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Introduction Primary hyperparathyroidism (PHPT) is characterized by hypercalcemia and an elevated level of serum parathyroid hormone (PTH). PHPT presents with a complex set of renal, skeletal, and neuropsychological symptoms. Parathyroidectomy (PTX) is a radical treatment that is recommended for all physically symptomatic patients with PHPT. However, psychiatric symptoms are not considered as an indication for surgery. There remains an important issue from the view of perioperative management of whether PTX should be performed with the presence of uncontrolled psychiatric symptoms or deferred until severe psychiatric symptoms have been controlled. We report a case of mild hypercalcemia that caused severe psychosis in PHPT, which improved dramatically following PTX and resulted in successful postoperative management. Patient concern Our patient was a 68-year-old Japanese woman. She was diagnosed with PHPT, which was triggered by mild hypercalcemia. She was due to receive an operation for osteoporosis and kidney stones. She had severe psychosis, despite medication. Blood examinations revealed mild hypercalcemia (10.4 mg/dL, 8.8-10.1 mg/dL) and elevated serum levels of intact PTH (184.0 pg/mL, 10-65 pg/mL). Diagnosis She was diagnosed with severe psychosis caused by mild hypercalcemia in PHPT. Interventions Although she was treated with 37.5 mg quetiapine and 2 mg risperidone daily, she was excessively sedated and rejected oral treatment. Therefore, we decided to perform the operation. Outcomes Immediately following surgery, serum levels of calcium, and intact PTH were normalized. Her psychotic symptoms ceased completely 5 days after surgery. Conclusion We emphasize that PHPT presents with various severe psychiatric symptoms, even in mild hypercalcemia. Psychiatric symptoms may be the only salient symptoms in PHPT, and thus clinicians should suspect PHPT in patients with psychiatric symptoms and mild hypercalcemia. Furthermore, PTX is recommended for PHPT-even in the presence of severe uncontrolled psychiatric symptoms, which carries risks for postoperative management-because psychiatric symptoms are expected to improve and good postoperative management is possible.

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.

Potassium release from blood cells is a contrast medium-induced phenomenon. The purposes of the study were to (1) assess the effect of hyperosmolality and of adding sodium ions and calcium ions to a solution on potassium release from human blood cells and (2) reevaluate the possibility of hemolysis as a cause of potassium elevation.Fresh human blood was mixed with a test solution to examine the temporal changes in the whole blood potassium levels and to calculate the potassium release rate. Test solutions included 5%, 20%, and 50% glucose; 0.9% and 10% NaCl; and 50% glucose mixed with various amounts of sodium and calcium ions. We also measured serum glutamine oxaloacetic acid transaminase (GOT) and serum lactate dehydrogenase (LDH) to evaluate the possibility of hemolysis.Hyperosmolality using glucose solutions promoted higher potassium release. The average +/- SD potassium release rates were 7.3 +/- 2.4 micromol/min with 5% glucose, 13.5 +/- 2.3 micromol/min with 20% glucose, and 128.4 +/- 44.9 micromol/min with 50% glucose. The solutions including sodium ions showed lower release rates. The addition of sodium and calcium ions into 50% glucose significantly lowered the potassium release rates. No significant elevation of GOT or LDH was observed, and the possibility of hemolysis was eliminated.Hyperosmolar glucose solution promoted potassium release, but the presence of sodium ions in the hypertonic solution inhibited it. In addition, there is no possibility of hemolysis as a cause of potassium release.

Bronchoscopic lung volume reduction (BLVR) for severe emphysema is less invasive than lung volume reduction surgery. Fibroblast growth factor-2 (FGF-2) has been reported to enhance fibrogenesis and angiogenesis. The aim of this study was to investigate the feasibility of BLVR with the FGF-2, and ability to reduce lung volume and promote recovery of lung function. The BLVR based on FGF-2 is less invasive than surgical procedures, and can be performed repeatedly if the effectiveness of volume reduction is inadequate. This simple bronchoscopic approach allows selective reduction in the volume of the emphysematous parenchyma, and intratracheal administration of FGF-2 induces an increase in pulmonary blood flow, thus allowing recovery of pulmonary function.

Study objectives Fibroblast growth factor (FGF)-2 is one of the most powerful angiogenic growth factors to be evaluated as an agent for the promotion of angiogenesis. The aim of this study is to investigate whether intratracheal administration of controlled-release FGF-2 microspheres restores pulmonary function in beagle dogs with emphysema Design Randomized, controlled, experimental animal study Subjects Eighteen Wister rats and 15 adult beagle dogs Methods In the rat study, we compared the time profiles of the radioactivity remaining after intratracheal injection of 125 I-labeled FGF-2, either incorporated with the controlled-release microspheres or as an aqueous solution. In the dog study, elastase-induced emphysema models were developed in 10 animals, classified into the following three groups: control group (n = 5), emphysema model with empty microspheres-treated group (FGF − group, n = 5), and emphysema model with FGF-2 containing microspheres-treated group (FGF + group, n = 5) Results In the rat study, controlled-release microspheres maintained higher whole-lung FGF-2 concentrations after intratracheal administration. In the dog study, Pao 2 in the FGF + group was significantly higher than in the FGF − group after treatment. Pulmonary perfusion dynamic MRI revealed significant improvement in the signal intensity of damaged lung with the FGF + group. Linear intercept of the FGF + group was significantly reduced than the FGF − group Conclusion Results indicate that intratracheal administration of FGF-2 induced an increase in pulmonary blood flow in the damaged lung and led to recovery of pulmonary function. The controlled-release microsphere system increased the effectiveness of FGF-2

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.

