Hiroko Komura

An animal model study for tissue-engineered trachea fabricated from a biodegradable scaffold using chondrocytes to augment repair of tracheal stenosis

INTRODUCTION We have designed an engineered graft fabricated from a biodegradable scaffold using chondrocytes and applied this construct to augment repair of tracheal stenosis. This study investigated the feasibility of using such tissue-engineered airways with autologous chondrocytes in a rabbit model. MATERIAL AND METHODS Chondrocytes were isolated and expanded from the auricular cartilage of New Zealand white rabbits, then seeded onto composite 3-layer scaffolds consisting of a collagen sheet, a polyglycolic acid mesh, and a copolymer (l-lactide/epsilon-caprolactone) coarse mesh. The engineered grafts were implanted into a 0.5 x 0.8-cm defect created in the midventral portion of the cervical trachea. Gelatin sponges that slowly released basic fibroblast growth factor (b-FGF) were then placed on the constructs, which were retrieved 1 or 3 months after implantation. RESULTS The biodegradable scaffold seeded with chondrocytes could maintain airway structure up to 3 months after implantation. Tracheal epithelial regeneration occurred in the internal lumen of this composite scaffold. Three months after implantation, staining of the sections showed cartilage accumulation in the engineered tracheal wall. CONCLUSION This composite biodegradable scaffold may be useful for developing engineered trachea. A gelatin sponge slowly releasing b-FGF might enhance chondrogenesis.

Study of mechanical properties of engineered cartilage in an in vivo culture for design of a biodegradable scaffold.

Introduction An engineered trachea with an absorbable scaffold should be used to augment the repair of a stenotic tracheal section in infants and children because this type of engineered airway structure can grow as the child grows. Our strategy for relief of tracheal stenosis is tracheoplasty by engineered cartilage implantation in accordance with the concept of costal cartilage grafting to enlarge the lumen. This study investigated the mechanical properties of regenerative cartilage with a biodegradable scaffold, Neoveil®, to aid in design of a composite scaffold that maintained semi-rigid properties until cartilage could be generated. Materials and methods New Zealand White rabbit (n=3) chondrocytes were isolated from auricular cartilage with collagenase type 2 digestion. Then 10×106/cm3 chondrocytes in atelocollagen solution were seeded onto polyglycolic acid (PGA) mesh. A total of 36 constructs, 12 from each rabbit, were implanted into athymic mice (3 constructs/mouse). Constructs were retrieved after 8 weeks and evaluated by measurements of mechanical and biochemical properties as well as histological examination. Thirty-six PGA mesh sheets of the same size but without cells were implanted in control mice. Results After 6 weeks of implantation, staining of sections with Safranin O revealed cartilage accumulation. Glycosaminoglycan was gradually produced from chondrocytes in the engineered constructs, correlating with the duration of implantation. Mechanical parameters had the same values as those for rabbit tracheal cartilage 8 weeks after implantation. Conclusions Biodegradable Neoveil® had good biocompatibility and was able to support extracellular matrix formation in engineered cartilage in an animal model.

Promotion of tracheal cartilage growth by intra-tracheal injection of basic fibroblast growth factor (b-FGF)

PURPOSE Basic fibroblast growth factor (b-FGF) is a very effective growth factor that induces the proliferation of chondrocytes. This study aimed to investigate whether intra-tracheally-injected b-FGF solution promotes the growth of tracheal cartilage. METHODS Group 1: 500 μl of distilled water was injected at the posterior wall of the cervical trachea of New Zealand white rabbits by using a tracheoscope (n=5). Group 2: 100 μg/500 μl of b-FGF solution was injected at the posterior wall of the cervical trachea (n=5). Group 3: Biodegradable gelatin hydrogel microspheres incorporating 100 μg/500 μl of b-FGF solution were injected at the posterior wall of the cervical trachea (n=5). All animals were sacrificed 4 weeks later, and the outer diameter and luminal area of the cervical trachea at the site of b-FGF injection were measured. RESULTS The cervical tracheas in the two b-FGF injection groups were spindle-shaped and had a maximum diameter at the injection site. The median outer diameter of the cervical trachea in Groups 1, 2, and 3 was 7.3, 8.0, and 8.0mm, respectively, showing a significant difference among Groups 1, 2, and 3 (P=0.04). The median luminal area in Groups 1, 2, and 3 was 27.4, 29.4, and 32.1mm(2), respectively. The ad hoc test showed a marginally significant difference only between groups 1 and 3 (p=0.056). CONCLUSION Intra-tracheal injection of slowly released b-FGF enlarged the tracheal lumen.

