Chondroitinase has been used as an important tool in the study of the structure, function and distribution of glycosaminoglycans for many years. Recently, the enzyme has been reported to be a potential enzyme for chemonucleolysis, an established treatment for intervertebral disc protrasion. In this paper, a chondroitinase had been purified from the culture supernatant of Aeromonas sobria YH311 using a simple purification procedure of ammonium sulfate precipitation, QAE-Sephadex A50 ion exchange chromatography and Sephadex G-150 gel filtration. The immobilization of purified chondroitinase using sodium alginate or cellulose as carriers has also been studied. The chondroitinase obtained from Aeromonas sobria YH311 was purified 55-fold to 95.3% pure, the specific activity of the purified enzyme was 31.86u/mg and the yield was 37%. The molecular weight of chondroitinase from Aeromonas sobria YH311 was determined by SDS-PAGE to be 80kD, which was almost the same as those chondroitinase AC from Arthrobacter aurescens, Aeromonas liquefaciens and Flavobacterium heparinum. But its isoelectric point was 4.3 - 4.6, which was far lower than the microbial chondroitinase AC. After the immobilization on sodium alginate or cellulose, the properties of chondroitinase changed greatly. The optimum temperature and pH of the free enzyme were 50 degrees C and 7.0 respectively, and about 10% activity remained after heat treatment at 80 degrees C for 20 minutes, and 47% activity remained after two weeks storage at 4 degrees C. The chondroitinase immobilized on sodium alginate had the optimum temperature and pH of 40 degrees C and 7.0 respectively, about 50% activity remained after 80 degrees C heat treatment for 120 minutes and 50% remained after 30 days storage at 4 degrees C. The chondroitinase immobilized on cellulose had the optimum temperature and pH of 70 degrees C and 6.0 respectively, and more than 70% activity remained after heat treatment at 80 degrees C and 30 days storage at 4 degrees C. The yield of the immobilization was very low, with 18.56% for alginate and 18.86% for cellulose.
Aeromonas ; enzymology ; Chondroitinases and Chondroitin Lyases ; isolation & purification ; metabolism ; Enzyme Stability ; Enzymes, Immobilized ; metabolism ; Temperature

Proteoglycan is highly hydrophilic and negatively charged which enable them attract the water. The objective of study was to investigate the effects of Proteoglycan on microtensile bond strength of dentin adhesives and on architecture of dentin collagen matrix of acid etched dentin by removing the chondroitin sulphate attached on Proteoglycan. A flat dentin surface in mid-coronal portion of tooth was prepared. After acid etching, half of the specimens were immersed in 0.1 U/mL chondroitinase ABC (C-ABC) for 48 h at 37degrees C, while the other half were stored in distilled water. Specimens were bonded with the dentin adhesive using three different bonding techniques (wet, dry and re-wet) followed by microtensile bond strength test. SEM examination was done with debonded specimen, resin-dentin interface and acid-etched dentin surface with/without C-ABC treatment. For the subgroups using wet-bonding or dry-bonding technique, microtensile bond strength showed no significant difference after C-ABC treatment (p > 0.05). Nevertheless, the subgroup using rewetting technique after air dry in the Single Bond 2 group demonstrated a significant decrease of microtensile bond strength after C-ABC treatment. Collagen architecture is loosely packed and some fibrils are aggregated together and relatively collapsed compared with normal acid-etched wet dentin after C-ABC treatment. Further studies are necessary for the contribution to the collagen architecture of noncollagenous protein under the various clinical situations and several dentin conditioners and are also needed about long-term effect on bond strength of dentin adhesive.
Adhesives ; Bisphenol A-Glycidyl Methacrylate ; Chondroitin ; Chondroitin ABC Lyase ; Chondroitin Sulfates ; Collagen ; Dentin ; Proteoglycans ; Tooth ; Water

