AAO-HNSF Resident Research Award (Travis Shiba).
Vocal fold scarring is a clinical problem without reliable treatment. Tissue engineering of a vocal fold replacement is an exciting potential treatment for vocal fold scars that involve multiple layers of the vocal fold. Human adipose-derived stem cells (ASC) were previously used to produce a promising vocal fold cover layer replacement. However, relevant
Translating
For example, tissue engineering of a vocal fold replacement is an exciting potential treatment for vocal fold scars that impair vibration and cause voice disturbance. Few reliable options currently exist to improve the dysphonia
In this work, 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 adequate laryngeal size for implantation surgery, histologic similarities to human larynges, and economical animal husbandry relative to larger mammals
Rabbit adipose-derived stem cells (rASC) were isolated from rabbit adipose tissue
Rabbit ASC in culture were harvested using trypsin-EDTA and re-seeded in 12-well culture plates. Once confluent, control medium was switched to induce differentiation. Osteogenic differentiation was induced with DMEM, 10% FBS, 0.01 uM 1,25-dihydroxyvitamin D3, 50 uM ascorbate-2-phosphate, 10 mM B-glycerophosphate, and 1% penicillin/streptomycin. Adipogenic differentiation was induced with DMEM, 10% FBS, 0.5 mM isobutyl-metylxanthine (IBMX), 1 µM dexamethasone, 10 µM insulin, 200 µM indomethacin, and 1% penicillin/streptomycin
Human cryoprecipitate, rabbit fibrinogen, and bovine fibrinogen were investigated as fibrin sources. Rabbit fibrinogen (Innovative Research) was obtained as a frozen liquid at 5mg/ml in 0.02M Sodium Citrate-HCl at pH 7.4. Bovine fibrinogen (Sigma-Aldrich) was dissolved in 20 mM HEPES-buffered saline at 5 mg/ml. Human cryoprecipitate, pooled from 10 donors, was obtained from the UCLA blood bank in accordance with institutional research policy. All were kept frozen at -80oC until use. Bovine thrombin (Sigma-Aldrich) was prepared at 2U/mL in 20mM HEPES-buffered saline with 7.5mM CaCl2 just prior to its use. The fibrinogen source was mixed in a 4:1:1 ratio with cell suspension and with thrombin solution, in that order, to form fibrin-rASC gels. Initial cell density was 1x106cells/mL to approximate the cell density in true vocal fold lamina propria. The fibrin solution was mixed in a Transwell (Corning) insert for 12-well culture plates, with a porous polycarbonate membrane (0.4µm pore size) at its base. Gelation was achieved within an hour of incubation at 37oC. After gelation was complete, an additional 500,000 rASC were pipetted directly on top of the gel surface to reproduce the cell density of a confluent epithelium
Tissue constructs were harvested with care to maintain their orientation. Tissue-Tek O.C.T. compound (Sakura Finetek) was used to embed the samples, and the blocks were flash-frozen in liquid nitrogen vapor. Sections were cut at 5 µm on a cryostat, and stored at -20oC until use. For staining, sections were thawed at room temperature and fixed in 4% paraformaldehyde. Hematoxylin and eosin staining was performed on one slide from each construct.
Rabbit larynges were collected within four hours of sacrifice for unrelated experiments, and were frozen at -80oc until use. After thawing, true vocal folds were dissected free from the surrounding cartilage for use as positive controls. Vocal folds were embedded, frozen, cryosectioned, fixed, and stained using the same methods as tissue constructs.
Lipid deposits, indicating adipose differentiation, were identified with Oil Red O staining. Stock solution was prepared using 300 mg Oil Red O (Sigma Aldrich) in 100 mL 99% isopropanol. Sections were rinsed in distilled water followed by 60% ethanol for five minutes each, then stained for five minutes with a working solution comprised of 15 mL Oil Red O stock solution mixed with 10 mL distilled water, incubated at room temperature for 10 minutes. Sections were washed with water and then counterstained with hematoxylin (Richard-Allan Scientific).
Von Kossa solution (5% AgNO3 in distilled water) was used to detect osteogenic differentiation by staining calcium phosphate or calcium carbonate. Sections were rinsed in distilled water for 5 minutes before applying Von Kossa solution and set beneath UV light for ten minutes. Sections were then rinsed in distilled water three times for 5 minutes each and counterstained with hematoxylin for one minute.
All samples were viewed and imaged the same day as staining using light microscopy with an EVOS XL inverted microscope (Life-Technologies).
