The proximate compositions of fresh and fermented skate skin were each 75.95% and 74.5% moisture, 22.7% and 21.8% protein, 0.5% and 0.7% lipid and 0.6% and 0.9% ash, respectively. The predominant minerals were potassium and phosphorus (i.e. 53.5 and 33.0 mg/100 g in fresh skin, and 10.46 and 10.51 mg/100 g in fermented skin, respectively). Amino acid concentrations were lower in the fermented skin compared with the fresh skin. Histidine, glycine, alanine and glutamic acid were the major free amino acids in both skins. Palmitic acid (C16:0) was the major fatty acid in both fresh (16.68%) and fermented (20.38%) skate skin. Omega-3 polyunsaturated fatty acids were higher in fresh skin (22.17%) and fermented skin (24.54%) compared with omega-6 polyunsaturated fatty acids. The predominant microflora present in the both fresh and fermented skin were Photobacterium sp. and Vibrio sp. Total plate counts for the fresh and fermented skin were 2.410⁴ CFU/g and 7.710⁷ CFU/g, respectively.

Skate cartilage is a fishery by-product, which contains chondroitin sulfate (CS), a glycosaminoglycan well known for its chondroprotective effect. Here described is a low-cost two-step process producing CS in non-denaturing conditions, consisting of an enzymatic extraction followed by tangential filtration to concentrate and purify CS. The performances of UF and MF membranes were compared in terms of flux and selectivity. The 0.1 μm-pore size membrane appeared to be the most efficient to separate CS from the other compounds.

ü Effects of exogenous glycosaminoglycans on human chondrocytes cultivated on type II collagen scaffolds.

Wu CH, Ko CS, Huang JW, Huang HJ, Chu IM.

(1)Department of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, 30013, Taiwan, ROC

(2)Biomedical Engineering Center, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, 30013, Taiwan, ROC

Cartilage extracellular matrix (ECM) is composed primarily of type II collagen (COL II) and large, networks of proteoglycans (PGs) that contain glycosaminoglycans such as hyaluronic acid (HA) and chondroitin sulfate (CS). Since cartilage shows little tendency for self-repair, injuries are kept unhealed for years and can eventually lead to further degeneration. During the past decades, many investigations have pursued techniques to stimulate articular cartilage repair or regeneration. The current study assessed the effects of exogenous glycosaminoglycans (GAGs) including CS-A, CS-B, CS-C, heparan sulfate and HA, administration on human chondrocytes in terms of proliferation and matrix synthesis, while the cells were seeded and grown on the genipin-crosslinked collagen type II (COL II) scaffold. DNA content was measured by Hoechst dye intercalation, matrix deposition was evaluated by DMMB dye. Expression of collagen II and aggrecan mRNAs was assessed by RT-PCR, followed by gel electrophoresis. In a 28-day in vitro culture, administration of 5 mug/ml CS-A, 50 mug/ml CS-B, 50 mug/ml CS-C, 5 mug/ml HS, and 500 kDa HA led to significant increase in biosynthesis rate of PGs. Gene expression of aggrecan and collagen II were upregulated by CS-A, CS-C and HA. These results showed considerable relevance of GAGs to the issue of in vitro/ex vivo neo-cartilage synthesis for tissue engineering and regenerative medical applications.

ü Serum levels of glycosaminoglycans, oxyproline, and fibronectin in patients with osteochondrosis of the vertebral column.

The content of the major components of the extracellular matrix was studied in 60 patients with spinal osteochondrosis over time during complex rehabilitative therapy including radon-containing mineral baths (before, 7-9 and 14-15 days after treatment). The complex rehabilitative treatment using radon baths in patients with spinal osteochondrosis was shown to result in a reduction in the serum level of glycosaminoglycans (GAG) and total oxyproline by the end of a course of therapy, which suggests the lower rate of destructive processes in the extracellular matrix. The determination of GAG concentrations and oxyproline forms in patients with asteochondrosis may be used to evaluate the efficiency of the treatment performed.

ü Cartilage outgrowth in fibrin scaffolds.

BACKGROUND: Fibrin glue has been a favorable hydrogel in cartilage tissue engineering, but implantation of chondrocyte-fibrin suspensions have resulted in volume loss. In this study, human septal cartilage chips were seeded onto a fibrin scaffold, and cellular proliferation and production of cartilaginous extracellular matrix (ECM) were evaluated. METHODS: Human septal cartilage was diced into cartilage chips and encased with and without fibrin glue. Four conditions were initially tested for DNA content and glycosaminoglycan (GAG) production: (1) control medium in tissue culture, (2) control medium with fibrin glue, (3) collagenase-supplemented medium in tissue culture, and (4) collagenase-supplemented medium seeded in fibrin glue. Cartilage chips cultured in collagenase-treated medium were then seeded onto either cell culture plates, suspended in alginate, or mixed with fibrin. Cellular proliferation, GAG production, and histochemistry were evaluated. RESULTS: Fibrin preparations increased cellular proliferation and DNA content. GAG levels were highest in collagenase-treated samples encased in fibrin. Cartilage chips treated with collagenase showed increased cellular proliferation in the fibrin preparations compared with preparations without fibrin. GAG increased with the addition of fibrin when compared with explant. Histochemistry revealed increased GAG accumulation in the regions between the cartilage chips with the addition of fibrin. CONCLUSION: Adding fibrin glue to collagenase-treated cartilage chips results in increased proliferation and maintains ECM production and, therefore, may facilitate generation of cartilaginous tissue for use in reconstructive surgery.

ü Glycosaminoglycans reduced inflammatory response by modulating toll-like receptor-4 in LPS-stimulated chondrocytes.

Lipopolysaccharide (LPS)-mediated activation of toll-like receptor-4 (TLR-4) complex induces specific signaling pathways, such as the myeloid differentiation primary response protein-88 (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), involving NF-kappaB activation. As previous data reported that hyaluronan (HA) and heparan sulfate (HS) may interact with TLR-4, the aim of this study was to investigate whether glycosaminoglycans (GAGs) may modulate the TLR-4 receptor in a model of LPS-induced inflammatory cytokines in mouse chondrocytes. LPS stimulation up-regulated all inflammation parameters. The GAG treatment produced various effects: HA reduced MyD88 and TRAF-6 levels and NF-kappaB activation at the higher dose only, and exerted a very low anti-inflammatory effect; chondroitin-4-sulfate (C4S) and chondroitin-6-sulfate significantly inhibited MyD88, TRAF-6 and NF-kappaB activation, the inflammation cytokines, and inducible nitric oxide synthase; HS, like C4S, significantly reduced MyD88, TRAF-6, NF-kappaB and inflammation. Specific TLR-4 blocking antibody confirmed that TLR-4 was the target of GAG action.

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