Soichiro Itoh

SELF-ORGANIZATION MECHANISM IN A BONE-LIKE HYDROXYAPATITE/COLLAGEN NANOCOMPOSITE SYNTHESIZED IN VITRO AND ITS BIOLOGICAL REACTION IN VIVO

When bone is lost due to injury and/or illness, the defects are generally filled with natural bone because artificial bone materials have problems of bioaffinity. However, natural bone also has supply and infection problems. If an artificial material has the same biological properties as bone, it can replace natural bone for grafting. We synthesized a hydroxyapaite (HAp) and collagen (Col) composite by a simultaneous titration coprecipitation method using Ca(OH)2, H3PO4 and porcine atelocollagen as starting materials. The composite obtained showed a self-organized nanostructure similar to bone assembled by the chemical interaction between HAp and Col. The consolidated composite by a cold isostatic pressure of 200 MPa indicated a quarter of the mechanical strength of bone. It also indicated the same biological properties as grafted bone: The material was resorbed by phagocytosis of osteoclast-like cells and conducted osteoblasts to form new bone in the surrounding area. This HAp/Col composite having similar nanostructure and composition can replace autologous bone grafts.

The biocompatibility and osteoconductive activity of a novel hydroxyapatite/collagen composite biomaterial, and its function as a carrier of rhBMP-2

A hydroxyapatite/type I collagen (HAp/Col) composite, in which the hydroxyapatite nanocrystals align along the collagen molecules, has been prepared. The biocompatibility, osteoconductive activity, and efficacy as a carrier of recombinant human bone morphogenetic proteins (rhBMPs) of this novel biomaterial were examined. The composite material was implanted in the backs of Wistar rats, and specimens were collected for histological observations until week 24. In a second experiment, other samples of the composite material (5 x 5 x 10 mm3) were drilled and immersed in a solution of rhBMP-2 (0, 200, 400 microg/mL), and subsequently grafted in radii and ulnae in beagle dogs. As a control, three unfilled holes were left in one radius and ulna. X-ray images were prepared, and specimens collected for histological observation at weeks 8 and 12. Histological findings of the composites grafted in rats showed that the surface of the material was eroded as a result of macrophage infiltration. X-ray images and histological findings for the composites implanted in dogs support the idea that HAp/ Col has a high osteoconductive activity and is able to induce bone-remodeling units. In cases where the implants are grafted at weight bearing sites, treatment with rhBMP-2 at a dose of 400 microg/mL may be useful to shorten the time needed until bone union has occurred.

Development of an artificial vertebral body using a novel biomaterial, hydroxyapatite/collagen composite.

Hydroxyapatite/collagen (HAp/Col) composites having a bone-like nanostructure were synthesized and shaped into implants. This study was designed to develop an artificial vertebra system using this novel implant for anterior fusion of the cervical spine. Anterior fusion was carried out on 6 beagle dogs with the implants adsorbing rhBMP-2 (400 microg/ml). and 9 dogs with the implants without rhBMP-2. In 3 dogs of the rhBMP-treated group, as well as 6 dogs of the non-rhBMP-treated group, the implant was fixed with a poly-L-lactide plate and 2 titanium screws. Implants were taken out after 13 weeks from each 3 dogs in the rhBMP(-):plate(-). rhBMP(-):plate(+) and rhBMP(+):plate(+) groups. Also, the implants were removed from each 3 dogs in the rhBMP(-):plate(+) and rhBMP(+):plate(+) groups after 24 weeks. Histological and radiographical analysis suggested that since the larger part of the composite material was absorbed within 13 weeks, reduction of the intervertebral distance was caused, and that enhancement of callus formation and bone bridging by rhBMP-treatment was effective to prevent collapse of the implant, even though an effect of anterior plate-fixation was not obvious. The HAp/Col implant adsorbing rhBMP-2 may be a suitable replacement for the existing ceramics in anterior interbody fusion of the cervical spine.

Hydroxyapatite-coated tendon chitosan tubes with adsorbed laminin peptides facilitate nerve regeneration in vivo.

