Yukio Nomoto

Clinical application of in situ tissue engineering using a scaffolding technique for reconstruction of the larynx and trachea.

Objectives: The objective of the present study was to demonstrate the efficacy of the clinical application of in situ tissue engineering using a scaffolding technique for laryngeal and tracheal tissue. Methods: We have developed a tissue scaffold made from a Marlex mesh tube covered by collagen sponge. Based on successful animal experimental studies, in situ tissue engineering with a scaffold implant was applied to repair the larynx and trachea in 4 patients. Results: In 1 patient with subglottic stenosis, the thyroid cartilage, cricoid cartilage, and cervical trachea with scarring and granulation were resected and reconstructed by use of the scaffold. In 3 patients with thyroid cancer, the trachea and cricoid cartilage with tumor invasion were resected and the scaffold was implanted into the defect. Postoperative endoscopy during the observation period of 8 to 34 months showed a well-epithelialized airway lumen without any obstruction. Conclusions: Our current technique of in situ tissue engineering using a scaffold shows great potential for use in the regeneration of airway defects.

A tissue-engineered trachea derived from a framed collagen scaffold, gingival fibroblasts and adipose-derived stem cells.

In some types of tracheal disease, tracheal resection is required. For patients with tracheal resection, artificial grafts, made from collagen sponge with a spiral polypropylene stent and mesh, have been clinically used by our group. However, epithelial regeneration was confirmed to be slow. In the present study, we investigated the potential of gingival fibroblasts (GFBs) and adipose-derived stem cells (ASCs) as autologous transplanted cells in combination with artificial graft for tracheal epithelial regeneration. In in vitro co-culturing with tracheal epithelial cells, GFBs stimulated epithelial cell differentiation and reconstruction of a pseudostratified epithelium. ASCs stimulated epithelial cell proliferation and reconstruction of a multi-layered epithelium. Subsequently, we prepared three kinds of bioengineered scaffolds from GFBs and/or ASCs and implanted them into rat tracheal defects. The bioengineered scaffolds containing GFBs were covered with tracheal epithelial cells after 1 week, and highly ciliated epithelium was formed after 2 weeks of transplantation. The bioengineered scaffold containing ASCs induced thick epithelium, and then pseudostratified epithelium containing goblet cells was formed. Furthermore, the application of both GFBs and ASCs had synergistic effects on tracheal epithelial regeneration, suggesting that bioengineered scaffolds containing GFBs and ASCs are useful for hastening tracheal epithelial regeneration.

Effect of Fibroblasts on Epithelial Regeneration on the Surface of a Bioengineered Trachea

Objectives: Our group applied a tracheal prosthesis, which was composed of polypropylene as the frame and collagenous sponge as the scaffold, to the first human case and had successful results. The objective of this study was to find a way to acquire more rapid re-epithelialization with fibroblasts on this tracheal prosthesis. Methods: Tracheal epithelial cells, which were isolated from the trachea of rats, were suspended in a collagenous gel. The collagenous gel with fibroblasts was layered on a collagenous sponge. The grafts of this “bioengineered trachea” were implanted into tracheal defects of rats, and the regenerated epithelium on the grafts was histologically examined. Results: Seven days after implantation, stratified squamous epithelium covered almost all of the surface of the gel, and some of the implanted fibroblasts in the gel were lined up just below the epithelium. Fourteen days after implantation, columnar and cuboidal ciliated epithelium covered almost all of the surface of the defects, and the implanted fibroblasts had disappeared. The numbers of regenerated epithelial cells at 14 days after implantation were larger than those of control models without fibroblasts, with statistical significance. Conclusions: The results suggested that the grafts of bioengineered trachea composed of collagenous sponge and collagenous gel with tracheal fibroblasts accelerated epithelial differentiation and proliferation in vivo.