PURPOSE: To create an experimental model of aortic dissection with a long-lasting patent false lumen as a proper animal model for development of less-invasive treatment for aortic dissection. MATERIALS AND METHODS: Fifteen adult beagle dogs (weight, 10–12 kg) were used. The descending aorta was exposed by a left thoracotomy at the sixth intercostal space. The entry for the aortic dissection was created surgically just distal to the origin of the left innominate artery and the reentry was 5 cm distal to the entry point. Normal saline solution was injected into the aortic wall (ie, media) between these two points to create the dissection. The dogs were followed up at 1 day, 3 months, 1 year, and 2 years. RESULTS: All 12 surviving dogs had completely patent true and false lumina without any thrombi. Microscopic examination showed that the dissection was created in the tunica media layer, making it identical to aortic dissection in humans. Color Doppler imaging confirmed the patency of the true and false lumina and the relatively narrowed true lumen. CONCLUSION: In this canine model of aortic dissection, the false lumen has excellent long-term patency and the dissection plane is histologically similar to that in human aortic dissection. This model may contribute to the development of new treatments for Stanford type B aortic dissection.

Many studies have been made to develop artificial tracheas with good biocompatibility and mechanical performance. We have designed a new type of mesh-type artificial tracheas. The basic frame of the specimens was composed of a mesh cylinder and stent attached to the outside of the cylinder. We have demonstrated from the mechanical tests that artificial tracheas with ring-type stents are stiffer than those with spiral-type stents. Artificial tracheas with arc-shaped shaped stents are half as stiff as artificial tracheas with whole ring stents. We have compared the mechanical properties of mesh-type artificial tracheas with nylon ring stents and those of canine tracheas, and have found the proper number of rings per unit length for the mesh-type artificial tracheas.

Primary pulmonary hypertension (PPH) is still a refractory disease, and patients deteriorate despite any treatment. We hypothesized that neovascularization in the lung could increase the volume of the vascular bed in the pulmonary circulation and thus reduce the development of pulmonary hypertension (PH). Endothelial progenitor cells (EPCs) might be a potential cell source for neovascularization. We examined the effects of EPC transplantation into the lungs of dogs with dehydromonocrotaline-induced PH. The lung parenchyma of PH model dogs was injected with ex vivo-expanded, autologous EPCs originated from peripheral blood (experiments, n=4) or culture medium (control, n=3), using a bronchoscope. EPC transplantation gave significant improvements in mean pulmonary artery pressure, cardiac output, and pulmonary vascular resistance. Histological evaluation revealed both improvement in the medial thickness of the small pulmonary artery and neovascularization of the lung tissue. These results indicate that EPC transplantation into the lung is effective at preventing the progression of dehydromonocrotaline-induced PH in dogs, and suggest a new therapeutic option for PPH.

The feasibility of an in situ tissue-engineering method employing cell-based therapy with autologous periodontal ligament-derived cells was investigated. Periodontal ligament cells were obtained from six beagle dogs. Periodontal fenestration defects (6 x 4 mm) were created bilaterally at a location 6 mm apical to the marginal alveolar crest in the maxillary canines. Alkaline phosphatase-positive periodontal ligament cells (3 x 10(5) cells) were seeded onto a collagen sponge scaffold just before implantation. One defect was filled with the cell-scaffold construct, and another was left empty as the control. All animals were killed 4 weeks after surgery, and specimens were evaluated histomorphometrically. All the histomorphometrical data were analyzed by three-way analysis of variance with the Bonferroni multiple comparisons test. Regeneration of apical tissue was faster than that of coronal and isolated tissues on the control side (apical > coronal > isolated; p < 0.0001). On the other hand, on the cell-seeded side, regeneration of the cementum was observed uniformly on the root surface. Our data suggest that the seeded cells induced cementum regeneration on the root surface, indicating the potential of in situ tissue engineering using autologous cells for the regeneration of periodontal tissues.

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.

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.

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.

We studied capillary endothelial injury, as demonstrated by fibrin deposition and changes in thrombomodulin (Tm) expression, in rats receiving continuous or bolus iv lipopolysaccharide (LPS). Rats were continuously infused with iv LPS (0.1, 0.2, 0.5, or 1.0 mg/kg/hr) for up to 6 hours. Others were given a bolus iv dose of LPS (20 mg/kg), and then the same dose of saline as a continuous infusion was administered for up to 3 hours. Harvested lungs, livers, and kidneys were examined immunohistochemically for thrombomodulin expression and fibrin deposition. Tm expression began to diminish dose- and time-dependently in lung, liver, and renal peritubular capillaries within 2 to 4 hours of the start of continuous LPS administration (1.0 mg/kg/hr) and had completely disappeared by 3 hours, although Tm remained in the glomerulus. The amount of fibrin deposition observed varied with the organ, dose, and duration of treatment in rats that received continuous LPS administration, but little was deposited in the lung. After bolus LPS administration, Tm in the endothelia of lung, liver, and peritubular capillaries diminished 20 to 40 minutes after treatment and then recovered 120 to 180 minutes after treatment, but the Tm activity of the glomerulus did not change. Fibrin deposition in the capillaries was observed in the liver, glomerulus, and peritubular capillaries, but not in the lung. Endothelial injury by LPS administration is dependent on the dose of LPS and the duration of treatment. The amount of fibrin deposition differs among organs and with the duration of contact between the endothelium and the endotoxin.

