Intravenous administration of anti-vascular endothelial growth factor humanized monoclonal antibody bevacizumab improves articular cartilage repair.

INTRODUCTION: In this study, we investigate the efficacy of repairing an osteochondral defect in rabbit knee joints by administering bevacizumab, a humanized monoclonal anti-vascular endothelial growth factor (VEGF) antibody. METHODS: An osteochondral defect was created on the patellar groove of 20 Japanese white rabbits that were classified into two recipient groups: group B, administration of bevacizumab (100-mg intravenous injection on the day of surgery and 2 weeks later), and a control group (defect only). Rabbits were killed 1 and 3 months postoperatively. Sections were stained with safranin O. Repair sites were evaluated using the modified O'Driscoll International Cartilage Repair Society grading system. The expression of chondromodulin (ChM)-I and VEGF was evaluated using immunohistochemical analyses. RESULTS: At 1 month postoperatively, the repair site in group B was filled with cartilaginous tissue. At 3 months, the repair site retained this cartilage phenotype. At 1 month in the controls, the defects were mainly filled with fibrous tissue. At 3 months, the defect was replaced by fibrous tissue and bone. Over the 3-month period, histological scores were significantly higher in group B than in the controls. At 1 month, group B showed intense positive results for ChM-I in the bottom of the repair tissue. VEGF was also identified in the same area. In the controls, no ChM-I was observed in the repair tissue. Conversely, the remodeling hypertrophic chondrocyte layer stained intensely for VEGF. CONCLUSIONS: Intravenous administration of bevacizumab contributes to better repair of articular cartilage in an osteochondral defect model. We suggest the possibility of facilitating articular cartilage repair with anti-VEGF antibody rather than using cultured cells or artificial scaffolds.

Jellyfish mucin may have potential disease-modifying effects on osteoarthritis.

BACKGROUND: We aimed to study the effects of intra-articular injection of jellyfish mucin (qniumucin) on articular cartilage degeneration in a model of osteoarthritis (OA) created in rabbit knees by resection of the anterior cruciate ligament. Qniumucin was extracted from Aurelia aurita (moon jellyfish) and Stomolophus nomurai (Nomura's jellyfish) and purified by ion exchange chromatography. The OA model used 36 knees in 18 Japanese white rabbits. Purified qniumucin extracts from S. nomurai or A. aurita were used at 1 mg/ml. Rabbits were divided into four groups: a control (C) group injected with saline; a hyaluronic acid (HA)-only group (H group); two qniumucin-only groups (M groups); and two qniumucin + HA groups (MH groups). One milligram of each solution was injected intra-articularly once a week for 5 consecutive weeks, starting from 4 weeks after surgery. Ten weeks after surgery, the articular cartilage was evaluated macroscopically and histologically. RESULTS: In the C and M groups, macroscopic cartilage defects extended to the subchondral bone medially and laterally. When the H and both MH groups were compared, only minor cartilage degeneration was observed in groups treated with qniumucin in contrast to the group without qniumucin. Histologically, densely safranin-O-stained cartilage layers were observed in the H and two MH groups, but cartilage was strongly maintained in both MH groups. CONCLUSION: At the concentrations of qniumucin used in this study, injection together with HA inhibited articular cartilage degeneration in this model of OA.

Recent technological advancements related to articular cartilage regeneration.

Some treatments for full thickness defects of the articular cartilage, such as the transplantation of cultured chondrocytes have already been performed. However, in order to overcome osteoarthritis, we must further study the partial thickness defects of articular cartilage. It is much more difficult to repair a partial thickness defect because few repair cells can address such injured sites. We herein show that bioengineered and layered chondrocyte sheets using temperature-responsive culture dishes may be a potentially useful treatment for the repair of partial thickness defects. We also show that a chondrocyte-plate using a rotational culture system without the use of a scaffold may also be useful as a core cartilage of an articular cartilageous defect. We evaluated the properties of these sheets and plates using histological findings, scanning electrical microscopy, and photoacoustic measurement methods, which we developed to evaluate the biomechanical properties of tissue-engineered cartilage. In conclusion, the layered chondrocyte sheets and chondrocyte-plates were able to maintain the cartilageous phenotype, thus suggesting that they could be a new and potentially effective therapeutic product when attached to the sites of cartilage defects.