As the components of proteoglycans, glycosaminoglycans (GAGs) are linear polysaccharides consisting of hexose and uronic acid units linked by β-1,3-glycosidic bond. GAGs mainly distribute in extracellular matrix and on cell surfaces. They guide many biological processes, such as proliferation of cells, transmission of signals and mediation of inflammation. Because of their large molecular weights, GAGs have limited biological functions in vitro. However, the appearance of chondroitinase ABC (ChSase ABC), which can lyse polysaccharides, solves the difficulties. Based on our work, we summarized the classification and the crystal structure of ChSase ABC, as well as other recent research progress on ChSase ABCs. The separation and purification methods of ChSase ABC and construction of engineering bacteria are illustrated. The stability and immobilization are also analyzed by taking account of the characterization of ChSase ABC. Finally, problems and future prospect of the ChSase ABC study are summarized.
Bacteria ; Chondroitin ABC Lyase ; chemistry ; Extracellular Matrix ; chemistry ; Glycosaminoglycans ; chemistry ; Proteoglycans ; chemistry

OBJECTIVE: This study is aimed to evaluate the effect of chondroitinase ABC on normal rabbit lumbar discs. MATERIALS AND METHODS: A series of intradiscal injections of chondroitinase ABC was performed in 9 young adult rabbits. A control series of intradiscal injections of iodine contrast medium was performed in 6 young adult rabbits. Roentgenograms were taken preoperatively and were repeated at one, three, five, seven days after injection of chondroitinase ABC. Roentgenograms also were taken preoperatiely and at seven days after injection of contrast dye. Magnetic resonance imagings(MRI) scan was performed pre-operatively and at seven days after injection. Light microscopic examination of both groups was done at 7 days postinjection. RESULTS: Roentgenographic evidence of disc space narrowing showed significant correlation with time course in the series of intradiscal injections of chondroitinase ABC compared with the control series. T2 weighted MRI of disc space demonstrated significantly decreased signal intensity in the series of intradiscal injections of chondroitinase ABC at seven days after injection, as compared with the control series. Histologic evaluation revealed the stainability of nucleus pulposus and annulus to toluidine blue which was quite decreased. The cytoplasm of notochordal cells of nucleus pulposus appeared to be shrunken, and the large cytoplasmic vacuoles in hematoxylin-eosin stain were decreased in the series of intradiscal injections of chondroitinase ABC, which were not evident in the control series. CONCLUSION: Intradiscal injections of chondroitinase ABC on normal rabbit lumbar disc proven to have chemonucleolytic effects.
Chondroitin ABC Lyase* ; Cytoplasm ; Humans ; Intervertebral Disc Chemolysis* ; Iodine ; Magnetic Resonance Imaging ; Notochord ; Rabbits ; Tolonium Chloride ; Vacuoles ; Young Adult

PURPOSE: To determine the effect of chondrocyte degeneration on chondrocyte adhesion, cell proliferation, proteoglycan and collagen synthesis and the effect of chondroitinase ABC on them. MATERIALS AND METHOD: Human cartilage explant and chondrocytes were harvested from patients underwent knee replacement arthroplasties for osteoarthritis. The articular surface was cut into a disc. Cartilage discs were grouped by grade of degeneration; normal (G0), superficial fissure (G1), and deep fissure (G2). Human chondrocytes were transferred onto cartilage discs pretreated with Chondroitinase ABC. The number of the attached chondrocyte and the cell proliferation and the amount of secreted proteoglycan and collagen was measured. The morphology of transplanted chondrocyte and cartilage surface was assessed using scanning electron microscopy (SEM). RESULTS: The number of chondrocytes attached to G1, G2 cartilage disc is greater than that of cells attached to G0 disc. The proliferation of chondrocyte attached to G1, G2 cartilage disc is greater than that of cells attached to G0 disc. Chondroitinase ABC treatment increases chondrocyte proliferation in G0 cartilage disc, but decreases chondrocyte proliferation in G1, G2 cartilage disc. The proliferation of transplanted chondrocyte is greater in G1, G2 group than G0 group. The amount of proteoglycan and collagen synthesized by transplanted chondrocyte is greater in G1, G2 group than G0 group. Chondroitinase ABC treatment decreases proteoglycan and collagen synthesis. At 21 days after transplantation, the degenerated surface of G1 or G2 cartilage disc was covered with the matrix synthesized by the transplanted chondrocyte. The degenerated surface of cartilage disc became very similar with normal articular cartilage surface with the new matrix made by transplanted chondrocyte under SEM. CONCOUSION: In this in vitro study, the transplanted chondrocytes onto osteoarthritic cartilage could repair the defects on the surface of osteoarthritic cartilage. The transplanted chondrocyte attached, proliferated, synthesized proteoglycan and collagen better on the surface of degenerated cartilage than on that of normal cartilage, and Chondroitinase ABC treatment of cartilage surface enhanced the cell attachment and inhibited proteoglycan and collagen synthesis. These findings may be applied to developing a new method of intraarticular chondrocyte injection for the treatment of osteoarthritis.
Arthroplasty, Replacement, Knee ; Cartilage ; Cartilage, Articular* ; Cell Adhesion ; Cell Proliferation ; Chondrocytes* ; Chondroitin ABC Lyase* ; Collagen ; Humans* ; Microscopy, Electron, Scanning ; Osteoarthritis ; Proteoglycans

