Yasuhiko Shimizu

Novel Approach to Regeneration of Periodontal Tissues Based on in Situ Tissue Engineering: Effects of Controlled Release of Basic Fibroblast Growth Factor from a Sandwich Membrane

To regenerate periodontal tissues, a sandwich membrane composed of a collagen sponge scaffold and gelatin microspheres containing basic fibroblast growth factor (bFGF) in a controlled-release system was developed according to the new concept of in situ tissue engineering. A three-walled alveolar bone defect (3 x 4 x 4 mm) was made bilaterally in edentulous regions created mesially to the canines in both the maxilla and mandible of nine beagle dogs. A sandwich membrane with or without bFGF (100 microg) was implanted in each defect (each group, n = 18). During weeks 1, 2, and 4, histologic evaluation and histometric analyses were performed on three dogs. Throughout the 4 weeks, vascularization and osteogenesis were active only in the bFGF-treated group (p < 0.01). New cementum was formed (2.4 +/- 0.9 mm) on the exposed root surface at 4 weeks, and functional recovery of the periodontal ligament was indicated in part by the perpendicular orientation of regenerated collagen fibers. In the control group, epithelial downgrowth and root resorption occurred and the defects were filled with connective tissue. Thus, our sandwich membrane induced successful regeneration of the periodontal tissues in a short period of time.

Use of collagen sponge incorporating transforming growth factor-β1 to promote bone repair in skull defects in rabbits

The objective of this study was to evaluate the potential of collagen sponge incorporating transforming growth factor-beta1 (TGF-beta1) to enhance bone repair. The collagen sponge was prepared by freeze-drying aqueous foamed collagen solution. Thermal cross-linking was performed in a vacuum at 140 degrees C for periods ranging from 1 to 48 h to prepare a number of fine collagen sponges. When collagen sponges incorporating 125I-labeled TGF-beta1 were placed in phosphate-buffered saline (PBS) solution at 37 degrees C, a small amount of TGF-beta1 was released for the first hour, but no further release was observed thereafter, irrespective of the amount of cross-linking time the sponges had received. Collagen sponges incorporating 125I-labeled TGF-beta1 or simply labeled with 125I were implanted into the skin on the backs of mice. The radioactivity of the 125I-labeled TGF-beta1 in the collagen sponges decreased with time; the amount of TGF-beta1 remaining dependent on the cross-linking time. The in vivo retention of TGF-beta1 was longer in those sponges that had been subjected to longer cross-linking times. The in vivo release profile of the TGF-beta1 was matched with the degradation profile of the sponges. Scanning electron microscopic observation revealed no difference in structure among sponges subjected to different cross-linking times. The TGF-beta1 immobilized in the sponges was probably released in vivo as a result of sponge biodegradation because TGF-beta1 release did not occur in in vitro conditions in which sponges did not degrade. We applied collagen sponges incorporating 0.1 microg of TGF-beta1 to skull defects in rabbits in stress-unloaded bone situations. Six weeks later, the skull defects were covered by newly formed bone, in marked contrast to the results obtained with a TGF-beta1 free empty collagen sponge and 0.1 microg of free TGF-beta1. We concluded that the collagen sponges were able to release biologically active TGF-beta1 and were a promising material for bone repair.

Tissue engineering of the small intestine by acellular collagen sponge scaffold grafting.

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.

A new acellular vascular prosthesis as a scaffold for host tissue regeneration

OBJECTIVEnIn situ tissue engineering using acellular xenografts is a new approach to autologous tissue regeneration. In the present study, we analyzed a new regeneration scaffold comprised of xenogenic acellular vessels in a dog model.nnnDESIGN OF STUDY AND RESULTSnXenogenic vascular conduits of porcine carotid arteries were acellularized by a 48-hour detergent incubation to extract all the cell components of the graft. The acellularisation procedure resulted in a complete removal of all cells. After this procedure, heparinization was performed sequentially to induce antithrombogenicity in the graft. The burst pressure of the graft was tested. The bursting pressure of the fresh vessels was 2.83 +/- 0.56 x 10(3) mmHg (377 +/- 75kPa, n = 6). After detergent treatment, the bursting pressure of the grafts was 2.50 +/- 0.48 x 10(3) mmHg (334 +/- 63 kPa, n = 6); after heparinization, it was 3.44 +/- 0.38 x 10(3) mmHg (459 +/- 51 kPa, n = 5). In vivo function was tested in a dog model by transplantation to the abdominal artery. By 18 weeks, endothelial cells were aligned as in normal, healthy artery over the surface of the entire graft. Fibroblasts and macrophages had infiltrated the graft from both inside and outside. The neointima contained normal layers of smooth muscle cells, which were identified by anti alpha-smooth muscle fiber antigen staining.nnnCONCLUSIONSnThe acellular heparinized xenograft has sufficient mechanical properties and was successfully replaced with host tissue, and is thus a promising new type of vascular prosthesis.

