In situ tissue engineering of the cricoid and trachea in a canine model.

OBJECTIVES: The purpose of the current study was to demonstrate the efficacy of in situ tissue engineering of the cricoid and trachea in a canine model. METHODS: Marlex mesh tube reinforced with polypropylene threads and covered by collagen sponge was used as a tissue scaffold for airway regeneration in 9 beagle dogs. The anterior half of the cricoid cartilage was resected in 5 dogs, whereas the cricoid cartilage and cervical trachea were simultaneously resected in 4 dogs. The tissue scaffold was implanted into the resultant defect. RESULTS: Endoscopic examination showed no airway obstruction for a postoperative period of 3 to 40 months in all dogs. Granulation tissue was observed in 2 dogs, and slight mesh exposure in 1 dog, although all were asymptomatic. Light microscopy and electron microscopy showed the endolaryngeal and endotracheal lumen to be covered by ciliated epithelium. According to strain-force measurement, the framework was firmly supported by regenerated tissue, as well as the normal cricoid and trachea. CONCLUSIONS: Our current tissue scaffold provides a rigid framework for the airway, and the collagen coating invites tissue regrowth around the tube. This study presents the possibility of successful reconstruction of the cricoid and trachea with epithelial regeneration by means of in situ tissue engineering.

Experimental regeneration of canine larynx: a trial with tissue engineering techniques.

CONCLUSION: Since this tissue engineering technique is cost-effective and is less invasive to patients, it may replace conventional approaches in laryngeal reconstructive surgeries. OBJECTIVE: Laryngeal cancer is one of the most prevalent cancers in the head and neck region, and frequently requires surgical resection. Although there are many ways to reconstruct the larynx after resection, donor tissue is usually required. Recently, tissue engineering techniques have become widely accepted in clinical medicine and have already been applied to some organs. This animal experiment was designed to elucidate the efficacy of laryngeal regeneration using tissue engineering technique. MATERIALS AND METHODS: A bioartificial scaffold was designed from a replica of a canine larynx. A dental cast was used to replicate the intricate inside shape of the larynx. After copying its shape on a polypropylene mesh sheet, this sheet was coated with spongy collagen from porcine skin. A hemilaryngectomy was performed on beagle dogs under general anesthesia. Then the scaffold, preclotted with a mixture of peripheral blood and bone marrow-derived stromal cells, was implanted and fixed. The postoperative status was examined fiberscopically. RESULTS: On the eighth day after the operation, the surface of the implant was covered with soft tissue. Finally, the implant was completely covered with regenerated mucosa.

Tracheal regeneration after partial resection: a tissue engineering approach.

OBJECTIVES: The aims of this study are to investigate the efficiency of a tissue engineering approach to partial tracheal reconstruction and to improve epithelialization of the reconstructed trachea. The trachea must be resected in some cases of cancer or trauma. Various restructuring techniques are used, with no consensus on the best approach. Two problems that arise when treating tracheal defects by conventional techniques are an inability to regenerate ciliated epithelium at the reconstructed site and having to perform multiple procedures to achieve the desired repair. This study is designed to address these problems. STUDY DESIGN: Preliminary, an animal experiment. METHODS: Surgery was performed on five adult beagles under anesthesia. After the making of a longitudinal cervical skin incision, the trachea was exposed and a circular defect created. A polypropylene and collagen scaffold preclotted with peripheral blood was inserted to the defect site. Postoperatively, the site was evaluated fiberscopically, histologically, and radiographically. RESULTS: All dogs did well postoperatively. Fiberscopic examination showed that the implanted scaffolds were completely covered with regenerated mucosa with capillaries in all cases. Histologic data showed ciliated epithelium regenerated at the operated site from 1 month postoperatively. Newly formed cartilage was detected in the specimens from 8 to 12 postoperative months. Computed tomography images revealed the fine luminal contour of the regenerated site. CONCLUSIONS: Good epithelial regeneration was observed after repair of a round tracheal resection using a simple tissue engineering technique, making the technique a good substitute for conventional approaches to tracheal reconstruction in patients with cancer or trauma.

Fine needle aspiration of the vocal fold lamina propria in an animal model.

OBJECTIVES: The development of a minimally invasive fine needle aspiration (FNA) technique for vocal fold (VF) biopsy would have far-reaching implications and applicability in laryngology. The objective of this study was to determine whether FNA of the VF lamina propria is feasible and whether it causes injury to the VF. METHODS: Unilateral VF FNA was performed with a 26-gauge needle on 20 rabbits. The FNA cell collection of the lamina propria was confirmed by cytology. Four weeks after the FNA, the rabbits were painlessly sacrificed and the larynges were harvested. Histologic analysis of the VF lamina propria included Masson's trichrome and elastin-van Gieson stains. The linear viscoelastic shear properties, elastic modulus, and viscous modulus of the tissue were measured. RESULTS: Compared to the contralateral normal VFs, those VFs that underwent FNA demonstrated no significant differences in levels of collagen (p = .17) and elastin (p = .94). Rheologically, the elastic shear modulus and viscous modulus did not significantly differ between the normal and FNA VFs (p = .9380 and p = .9359, respectively). CONCLUSIONS: Fine needle aspiration of the VF lamina propria is feasible without injury and offers a potentially promising, less invasive alternative to be developed for future diagnostic and therapeutic management of VF lesions.