BACKGROUND A previous report has noted that tracheal allotransplantation is possible using allografts without immunosuppressants. In the present study, we investigated whether blood typing is necessary in immunosuppressant-free tracheal allotransplantation in dogs. METHODS In group 1, the blood types of paired dogs were mismatched (n=6, three pairs), while the blood types of paired dogs were matched in group 2 (n=6, three pairs). In paired dogs, an intrathoracic 5-ring tracheal segment was exchanged. No immunosuppressants were used. RESULTS All animals survived for more than 8 weeks. In each group, different degrees of airway stenosis were observed in four dogs. However, no stenosis was observed in the other two dogs. Histologically, no vascular rejection was detected. Ciliated columnar epithelium covered the inner surface of the implanted graft in the long-term survival cases. CONCLUSIONS In immunosuppressant-free tracheal allotransplantation, no hyperacute rejection occurred in any recipient dog whose blood type was mismatched with the donor. Blood typing was not the key factor to successful immunosuppressant-free tracheal allotransplantation. Immunosuppressant-free tracheal allotransplantation may be possible using mismatched blood type tracheal grafts.

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.

We have recently demonstrated that a collagen sponge incorporating TGF-β1 was effective in inducing bone repair at the skull defect of rabbits. In this study, the bone repairing of rabbit skulls was tried by TGF-β1-incorporated collagen sponges of different thickness to assess the influence of the size of osteoinductive materials on the bone repairing. The collagen sponge used was prepared by foaming and freeze-drying 1 % aqueous solution of pepsin-processed porcine atelo-collagen, followed by dehydrothermal crosslinking for 6 hr in vacua. Collagen sponges with different thickness were prepared by changing the volume of collagen solution for freeze-drying and changing the number of sponges piled up. An aqueous solution of TGF-β1 or 125 I -labeled TGF-β1 was dropped onto the freeze-dried sponge to prepare collagen sponges incorporating TGF-β1 or 125 I -TGF-β1, respectively. Collagen sponges were also radioiodinated with Bolton Hunter Reagent. Following implantation of the collagen sponges incorporating 125 I -TGF-β1 and the 125 I -labeled collagen sponges into the mice subcutis, each radioactivity remaining was measured to compare the time profile of in vivo retention of TGF-β1 with that of the sponge. The TGF-β1 incorporated collagen sponges of different thickness were implanted into full-thickness defects of rabbit skulls with 6 mm in diameter. The bone repairing at the defect was evaluated in terms of histological and DEXA examinations 6 weeks later. The remaining radioactivity of 125 I -labeled collagen sponges and 125 I -labeled TGF-β1 incorporated in collagen sponges decreased with time. The remaining periods of radioactivities of the sponges prolonged with an increase in the sponge thickness, but those of TGF-β1 were independent with the sponge thickness. The collagen sponge with 2 mm thickness was the most effective in increasing the bone mineral density at the bone defect. This finding indicated that the sponge thickness is one of the key factors contributing to successful bone repairing.

We investigated whether saber sheath-type tracheomalacia could be treated by the slow release of bone morphogenetic protein (BMP)-2 from a gelatin sponge. A 1 cm gap was made in the middle portion of each of 10 consecutive tracheal cartilage rings in the canine cervix (control group, n = 3), then a gelatin sponge containing 12 microg of BMP-2 solution was implanted in the gap (12 microg group, n = 3). In another group (120 microg + P group, n = 3), the implanted gelatin sponge contained 120 microg of BMP-2 solution, and the gap was covered with periosteum. All of the control dogs developed saber sheath-type tracheomalacia, whereas tracheomalacia was not observed in the 12 microg and 120 microg + P groups. In the 12 microg group, fibrous cartilage was observed at the ends of the cartilage stumps. In the 120 microg + P group, newly formed bone and cartilage were observed to form a bridge between the cartilage stumps. The regeneration of cartilage or bone induced by the slow release of BMP-2 from a gelatin sponge might be useful for treatment of tracheomalacia.

Biodegradability of new types of polyglycolic acid (PGA)-collagen composite tubes for nerve regeneration was evaluated in the peritoneal cavity. Hollow PGA mesh tubes were coated with atelocollagen solution, dried at room temperature, and then subjected to dehydrothermal treatment (hollow composite tubes). Hollow composite tubes filled with collagen sponge were also investigated in this study (sponge tubes). Tubes were fixed at the parietal peritoneum of BALB/c mice, and excised 0.5, 1, 2 and 3 months after the insertion. The inner areas of the excised tubes were measured microscopically. The rate of remained inner areas were calculated and analyzed statistically by one-way ANOVA and Fisher's PLSD test. 1 month after the insertion, the inner areas of the sponge tubes were well maintained, though they were not maintained in the hollow composite tubes. The rate of remained inner areas of the sponge tubes was significantly larger than that of the hollow composite tubes until 2 months after insertion. These results suggest that sponge tubes are more suitable than hollow composite tubes for nerve regeneration in the peritoneal cavity.