Capillary endothelial thrombomodulin expression and fibrin deposition in rats with continuous and bolus lipopolysaccharide administration.

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.

Chromosome 13 locus, Pbd2, regulates bone density in mice.

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.

Recurrent laryngeal nerve regeneration by tissue engineering

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

Mechanism of burn injury during magnetic resonance imaging (MRI)--simple loops can induce heat injury.

To determine the mechanism of burn injury associated with magnetic resonance imaging (MRI), the induced current in the loops of a conductive lead was measured. Seven types of loops with effective areas within the range from 100 to 12 000 cm2 were made and then each loop was placed in the bore of a whole-body MR system at 0.5 T and MRI was carried out. During radio frequency (RF) irradiation, an induced voltage was observed in loops that were placed with their axes parallel to the linearly polarized transmitting RF field. The voltage had a sincfunction characteristic and was within the range 55-235 V at the RF pulse sequence for usual MR imaging. When the axis was vertical, negligible current was observed. A resistor inserted into the circuit of a 30 x 40 cm loop sparked and burned out. Simple loops of conductive material may result in the induction of a large and potentially hazardous voltage in the imaging system. Self-resonance of such a loop may add greatly to hazards by increasing the effective coupling to the RF transmitter. Also, impedance matching of the inserted resistor with the impedance of the loop increases the dissipated power at the resistor.

Mechanical properties of artificial tracheas composed of a mesh cylinder and a spiral stent.

Much work has been done on the materials used for mesh-type artificial tracheas, but a precise mechanical evaluation of these structures has not yet been performed. In the present study, we determined the mechanical properties of typical mesh-type artificial tracheas and compared them with those of native trachea. Four types of artificial trachea were made and used for the mechanical tests. The basic frame of all the specimens was composed of a mesh cylinder and a spiral stent. The specimen whose mesh was sealed with collagen sponge showed almost the same behavior in the force-strain curve under compression, suggesting that collagen sealing has little effect on mechanical properties. Agreement between measured and estimated mechanical properties was good, especially in the low strain region, suggesting that artificial tracheas can be designed in terms of mechanical properties by mainly considering the basic frame structure.

Repairing of rabbit skull defect by dehydrothermally crosslinked collagen sponges incorporating transforming growth factor β1

Collagen sponges of various biodegradabilities were prepared by dehydrothermal crosslinking at 140 degrees C for different time periods. When the collagen sponges were radioiodinated and implanted subcutaneously into the back of mice, the radioactivity remaining at the implanted site decreased with time; the longer the time of dehydrothermal crosslinking, the slower the radioactivity decrement. The radioactivity following the subcutaneous implantation of collagen sponges incorporating (125)I-labeled transforming growth factor (TGF)-beta1 also decreased with time. The time profile of both the radioactivity remainings was in good accordance to each other, irrespective of the crosslinking time. This indicates that the TGF-beta1 incorporated in the sponges was released as a result of sponge biodegradation. Potential of collagen sponges incorporating 0.1 micro g of TGF-beta1 in repairing the defect of rabbit skulls was evaluated in a stress-unloaded state. Bone repairing was induced by application of the collagen sponges incorporating 0.1 micro g of TGF-beta1 whereas that of free TGF-beta1 at the same dose and TGF-beta1-free, empty collagen sponges were ineffective. The bone defect was histologically closed by the bone tissue newly formed 6 weeks after application. Bone mineral density (BMD) analysis revealed that the collagen sponge incorporating TGF-beta1 enhanced the BMD value at the bone defect to a significantly great extent compared with other agents. A maximum enhancement of BMD was observed for the collagen sponge incorporating TGF-beta1 which was prepared by dehydrothermal crosslinking for 6 h. It was concluded that the TGF-beta1 incorporated in the collagen sponge was released in a biologically active form as a result of sponge biodegradation, resulting in enhanced bone repairing at the skull defect. It is possible that for too slowly degraded sponges, the remaining physically impairs the bone repairing at the skull defect. Induction of bone repairing would not be achieved through a rapid release of TGF-beta1 from too fast-degraded sponge.