Adipose-Derived Mesenchymal Stromal Cells Persist in Tissue-Engineered Vocal Fold Replacement in Rabbits.

OBJECTIVES:: Cell therapies using mesenchymal stromal cells (MSCs) have been proposed as a promising new tool for the treatment of vocal fold scarring. However, the mechanisms by which MSCs promote healing as well as their duration of survival within the host vocal fold have yet to be defined. The aim of this work was to assess the persistence of embedded MSCs within a tissue-engineered vocal fold mucosal replacement in a rabbit model of vocal fold injury. METHODS:: Male rabbit adipose-derived MSCs were embedded within a 3-dimensional fibrin gel, forming the cell-based outer vocal fold replacement. Four female rabbits underwent unilateral resection of vocal fold epithelium and lamina propria and reconstruction with cell-based outer vocal fold replacement implantation. Polymerase chain reaction and fluorescent in situ hybridization for the sex-determining region of the Y chromosome (SRY-II) in the sex-mismatched donor-recipient pairs sought persistent cells after 4 weeks. RESULTS:: A subset of implanted male cells was detected in the implant site at 4 weeks. Many SRY-II-negative cells were also detected at the implant site, presumably representing native female cells that migrated to the area. No SRY-II signal was detected in contralateral control vocal folds. CONCLUSIONS:: The emergent tissue after implantation of a tissue-engineered outer vocal fold replacement is derived both from initially embedded adipose-derived stromal cells and infiltrating native cells. Our results suggest this tissue-engineering approach can provide a well-integrated tissue graft with prolonged cell activity for repair of severe vocal fold scars.

Mesenchymal stromal cell injection promotes vocal fold scar repair without long-term engraftment.

BACKGROUND: Regenerative medicine holds promise for restoring voice in patients with vocal fold scarring. As experimental treatments approach clinical translation, several considerations remain. Our objective was to evaluate efficacy and biocompatibility of four bone marrow mesenchymal stromal cell (BM-MSC) and tunable hyaluronic acid based hydrogel (HyStem-VF) treatments for vocal fold scar using clinically acceptable materials, a preclinical sample size and a dosing comparison. METHODS: Vocal folds of 84 rabbits were injured and injected with four treatment variations (BM-MSC, HyStem-VF, and BM-MSC in HyStem-VF at two concentrations) 6 weeks later. Efficacy was assessed with rheometry, real-time polymerase chain reaction (RT-PCR) and histology at 2, 4 and 10 weeks following treatment. Lung, liver, kidney, spleen and vocal folds were screened for biocompatibility by a pathologist. RESULTS AND DISCUSSION: Persistent inflammation was identified in all hydrogel-injected groups. The BM-MSC alone treatment appeared to be the most efficacious and safe, providing an early resolution of viscoelasticity, gene expression consistent with desirable extracellular matrix remodeling (less fibronectin, collagen 1α2, collagen 3, procollagen, transforming growth factor [TGF]ß1, alpha smooth muscle actin, interleukin-1ß, interleukin-17ß and tumor necrosis factor [TNF] than injured controls) and minimal inflammation. Human beta actin expression in BM-MSC-treated vocal folds was minimal after 2 weeks, suggesting that paracrine signaling from the BM-MSCs may have facilitated tissue repair.

The importance of stem cell engineering in head and neck oncology.

Head and neck squamous cell carcinoma is the sixth leading cause of cancer worldwide. The most common risk factors are carcinogens (tobacco, alcohol), and infection of the human papilloma virus. Surgery is still considered as the treatment of choice in case of head and neck cancer, followed by a reconstructive surgery to enhance the quality of life in the patients. However, the widespread use of artificial implants does not provide appropriate physiological activities and often cannot act as a long-term solution for the patients. Here we review the applicability of multiple stem cell types for tissue engineering of cartilage, trachea, vocal folds and nerves for head and neck injuries. The ability of the cells to self-renew and maintain their pluripotency state makes them an attractive tool in tissue engineering.

Bioengineered vocal fold mucosa for voice restoration.

Patients with voice impairment caused by advanced vocal fold (VF) fibrosis or tissue loss have few treatment options. A transplantable, bioengineered VF mucosa would address the individual and societal costs of voice-related communication loss. Such a tissue must be biomechanically capable of aerodynamic-to-acoustic energy transfer and high-frequency vibration and physiologically capable of maintaining a barrier against the airway lumen. We isolated primary human VF fibroblasts and epithelial cells and cocultured them under organotypic conditions. The resulting engineered mucosae showed morphologic features of native tissue, proteome-level evidence of mucosal morphogenesis and emerging extracellular matrix complexity, and rudimentary barrier function in vitro. When grafted into canine larynges ex vivo, the mucosae generated vibratory behavior and acoustic output that were indistinguishable from those of native VF tissue. When grafted into humanized mice in vivo, the mucosae survived and were well tolerated by the human adaptive immune system. This tissue engineering approach has the potential to restore voice function in patients with otherwise untreatable VF mucosal disease.

