Analytical method to determine keratan sulfate in the serum using HPLC.

There is an established method to assess the extent of articular cartilage damage that is based on the determination of keratan sulfate (KS) in synovial fluid by high performance liquid chromatography (HPLC). However, since it is easier to collect serum than synovial fluid, and the serum concentration of KS might reflect the extent of degeneration and injury of the articular cartilage, we aimed at developing a method to determine KS in serum by HPLC. Human serum was spiked with low-sulfated KS or poly-sulfated KS. After digestion of serum protein with a protease, the KS fraction was extracted by anion exchange column chromatography and then desalted. The KS fraction was digested with keratanase II and determined by HPLC. The recovery of KS added to the serum was 98% or more and did not depend on the degree of KS sulfation. A good recovery rate was obtained within a range of 50-100,000 ng/ml. The inter-day assay variation was 6.0% for 10 days. This method enables the determination of serum KS concentration without being influenced by the degree of KS sulfation.

Evaluation of in vivo biocompatibility and biodegradation of photocrosslinked hyaluronate hydrogels (HADgels).

HADgels are newly developed photocrosslinked hyaluronate hydrogels. They are produced from an aqueous solution of a hyaluronan derivative (HAD) in which cinnamic acid is introduced into the carboxyl moiety of hyaluronan using 3-aminopropanol as a spacer. High-energy ultraviolet irradiation of the HAD solution induces photodimerization of cinnamic acid, resulting in the development of a macromolecular network of each hyaluronan to water-insoluble hydrogels. The biocompatibility and biodegradation of HADgels were evaluated by guinea pig intracutaneous injection testing for up to 28 days. By macroscopic and histological observations, HADgels showed good tissue compatibility and did not induce excess inflammation at the injection sites. Biodegradation of the HADgels clearly depended on the degree of crosslinking at the fixed weight concentrations of HAD (0.5% and 1.0%). In addition, serum analyses showed that the injected guinea pigs did not produce specific antibodies against HADgels. These results indicate that HADgels have preferable biocompatibility and can be used as a new class of injectable, absorbable biomaterial, especially for preventing postsurgical adhesion formations.

Chondroitinase injection improves keloid pathology by reorganizing the extracellular matrix with regenerated elastic fibers.

Keloids are a proliferative fibrotic disease characterized by abnormal accumulation of extracellular matrix in the dermis. Keloid lesions lack skin plasticity due to deficiencies in elastic fiber formation in the extracellular matrix. The loss of elastic fiber is caused by excessive accumulation of chondroitin sulfate (CS), a sulfated glycosaminoglycan. However, there is no radical cure for keloids. Using a model system, we show herein that treatment of keloid tissues with chondroitinase ABC, an enzyme that specifically digests CS, improves clinical features of keloids. Keloid tissues obtained from patients were grafted on nude mice, and chondroitinase ABC was injected into the grafted keloid tissues. Chondroitinase ABC treatment significantly reduced the volume of keloid implants concomitant with recovery of elastic fiber formation. These results suggest that chondroitinase ABC injection is an effective therapy for keloid.

Role of GalNAc4S-6ST in astrocytic tumor progression.

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) is the sulfotransferase responsible for biosynthesis of highly sulfated chondroitin sulfate CS-E. Although involvements of CS-E in neuronal cell functions have been extensively analyzed, the role of GalNAc4S-6ST in astrocytic tumor progression remains unknown. Here, we reveal that GalNAc4S-6ST transcripts were detected in astrocytic tumors derived from all 30 patients examined using quantitative reverse transcription-PCR analysis. Patients with high GalNAc4S-6ST mRNA expression had significantly worse outcome compared with patients with low expression, and multivariate survival analysis disclosed that GalNAc4S-6ST is an independent poor prognostic factor for astrocytic tumors. We then tested whether CS-E enhanced haptotaxic migration of glioblastoma U251-MG cells that endogenously express both the CS-E's scaffold tyrosine phosphatase ζ (PTPζ) and GalNAc4S-6ST, in the presence of CS-E's preferred ligands, pleiotrophin (PTN) or midkine (MK), using a modified Boyden chamber method. Haptotaxic stimulation of cell migration by PTN was most robust on control siRNA-transfected U251-MG cells, while that enhancing effect was cancelled following transduction of GalNAc4S-6ST siRNA. Similar results were obtained using MK, suggesting that both PTN and MK enhance migration of U251-MG cells by binding to CS-E. We also found that PTPζ as well as PTN and MK were frequently expressed in astrocytic tumor cells. Thus, our findings indicate that GalNAc4S-6ST mRNA expressed by astrocytic tumor cells is associated with poor patient prognosis likely by enhancing CS-E-mediated tumor cell motility in the presence of PTN and/or MK.

Comparison of various mixtures of beta-chitin and chitosan as a scaffold for three-dimensional culture of rabbit chondrocytes.

With the use of a recently created chitosan neutral hydrogel, we have been able to create various mixtures of chitin and chitosan without changing their characteristics even at room temperature. The aim of this study was the initial comparison of various mixtures of beta-chitin and chitosan as a scaffold for rabbit chondrocyte culture. We created five types of sponges: pure beta-chitin, pure chitosan, 3:1, 1:1, and 1:3 beta-chitin-chitosan. The absorption efficiencies of chondrocytes in all five types of sponges were found to be around 98%. The mean concentrations of chondroitin sulfate were statistically different neither at week 2 nor at week 4 postculture between the types of sponges. The content of hydroxyproline in the beta-chitin sponge was significantly greater than in other sponges at week 4 postculture. From the histochemical and immunohistochemical findings, the cartilage-like layer in the chondrocytes-sponge composites of all five types of sponges was similar to hyaline cartilage. However, only immunohistochemical staining of type II collagen in the pure beta-chitin sponge was closer to normal rabbit cartilage than other types of sponges. The pure beta-chitin sponge was superior to other sponges concerning the content of extracellular matrices of collagen.