OBJECTIVETo investigate the possibility of repairing spinal cord injury by bone marrow stromal cell (MSC) transplantation and microinjection of chondroitinase ABC (ChABC) in adult rats.

METHODSMSCs isolated from the femoral and tibial bones of new-born Wistar rats were cultured and the cell density was adjusted to 1×10(5)µl before transplantation. SD rats with T8 spinal cord crush injury were divided into 4 groups, namely group A (control) and groups B, C and D with injections of the MSCs, ChABC and MSCs+ChABC at the injury site, respectively. At 0 h, 1 day, 2 days, 3 days, 1 week, and 2 weeks after the injury, the BBB score system was used to evaluate the motion function. At 14 days after the injury, the maximal transverse diameter and actual area of necrosis were evaluated on HE stained sections. GFAP-CS56, GFAP-GAP43 and GFAP-NF160 immunofluorescence double labeling staining were carried out to evaluate the regeneration of the nerve fibers.

RESULTSAt the 14th day after the injury, BBB scores showed significant differences between group A and groups B, C and D (P<0.05), between the groups B and D and between groups C and D, butnot between groups B and C. HE staining showed cavity formation at the injury site in each group, with significant differences between group A and groups B, C and D and also between the latter 3 groups. Immunofluorescence staining revealed more intense expression of GFAP in group A than in the other groups with apparent cavity formation at the lesion site, which was only moderate in groups B and C. The expression of GAP-43 was more intense in group D than in groups B and C. The expression of NF-160 was more intense in group D than in the other 3 groups.

CONCLUSIONThe strategy of MSC transplantation combined with ChABC can be effective for repairing spinal cord injury in adult rats.

Animals ; Bone Marrow Transplantation ; methods ; Chondroitin ABC Lyase ; administration & dosage ; Combined Modality Therapy ; Male ; Microinjections ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; therapy ; Stromal Cells ; transplantation

Chymopapain and collagenase are well known chemonucleolytic agents for lumbar disc herniation. However, these enzymes have serious problems occasionally, such as severe neurotoxicity or anaphylaxis even fatal to patients. Chondroitinase ABC, a metabolic product of Proteus vulgaris, has a specific action on the proteoglycans of the nucleus pulposus, but rarely no effect on the intrathecal nerve tissues of vessels. Seventy eight rabbit lumbar discs were evaluated radiographically and histologically after injection of chondroitinase ABC 40U/ml per disc and compared with buffer injected group and nonigected control group. There was considerable disc space narrowing of the chondroitinase ABC injected group which was verified radiographically and histologically(p < 0.01). A zone of Safranin 0 depletion was present in the ventral anulus fibrosus adjacent to the nucleus pulposus in all treated discs, indicating proteoglycan loss. On electron microscopic findings there were collapse of chondrocytes and notochordal cells. All of these findings are corresponding to the evidence that chondroitinase ABC may be another chemonucleolytic agent by decreasing disc volume and thereby decompressing spinal cord or nerve roots. All histologic effects of chondroitinase ABC were confined to intervertebral disc tissues. Chondroitinase ABC deserves to be a study object for the alternative of chemonucleolysis.
Anaphylaxis ; Chondrocytes ; Chondroitin ABC Lyase ; Chymopapain ; Collagenases ; Humans ; Intervertebral Disc ; Intervertebral Disc Chemolysis ; Nerve Tissue ; Notochord ; Proteoglycans ; Proteus vulgaris ; Spinal Cord