Tissue Engineering for Regeneration of the Tracheal Epithelium

Objectives: The slowness of epithelialization on the artificial trachea that has been successfully used in humans is a problem. The purpose of this study was to develop a way to regenerate the epithelium on the surface of this artificial trachea. Methods: In an in vitro study, isolated rat tracheal epithelial cells were seeded on a collagenous gel that was stratified on a collagenous sponge. Histologic and immunohistochemical examinations were made. In an in vivo study, we transplanted grafts with green fluorescent protein–positive tracheal epithelial cells onto the tracheal defects of normal rats. At 3, 7, 14, and 30 days after the operation, histologic and immunohistochemical examinations were made. Results: In the in vitro study, the 3 layers — the epithelium, gel, and sponge — could be observed. The epithelium expressed cytokeratin 14, cytokeratin 18, and occludin. In the in vivo study, the artificial trachea was covered with epithelium at 3 days after operation, and then the epithelium differentiated from single- or double-stratified squamous epithelium into columnar ciliated epithelium. Green fluorescent protein–positive cells were found 3 days after operation. Conclusions: We believe that the method used in our experiment is an effective way to regenerate the epithelium on the surface of an artificial trachea. With further experimentation, this method should be suitable for clinical application.

Regeneration of Tracheal Epithelium Utilizing a Novel Bipotential Collagen Scaffold

Objectives: The purpose of the present study was to evaluate the effectiveness of a novel bipotential collagen scaffold as a bioengineered trachea for the regeneration of the tracheal epithelium. Methods: The bipotential collagen scaffold was developed by conjugating a collagen vitrigel membrane to a collagen sponge in order to promote both epithelial cell growth and mesenchymal cell infiltration. The bipotential collagen scaffold was transplanted into tracheal defects in rats, and a conventional collagen sponge was implanted as a control model. Histologic examinations were undertaken to evaluate the results. Results: The bioengineered trachea was covered with epithelium in the vitrigel model, but not in the control model, at 7 days after implantation. At 14 days after implantation, the bioengineered trachea was covered with epithelium involving the basal cell layer in the vitrigel model. At 28 days after implantation, a columnar ciliated epithelium was observed only in the vitrigel model. Conclusions: Our technique for trachea reconstruction using a novel bipotential collagen scaffold affords a feasible approach for accelerating epithelial regeneration on the intraluminal surface of the host tracheal defect.

Stiffness of salivary gland and tumor measured by new ultrasonic techniques: Virtual touch quantification and IQ.

OBJECTIVE To evaluate normal salivary gland stiffness and compare the diagnostic performance of virtual touch quantification (VTQ) and virtual touch imaging quantification (VTIQ) for head and neck tumor. METHODS A total of 92 measurements were examined, comprising 77 normal salivary glands, 11 benign tumors and four malignant tumors. Examinations were made to evaluate normal salivary gland stiffness and compare the diagnostic performances of new ultrasonic techniques regarding head and neck tumor. RESULTS The mean values of VTQ and VTIQ for the normal salivary group (NSG) were 1.92 and 2.06m/s, respectively. The VTQ and VTIQ values were correlative, and there were no statistical differences in each mean value between the normal parotid glands and submandibular glands. For the benign tumor group (BTG), four of the 11 values were non-numeric and were considered above the measurable range. The mean VTIQ value for the BTG was 4.24m/s. For the malignant tumor group (MTG), all four VTQ values were non-numeric. The mean VTIQ value for the MTG was 6.52m/s. For the mean VTIQ values, significant differences were observed among the three groups. The optimum VTQ cutoff value to detect malignant tumors was above the measurable range, and that of VTIQ was 4.83m/s. CONCLUSION The VTQ and VTIQ values were correlative for the salivary glands, and the stiffnesses of normal parotid glands were almost same as those of submandibular glands. VTQ and VTIQ values could be applied for the preoperative diagnosis in salivary gland lesions.

Age-dependent degeneration of the stria vascularis in human cochleae.

Objective: Aging is a common cause of acquired hearing impairments. This study investigated age‐related morphologic changes in human cochleae, with a particular focus on degeneration of the stria vascularis (SV) and the spiral ganglion (SG).

Regeneration of tracheal epithelium using a collagen vitrigel-sponge scaffold containing basic fibroblast growth factor.