Background/Purpose: Circular myotomy often has been used to elongate the esophagus in end-to-end anastomosis for esophageal atresia with a long gap. Defects of the muscular layer often have led to the ballooning of the esophagus during the postoperative course. Recently, successful regeneration of the neo-esophagus using collagen sponge scaffold (CSS) for self-tissue regeneration has been reported. The authors applied this CSS to the muscular defect after circular myotomy in animal experiments. Methods: Myectomy or myotomy of the cervical or thoracic esophagus was performed in 11 piglets. CSS was applied to the amuscular site, which was 20 to 30 mm in length, to examine the regeneration of the muscular layer in 8 piglets (CSS group). The amuscular site was not wrapped by CSS in 3 piglets as a control (control group). Two months after operation, the animals were killed, and their operated esophagi were examined macroscopically and microscopically. Results: All 11 piglets increased in weight normally without any sign of dysphagia or vomiting. The amuscular site onto which CSS was implanted was covered with loose connective tissue, but muscle regeneration was not found microscopically. The amuscular site, which had been wrapped by CSS, was not distended in any of the 8 piglets when inflated, and ballooning was observed in all 3 of the control group. Conclusion: The results suggested that application of CSS to the site of circular myotomy for esophageal atresia with a long gap might not induce muscle regeneration but could prevent postoperative ballooning by inducing connective tissue growth. J Pediatr Surg 37:1409-1413. Copyright 2002, Elsevier Science (USA). All rights reserved.

The present investigation examined the key factors in the preparation of low-antigenic tracheal allografts by detergent treatment in dogs. In group 1 (n = 5), the grafts were treated by detergent at room temperature for 48 h and rinsed with running water until use. In group 2 (n = 4), detergent treatment was performed at 4 degrees C for 48 h, but the rinsing step was omitted. In group 3 (n = 6), the grafts were treated at 4 degrees C for 48 h, rinsed thoroughly with physiologic saline, and stored at 4 degrees C. The grafts were then used for tracheal replacement without immunosuppressant in dogs. The epithelium and mixed glands had been removed completely from the grafts in groups 1 and 2, and in five of the six grafts of group 3. In groups 1 and 2, cartilage viability appeared to have been lost and all animals died of airway stenosis within 38 days. In group 3, the chondrocytes were viable and all animals survived uneventfully. These results suggest that maintaining cartilage viability in a tracheal graft is necessary for successful immunosuppressant-free allotransplantation and, consequently, to maintain an open airway. The treatment temperature and sufficient rinsing are key factors for maintaining cartilage viability of grafts.

Recently, we have demonstrated canine peroneal nerve regeneration with functional recovery across an 80 mm gap using a polyglycolic acid (PGA) -collagen tube filled with laminin coated collagen fibers. In that study, the laminin coating was applied before a dehydrothermal (DHT) treatment designed to extend preservation of laminin in situ. To address concerns that the biological activity of laminin might consequently be reduced, the present investigation examined the influences of DHT crosslinking on the activity of laminin in terms of neural cell growth in vitro. DHT crosslinking was performed on collagen (type I or IV) spread on glass in three groups: (1) before coating, (2) after coating, and (3) both before and after coating. PC-12 cells were disseminated in each of the three groups. All three groups were cultured, and the number of cells were compared statistically. Cell growth achieved through application of laminin coating after DHT crosslinking was statistically greater than that achieved when laminin coating was performed before crosslinking. A reduction in laminin activity induced by DHT crosslinking was demonstrated. The optimal timing for the crosslinking of biornaterials treated with trophic factors such as-laminin should be examined in terms of the effects of crosslinking on the activity of the trophic factors.

Bone density is inherited as a complex polygenic trait. Previously, we identified two quantitative trait loci (QTLs) specifying the peak relative bone mass (bone mass corrected by bone size) on chromosomes (Chrs) 11 and 13 by interval mapping in two mouse strains: SAMP2 and SAMP6. The latter strain is an established murine model of senile osteoporosis and exhibits a significantly lower peak relative bone mass than SAMP2 mice. In this study, we report the effects of the Chr 13 QTL on peak bone density (Pbd2). First, we constructed a congenic strain P6.P2-Pbd2b, which carried a single genomic interval from the Chr 13 of SAMP2 on an SAMP6-derived osteoporotic background, to dissect this polygenic trait into single gene factors. This congenic strain had a higher bone density than the background strain using three measurement methods with different principles for bone density. Next, we measured the peak relative bone mass of the AKR/J strain and the 13 senescence-accelerated mouse (SAM) strains, which are considered to be a series of recombinant-like inbred (RI) strains derived from the AKR/J strain and other unspecified strains. We then determined the microsatellite marker haplotypes of these strains around the Pbd2 locus, in which three strains with a high relative bone mass shared the same haplotype over the 26-centimorgan (cM) region. In the Pbd2 locus, a high relative bone mass was associated with alleles of the unknown strain, whereas a low relative bone mass was associated with the alleles from the AKR/J strain. These results confirmed the existence of a Pbd2 locus regulating bone density in the SAM strains.