Acid Etching, Dental ; adverse effects ; Chondroitin ABC Lyase ; chemistry ; Dental Bonding ; Dentin ; chemistry ; ultrastructure ; Dentin-Bonding Agents ; chemistry ; Humans ; Hydrolysis ; Matrix Metalloproteinase Inhibitors ; pharmacology ; Matrix Metalloproteinases ; metabolism ; Microscopy, Electron, Transmission ; Surface Properties

BACKGROUNDSeveral studies have revealed that adipose-derived mesenchymal stem cells (ADSCs) can be used as seed cells for the treatment of spinal cord injury (SCI). Chondroitinase ABC (ChABC) decomposes chondroitin sulfate proteoglycans in the glial scar that forms following SCI, allowing stem cells to penetrate through the scar and promote recovery of nerve function. This study aimed to establish ADSCs that stably express ChABC (ChABC-ADSCs) and evaluate the migratory capability of ChABC-ADSCs in vitro.

METHODSADSCs were obtained from Sprague-Dawley rats using secondary collagenase digestion. Their phenotypes were characterized using flow cytometry detection of cell surface antigens and their stem cell properties were confirmed by induction of differentiation. After successful culture, ADSCs were transfected with lentiviral vectors and ChABC-ADSCs were obtained. Proliferation curves of ChABC-ADSCs were determined using the Cell Counting Kit-8 method, ChABC expression was verified using Western blotting, and the migration of ChABC-ADSCs was analyzed using the transwell assay.

RESULTSSecondary collagenase digestion increased the isolation efficiency of primary ADSCs. Following transfection using lentiviral vectors, the proliferation of ChABC-ADSCs was reduced in comparison with control ADSCs at 48 h (P < 0.05). And the level of ChABC expression in the ChABC-ADSC group was significantly higher than that of the ADSC group (P < 0.05). Moreover, ChABC-ADSC migration in matrigel was significantly enhanced in comparison with the control (P < 0.05).

CONCLUSIONSSecondary collagenase digestion can be used to effectively isolate ADSCs. ChABC-ADSCs constructed using lentiviral vector transfection stably express ChABC, and ChABC expression significantly enhances the migratory capacity of ADSCs.

Adipocytes ; cytology ; metabolism ; Adipose Tissue ; cytology ; Animals ; Cell Differentiation ; physiology ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Cells, Cultured ; Chondrocytes ; cytology ; metabolism ; Chondroitin ABC Lyase ; metabolism ; Flow Cytometry ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Osteoblasts ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley

The aim of the study was to investigate the effect of chondroitinase ABC (ChABC) on ephrin A4 (EphA4) expression after spinal cord impairment (SCI) in rats. Adult female SD rats were randomly divided into three groups: ChABC group, normal saline (NS) group and sham group. In the ChABC and NS group, the SCI model was produced by the spinal cord hemisection. The rats in sham group received sham operation without the spinal hemisection. ChABC and NS groups were intrathecally injected with ChABC and normal saline, respectively. At different time points after SCI, injured region of spinal cord was taken out as sample. The levels of EphA4 expression were measured by immunofluorescence technique and Western blot. And the expressions of growth associated protein 43 (GAP-43) and glial fibrillary acidic protein (GFAP) were detected using double immunofluorescent staining. Immunofluorescent results showed that, compared with that in sham group, the EphA4 expression was significantly down-regulated on 1, 3 and 7 d after SCI, then up-regulated on 14 and 21 d after SCI in NS group. In ChABC group, the level of EphA4 expression was significantly less than that in the NS group during the whole time after SCI. Western blot showed an identical result to that of immunofluorescent staining. The double labeling results showed that on 3 d after SCI, the number of GFAP, glial cells marker, positive cells in NS group was lower than that in sham group, but higher than that in ChABC group. Moreover, GAP-43 was not detected in all three groups. These results suggest that ChABC can decrease the expression level of EphA4 and reduce the number of astrocytes after SCI, thus improving microenvironment of the injured region and promoting axonal growth and extension.
Animals ; Astrocytes ; pathology ; Chondroitin ABC Lyase ; pharmacology ; Ephrin-A4 ; metabolism ; Female ; Neurons ; metabolism ; Neuroprotective Agents ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; metabolism ; pathology ; Spinal Cord Injuries ; metabolism