The junction between hyaline cartilage and engineered cartilage in rabbits

Tracheoplasty using costal cartilage grafts to enlarge the tracheal lumen was performed to treat congenital tracheal stenosis. Fibrotic granulomatous tissue was observed at the edge of grafted costal cartilage. We investigated the junction between the native hyaline cartilage and the engineered cartilage plates that were generated by auricular chondrocytes for fabricating the airway.

Tracheoplasty with cartilage-engineered esophagus environments

PURPOSE Our objective was to investigate the feasibility of engineering cartilage on the esophagus layer and outside the esophagus. Moreover, we investigated the feasibility of tracheoplasty with cartilage engineered on the esophagus in rabbits. METHODS Chondrocytes were isolated from auricular cartilages. 1. Engineered cartilage formation by histological findings on/into the esophageal layer was compared with that of injectable scaffold and preformed scaffold with chondrocytes. 2. Chondrocytes adhered to gelatin+vicryl mesh™ and b-FGF, were implanted on the outer esophageal surface. Four weeks after seeding, we found that cartilage was implanted in the midposterior portion of the cervical trachea (n=5), and it was retrieved 8weeks after seeding. RESULTS 1. A gelatin sponge incorporating β-TCP with vicryl mesh™ showed the best performance for fabricating engineered cartilage on the outer side of the esophagus. 2. Two of 5 rabbits died due to obstructed esophagus. Cartilage engineered outside the esophagus by a composite scaffold as the main material in the gelatin sponge, maintained the airway structure for up to 1month after implantation. Tracheal epithelial regeneration occurred in the internal lumen of this engineered cartilage. CONCLUSION Tracheoplasty with cartilage engineered outside the esophagus may be useful for reconstructing airways.

Slow release of basic fibroblast growth factor (b-FGF) enhances mechanical properties of rat trachea

AIM Severe tracheomalacia is a life-threatening disease, but symptoms usually improve with growth. The aims of this study were to investigate how slow release basic-Fibroblast Growth Factor (b-FGF) acts on tracheal cartilage, and whether growth-promoted trachea is more resistant against an increase in externally-applied pressure. METHODS Biodegradable gelatin hydrogel sheets soaked in 10 μl of distilled water (sham) or 0.5 or 5 μg/10 μl of b-FGF solution were inserted behind the cervical trachea of three-week-old male Wistar rats. The cervical trachea was harvested 4 weeks later. Extratracheal pressure was increased from 0 to 40 cmH2O in a chamber, while video-recording the internal lumen. The luminal area at each pressure was expressed as a proportion to that at 0 cmH2O. The amounts of collagen type II and glycosaminoglycan were measured by ELISA. RESULTS The luminal areas at 40 cmH2O in the control (no intervention), sham, and each of the b-FGF groups were 0.65, 0.62, 0.72, and 0.73, respectively. The amounts of collagen type II and glycosaminoglycan in each group were 127, 136, 193, 249 μg/mg, respectively, and 15, 16, 19, 33 μg/mg, respectively. There were significant differences between the control group and the FGF 5 group (P=0.02, 0.01, 0.01, for luminal area, collagen, and glycosaminoglycan, respectively). CONCLUSION 5 μg of slow-release b-FGF promotes matrix production (collagen type II and glycosaminoglycan). The growth-enhanced trachea was more resistant to collapse, suggesting that slowly released b-FGF might be useful in patients with severe tracheomalacia.

Slow release of basic fibroblast growth factor (b-FGF) promotes growth of tracheal cartilage ☆

PURPOSE Tracheomalacia is a major cause of morbidity in conditions such as oesophageal atresia. However, symptoms usually improve with age. A more rapid growth of tracheal cartilage can be induced by basic-Fibroblast Growth Factor (b-FGF). This study aimed to investigate whether slow-release b-FGF could act as a novel treatment for tracheomalacia. METHODS Biodegradable gelatin hydrogel sheets incorporating 0.5, 5, or 50 μg/20 μl of b-FGF solution were inserted between the cervical trachea and esophagus of rats. No intervention was performed in rats in a control group. All animals were sacrificed 4 weeks later, and the luminal area of the cervical trachea and the thickness of the cartilage were measured. RESULTS The mean luminal areas in the control group and in the b-FGF groups were 3.1, 3.2, 3.8, and 2.6mm(2), respectively, and showed a peak area at 5 μg of b-FGF. A significant difference was seen only between the control group and the b-FGF 5 μg group (p<0.05). The mean thickness of the tracheal cartilage was 0.12, 0.13, 0.19, and 0.32 mm in the control and the b-FGF groups, respectively, and showed a dose-dependent increase, which was statistically significant between the b-FGF 5 μg or 50 μg groups and the control group (p<0.01). CONCLUSION This study showed that slow-release b-FGF enlarges the tracheal lumen and thickens the cartilage in a dose-dependent fashion.