Regenerative medicine of the trachea: the first human case.

OBJECTIVES: The objective of the present study was to demonstrate regenerative medicine of the tracheal tissue by using an in situ tissue engineering technique for airway reconstruction. METHODS: Based on the previous successful experimental animal studies, the current regenerative technique was applied to repair of the trachea of a 78-year-old woman with thyroid cancer. A Marlex mesh tube covered by collagen sponge was used as a tissue scaffold. The operative intervention included right hemithyroidectomy, resection of the trachea, and tracheoplasty using the scaffold. The right half of three rings of the trachea was resected, and the scaffold material was sutured to the defect of the trachea. RESULTS: After 2 weeks, the mesh collagen structure of the artificial material could be seen with endoscopy in most of the implanted area. The artificial material was covered with epithelial growth after 2 months. Epithelialization continued to cover the artificial material completely for 2 years without any complications. CONCLUSIONS: The current regenerative technique avoided tracheotomy, a second operation, and deformity. Good epithelialization has been observed on the tracheal luminal surface without any complications for 2 years. Although long-term observation is required, regenerative medicine of the tracheal tissue appears feasible for airway reconstruction.

Regeneration of mastoid air cells in clinical applications by in situ tissue engineering.

OBJECTIVES: To regenerate of the mastoid air cells and their functions for the treatment of incurable otitis media. MATERIALS AND METHOD: In situ tissue-engineered mastoid air cells using three-dimensional hydroxy apatite (3D-HA) of honeycomb-like structure were used as artificial pneumatic bones. This 3D-HA is made of calcium phosphate and has a high ratio of micropores, 90%. Its surface is coated with collagen. Ten patients (4 males, 6 females) for this clinical study were randomly selected among the patients with severe cholesteatoma, adhesive otitis media, and purulent chronic otitis media, and they were to be received a staged operation. At the first stage of tympanoplasty, collagen-coated 3D-HA was put into the newly opened mastoid cavity, and it was fixed by fibrin glue. Recovery of mastoid aeration and regeneration of the pneumatic air cells of the mastoid cavity were estimated by images of high-resolution computed tomography (HRCT) after the first operation. At the second stage of operation, histopathologic examinations of specimens of HA taken from the reopened mastoid cavity were performed. RESULTS: Aerations in the mastoid cavity were observed in 9 of the 10 patients within 12 months after the second operation. Moreover, the pneumatic structure in the mastoid cavity was partially regenerated in five patients. In these successful cases, mucosa with newly formed capillaries was observed on the surface of the implanted HA at the second stage of operation. In the failure cases, however, connective tissues and granulations invaded the space of the 3D-HA. They were observed as soft-tissue density areas in the HRCT scan images. CONCLUSIONS: This study indicates that mucosa can grow on the surface of implanted artificial 3D-HA and can have a gas exchange function in the newly opened mastoid cavity. These tissue-engineered mastoid air cells may be a possible treatment for intractable otitis media.

Destiny of autologous bone marrow-derived stromal cells implanted in the vocal fold.

OBJECTIVES: The aim of this study was to investigate the destiny of implanted autologous bone marrow-derived stromal cells (BSCs) containing mesenchymal stem cells. We previously reported the successful regeneration of an injured vocal fold through implantation of BSCs in a canine model. However, the fate of the implanted BSCs was not examined. In this study, implanted BSCs were traced in order to determine the type of tissues resulting at the injected site of the vocal fold. METHODS: After harvest of bone marrow from the femurs of green fluorescent transgenic mice, adherent cells were cultured and selectively amplified. By means of a fluorescence-activated cell sorter, it was confirmed that some cells were strongly positive for mesenchymal stem cell markers, including CD29, CD44, CD49e, and Sca-1. These cells were then injected into the injured vocal fold of a nude rat. Immunohistologic examination of the resected vocal folds was performed 8 weeks after treatment. RESULTS: The implanted cells were alive in the host tissues and showed positive expression for keratin and desmin, markers for epithelial tissue and muscle, respectively. The implanted BSCs differentiated into more than one tissue type in vivo. CONCLUSIONS: Cell-based tissue engineering using BSCs may improve the quality of the healing process in vocal fold injuries.

Cricoid regeneration using in situ tissue engineering in canine larynx for the treatment of subglottic stenosis.