Optimization of allograft implantation using scaffold-free chondrocyte plates.

If a tissue-engineered cartilage transplant is to succeed, it needs to integrate with the host tissue, to endure physiological loading, and to acquire the phenotype of the articular cartilage. Although there are many reported treatments for osteochondral defects of articular cartilage, problems remain with the use of artificial matrices (scaffolds) and the stage of implantation. We constructed scaffold-free three-dimensional tissue-engineered cartilage allografts using a rotational culture system and investigated the optimal stage of implantation and repair of the remodeling site. We evaluated the amounts of extracellular matrix and gene expression levels in scaffold-free constructs and transplanted the constructs for osteochondral defects using a rabbit model. Allografted 2-week constructs expressed high levels of proteoglycan and collagen per DNA content, integrated with the host cartilage successfully, and were able to counter physiological loads, and the chondrocyte plate contributed reparative mesenchymal stem cells to the final phenotype of the articular cartilage.

Exfoliation of the epidermal cells and defecation by amphibian larvae in response to coelomic fluid and lysenin from the earthworm Eisenia foetida.

Coelomic fluid (CF) and lysenin from the earthworm Eisenia foetida induced heavy epidermal exfoliation in the larvae of Bufo japonicus formosus at developmental stages from hatching (stage 22) to operculum completion (stage 34). In experiments with Xenopus laevis, we observed that exfoliated cells were not stained by trypan blue. Thus, it appeared that these cells were still alive. It is likely, therefore, that both CF and lysenin might disrupt the adhesion between epidermal cells of larvae prior to stage 34. Since it is known that lysenin exerts its toxic effects through its specific binding to sphingomyelin (SM), SM might be involved in such adhesion. This hypothesis was supported by the observations that CF and lysenin which had been incubated with SM-liposomes lost their exfoliative activity. In larvae after stage 34, the mechanism of adhesion between epidermal cells seemed to change and the adhesion was no longer disrupted by CF and lysenin. In larvae at around stage 34, a collagen layer started to form beneath the basement membrane of the epidermis. Furthermore, larvae at around this stage started to eat solid food. The developing collagen layer and food intake might be related indirectly to the chemical change in epidermal adhesion. The induction of exfoliation by CF and lysenin was also observed in other amphibian species. In Bufo larvae, defecation was induced both by CF and by lysenin but this effect was independent of exfoliation.

Sphingomyelin levels in the plasma membrane correlate with the activation state of muscle satellite cells.

Satellite cells are responsible for postnatal growth, hypertrophy, and regeneration of skeletal muscle. They are normally quiescent, and must be activated to fulfill these functions, yet little is known of how this is regulated. As a first step in determining the role of lipids in this process, we examined the dynamics of sphingomyelin in the plasma membrane. Sphingomyelin contributes to caveolae/lipid rafts, which act to concentrate signaling molecules, and is also a precursor of several bioactive lipids. Proliferating or differentiated C2C12 muscle cells did not bind lysenin, a sphingomyelin-specific binding protein, but noncycling reserve cells did. Quiescent satellite cells also bound lysenin, revealing high levels of sphingomyelin in their plasma membranes. On activation, however, the levels of sphingomyelin drop, so that lysenin did not label proliferating satellite cells. Although most satellite cell progeny differentiate, others stop cycling, maintain Pax7, downregulate MyoD, and escape immediate differentiation. Importantly, many of these Pax7-positive/MyoD-negative cells also regained lysenin binding on their surface, showing that the levels of sphingomyelin had again increased. Our observations show that quiescent satellite cells are characterized by high levels of sphingomyelin in their plasma membranes and that lysenin provides a novel marker of myogenic quiescence.

