L-Asparaginase from Escherichia coli was coupled with two types of comb-shaped copolymer of poly-(ethylene glycol) derivative and maleic anhydride (activated PM), having molecular weights of 13,000 and 100,000 (activated PM13 and PM100, respectively) with multivalent reaction sites. After single intravenous injections of PM100-asparaginase and nonmodified asparaginase into rats, the enzymic activity of PM100-asparaginase in serum was well retained for at least 11 days, and the serum L-asparagine concentration remained undetectable for 27 days. The half-lives of PM100-asparaginase and nonmodified asparaginase were 50 and 1.5 h, respectively. Stabilization of L-asparaginase toward heat, urea, and acidity was caused by modifying the enzyme with activated PM13 and PM100. Especially, PM100-asparaginase retained high enzymic activity toward heat and urea, compared with PM13-asparaginase. It was suggested that these modifiers with a comb-shaped form and with multivalent reactive sites cover the whole surface of the asparaginase molecule and stabilize its conformation possibly through multiple covalent bindings and through various noncovalent interactions.

Animals↗

PubMedDate not supplied

The essential light chains constitute part of the active site of smooth muscle myosin.

Myosin, a major contractile protein, characteristically possesses a long coiled-coil alpha-helical tail and two heads. Each head contains both an actin binding site and an ATPase site and is formed from the NH2-terminal half of one of the two heavy chains (relative molecular mass, Mr, 200,000) and a pair of light chains; the so-called regulatory and essential light chains of approximately Mr 20,000 each. Recently we have identified Trp 130 of the myosin heavy chain from rabbit skeletal muscle as an active-site amino-acid residue after labelling with a new photoaffinity analogue of ADP, N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate (NANDP). Nonspecific labelling was eliminated by first trapping NANDP at the active site with thiol crosslinking agents. Exclusive labelling of the heavy chains with no labelling of the light chains agreed with previous findings that the heavy chains alone contain the actin-activated Mg-ATPase activity of rabbit skeletal myosin. Here we report similar photolabelling experiments with smooth muscle myosin (chicken gizzard) in which 3H-NANDP is trapped at the active site with vanadate and which show that both the heavy chains and the essential light chains are labelled. The results indicate that both chains contribute to the ATP binding site and represent the first direct evidence for participation of the essential light chains in the active site of any type of myosin.

Animals↗

PubMedDate not supplied

Replacement of the tracheobronchial bifurcation by a newly developed Y-shaped artificial trachea.

We have previously reported on a straight artificial trachea developed by our group that can be applied to the cervical and thoracic trachea. In this study, a new Y-shaped artificial trachea was designed and adapted for experimental replacement of defective tracheal bifurcations in 20 dogs. The Y-shaped Marlex mesh tube was reinforced with a polypropylene spiral and coated with collagen made from porcine skin. This coating process makes the prosthesis biocompatible and airtight. Replacement of the tracheobronchial bifurcation was performed through a right thorachotomy, and omentopexy was added in all 20 dogs. Six of the 20 dogs have survived. The causes of death of the other 14 dogs were obstruction of the main bronchus (one dog), omental necrosis (three dogs), and air leakage from the prostheses and suture points (10 dogs). In all six surviving dogs, the artificial tracheas are covered with regenerated tissue and there is no evidence of stenosis or dehiscence. These results indicate that the prosthesis can be effective and safe in the long-term; however, air leakage from the prosthesis is a serious complication. Therefore, a better method is needed to make the prosthesis airtight to improve the postoperative result.

Animals↗

PubMedDate not supplied

A novel surgical material made from collagen with high mechanical strength: a collagen sandwich membrane.

We have developed a novel collagen membrane with excellent mechanical properties using a new method and examined its breaking strength, microstructure, and absorption behavior in vivo. Types I and III atelopeptide collagen extracted from porcine skin by treatment with pepsin were used. One volume percent collagen hydrochloride solution, pH 3.0, was frozen in a cast, then freeze-dried and pressed. Both surfaces of the resulting membrane were coated with 2% w/v collagen hydrochloride solution, pH 3.0, and dried. This procedure was then repeated 10 times, and the membrane was finally heated in vacuo. Microstructural observation showed that the inner part of the membrane consisted of piles of thin collagen nonwoven fabric layers, whereas the coated surface consisted of an amorphous collagen layer. The breaking strength of this membrane with a thickness of 0.5 mm was 73.5 N/cm in dry conditions. The mechanical strength of the membrane was 3.4 to 10.8 times greater than that of control membranes that had not been subjected to coating procedures. The absorption of this membrane was investigated by an implantation test in rabbits. The absorption rate of the membrane in vivo was similar to that of the noncoated control material, and only minimal tissue reaction was evident. Because the mechanical properties of this membrane, made from only collagen, are adequate for suturing without reinforcement by other materials, and its absorption in vivo has so far been equal to that of collagen membranes, the membrane is potentially applicable for various surgical uses such as replacement of tissue defects.