A novel artificial nerve conduit was developed and its efficiency was evaluated on the basis of promotion of peripheral nerve regeneration across an 80-mm gap in dogs. The nerve conduit was made of a polyglycolic acid-collagen tube filled with laminin-soaked collagen sponge. Conduits filled with either sponge- or fiber-form collagen were implanted into an 80-mm gap of the peroneal nerve (five dogs for each form). Twelve months postoperatively nerve regeneration was superior in the sponge group both morphometrically (percentage of neural tissue: fiber: 39.7 +/- 5.2, sponge: 43.0 +/- 4.5, n=3) and electrophysiologically (fiber: CMAP 1.06 +/- 0.077, SEP 1.32 +/- 0.127 sponge: CMAP 1.04 +/- 0.106, SEP 1.24 +/- 0.197, n=5), although these differences were not statistically significant. The observed regeneration was complementary to successful results reported previously in the same model, in which collagen fibers exclusively were used. The results indicate a possible superiority of collagen sponge over collagen fibers as filling materials. In addition, the mass-producibility, superior scaffolding potential, and capacity for gradual release of soluble factors of the sponge provide make it an attractive alternative to fine fibers, which are both technologically difficult and costly to produce. This newly developed nerve conduit has the potential to enhance peripheral nerve regeneration across longer gaps commonly encountered in clinical settings.

Tissue engineering of the small intestine will prove a great benefit to patients suffering from short bowel disease. However cell seeding in tissue engineering, such as fetal cell use, is accompanied by problems of ethical issues, rejection, and short supply. To overcome these problems, we carried out an experimental study on tissue engineering of the small intestine by acellular collagen sponge scaffold grafting. We resected the 5 cm long jejunum from beagle dogs and reconstructed it by acellular collagen sponge grafting with a silicon tube stent. The graft was covered with the omentum. At 1 month after operation, the silicon stent was removed endoscopically. Animals were sacrificed 1 and 4 months after operation, and were examined microscopically. Neo-intestinal regeneration was observed and the intestinal mucosa covered the luminal side of the regenerated intestine across the anastomosis. Thus, the small intestine was regenerated by tissue engineering technology using an acellular collagen sponge scaffold.

Two types of flat plates made from a polyvinyl alcohol (PVA) hydrogel with a water content of 80 and 20 (PVA-H80, PVA-H20), 20 × 10 × 1 mm in size, were subcutaneously implanted into each of 50 young, male Wistar rats. As a control, a sham operation was done on another set of 50 rats (Sham Op group). The shape and transparency of the PVA hydrogel were unchanged for up to 24 months. Tumors arose in 14 rats from the PVA-H80 group. In the PVA-H20 group, tumors appeared in 15 rats. The average tumor latency was 598 ± 109 days in the PVA-H80 and 637 ± 94 days in the PVA-H20. There was no difference in tumor incidence between the PVA-H20 and PVA-H80 groups (p < 0.05). In the Sham Op group, no malignant tumors appeared. Histopathologically, the tumors induced by hydrogel plates were malignant tumors resembling fibrosarcoma or malignant fibrous histiocytoma. This indicates that PVA hydrogel implants also induce solid state carcinogenesis at a similarly high rate to medical grade hydrophobic material reported in a previous study. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 56: 289–296, 2001

Recently, the new interdisciplinary studies of the tissue engineering are progressing remarkably. These technologies are expected as a breakthrough to overcome the defects and the disadvantages of artificial organs and organ transplantations.The strategy of the tissue engineering is divided to two groups. One is the in vitro method, and the other is in vivo method.In the in vitro method, the living cells are picked out and cultured to make some tissues and organs. After that, the tissue or organs are implanted into patient's body.In the in vivo method, the extracellural matrix which cells can use to make their own environment is settled in the internal body. At that time the stem cells, the precursor cells, and the DDS of cell growth factors to acceralate the growth speed are administrated into the artificial extracellural matrix. Regeneration of almost all of the tissues and organs have already been studied and some of them are tried to apply in clinical patients.Applying this new technology, the medical therapy will be able to progress and change the thought of the medical therapeutology.

Background Poor healing of the sternum often limits the use of bilateral internal thoracic arteries (BITAs) after coronary bypass surgery in diabetic patients. We have reported that a gelatin sheet that incorporates basic fibroblast growth factor (bFGF) accelerates sternal healing after BITA removal in normal rats. This study evaluated the effects of the above method for sternal healing in diabetic animals. Methods and Results Diabetic Wistar rats with blood glucose levels >400 mg/dL and body-weight loss >20 g were established by a single intravenous injection of streptozotocin (55 mg/kg). After median sternotomy and BITA removal, 16 diabetic rats received either a gelatin sheet that incorporated bFGF (100 μg/sheet) on the posterior table of the sternum (FGF group, n=9) or no gelatin sheet (control, n=7). Peristernal blood flow, as measured by a noncontact laser Doppler 4 weeks after surgery in the FGF group, recovered to the preoperative level (106±10% versus 82±9%, P P P P Conclusions A gelatin sheet that incorporates bFGF may offset sternal ischemia and accelerate sternal bone regeneration and healing, even in diabetic patients.

Background . We have shown that a gelatin sheet incorporating basic fibroblast growth factor enhanced bone regeneration of the devascularized sternum. The purpose of this study was to determine if topical use of the gelatin sheet accelerated normal sternal regeneration and bone remodeling. Methods . Thirty Wistar rats had median sternotomy and were divided into 3 groups: 10 had the bilateral internal thoracic arteries removed and basic fibroblast growth factor sheet applied on the sternum (group A), 10 had just the bilateral internal thoracic arteries removed (group B), and 10 had intact bilateral internal thoracic arteries (group C). Results . Four weeks later the peristernal blood flow significantly increased and marked angiogenesis was seen around the sternum in group A. Histologically, the sternum was almost completely healed only in group A. In group A the bone mineral content was highest, but the bone mineral density was similar to that in other groups. The osteoclast index in group A was highest at the border zone of bone formation and remained high in regenerated bone. Conclusions . The basic fibroblast growth factor sheet offset sternal ischemia and accelerated normal sternal bone regeneration and remodeling, not only by callus formation but also by callus resorption.