Biology of lysenin, a protein in the coelomic fluid of the earthworm Eisenia foetida.

Lysenin is a protein of 33?kDa in the coelomic fluid (CF) of the earthworm Eisenia foetida. It differs from other biologically active proteins, such as fetidins, eiseniapore, and coelomic cytolytic factor (CCF-1), that have been found in Eisenia foetida, in terms of both its biochemical and its biological characteristics. The large coelomocytes and free chloragocytes in the typhlosole of Eisenia foetida appear to be the cells that produce lysenin since the mRNA for lysenin and immunoreactive lysenin have been found in these cells. Lysenin binds specifically to sphingomyelin (SM) but not to other phospholipids in cell membranes. After binding to the cell membranes of target cells, lysenin forms oligomers in an SM-dependent manner, with subsequent formation of pores with a hydrodynamic diameter of approximately 3?nm. The biochemical interactions between lysenin and SM in cell membranes are responsible for the pharmacological activities of lysenin and of CF that contains lysenin in vertebrates, such as hemolysis, cytotoxicity, and contraction of smooth muscle in vitro and vasodepressor activity and lethality in vivo. When incubated with SM-liposomes, CF and lysenin lost some or all of their activity, an observation that suggests that SM might be involved in the induction of the various activities of lysenin and CF. However, in general, lysenin is neither cytotoxic nor lethal to invertebrates. An attempt has been made to explain the differences in the responses to lysenin and CF between vertebrates and invertebrates in terms of the presence or absence of SM in the various animals. Among Protostomia, SM is absent in Lophotrochozoa, with the exception of some molluscan species, but it is present in Ecdysozoa, with the exception of Nematomorpha and flies. Among Deuterostomia, Echinodermata and Hemichordata lack SM but SM is found in Chordata. Thus, the difference in terms of the response to lysenin between invertebrates and vertebrates cannot be fully explained by reference to the presence or absence of SM in the organism. Lysenin and its antiserum have made it possible to localize SM in the cell membranes. They should be a useful tool for studies of membrane physiology and the role of SM.

Damage to the gills, skin and other tissues by lysenin and the coelomic fluid of the earthworm Eisenia foetida in two teleosts, Tanichthys albonubes and Oreochromis mossambicus.

Lysenin is a 33-kDa protein found in the coelomic fluid (CF) of the earthworm Eisenia foetida. Purified lysenin binds specifically to sphingomyelin (SM). In the present studies, we found that the white cloud mountain minnow Tanichthys albonubes and the Mozambique tilapia Oreochromis mossambicus died in solutions of lysenin (at concentrations above 2.5 microg/ml) and CF (0.6%, v/v) within 2 h. The gills of both species of fish were damaged similarly by lysenin and by CF. Most gill lamellae became irregularly bent or curled, with swelling of the epithelial cells of the lamellae. Red blood cells in the lamellar vascular sinuses, in the central venous sinuses, and in the blood vessels of the entire body became swollen and lysed, choking the sinuses. Epithelial cells in the skin were also damaged. When fish of both species were treated with lysenin or CF that had been incubated with SM-liposomes, they did not die. Their behavior remained normal and there was no damage to any cells or tissues. These findings suggest that SM might be involved in the lethal effects of lysenin and CF. It is likely that purified lysenin and lysenin in CF bound to SM in the cell membranes of the tissues mentioned above, damaging the cells. The presence of SM in the gills and skin was confirmed, supporting this hypothesis. The damage to gills and hemolysis might have resulted in lethal respiratory problems. Damage to the skin might disturb the exchange of ions through the skin, hastening death. Damage by lysenin and CF to epithelial cells of the cornea and the wall of the oral cavity was also recognized, but there was no such damage to the intestine.