Satoshi Matsuzaka

Oversulfated chondroitin sulfate‐E binds to BMP‐4 and enhances osteoblast differentiation

Small leucine‐rich proteoglycans, such as biglycan, and their side chain sulfated glycosaminoglycans (GAGs), have been suggested to be involved in bone formation and mineralization processes. The present study was designed to investigate whether chondroitin sulfate (CS), one of the GAG, and its oversulfated structures coupled with bone morphogenetic protein‐4 (BMP‐4) alter the differentiation and subsequent mineralization of MC3T3‐E1 osteoblastic cells. CS‐E, one of the oversulfated CS structure, enhanced cell growth, alkaline phosphatase (ALP) activity, collagen deposition, and mineralization whereas heparin enhanced only ALP activity and mineralization. As well as CS‐E, CS‐H, and CPS also enhanced the mineralization of the cells. CS‐E enhanced the mineralization of the cells by interacting with protein in the conditioned medium. CS‐E induced mineralization was significantly inhibited by an antibody against BMP‐4. The addition of exogenous BMP‐4 further increased the capacity of CS‐E to enhance mineralization. Fluorescence correlation spectroscopy method using fluoresceinamine‐labeled GAG revealed that the oversulfated GAGs have a high affinity for BMP‐4. The disaccharide analysis of the cells indicated that MC3T3‐E1 cells are capable of producing oversulfated structures of CS by themselves. The lack of CS from the cells after chondroitinase treatment resulted in the inhibition of mineralization. These results in the present study indicate that oversulfated CS, which possesses 4,6‐disulfates in N‐acetyl‐galactosamine, binds to BMP‐4 and promotes osteoblast differentiation and subsequent mineralization. J. Cell. Physiol. 217: 769–777, 2008.

Chemonucleolytic effects of chondroitinase ABC on normal rabbit intervertebral discs : Course of action up to 10 days postinjection and minimum effective dose

Study Design This study demonstrated the chemonucleolytic effects of chondroitinase ABC and its histologic and biochemical background. Objectives To determine the course of chondroitinase ABC action on normal rabbit discs, and to find its minimum effective dosage. Summary of Background Data No previous study has assessed the chemonucleolytic action of chondroitinase ABC in a time‐ and dose‐dependent manner. This study also investigated the biochemical causes of radiologic and histologic changes in the discs. Methods Rabbits were injected with 4 U of pharmaceutical‐grade chondroitinase ABC intradiscally. They were radiologically and histologically observed, and biochemical analyses of the discs were conducted on days 1, 3, 5, 7, and 10 postinjection in the time course study. Different doses of chondroitinase ABC were injected, and radiologic observations and water content of the discs were measured in the dose‐finding study. Results The time course study revealed that the chondroitin sulfate content of discs significantly decreased from day 1 postinjection until the end of the experimental period. The weight and water content of the nucleus pulposus decreased on day 3, and disc space narrowing was observed from the day after injection. The dose‐finding study showed that a dose of 0.0002 U/disc still induced disc space narrowing and a decrease in water content. Conclusions Chondroitinase ABC is estimated to have a chemonucleolytic effect at least by day 3 postinjection at a dose level of 0.0002 U/disc or higher in rabbits.