[Vocal fold augmentation by injection of autologous fascia and fat].

OBJECTIVE: To evaluate the effect of combination of autologous fascia and fat injection into vocal fold for the treatment of patients with unilateral vocal fold paralysis and to observe the long-term effectiveness of this procedure. METHODS: A total of 26 unilateral vocal fold paralysis patients underwent vocal fold injection under general anesthesia, meanwhile, the mucosa of the injected point was sutured through laryngoscope under direct vision. There were 6 patients underwent autologous fat injection into vocal fold (group A), and 20 patients underwent autologous anterior rectus sheath fascia and fat injection (group B). Therapeutic efficacy were evaluated by videostroboscopy, voice-related parameters analysis and voice evaluation before and after treatment. Clinical analysis of this procedure was retrospectively performed in this serial of patients. RESULTS: All patients were followed up for 24 months. On the third day after operation, there was an acute inflammatory reaction induced by the graft. This reaction disappeared three months later. In all 20 cases, videolaryngostroboscopy showed significant improvement of the glottic closure, the improvement in acoustical parameters was statistically significant (P < 0.01). Perceptual evaluation of GRBAS scale showed significant improvement of phonatory function on G, B, A scale. The results remained stable 6 - 24 months after operation and were not changed by the length of follow-up. And in the 6 cases, videolaryngostroboscopy showed significant improvement of the glottic closure at 3 months compared with preoperative observation, a little spindle-shaped disclosure. The improvement in acoustical parameters was significant statistically at 3, 6 and 24 months (P < 0.05 or < 0.01), the voice quality decreased significantly at 6 and 24 months compared with 3 months (P < 0.05 or < 0.01). The significant differences were not observed between 6 and 24 months (P > 0.05). No complications were observed in all patients perioperatively or during the follow-up period. Voice-related parameters jitter, normalized noise energy and maximum phonation time showed significant differences between Group A and Group B on 24 months (P < 0.05 or < 0.01). CONCLUSION: The combination of autologous fascia and fat vocal fold injection is an effective procedure for the treatment of unilateral vocal fold paralysis, and the stable results can be achieved during the follow-up period for 24 months.

A biodegradable, acellular xenogeneic scaffold for regeneration of the vocal fold lamina propria.

A novel method for preparing an acellular xenogeneic extracellular matrix scaffold for tissue engineering was developed. Bovine vocal fold lamina propria specimens were treated with high-concentration sodium chloride, nucleic acid digestion, and ethanol dehydration for decellularization and removal of immunogenic foreign epitopes. Human vocal fold fibroblasts from primary culture were seeded onto the acellular scaffolds and cultured for 21 days. The decellularized and the recellularized scaffolds were examined by light microscopy, fluorescent microscopy, and scanning electron microscopy. Collagen synthesis and release by fibroblasts were quantified by the Sircol assay, whereas the synthesis and release of hyaluronic acid, decorin, and fibronectin were assessed by enzyme-linked immunosorbent assays. Viscoelastic shear properties of the scaffolds were quantified by a simple-shear rheometer at frequencies of up to 250 Hz. Preliminary results showed that a biodegradable, acellular extracellular matrix scaffold with an intact basement membrane and 3-dimensional structure of the matrix proteins was engineered. Vocal fold fibroblasts readily attached to and infiltrated the scaffold with high viability and active protein synthesis, demonstrating the biocompatibility. The elastic shear modulus and dynamic viscosity of the acellular scaffold and the fibroblast-repopulated scaffold were comparable to those of the human vocal fold cover. These findings support the potential of the scaffold as a xenograft for vocal fold reconstruction and regeneration.

In vitro and in vivo organ cultures containing respiratory epithelium.

A combination of two methods, organ culture and transplantation under the kidney capsule of nude mice, can be a suitable tool to study the mechanism of carcinogenesis of the human respiratory epithelium and to detect risks in environmental pollution. We used two tissues containing the respiratory epithelium--rat lung and human vocal cord. Gelita, a porous material used in surgery for haemostasis, produces a good support for the growing organ cultures of both tissues. These tissues continue to grow and to differentiate after transplantation under the kidney capsule. No pronounced immunological reaction of the nude mouse was detected. These conditions not only allow us to test various carcinogenic substances and their combinations, but as well enable us to detect an eventual malignant growth.