Objectives: Our group has had good results in tracheal mucosal regeneration using a collagen vitrigel–sponge scaffold in an animal model. In this study, the effectiveness of this scaffold with the application of basic fibroblast growth factor (b-FGF) was investigated. Methods: A collagen vitrigel–sponge scaffold was fabricated with simultaneous addition of b-FGF. Three types of collagen vitrigel–sponge scaffolds were made: No b-FGF, 10 ng of b-FGF, and 100 ng of b-FGF. At 3, 5, 7, and 14 days after implantation in rats, the tracheas were removed and histologically evaluated. The regeneration of mucosal epithelium and the subepithelial layer was evaluated. Results: Mucosal epithelium, including pseudostratified epithelium and ciliated cells, regenerated earlier in the scaffolds when b-FGF was applied than when b-FGF was not applied. Regeneration of the subepithelial layer, infiltration of inflammatory cells and fibroblasts, and angiogenesis were promoted earlier in the scaffolds with b-FGF application. Conclusions: Our technique for tracheal reconstruction using collagen vitrigel–sponge scaffolds with b-FGF application affords a feasible approach for accelerating the regeneration of the intraluminal surface and subepithelial layer of tracheal tissue.

Bioengineered Trachea with Fibroblasts in a Rabbit Model

Objectives: Although our group has had mostly successful results with clinical application of a tracheal prosthesis, delayed epithelial regeneration remains a problem. In our previous studies using rats, it was demonstrated that tracheal fibroblasts accelerated proliferation and differentiation of the tracheal epithelium in vitro and in vivo. The purpose of this study was to evaluate the effects of fibroblasts on epithelial regeneration in larger tracheal defects in rabbits. Methods: We developed a bioengineered scaffold, the luminal surface of which was coated with fibroblasts. This scaffold was implanted into tracheal defects in 12 rabbits (bioengineered group), and scaffolds without fibroblasts were implanted in 12 rabbits (control group). The regenerated epithelium was histologically examined by light microscopy, scanning electron microscopy, and immunohistochemical studies. Results: In the bioengineered group, a stratified squamous epithelium was observed on the surface 7 days after transplantation. However, in the control group, the scaffolds were exposed. Fourteen days after implantation, a columnar ciliated epithelium was observed in the bioengineered group. The average thickness of the regenerated epithelium in the bioengineered group was significantly greater than that in the control group. Conclusions: This study indicated that fibroblasts had a stimulatory effect that hastened regeneration of the epithelium in large tracheal defects.

Evaluation of the use of induced pluripotent stem cells (iPSCs) for the regeneration of tracheal cartilage.

The treatment of laryngotracheal stenosis remains a challenge as treatment often requires multistaged procedures, and successful decannulation sometimes fails after a series of operations. Induced pluripotent stem cells (iPSCs) were generated in 2006. These cells are capable of unlimited symmetrical self-renewal, thus providing an unlimited cell source for tissue-engineering applications. We have previously reported tracheal wall regeneration using a three-dimensional (3D) scaffold containing iPSCs. However, the efficiency of differentiation into cartilage was low. In addition, it could not be proven that the cartilage tissues were in fact derived from the implanted iPSCs. The purpose of this study was to evaluate and improve the use of iPSCs for the regeneration of tracheal cartilage. iPSCs were cultured in vitro in a 3D scaffold in chondrocyte differentiation medium. After cultivation, differentiation into chondrocytes was examined. The ratio of undifferentiated cells was analyzed by flow cytometry. The 3D scaffolds were implanted into tracheal defects, as an injury site, in 24 nude rats. Differentiation into chondrocytes in vitro was confirmed histologically, phenotypically, and genetically. Flow cytometric analysis demonstrated that the population of undifferentiated cells was decreased. Cartilage tissue was observed in the regenerated tracheal wall in 6 of 11 rats implanted with induced iPSCs, but in none of 13 rats implanted with the control and noninduced iPSCs. The expression of cartilage-specific protein was also demonstrated in vivo in 3D scaffolds containing iPSCs. The presence of the GFP gene derived from iPSCs was confirmed in samples of cartilage tissue by the combination of laser microdissection (LMD) and polymerase chain reaction (PCR) techniques. Our study demonstrated that iPSCs have the potential to differentiate into chondrogenic cells in vitro. Cartilage tissue was regenerated in vivo. Our results suggest that iPSCs could be a new cell source for the regeneration of tracheal cartilage.