Makoto 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.

Early stage foreign body reaction against biodegradable polymer scaffolds affects tissue regeneration during the autologous transplantation of tissue-engineered cartilage in the canine model.

To overcome the weak points of the present cartilage regenerative medicine, we applied a porous scaffold for the production of tissue-engineered cartilage with a greater firmness and a 3D structure. We combined the porous scaffolds with atelocollagen to retain the cells within the porous body. We conducted canine autologous chondrocyte transplants using biodegradable poly-l-lactic acid (PLLA) or poly-dl-lactic-co-glycolic acid (PLGA) polymer scaffolds, and morphologically and biochemically evaluated the time course changes of the transplants. The histological findings showed that the tissue-engineered constructs using PLLA contained abundant cartilage 1, 2, and 6 months after transplantation. However, the PLGA constructs did not possess cartilage and could not maintain their shapes. Biochemical measurement of the proteoglycan and type II collagen also supported the superiority of PLLA. The biodegradation of PLGA progressed much faster than that of PLLA, and the PLGA had almost disappeared by 2 months. The degraded products of PLGA may evoke a more severe tissue reaction at this early stage of transplantation than PLLA. The PLLA scaffolds were suitable for cartilage tissue engineering under immunocompetent conditions, because of the retarded degradation properties and the decrease in the severe tissue reactions during the early stage of transplantation.

A Female Infant Who Had both Complete VACTERL Association and MURCS Association : Report of a Case

A 41-day-old female infant with VACTERL association was transferred to the pediatric intensive care unit of our hospital. She had been delivered at 36 weeks gestation by spontaneous vaginal delivery and weighed 2340 g. Esophageal atresia type A with long gap, anal atresia, cardiac anomaly (atrial septal defect and patent ductus arteriosus), thoracic vertebral dysplasia, left renal agenesis, and minor anomalies (left-side facial nerve palsy, left-side difficulty in hearing, and the absence of the right thenar) had been diagnosed by various examinations. She was transferred to our hospital to receive treatment for heart failure due to a cardiac anomaly. We recognized vaginal atresia during a radical operation for anal atresia (rectovestibular fistula) at 8 months of age. Furthermore, magnetic resonance imaging (MRI) revealed agenesis of the uterus. MURCS association includes Mullerian duct aplasia or hypoplasia, renal aplasia, and cervicothoracic somite dysplasia. This is the first case of complete VACTERL association combined with MURCS association.

ERas is Expressed in Primate Embryonic Stem Cells but not Related to Tumorigenesis

The ERas gene promotes the proliferation of and formation of teratomas by mouse embryonic stem (ES) cells. However, its human orthologue is not expressed in human ES cells. This implies that the behavior of transplanted mouse ES cells would not accurately reflect the behavior of transplanted human ES cells and that the use of nonhuman primate models might be more appropriate to demonstrate the safety of human ES cell-based therapies. However, the expression of the ERas gene has not been examined in nonhuman primate ES cells. In this study, we cloned the cynomolgus homologue and showed that the ERas gene is expressed in cynomolgus ES cells. Notably, it is also expressed in cynomolgus ES cell-derived differentiated progeny as well as cynomolgus adult tissues. The ERas protein is detectable in various cynomolgus tissues as assessed by immunohistochemisty. Cynomolgus ES cell-derived teratoma cells, which also expressed the ERas gene at higher levels than the undifferentiated cynomolgus ES cells, did not develop tumors in NOD/Shi-scid, IL-2Rγnull (NOG) mice. Even when the ERas gene was overexpressed in cynomolgus stromal cells, only the plating efficiency was improved and the proliferation was not promoted. Thus, it is unlikely that ERas contributes to the tumorigenicity of cynomolgus cells. Therefore, cynomolgus ES cells are more similar to human than mouse ES cells despite that ERas is expressed in cynomolgus and mouse ES cells but not in human ES cells.

Laparoscopic Fundoplication for Gastroesophageal Reflux Disease in Infants and Children

The number and types of minimally invasive surgical procedures being performed in children have increased exponentially in the last 15 years. Laparoscopic fundoplication is commonly performed for gastroesophageal reflux disease (GERD), although the population of patients who undergo this procedure is different in adults and children. In Japan, laparoscopic fundoplication has become a standard procedure, even for children with neurological impairment; however, its indications remain controversial. In this article we review the status of laparoscopic antireflux surgery for infants and children, looking at its indications, the procedures available, the complications, and the training required to perform the procedure safely and effectively.

Survival in a Neonate with Complete Urorectal Septum Malformation Sequence after Fetal Vesico-Amniotic Shunting for a Prominently Dilated Cloaca

Complete urorectal septum malformation sequence (URSMS) is usually a lethal anomaly that is characterized by urethral obstruction, imperforate anus, ambiguous genitalia, renal agenesis or dysplasia, and mullerian duct maldevelopment. This anomaly is thought to be caused by the cessation of urorectal septum migration toward the caudal cloacal membrane. Teratogenic factors or a genetic abnormality is postulated as the etiology. To date, only 4 patients with URSMS have survived the neonatal period; however, 2 of these infants died before the age of 1 year. We report the survival in a case with complete URSMS who had moderate pulmonary hypoplasia and preserved left renal function. The cloacal remnant wasdilated more than expected because the wall of the muscle layer was torn, perhaps in early fetal life, and timely placement of vesico-amniotic shunts prevented severe pulmonary hypoplasia caused by oligohydramnios.

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.

Tissue responses against tissue-engineered cartilage consisting of chondrocytes encapsulated within non-absorbable hydrogel

To disclose the influence of foreign body responses raised against a non‐absorbable hydrogel consisting of tissue‐engineered cartilage, we embedded human/canine chondrocytes within agarose and transplanted them into subcutaneous pockets in nude mice and donor beagles. One month after transplantation, cartilage formation was observed in the experiments using human chondrocytes in nude mice. No significant invasion of blood cells was noted in the areas where the cartilage was newly formed. Around the tissue‐engineered cartilage, agarose fragments, a dense fibrous connective tissue and many macrophages were observed. On the other hand, no cartilage tissue was detected in the autologous transplantation of canine chondrocytes. Few surviving chondrocytes were observed in the agarose and no accumulation of blood cells was observed in the inner parts of the transplants. Localizations of IgG and complements were noted in areas of agarose, and also in the devitalized cells embedded within the agarose. Even if we had inhibited the proximity of the blood cells to the transplanted cells, the survival of the cells could not be secured. We suggest that these cytotoxic mechanisms seem to be associated not only with macrophages but also with soluble factors, including antibodies and complements. Copyright

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.