Absorption↗

PubMedDate not supplied

Experimental replacement of the thoracic esophagus with a bioabsorbable collagen sponge scaffold supported by a silicone stent in dogs.

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.

Animals↗

PubMedDate not supplied

Use of a newly developed artificial nerve conduit to assist peripheral nerve regeneration across a long gap in dogs.

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.

Action Potentials↗

PubMedDate not supplied

Collagen coated Y-shaped prosthesis for carinal replacement promotes regeneration of the tracheal epithelium.

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.

Animals↗

PubMedDate not supplied

A new tracheal bioartificial organ: evaluation of a tracheal allograft with minimal antigenicity after treatment by detergent.

The aim of this study was to reduce the antigenicity of tracheal allografts by detergent treatment. We attempted to apply these grafts to tracheal immunosuppressant-free allotransplantation. Fresh tracheal grafts were harvested from donor beagle dogs and treated with a detergent at 4 degrees C for 48 hours. By using treated grafts, we have performed tracheal immunosuppressant-free transplantation in six dogs at an intrathoracic five-ring defect. Implanted grafts were covered with an omental pedicle. In five of the six grafts, complete removal of the epithelium and mixed glands was recognized with both light microscopy and scanning electron microscopy. The appearance of the cartilage cells in the grafts was similar to those in fresh trachea. Five dogs that received detergent treated grafts survived uneventfully. The grafts had been incorporated by the host trachea without stenosis. On the one tracheal graft in which removal of the epithelium was incomplete (noted after implantation), moderate stenosis occurred 1 month after placement. These results suggest that removal of the tracheal epithelium and mixed glands can remarkably reduce tracheal antigenicity. A tracheal graft that has its epithelium and mixed glands removed can be used in tracheal immunosuppressant-free allotransplantation.

Animals↗

PubMedDate not supplied

Intrathoracic esophageal replacement with a collagen sponge--silicone double layer tube: evaluation of omental-pedicle wrapping and prolonged placement of an inner stent.

In a previous study, we replaced a 5 cm gap created in the canine intrathoracic esophagus with an artificial esophagus. However, although newly formed esophageal tissue subsequently bridged the gap, mild stenosis occurred, and this seemed to be caused by inadequate regeneration of the skeletal muscle. In the present study, we evaluated whether omental pedicle wrapping (OMPx) of the prosthesis could promote tissue regeneration and whether prolonged retention of the silicone tube within the prosthesis could prevent stenosis. A gap was created in 14 dogs, and the defect was repaired by our prosthesis. OMPx was performed in 5 of the 14 dogs (OMPx group) but not in the rest (control group). The silicone tube was retained for 4 weeks in the control group and for 8 weeks in the OMPx group. All of the dogs in the control group survived for more than 3 months, except for those that were killed. Four dogs in the OMPx group died within 3 months, one caused by perforation at 7 months. Only the thin epithelial and submucosal layer regenerated in the OMPx group. OMPx is not effective for promoting tissue regeneration, and prolonged retention of the silicone tube interrupts epithelial regeneration.

Animals↗

PubMedDate not supplied

A gelatin coated collagen-polyglycolic acid composite membrane as a dural substitute.

We designed a new biodegradable composite membrane for use as a dural substitute. This membrane is composed of polyglycolic acid (PGA) mesh, collagen sponge, and gelatin sponge. Collagen layers are attached on both sides of the PGA mesh. One side of the membrane is coated with freeze-dried gelatin to prevent tissue adhesion. We examined the mechanical properties of the membrane and evaluated its efficacy in vivo by implanting it into dogs. When the mechanical properties of the membrane were measured under wet conditions, tensile strength and resistance against cutting by a suture were approximately 20 N/cm and 13 N, respectively, which are adequate for a surgical material. In the in vivo experiment, our dural substitute was implanted into six adult beagle dogs to repair a 25 x 15 mm defect in the dura mater. Two dogs each were killed at 2 and 4 months after implantation and two were observed over 6 months. At 4 months after implantation, the implanted dural substitute was almost absorbed and a fibrous membrane resembling native host dura had regenerated at the site. No foreign body reaction or significant adhesion to the cerebral cortex was observed. These results indicate the clinical potential of our novel dural substitute.

Absorbable Implants↗