Kyoko Suzuki

Alginate hydrogel linked with synthetic oligopeptide derived from BMP-2 allows ectopic osteoinduction in vivo

Bone morphogenetic proteins (BMP) are unique molecules with a specific biological activity for inducing ectopic bone formation when implanted with a suitable carrier matrix. However, incorporation of BMP into the carrier has disadvantages, including early burst release and protein degradation in biological environments. Therefore, we considered that the next greatest challenge in achieving successful clinical use was the development of a carrier system for site-specific delivery of the morphogenetic signal of BMP. In this study, a novel BMP-2-derived oligopeptide, NSVNSKIPKACCVPTELSAI, was coupled covalently to alginate. Then NSVNSKIPKACCVPTELSAI-linked alginate hydrogel composites were implanted into the calf muscle of rats and harvested 3 or 8 weeks after surgery. Ectopic bone formation was observed in alginate hydrogel linked with BMP-2-derived peptide. It is suggested that alginate hydrogel linked with an oligopeptide derived from BMP-2 might provide an alternative system for topical delivery of the morphogenetic signal of BMP-2.

Cat peripheral nerve regeneration across 50 mm gap repaired with a novel nerve guide composed of freeze-dried alginate gel

We have developed a novel artificial nerve guide composed of biodegradable freeze-dried alginate gel covered by polyglycolic acid mesh, and evaluated its effect on peripheral nerve regeneration, using a 50-mm gap cat sciatic nerve model. Functional reinnervation of motor and sensory nerves occurred 13 weeks after implantation, as demonstrated by recovery of compound muscle action potential (CMAP) and somatosensory evoked potential (SEP). For histologic evaluation, samples of tissue were harvested from the grafted material segment 7 months after operation. Many newly developed nerve fasciculi were found, and the implanted nerve guidance material had completely disappeared with little inflammation. These results indicate that freeze-dried alginate gel allows the nerve to regenerate across longer gaps than described in previous literature.

Evaluation of a novel alginate gel dressing : Cytotoxicity to fibroblasts in vitro and foreign-body reaction in pig skin in vivo

Calcium alginate dressings have beneficial effects on wound healing by providing a moist wound environment. However, cytotoxicity and the nonbiodegradable nature of calcium alginate dressings induce unresolved chronic foreign-body reaction. In this study, a novel freeze-dried alginate gel dressing (AGA-100) low in calcium ions was evaluated for cytotoxicity to L929 cells in vitro and in full-thickness pig wounds in vivo. Cytotoxicity testing on L929 cells showed the cytocompatibility of AGA-100 extracts, while extracts from Kaltostat, a well-established alginate dressing, induced cytopathic effects. In an in vivo study using pigskin, AGA-100, Kaltostat, and gauze were applied on 1-in-diameter circular full-thickness wounds on the back of pigs and the time course of wound closure was evaluated. Kaltostat and gauze dressings were used as controls. For histologic evaluation, wound tissue was harvested on day 18. AGA-100-treated wounds showed rapid wound closure compared to control wounds on day 15. Foreign-body reaction was marked in Kaltostat- and gauze-treated wounds, and differed significantly from AGA-100-treated wounds. Based on these data, AGA-100 could reduce the cytotoxicity to fibroblasts and foreign-body reaction that have been observed with currently available calcium alginate dressings; it was also found to be useful as an alginate dressing.

A new drug delivery system with controlled release of antibiotic only in the presence of infection.

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.

Alginate, a bioresorbable material derived from brown seaweed, enhances elongation of amputated axons of spinal cord in infant rats

Freeze-dried alginate sponge crosslinked with covalent bonds was developed in our laboratory and has been demonstrated to enhance peripheral nerve regeneration. In this study, we examined spinal cord repair using alginate sponge in infant rats. On postnatal day 8-12, the spinal cord was transversely resected at Th7-Th8 to produce a 2-mm gap. The gap was filled with alginate sponge in the alginate group. For the control group, the gap was left empty. In the alginate group, the recovery of evoked electromyogram and sensory-evoked potentials 6 weeks after surgery indicated that elongation of axons could establish electrophysiologically functional projections through the gap. A histological study revealed that myelinated and unmyelinated axons, surrounded by a perineurial-like structure, had elongated across the gap. An immunohistochemical examination revealed that elongation of astrocytic processes and/or migration of astrocytes into the alginate sponge was induced, whereas astrocyte gliosis was reduced at the interface between the implanted alginate and the host spinal cord, compared with the control group. However, a horseradish peroxidase tracing study revealed ascending and descending fibers had also elongated into the gap and reentered the other stump of the transected spinal cord beyond the gap. These results suggest that alginate might provide a permissive microenvironment for elongation of spinal cord axons.

In vivo evaluation of a novel alginate dressing.