There is now considerable evidence that peripheral nerves have the potential to regenerate if an appropriate microenvironment is provided. However, there are only a few reports of the successful use of artificial nerve conduits to repair major nerve defects more than 30 mm in length. In this study, we examined nerve regeneration across a long gap in the dog peroneal nerve using a novel artificial nerve conduit developed by our group. The conduit consists of a polyglycolic acid (PGA) collagen tube filled with laminin coated collagen fibers. In 12 dogs, the nerve conduit was implanted across an 80 mm gap in the left peroneal nerve. Three months after surgery, compound muscle action potentials (CMAPs) and somatosensory evoked potentials (SEPs) were detected. Evaluation of locomotor function revealed obvious limping for up to 3 months, but no marked difficulty in walking by 6 months. Microscopic observation of the regenerated nerve segment at 12 months showed numerous myelinated nerve fibers, which were smaller in diameter and enclosed in a thinner myelin sheath than normal axons. These results suggest that our artificial nerve conduit has potential usefulness in enhancing peripheral nerve regeneration, even across large gaps.

Object. Facial nerve paralysis due to a surgical procedure or trauma is a frequently observed complication. The authors evaluated facial nerve repair achieved by the interposition of a collagen nerve guide.Methods. Ten cats were divided into three groups. Group 1 consisted of six animals in which a 5-mm facial nerve segment on one side was resected and replaced by a collagen tube that was sutured to bridge both nerve stumps. On the opposite side a 5-mm segment of facial nerve was resected, reversed 180°, and sutured to the stumps as an autograft nerve. Group 2 consisted of two cats in which the collagen nerve guide was interposed on one side and the nerve on the other side was left intact. Group 3 consisted of two cats in which a reversed autograft nerve was placed on one side and the nerve on the other side was left intact. Histological, electrophysiological, and horseradish peroxidase labeling examinations were performed starting 3 weeks after surgery.Light and electron microscopic examinations of collagen tube—implanted specimens revealed a well-vascularized regenerated nerve. The electrophysiological study confirmed the recovery of electrical activity in regenerated axons. Horseradish peroxidase labeling also confirmed restoration of the whole facial nerve tract.Conclusions. The collagen nerve guide shows great promise as a nerve conduit.

Objective: To develop a method for eliminating the epithelium and mixed glands from tracheal grafts by detergent treatment and evaluate these grafts for immunosuppressant-free allotransplantation in dogs. Methods: Fresh canine tracheal grafts were treated with a detergent (1% Triton X-100 t-octylphenoxypolyethoxyethanol; T-9284; Sigma Chemical Co, St Louis, Mo) at 4°C for 48 hours. The grafts were then used for intrathoracic 5-ring tracheal replacement in other dogs without immunosuppressant treatment (n = 6, detergent treatment group). In the control group (n = 6) fresh untreated canine tracheal segments were implanted as allografts. All the implanted grafts were covered with an omental pedicle. Results: In the detergent treatment group the chondrocytes in the graft had a similar appearance to those in the fresh trachea, indicating that the chondrocytes remained viable after the detergent treatment. In 5 of the 6 grafts, the epithelium and mixed glands had been removed completely. After transplantation, these 5 grafts were incorporated by the host trachea without stenosis. In the remaining treated tracheal graft, in which removal of the epithelium was incomplete, moderate stenosis was observed at the fourth week after implantation, although this was not progressive. In the control group, granulation tissue of the graft and significant stenosis were observed after transplantation. Conclusion: The antigenicity of tracheal grafts can be greatly reduced by removing the epithelium and mixed glands by the use of detergent treatment. The epithelium and mixed glands of the graft appear to be the determining elements involved in rejection after tracheal allotransplantation. (J Thorac Cardiovasc Surg 2000;120:108-14)

Carinal reconstruction has the highest operative mortality rate in pulmonary surgery, and high tension at the anastomoses is one of the reasons. We have reported previously that a new Y-shaped artificial trachea gives acceptable short-term results. Here, we report our long-term observations and histologic findings. A Y-shaped Marlex mesh tube reinforced by a polypropylene spiral was coated with collagen, which was extracted from porcine skin, to increase the biocompatibility and airtightness of the prosthesis. Replacement of the carina was performed in 20 beagle dogs. The prosthesis was covered by the omental pedicle in all dogs. Six of the 20 dogs survived past 6 months, and 4 were in good condition when they were killed. The other 13 dogs died within 11 days of the operation. The main cause of death was air leakage from the prosthesis. Bronchoscopic examination of the surviving dogs 12 months after surgery revealed that the prosthesis was covered with regenerated tissue, and neither stenosis nor dehiscence was recognized; 15 months after implantation microscopic examination showed the growth of ciliated epithelial cells over the lumen of the prosthesis. These results suggest that our Y-shaped artificial trachea might be useful for reconstruction of the carina with tissue regrowth, although some improvement of the prosthesis is necessary.