On the inner surface of tendon chitosan tubes having a triangular shape and a hydroxyapatite coating (t-chitosan/HAp tube), laminin-1 and laminin peptides (YIGSR, IKVAV) have been adsorbed in order to develop nerve growth conduits. The mechanical property, biocompatibility and efficacy of these tubes for nerve regeneration were examined. Step-1: bridge grafting (15 mm) into the sciatic nerve of Sprague-Dawley (SD) rats was carried out using either t-chitosan or t-chitosan/HAp tubes having either a circular or triangular cross section (N=12 in each group). Specimens were taken after 2-, 4-, 6- and 8-week post-implantation (N=3 in each group) for histology determinations. Step-2: t-chitosan/HAp tubes having a triangular cross section with adsorbed laminin-1, CDPGYIGSR or CSRARKQAASIKVAVSAD, as well as control tubes without pre-adsorption were used for implantation (N=18 in each group). Isografting was also carried out (N=6). Histological evaluation was carried out similarly as in Step-1. Furthermore, evoked muscle and sensory nerve action potentials were recorded, and the percentage of myelinated axon area measured at 10 mm distance of the distal anastomosed site in the experimental, control and isograft groups after 12 weeks (N=6 in each group). The results of histological findings, as well as mechanical properties, suggest that a triangular tube shape with a HAp coating benefits nerve regeneration. The effect of laminin peptides (YIGSR, followed by IKVAV) to enhance the growth of regenerating axons has been found comparable with intact laminin-1. Although histological regeneration in both the YIGSR- and laminin-1-treated t-chitosan/HAp tubes matches the isografts, the functional recovery is however delayed.

Evaluation of cross-linking procedures of collagen tubes used in peripheral nerve repair.

Three different cross-linking methods were compared to prepare collagen tubes, namely irradiation by ultraviolet (UV), heating and immersing in glutaraldehyde (GA). Bridge grafting of 15 mm was carried out with these tubes, as well as with non-cross-linked collagen tubes for comparison, in a defect of rat sciatic nerves (N = 21 in each group). As a control, isografting was carried out (N = 6). The specimens were taken from the grafted site in each experimental group for histological observation after, respectively 1, 2, 4, 6 and 8 weeks (N = 3 each). Evoked muscle action potentials were recorded on the calf muscle in the experimental and control groups after 12 weeks, and the grafted material and tibial nerve were harvested for histological analysis (N = 6). The inner space of UV-irradiated tubes was preserved with almost no cell infiltration and nerve regeneration matching for isograft was obtained. Rapid degradation of the heat-treated tubes occurred and many macrophages were mobilized to remove the collagen debris. The non-treated tube swelled and the regenerated nerve tissue in the tube was constricted with time. The GA-immersed tubes showed less cellular activity and poor regenerated nerve tissue compared with the other cross-linking methods. Therefore, UV irradiation to collagen tubes is recommended as a cross-linking method for nerve conduit.

The chitosan prepared from crab tendons: II. The chitosan/apatite composites and their application to nerve regeneration

The chitosan tubes derived from crab tendons form a hollow tube structure, which is useful for nerve regeneration. However, in order to use the chitosan tubes effectively for nerve regeneration, there remain two problems to be solved. First, the mechanical strength of the tubes is quite high along the longitudinal axis, but is somewhat low for a pressure from side. Second, the chitosan tube walls swell to reduce the inner space of the tubes in vivo. These two problems limit the clinical use of the chitosan tubes. In this study, to solve the problems, apatite was made to react with the chitosan tubes to enhance the mechanical strength of the tube walls. Transmission electron microscopy showed that apatite crystals were formed in the walls of the chitosan tubes. The c-axis of the crystals aligned well in parallel with chitosan molecules. These results indicate that the apatite crystals grow in the tubes starting from the nucleation sites of the chitosan molecules, probably by forming complexes with amino groups of chitosan and calcium ions. Further, the tubes were thermally annealed at 120 degrees C to prevent from swelling, and simultaneously formed into a triangular shape to enhance the stabilization of the tube structure. By these treatments, the hollow tubes could keep their shape even in vivo after implantation. Animal tests using SD rats further showed that the chitosan tubes effectively induced the regeneration of nerve tissue, and were gradually degraded and absorbed in vivo.

Effect of Electrical Polarization of Hydroxyapatite Ceramics on New Bone Formation

Large surface charges can be induced on hydroxyapatite (HAp) ceramics by proton transport polarization, but this does not affect β-tricalcium phosphate (TCP) because of its low polarizability. We wished to examine differences in osteogenic cell activity and new bone growth between positively or negatively surface-charged HAp and HAp/TCP plates using a calvarial bone defect model. In the first group of rats, test pieces were placed with their positively charged surfaces face down on the dura mater. In the second group, test pieces were placed with their negatively charged surfaces face down on the dura mater. A third group received noncharged test pieces. Histological examination, including enzymatic staining for osteoblasts and osteoclasts, was carried out. While no bone formation was observed at the pericranium, direct bone formation on the cranial bone debris and new bone growth expanded from the margins of the sites of injury to bridge across both the positively and negatively charged surfaces of HAp and HAp/TCP plates occurred. Electrical polarization of implanted plates, including positive charge, led to enhanced osteoblast activity, though decreased osteoclast activity was seen on the positively charged plate surface. Thus, polarization of HAp ceramics may modulate new bone formation and resorption.