Alginate dressings are currently used in the management of epidermal and dermal wounds, and provide a moist environment that leads to rapid granulation and reepithelialization. However, a cytotoxic effect on proliferation of fibroblasts and residual material with inflammation in healing wounds have been reported recently. We have developed a new alginate dressing (AGA-100), which does not have an inhibitory effect on proliferation of fibroblasts. The purpose of this study was to evaluate the new alginate dressing with respect to wound healing in full- and partial-thickness pig wounds and with respect to biodegradation following implantation into rabbit muscle. Kaltostat and Sorbsan, both well-established commercial dressings, were used as control. The closure rate of full-thickness wounds treated with AGA-100 was significantly higher on day 15 compared with that with Kaltostat and Sorbsan. Reepithelialization rate of partial-thickness wounds treated with Sorbsan was statistically significantly lower on day 3 than those with the other two dressings. As to dressing debris remained in the healing wound, a large amount of foreign debris was noted in all the full-thickness wounds treated with Kaltostat or Sorbsan, while only about one-third of wounds treated with AGA-100 showed a little dressing debris. AGA-100 implanted into the muscle of rabbits was bioresorbed completely within 3 months. Therefore, dressing residue in AGA-100-treated full-thickness wounds might be fully absorbed in a few months. In conclusion, it is shown that our newly developed AGA-100 possesses superior properties compared with typical alginate dressings.

Peripheral nerve regeneration through alginate gel : Analysis of early outgrowth and late increase in diameter of regenerating axons

Our previous study revealed that alginate gel cross-linked with covalent bonds promoted peripheral nerve regeneration in the cat and rat. The present study analyzed nerve regeneration through alginate gel in the early stages within 2 weeks and the late stages up to 21xa0months after implantation. Four days after surgery, regenerating axons grew without Schwann cell investment through the partially degraded alginate gel, being in direct contact with the alginate without a basal lamina covering. Numerous mast cells infiltrated into the alginate. One to 2 weeks after surgery, regenerating axons were surrounded by common Schwann cells to form small bundles, with some axons at the periphery being partly in direct contact with alginate. At the distal stump, numerous Schwann cells had migrated into the alginate 8–14 days after surgery. They had no basal laminae. The diameter of regenerated myelinated fibers was small (approximately 1xa0μm) at 8 weeks, but increased in diameter, having a distribution pattern similar to that of normal nerve 21xa0months after surgery. Much better nerve regeneration was found in alginate gel-, than collagen sponge-, and fibrin glue-implanted distal stump 12xa0months after surgery. These results indicate that alginate gel has good biocompatibility for regenerating axon outgrowth and Schwann cell migration, and that regenerated fibers can have a diameter as thick as that of normal fibers in the long term. Alginate gel is a promising material for use as an implant for peripheral nerve regeneration.

Reconstruction of rat peripheral nerve gap without sutures using freeze-dried alginate gel†

Many materials have been used for artificial tubular prostheses to assist peripheral nerve gap reconstruction. However, the clinical use of these devices has been restricted because a microsurgical procedure requires specialized techniques and expensive equipment, such as operating microscope systems. Therefore the authors developed a new gluing method, without sutures, that uses freeze-dried alginate gel. A 7-mm gap in the sciatic nerve of rats was bridged with freeze-dried alginate gel. Regeneration was evaluated by electrophysiologic testing and histologic study. Eighteen weeks after surgery, functional reinnervation of motor and sensory nerves had occurred, as demonstrated by recovery of compound muscle action potentials (CMAP), compound nerve action potentials (CNAP), and somatosensory-evoked potentials (SEP). Histologically, many regenerated nerve fasciculi, including myelinated and unmyelinated fibers, were observed and the implanted alginate gel had disappeared. In conclusion, a gluing technique using alginate gel is a potential alternative to the conventional nerve autograft technique. Advantages include simple application and rapid repair. Freeze-dried alginate gel is a promising material for artificial nerve guides for peripheral nerves and also could be used for repair of disrupted pathways in central nervous tissue that is amorphous and cannot be sutured.

Electrophysiological and horseradish peroxidase-tracing studies of nerve regeneration through alginate-filled gap in adult rat spinal cord

The spinal cord segments at T(9-10) were totally excised and the resulting gap was filled by implantation of alginate sponge in adult rats. A horseradish peroxidase-tracing study at 21 weeks after operation showed that numerous ascending and many but less numerous descending regenerating fibres traversed the alginate-filled gap, and that after re-entering the distal stump of the transected spinal cord, they extended randomly over a long distance away from the gap. Intracellular electrophysiological recording at the same postoperative time showed that both ascending and descending regenerating axons formed functional synapses with host neurons located beyond the gap. These findings suggest that alginate could be a promising material for the support of regenerating axons in the spinal cord.

Thrombin-Sensitive Peptide Linkers for Biological Signal-Responsive Drug Release Systems

We have previously reported an elevation of thrombin-like activity in infected wound exudates. Therefore, using this enzymatic activity as a biological signal, a system which can release an antimicrobial drug at infected wounds was investigated. In this paper, we report thrombin-sensitive peptide linkers, the key component of this system. Starting from amino acid sequences of the cleavage site in fibrinogen, which is the substrate of thrombin, we synthesized some thrombin-sensitive peptide linkers. We constructed devices in which the thrombin-sensitive peptide linker interconnected between polyvinyl alcohol hydrogel and gentamicin. The device was able to release gentamicin in response to thrombin.