We have developed a new adhesive for surgical use. The new adhesive is made of three components: porcine collagen, poly(L-glutamic acid) and water-soluble carbodiimides (WSC). The optimum concentration of each component was determined by measuring the time required for gel formation in experiments in vitro. Using these optimum concentrations, we applied the adhesive to wounds made on rats. A conventional fibrin glue was used as a control. Measurement of tensile strength and histological examination were performed 5, 7, 10, and 14 days after the operation. The tensile strength of wounds treated with 2.5 mg/mL collagen glue was not significantly different from that of wounds treated with fibrin glue except at 7 days after the operation (p < 0.05 by Student's t-test). Histological examination revealed that the speed of cell infiltration into, and absorption of 2.5 mg/mL collagen glue was slower than for fibrin glue, but faster than for 5.0 mg/mL collagen glue. One of the important advantages of our collagen glue is that the absorption rate of it can be controlled by the collagen concentration. Therefore, it seems to be adequate for sealing air leakage from the lung, which takes a relatively long period for recovery. Moreover it does not contain human serum, and, hence, it requires no blood donation and can be obtained with low cost.

Background . Human amnion has useful biomedical applications because it contains a large amount of human collagen fibers. We prepared purified human collagen membrane (HCM) from human amnion and used it to develop a new sheet by combining it with synthetic bioabsorbable polyglycolic acid (PGA) mesh. We evaluated its efficacy in preventing air leakage from the lungs of dogs. Methods . In 20 dogs, HCM-PGA sheet (n = 5), sheets using fibrin glue with a separate application method (n = 5), a mixed application method (n = 5), and fibrin glue alone (n = 5), were used as dressing materials after partial lung resection. Results . The HCM-PGA sheet using fibrin glue with a separate application method was shown to be significantly more effective by an air leakage pressure test than the other three methods. These results indicate that the HCM-PGA sheet is useful for preventing air leakage from the lung. Conclusions . The HCM-PGA sheet is more effective than conventional fibrin glue for controlling postoperative air leakage.

Objectives: Intrathoracic esophageal replacement with an artificial esophagus is considered difficult. We attempted to replace the intrathoracic esophagus with an artificial esophagus composed of a collagen sponge with a double-layered silicone tube and examined the state of host tissue regeneration. Methods: A 5-cm long gap was created in the intrathoracic esophagus in 9 dogs and repaired by interposition of our prosthesis. The dogs were fed only by intravenous hyperalimentation for 28 days. The silicone tube was removed at 29 days after the operation, and oral feeding was reintroduced. Results: One dog was put to death at each of the following times: 1, 2, 3, 3, 6, 12, and 24 months after the operation. One dog is still surviving without problems after more than 26 months. One dog died of malnutrition at 10 months. In all dogs, the host regenerated tissue had replaced the resulting gap at the time of silicone tube removal. The mucosa had fully regenerated within 3 months and the glands within 12 months. The process of stenosis and shrinkage was complete within 3 months and did not advance thereafter. The lamina muscularis mucosae were observed as islets of smooth muscle within 12 months. Although the skeletal muscle regenerated close to the anastomoses, it did not extend to the middle of the regenerated esophagus even after 24 months. Conclusions: Use of a collagen sponge with a double-layered silicone tube was shown to be feasible even in the thorax and to allow the regenerated host tissue, consisting of the mucosa, glands, and lamina muscularis mucosae, to replace the esophageal gap. (J Thorac Cardiovasc Surg 1999;118:276-86)

We manufactured an esophageal prosthesis made of a collagen sponge supported by an inner silicone tube. The collagen was derived from porcine skin and was crosslinked by dehydrothermal treatment. We implanted our prosthesis in nine dogs after resecting a 5 cm length of the thoracic esophagus. Intravenous hyperalimentation was performed for 4 weeks after the surgery. After 29 days, the inner silicone tube was removed endoscopically and oral feeding was restarted immediately. All nine dogs survived more than 1 month, and three of the nine dogs survived for more than 1 year. Endoscopically, the luminal surface of the regenerated esophagus was covered with a lustrous, smooth mucosa, but stenosis was observed in the midregion of the regenerated esophagus. Microscopically, no foreign material remained at the reconstructed site 1 month after surgery; the collagen sponge was absorbed and replaced by host tissue. Complete epithelization was observed within 3 months. Regeneration of the lamina muscularis mucosae, esophageal glands, and skeletal muscle were observed near the anastomoses. Our artificial esophagus holds promise for esophageal reconstruction in a clinical setting, even in the thorax.

We cloned a full-length cDNA encoding a novel mouse protein, A-C2, by differential display method using mouse embryonic fibroblast C3H10T1/2 cells and mouse chondroprogenitor-like EC cells, ATDC5. The deduced amino acid sequence of A-C2 consisted of 106 amino acids with no significant homology to the sequences previously reported. Northern blot analysis showed two major bands of 2.1 and 1.8 kb sizes. Expression of A-C2 mRNA was exclusive to ATDC5 cells at their undifferentiated stage. None of ATDC5 cells at their differentiated stage and adult mice tissues examined expressed A-C2 gene.