Tendon chitosan tubes covalently coupled with synthesized laminin peptides facilitate nerve regeneration in vivo

We have developed tendon chitosan tubes having the ability to bind peptides covalently, and the effectiveness of laminin peptides coupled to these tubular wall on nerve regeneration was examined in vivo. Bridge graft implantation (15 mm) into the sciatic nerve of SD rats was carried out using chitosan tubes having a triangular cross section containing either covalently bound intact laminin or the laminin peptides CDPGYIGSR or CSRARKQAASIKVAVSAD or being nontreated (N = 20 in each group). As a control, isografting (N = 5) was carried out. Three rats in each experimental group were sacrificed for histology observations after 1, 2, 4, 6, and 8 weeks. The total area of regenerating tissue in the tube and the length of the area where regenerating tissue attached to the inner surface of the tube were measured. In five rats from each experimental and control group, the latency quotient between the implanted and the nontreated site was determined 12 weeks after implantation. Furthermore, the percentage of myelinated axon area was measured at a 10‐mm distance from the distal anastomosed site. Histological findings suggest that the immobilized laminin, confirmed by immunostaining as long as 12 weeks postoperatively, as well as laminin oligopeptides may effectively assist nerve tissue extension. According to statistical analysis of the percentage neural tissue found in relation to evoked action potentials, the sequential treatments with YIGSR first followed by IKVAV matched the effectiveness of intact laminin in enhancing nerve regeneration. However, when compared with that after isografting, the enhancement of regenerated axon growth was less sufficient.

The chitosan prepared from crab tendon I: the characterization and the mechanical properties

Crystalline chitosan was prepared from crab tendon consisting mainly of chitin, including various proteins and calcium phosphates. The crab tendon has high mechanical properties due to its aligned molecular structure. Crab tendon components, i.e. proteins and calcium phosphates, were removed by deacetyl treatment using 50wt% NaOH aqueous solution at 100 degrees C, and a subsequent ethanol treatment. As judged from microscopic observations using an optical polarizer, the treated chitosan remained intact regarding its aligned molecular structure, and had a high tensile strength of 67.9+/-11.4MPa. The tensile strength was further enhanced to 235+/-30MPa by a thermal treatment at 120 degrees C, corresponding to the formation of the intermolecular hydrogen bonds.

Development of a hydroxyapatite/collagen nanocomposite as a medical device.

The effect of cross-linking of a hydroxyapatite/collagen (HA/Col) nanocomposite, in which HA nanocrystals and collagen fibers are aligned like natural bone by a self-organization mechanism between HA and collagen in vitro, on mechanical properties was examined. The influence of degree of cross-linking, as well as rhBMP-2 preadsorption to the composite on the substitution pattern and rate with bone, was examined. In Experiment 1, anterior fusion was carried out at the C3–C4 vertebrae on 10 dogs and they were implanted as follows: without cross-linking and without adsorbed rhBMP-2 (three dogs), with cross-linking and without adsorbed rhBMP-2 (three dogs), without cross-linking and with adsorbed rhBMP-2 (two dogs), and with cross-linking and adsorbed rhBMP-2 (two dogs). Implants were removed from each dog for histology determinations after 12, 16, and 24 weeks in the non-rhBMP-treated groups, and after 16 and 24 weeks in the rhBMP-treated groups. In Experiment 2, the HA/Col composites with cross-linking and both with and without rhBMP-2 pretreatment were implanted into a bone defect of 20 mm made in the central part of tibiae in dogs (N = 3 in each group). As a control, bone defects of 20 mm remained without implantation (N = 3). The dogs were allowed to walk using an Ilizarov extra skeletal fixator. The implants were removed after 12, 16, and 24 weeks from one dog in each group. The cross-linking of the HA/Col composite was effective in controlling both the mechanical strength and bioresorbability. A “self-organization process” on the HA/Col implant surface resulted in the formation of bone remodeling units in and around the implant. Radiographic and histological findings suggest that a combined treatment of cross-linking of the HA/Col composite with preadsorption of rhBMP-2 molecules may be a very suitable replacement of existing ceramic systems in the anterior fusion of the cervical spine, as well as inlay grafting of bone defects in weight-bearing sites.