The purpose of the present study was to evaluate the efficacy of cricoid regeneration via in situ tissue engineering in a canine larynx for the treatment of subglottic stenosis. As the tissue scaffold, a Marlex mesh tube coated by collagen sponge was used for a rigid airway framework and for tissue regrowth around the tube. On 5 dogs, the larynx was exposed and the anterior third of the cricoid cartilage was resected. The tube was anastomosed to the lower edge of the thyroid cartilage and to the first tracheal cartilage. By postoperative endoscopic examination at 3 to 7 months, no airway obstruction was observed in any of the dogs. There was granulation tissue in 2 dogs and slight mesh exposure in 1 dog, but they were asymptomatic. Confluent regeneration of the epithelium over the scaffold and good incorporation of the scaffold mesh into the host tissue were observed after surgery.

Tissue engineering for the regeneration of the mastoid air cells: a preliminary in vitro study.

Mastoid is a pneumatic bone, composed of small interconnecting chambers covered by a mono-layer of mucosa with an abundant blood supply. One of its main functions is gas exchange according to the concentration/pressure gradient. The final goal of our research project is to regenerate mastoid air cells and their unique physiologic functions. The aim of the present study is to determine appropriate cultivating conditions for the cells cultured on the surface of artificial hydroxyapatite. In our in vitro experiment, to imitate the skeleton of mastoid bone, we used two types of three-dimensional hydroxyapatite (3D-HA), i.e. with a high (90%) and low (60%) percentage of micropores. The former type was divided into two groups: collagen-coated and non-coated. Canine mucosal- and bone marrow-derived stromal cells (BSCs), from the oral floor and femur respectively, were harvested and cultured on the 3D-HA under different conditions. To estimate the proliferation/distribution of the cultured cells over the surface of the 3D-HA, these cells were stained with the dye DiI and hematoxylin-eosin. There were no significant differences in the proliferation of cultured cells on the 3D-HA with high and low percentages of micropores. Collagen-coated HA was a better material for the cultured cells compared with the non-coated HA. Co-cultured mucosal and BSCs proliferated better than those cultured separately. In conclusion, this tissue engineering technique may be applied for the regeneration of mastoid air cells.

Regeneration of mastoid air cells: clinical applications.

The objective of this study was to establish a method for regenerating mastoid air cells and their functions for clinical use in incurable otitis media. For this clinical study three patients (one male, two female) were randomly selected from patients with severe cholesteatoma about to undergo staged operations. Hydroxy-apatite in three-dimensional, honeycomb-like structures (3D-HA) were used as artificial pneumatic bones. This 3D-HA is made of calcium phosphate and has a high percentage of micropores (90%). Its surface is coated with collagen. At the first stage of tympanoplasty, collagen-coated 3D-HA was put into the opened mastoid cavity and fixed by fibrin glue. Recovery of mastoid aeration and regeneration of the pneumatic air cells of the mastoid cavity were estimated on CT scan images after the first operation. Aeration was recovered in all cases. The mastoid air cells were regenerated in two cases. In the failed case, subcutaneous connective tissues and granulations invaded into the spaces of the 3D-HA. This study demonstrated that mucosa would grow on the surface of a 3D-HA implant and could provide gas exchange functions in the newly opened mastoid cavity. This tissue engineering method may be a possible treatment for intractable otitis media.

Regeneration of the vocal fold using autologous mesenchymal stem cells.

The aim of this study was to regenerate the injured vocal fold by means of selective cultured autologous mesenchymal stem cells (MSCs). Eight adult beagle dogs were used for this experiment. Selective incubation of MSCs from bone marrow was done. These MSCs were submitted to 3-dimensional incubation in 1% hydrochloric acid atelocollagen. Three-dimensional incubated MSCs were injected into the left vocal fold, and atelocollagen only was injected into the right vocal fold of the same dog as a control. Four days after injection, the posterior parts of the vocal folds were incised. The regeneration of the vocal fold was estimated by morphological and histologic evaluations. Our results showed that 3-dimensional incubated MSCs were useful in the regeneration of the injured vocal fold. This study shows that damaged tissues such as an injured vocal fold would be able to be regenerated by tissue engineering.

Recurrent laryngeal nerve regeneration by tissue engineering.

The recurrent laryngeal nerve (RLN) does not regenerate well after it has been cut, and no current surgical methods achieve functional regeneration. Here, we evaluate the functional regeneration of the RLN after reconstruction using a biodegradable nerve conduit or an autologous nerve graft. The nerve conduit was made of a polyglycolic acid (PGA) tube coated with collagen. A 10-mm gap in the resected nerve was bridged by a PGA tube in 6 adult beagle dogs (group 1) and by an autologous nerve graft in 3 dogs (group 2). Fiberscopic observation revealed functional regeneration of the RLN in 4 of the 6 dogs in group 1. No regeneration of the RLN was observed in any dog in group 2. We also tested for axonal transport, and measured the compound muscle action potential. The RLN can be functionally regenerated with a PGA tube, which may act as a scaffold for the growth of regenerating axons.