Flat plates made from a copolymer of epsilon-caprolactone and L-lactide (P-CL-LA) [50:50 (w/w), molecular weight 1.62 x 10(5); 20 x 10 x 1 mm size] were subcutaneously implanted into 50 young, male Wistar rats (P-CL-LA group). After 24 months the plates had become a mass of small pieces, which were concentrated in an area of 3 x 2 x 1 mm. For comparison, 50 rats were implanted with medical-grade polyethylene plates (PE group) while another set of 50 rats was subjected to the same operation but without an implant (Sham Op group). Tumors arose in 25 rats from the P-CL-LA group: 24 were malignant mesenchymal tumors at the implant sites. In the PE group, tumors appeared in 16 rats (14 at the implant sites and two ectopically). The average tumor latency was 578+/-84 days in the P-CL-LA group and 452+/-102 days in the PE group. There was no difference in tumor incidence between the P-CL-LA and PE groups (p < 0.05). In the Sham Op group, two malignant tumors appeared over 2 years. Pathologically, these induced tumors arose from the inflammatory cells surrounding the degrading fragments of P-CL-LA within the tissue capsule. This indicates that relatively slowly degrading material can induce malignant tumors at a similarly high rate to nonabsorbable medical grade PE, at least in this animal model.

An ideal drug delivery system (DDS) releases an appropriate drug at specific locations and times. We tried to create a new antibiotic delivery system that releases gentamicin only when wounds are infected by Pseudomonas aeruginosa (P.A.). Exudate from the dorsal pouch of rats infected with P.A. showed significantly higher hydrolytic activity-thrombin-like activity-toward Boc-Val-Pro-Arg-MCA than exudate from noninfected wounds. We therefore constructed a device for controlled release of an antimicrobial drug triggered by thrombin-like activity. Briefly, gentamicin was bound to a polyvinyl alcohol derivative (PVA) hydrogel through a newly developed peptide linker cleavable by the proteinase, PVA-(linker)-gentamicin. In vitro experiments showed that proteinases from wounds infected with P.A. cleaved the linker and gentamicin was released while the exudate from noninfected wounds had no hydrolytic activity toward the linker. This device shows potential as an occlusive dressing with an effective antibiotic delivery system for treating infected wounds.

Objective: Attempts have been made to replace esophageal defects with a variety of artificial materials. However, because of the artificial nature of the materials, problems such as infection, leakage, stricture, or dislocation could not be avoided. Therefore we have designed a new type of artificial esophagus that is gradually replaced by host tissue. Methods: Our artificial esophagus was a two-layered tube consisting of a collagen sponge matrix and an inner silicone stent. We used it to replace 5 cm esophageal segmental defects in 43 dogs, and the inner silicone stent was removed endoscopically at weekly intervals from 2 to 4 weeks. Results: In the 27 dogs from which the silicone stent was removed at 2 or 3 weeks, constriction of the regenerated esophagus progressed and the dogs became unable to swallow within 6 months. In the 16 dogs from which the silicone stent was removed at 4 weeks, highly regenerated esophageal tissue successfully replaced the defect, leaving no foreign body in the host. Moreover, the regenerated esophagi had stratified flattened epithelia, striated muscle tissue composed of an inner circular and an outer longitudinal muscle layer, and esophageal glands. Conclusions: Even in mature adult higher mammals, esophageal high-order structures can be regenerated provided that an adequate three-dimensional extracellular structure is put in place for a sufficient period. (J Thorac Cardiovasc Surg 1998;116:98-106)

We prepared a complex membrane consisting of two collagen membranes purified from human amnion and a polyglycolic acid (PGA) mesh. Using this membrane, we produced two types of sheets for pericardial replacement by glutaraldehyde cross-linking followed by succinylation [PGA-amnion (GA-S) sheet] or by dehydrothermal cross-linking followed by hyaluronic acid fixation [PGA-amnion (DHT-H) sheet]. We used these sheets to replace part of the pericardium in dogs, and the state of pericardial repair and regeneration were evaluated. As a control sheet, expanded polytetrafluoroethylene (EPTFE) with a thickness of 0.1 mm was used. After implantation of the PGA-amnion (DHT-H) sheet, soft connective tissue with abundant capillaries was observed, and its surface was covered with a mesothelial cell layer with many microvilli. The basement membrane was nearly normal. After implantation of the PGA-amnion (GA-S) sheet, dense connective tissue was observed, and its surface was covered with mesothelial cells. However, there ...

Facial nerve paralysis due to resection of tumors or as a consequence of trauma is a frequently observed complication. Thus, in the present study, we evaluated a collagen nerve guide in facial nerve regeneration across a 5-mm nerve gap. This biological tube was manufactured from 3% collagen, coated over a Teflon tube used only as a template and submitted to thermal dehydration at 105°C for 24h. The collagen tube was implanted at the dorsal ramous of the facial nerve of five adult cats over a gap of 5mm. The facial nerve of the contralateral side was kept intact and used as control. Electrophysiological study was performed from 3 weeks after surgery, and histological and horseradish peroxidase labeling examination was carried out 8 weeks after implantation. Electrophysiological study confirmed the recovery of electrical activity of the collagenimplanted regenerated nerve. Light-microscopic examination of collagen tube-implanted specimens revealed a well vascularized regenerated nerve, which under an electron microscope showed many myelinated axons surrounded by Schwann cells and unmyelinated axons. Horseradish peroxidase staining demonstrated labeling of facial motoneurons in the brainstem and facial nerve terminals in the neuromuscular junction, also confirming restoration of the whole facial nerve tract from the reinnervated muscles, passing through the regenerated site to the brainstem. The collagen tube was very efficient as a nerve guide over a 5mm facial nerve gap and shows great promise as a nerve conduit.