For fluorescent immunohistochemistry, constructs were fixed, embedded in paraffin, and sectioned. After de-paraffinization, Trypsin EDTA was added to each section and incubated for 20 minutes at 37oC. Triton X-100 at 0.5% in PBS was applied 3 times for 5 minutes each application. Nonspecific binding was blocked with 10% goat serum (Pierce Biotechnologies) for 60 min and then washed with PBS for 5 minutes. Mouse monoclonal primary antibodies were applied overnight at 4oC in a humidified chamber. Primary antibodies included vimentin (clone AMF17B, Developmental Studies Hybridoma Bank, University of Iowa) at a dilution of 1:100 and prediluted pan-cytokeratin (Abcam ab961). BSA 1% was applied to negative control slides. After washing with PBS 3 times for 5 minutes each, goat anti-mouse secondary antibody tagged with rhodamine was applied for 30 minutes at room temperature at a dilution of 1:400 in PBS. Sections were washed again with 0.5% Triton to remove excess secondary antibody and cover-slipped with VectaShield mounting agent with diamidino-2-phenylindole (DAPI) nuclear stain (Vector Labs, Burlingame, CA).
Fluorescence was viewed and imaged the same day as staining, with an Olympus microscope. Rabbit vocal fold sections were used as positive controls to confirm expected antibody reactivity.
Light microscopy demonstrated that cultures induced to differentiate into adipogenic and osteogenic cell types showed morphological changes after 2 weeks in culture (
Rabbit ASC embedded within fibrin gels attached and survived. Constructs formed with rabbit or bovine fibrinogen could withstand handling, manipulation, and placement of 5-0 sutures, irrespective of treatment group or culture period. Constructs formed with human cryoprecipitate degenerated rapidly, could not withstand manipulation past day four, and were not pursued further (
For the constructs made with rabbit fibrinogen, hematoxylin and eosin microscopy showed a fibrin lattice with similar gross morphology to the vocal fold lamina propria and epithelium. Cell nuclei were identified throughout the construct (
Spontaneous differentiation to mesenchymal phenotypes did not occur. Histological staining using Oil Red O and Von Kossa on tissue-engineered constructs in cultured for 14 days, with and without EGF treatment, showed negative staining for lipid and mineral (
Epithelial differentiation was not induced by EGF and an air interface in this rabbit ASC culture, as indicated by immunohistochemistry. Immunohistochemistry at 14 days was negative for cytokeratin. Vimentin, a general mesenchymal cell marker, was preserved in all cells with and without EGF exposure (
Tissue engineering and regenerative medicine hold promise for diseases that are currently difficult to treat because of lack of suitable therapies or self-repair mechanisms. Vocal fold scarring is an excellent example. The specialized extracellular matrix of the vocal fold lamina propria and its attached epithelium are, thus far, irreplaceable after injury. Its geometry also makes the vocal fold mucosa a good candidate for tissue engineering in the laboratory. It is a small, thin structure with low metabolic activity due to sparse cellularity; these factors ensure adequate nutritional perfusion without need for neovascularization.
Befitting the label “stem cells”, adipose-derived stem cells possess the capabilities of self-renewal and multiple differentiation potential
We focused our attention here on rabbit ASC, since rabbits are an ideal small animal model for studies of the larynx. The larynx is accessible, adequately sized, able to phonate in an excised setting; and histologically and structurally similar to the human larynx. Furthermore, the inherently silent behavior of rabbits minimizes disruption to the healing implant. Multi-potent rabbit ASC are capable of differentiating into bone and fat-like lineages, as demonstrated here and in prior studies
Fibrin was chosen as the biological scaffold for this study due to its importance in normal wound healing, its biodegradable nature, and its potential for autologous sourcing from plasma. As the principal proteinaceous component of blood clots, fibrin is formed when the enzyme thrombin cleaves end-terminal peptides from circulating fibrinogen protein. Exposing the fibrinogen terminals allows rapid self-polymerization, and long fibrin chains result. Initial attempts at embedding the rabbit ASC used human cryoprecipitate as fibrinogen source, as was previously used for human ASC
For eventual human implantation, a fibrin scaffold could be produced from either autologous cryoprecipitate or pooled human donor cryoprecipitate. The risk of viral transmission or inflammatory reaction from allogeneic human cryoprecipitate implantation in the solid form is not well delineated, but is likely to be low since human pooled fibrin and thrombin-based products have been safely used for surgical hemostasis for many years
We did investigate epithelial differentiation in this system, but found that all cells primarily maintained a mesenchymal phenotype. Unwanted mesenchymal differentiation to produce lipid or mineral deposits did not occur. Li et al demonstrated differentiation of rabbit ASC to epithelial-like cells in a monolayer air liquid interface system, with nearly 70% epithelial-like differentiation
The construct developed here, even without significant epithelial differentiation, is suitable for implantation as a vocal fold mucosa graft in rabbits. The
Rabbit adipose-derived stem cells were used to create a three-dimensional tissue-engineered construct suitable for vocal fold cover replacement. The construct resembled the vocal fold lamina propria and epithelium 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. Notable differences were found in cell differentiation and scaffold degradation between human and rabbit ASC. This construct will be used for future
Vimentin antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242.