Seijun Fukuda

Experimental study on the regeneration of peripheral nerve gaps through a polyglycolic acid–collagen (PGA–collagen) tube

We have developed a bioabsorbable polyglycolic acid (PGA) tube filled with collagen sponge (PGA-collagen tube) as a nerve connective guide, and compared its effectiveness with that of autograft in terms of nerve regeneration across a gap. The PGA-collagen tube was implanted into 24 beagle dogs across a 15-mm gap in the left peroneal nerve. The right peroneal nerve was reconstructed with the autograft harvested from the left side, as a control. After the surgery, the connective tissue extended from both cut ends in the PGA-collagen tube and connected again at the center. Pathologically, the collagen sponge in the tube provided adequate scaffolding for nerve tissue extension, and the nerve tissue reconnected within 3 weeks. Electrophysiologically, muscle-evoked potentials (MEPs) and compound nerve action potentials (CNAPs) were detected 18 days after the surgery. For up to 6 months postsurgery, CNAPs and somatosensory-evoked potentials (SEPs) on the PGA-collagen side had a shorter latency and larger peak voltage than those on the autograft side. The myelinated axons on the PGA side were larger in diameter than those on the autograft side. It is suggested that the PGA-collagen tube has the potential to be an effective alternative to conventional autografting for the repair of some peripheral nerve defects.

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.

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 × 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.

Regeneration of Central Nervous Tissue Using a Collagen Scaffold and Adipose-Derived Stromal Cells

Adipose-derived stromal cells (ASCs) include stem cells, which have the potential to differentiate into a variety of cell lineages. The regeneration of central nerves was examined using ASCs and a collagen scaffold. A cerebral cortex defect (3 × 4 × 3 mm3) was created in the left frontal lobe of 16 male rats. In one group (n = 8), collagen (3 × 4 × 3 mm3) seeded with DiI-labeled ASCs was implanted in the defect. In order to seed the ASCs, a combination of the rotary cell culture system and pressing the collagen scaffold gently several times with a glass rod was applied. In the control group (n = 8), collagen was implanted without ASCs. The rats were sacrificed at 1 month after the scaffold implantation. Histologically, 0.2% of the implanted ASCs were positive for anti-human/rat microtubule-associated protein 2 (MAP2) antibody and microvessels were present at a density of 4.6 ± 1.2/mm2 within the collagen scaffold-implanted area in each coronal section. In the control group, no MAP2-positive cells were detected and the microvessel density was 0.6 ± 0.4/mm2. These data suggest that ASCs seeded into a collagen scaffold may have the potential to promote regeneration of nervous tissue after cerebral cortex injury.

Influence of dehydrothermal crosslinking on the growth of PC-12 cells cultured on laminin coated collagen.

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 biomaterials treated with trophic factors such as laminin should be examined in terms of the effects of crosslinking on the activity of the trophic factors.

Tracheal allotransplantation in beagle dogs without immunosuppressants.

BACKGROUND The antigenicity of tracheal grafts is still unclear. We investigated the possibility of performing tracheal allotransplantation without immunosuppressants. METHODS Intrathoracic five-ring tracheal replacements were performed in beagle dogs without immunosuppressants (n = 18). The dogs were divided into 9 pairs, and grafts were exchanged within the pairs. In group 1 (n = 6), the paired dogs were blood relatives, whereas in group 2 (n = 12), the paired dogs were not related. Full-thickness skin transplantation was also performed in both groups. RESULTS In group 1, 5 animals survived uneventfully for more than 3 months. No stenosis was observed in any of the dogs. In group 2, the grafts were incorporated by the host trachea in 2 dogs. Four animals died of airway obstruction within 3 months. Moderate or slight airway stenosis was observed in 6 dogs. Rejection was confirmed by histologic examination. In both groups, all of the skin allografts were destroyed within 2 weeks. CONCLUSIONS After tracheal allotransplantation, long-term survival was achieved, especially in recipient dogs that were blood relatives of donors. We conclude that it is possible to perform tracheal allotransplantation using histocompatible matched grafts without immunosuppressants.

Regeneration of the junctional epithelium and connective tissue after transplantation of detergent-processed allo-teeth.

The authors have developed a new artificial dental implant and evaluated it in a dog model in terms of its potential to produce: I) regeneration of junctional epithelium; II) regeneration and attachment of connective tissue. The implants were constructed from allo-teeth. We removed the cell components from the periodontal ligaments of these teeth with a detergent (1% TritonX-100); the remaining acellular periodontal ligament acted as an extracellular matrix upon which regeneration and attachment could proceed. We placed 10 of these implants in the just-extracted sites of three beagle dogs. We observed regeneration of both junctional epithelium and connective tissue at all implant sites after 3 months. The connective tissue was attached in all cases. Use of the acellular periodontal ligament as an extracellular matrix may facilitate regeneration of host periodontal ligament tissue, thus contributing to recovery of host immunological defense and long-term oral function.

Contents Vol. 190, 2009

F. Beck, Leicester A.L. Boskey, New York, N.Y. R.C. Burghardt, College Station, Tex. G. Burnstock, London F. Eckstein, Salzburg A.C. Enders, Davis, Calif. C. Farnum, Ithaca, N.Y. R.H.W. Funk, Dresden N.E. Fusenig, Heidelberg A. Gibson, Phoenix, Ariz. M. Glickstein, London J.W. Hermanson, Ithaca, N.Y. C.J. Kirkpatrick, Mainz P. Köpf-Maier, Berlin W. Kummer, Giessen J.W. Lichtman, Cambridge, Mass. K.G. Marra, Pittsburgh, Pa. O. Ohtani, Toyama P.J. Reier, Gainesville, Fla. R. Roy, Los Angeles, Calif. R. Segal, Chapel Hill, N.C. F. Sinowatz, Munich M. Sittinger, Berlin T. Skutella, Tübingen G.B. Stark, Freiburg i.Br. E. Th ompson, Melbourne C.G. Widmer, Gainesville, Fla. in vivo, in vitro