Tracheal cartilage growth by intratracheal injection of basic fibroblast growth factor.

BACKGROUND/PURPOSE We have previously shown that intratracheal injection of slowly released (in gelatin) basic fibroblast growth factor (bFGF) significantly enlarged the tracheal lumen by a slight margin. This study aimed to investigate differences in tracheal cartilage growth by the intratracheal injection of bFGF doses in a rabbit model. METHODS Water (group 1; n=7; control) or 100μg (group 2; n=8) or 200μg (group 3; n=8) of bFGF dissolved in water was injected into the posterior wall of the cervical trachea of New Zealand white rabbits using a tracheoscope. All animals were sacrificed four weeks later. RESULTS The mean circumferences of cervical tracheas for groups 1, 2, and 3 were 18.8±0.83, 21.1±2.0, and 22.1±1.3mm, respectively. A significant difference was found between groups 1 and 2 (P=0.034) and groups 1 and 3 (P=0.004). The mean luminal areas of cervical tracheas for groups 1, 2, and 3 were 27.0±2.1, 32.2±4.8, and 36.3±4.6mm2, respectively. A significant difference was found between groups 1 and 3 (P=0.001). CONCLUSION Intratracheal injection of bFGF in the dose range used significantly promoted the growth of tracheal cartilage in a rabbit model. LEVELS OF EVIDENCE Level II at treatment study (animal experiment).

Engineering and repair of diaphragm using biosheet (a collagenous connective tissue membrane) in rabbits

BACKGROUND Prosthetic patches can be used to repair large congenital diaphragmatic hernia defects but may be associated with infection, recurrence, and thoracic deformity. Biosheets (collagenous connective tissue membranes) have been used in regenerative medicine. We evaluated the efficacy of Biosheets in a rabbit model. METHODS Biosheets were prepared by embedding silicone plates in dorsal subcutaneous pouches of rabbits for 4weeks. In group 1 (n=11), Gore-Tex® sheets (1.8×1.8cm) were implanted into a diaphragmatic defect. In group 2 (n=11), Seamdura®, a bioabsorbable artificial dural substitute, was implanted in the same manner. In group 3 (n=14), biosheets were autologously transplanted into the diaphragmatic defects. All rabbits were euthanized 3months after transplantation to evaluate their graft status. RESULTS Herniation of liver was observed in 5 rabbits (45%) in group 1, 8 (73%) in group 2, and 3 (21%) in group 3. A significant difference was noted between groups 2 and 3 (P=0.017). Biosheets had equivalent burst strength and modulus of elasticity as native diaphragm. Muscular tissue regeneration in transplanted biosheets in group 3 was confirmed histologically. CONCLUSION Biosheets may be applied to diaphragmatic repair and replacement of diaphragmatic muscular tissue. LEVEL OF EVIDENCE Level III.

Long-term follow-up of tracheal cartilage growth promotion by intratracheal injection of basic fibroblast growth factor

BACKGROUND Intratracheal injection of basic fibroblast growth factor (b-FGF) has been shown to enlarge the tracheal lumen 4 weeks after treatment. The objective of this study was to investigate the long-term effect of tracheal cartilage growth promotion by intratracheal injection of b-FGF. MATERIALS AND METHODS New Zealand white rabbits were classified into four groups to receive either distilled water alone (Group 1; n = 16; control), 40 μg (Group 2; n = 10), 100 μg (Group 3; n = 13), or 200 μg (Group 4; n = 16) of b-FGF dissolved in water. The treatment was injected into the posterior wall of the cervical trachea using a tracheoscope. The animals were sacrificed 4 or 12 weeks later. RESULTS Four weeks after treatment, the mean luminal areas of tracheas for Groups 1, 2, 3, and 4 were 27.2, 25.6, 32.2, and 36.2 mm2, respectively. At 12 weeks, these were 29.3, 37.9, 42.5, and 56.0 mm2, respectively. The levels of glycosaminoglycan at 12 weeks were 93.9, 152.5, 123.2, and 210.6 μg/mg, respectively. At 12 weeks, the levels of type II collagen were 77.2, 133.1, 99.2, and 148.9 μg/mg, respectively. CONCLUSION Twelve weeks after a single injection of b-FGF, the mean luminal area of the trachea continued to increase.