Benefícios da condroitinase abc associada a células-tronco mesenquimais na lesão espinhal aguda em ratos / Benefits of chondroitinase abc associated with mesenchymal stem cells in acute spinal cord injury in rats

Com o objetivo de estudar o efeito da condroitinase associada às células-tronco mesenquimais na lesão aguda da medula espinhal, utilizaram-se 50 ratos Lewis, distribuídos igualmente nos grupos: controle negativo (CN), tratamento com placebo (PLA), condroitinase (CDN), células-tronco mesenquimais (CTM) e condroitinase mais células-tronco mesenquimais (CDN+CTM). Todos os animais tiveram a medula espinhal exposta por laminectomia, e os grupos PLA, CDT, CTM e CDT+CTM sofreram também trauma medular compressivo. Após sete dias, procedeu-se à reexposição da medula espinhal, quando os grupos PLA e CTM receberam 4µL de líquido cefalorraquidiano artificial via intralesional, e os grupos CDT e CDT+CTM receberam o mesmo líquido contendo 2,2U de condroitinase. Após 14 dias da cirurgia inicial, todos os animais receberam 0,2mL de PBS via endovenosa, contudo, nos grupos CTM e CDT+CTM, esse líquido continha 1x106 CTM. Avaliou-se a capacidade motora até o 28o dia pós-trauma e, posteriormente, as medulas espinhais foram analisadas por RT-PCR, para quantificação da expressão gênica para BDNF, NT-3, VEGF, KDR e PECAM-1, e por imunoistoquímica, para detecção das células-tronco GFP injetadas (anti-GFP), quantificação dos neurônios (anti-NeuN) e da GFAP e vimentina, para avaliação da cicatriz glial. As análises estatísticas foram realizadas com o auxílio do Prism 5 for Windows, com o nível de significância de 5%. Não houve diferença entre os grupos quanto à capacidade motora. O grupo CDT+CTM apresentou maior imunoexpressão de neurônios viáveis do que o placebo. No CTM, houve maior expressão dos fatores neurotróficos BDNF e VEGF. E no CDT, houve menor imunoexpressão de vimentina. Concluiu-se que a associação CDT+CTM favorece a viabilidade neuronal após o trauma, que o tratamento com CTM promove aumento na expressão dos fatores tróficos BDNF e VEGF e que o tratamento com condroitinase é efetivo na redução da cicatriz glial.(AU)

The aim of this work was to study the effect of chondroitinase associated with mesenchymal stem cells in acute spinal cord injury. Therefore, 50 Lewis rats were distributed in the following groups: negative control (NC), treatment with placebo (PLA), chondroitinase (CDT), mesenchymal stem cells (MSC), and chondroitinase associated with mesenchymal stem cells (CDT + MSC). All animals had their spinal cord exposed by laminectomy, and the groups named PLA, CDT, MSC and CDT + MSC also suffered compressive spinal cord trauma. After seven days, the spinal cord was re-exposed, when the PLA and MSCs groups received 4uL of artificial cerebrospinal fluid through the lesion, and the CDT group and CDT + MSC received the same fluid containing 2,2U of chondroitinase. 14 days after the first surgery, all animals received 0.2ml of PBS intravenously; however, the MSC and CDT + MSC groups received the same liquid also containing 1x106 MSCs. The motor skills were evaluated up to 28 days post-injury and, subsequently, the spinal cords were analyzed by RT-PCR for BDNF, NT-3, VEGF, PECAM-1 and KDR gene expression quantification, immunohistochemistry to detect injected stem cells GFP (anti-GFP), to quantify neurons (anti-NeuN), GFAP and detect vimentin in order to evaluate the glial scar. Statistical analyzes were performed by Prism 5 for Windows using a 5% level of significance. There was no difference between groups with regarding motor capacity. The CDT + MSC group showed increased immunoreactivity of viable neurons than placebo. In MSC, there was a greater expression of neurotrophic factors BDNF and VEGF. Also, there was less vimentin immunostaining in group CDT. It was concluded that CDT + MSC association promotes neuronal viability after trauma, in which treatment with MSC promotes increased expression of BDNF and VEGF trophic factors, and also that treatment with chondroitinase is effective in reducing the glial scar.(AU)

Migration of Adipose-derived Mesenchymal Stem Cells Stably Expressing Chondroitinase ABC In vitro / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Several 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.</p><p><b>METHODS</b>ADSCs 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.</p><p><b>RESULTS</b>Secondary 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).</p><p><b>CONCLUSIONS</b>Secondary 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.</p>

Research progress in chondroitinase ABC / 生物工程学报

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.

Effect of dentin proteoglycans on the bonding properties of dentin / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the potential effect of proteoglycan (PG) and glycosaminoglycan (GAG) on the bonding of etch and rinse adhesive to dentin, in order to improve the bonding effect of dentin.</p><p><b>METHODS</b>Forty-two extracted molars were used to obtain standard dentin bonding surface, and the specimens were etched for 15 s with 37% phosphoric acid and divided into three groups using a table of random numbers. Then the three groups undergone different incubating procedures as follow: specimens in chondroitinase ABC (C-ABC) group were incubated with C-ABC at 37 °C for 48 h in vibrator. Specimens in trypsin (TRY) group were incubated with trypsin, and specimens in the control group were incubated with deionized water for 48 h in the oscillators. Then specimens in each group were randomly assigned into two subgroups, A (Adper(TM) Single Bond 2) and B (Prime & Bond NT) (n = 7). The microtensile bond strength (µTBS), fracture mode and bonding interface morphology of the specimens were evaluated via microtensile testing, stereo microscope and field emission scanning electron microscopy (FE-SEM) respectively after specimens being incubated in 37 °C water for 24 h.</p><p><b>RESULTS</b>The immediate µTBS of C-ABC group bonding with adhesive A and B [(32.9±2.5) and (26.8±2.2) MPa] were significantly lower than that of the control group [(40.7±3.3) and (34.6±3.7) MPa] (P < 0.05). While the immediate µTBS of TRY group [(49.0 ± 3.6) and (44.5 ± 3.0) MPa] were significantly higher than that of the control group(P < 0.05).</p><p><b>CONCLUSIONS</b>Dentin PG participates in the dentin bonding process. Removal of PG increased the immediate µTBS of dentin and total etching adhesives, while removal of GAG decreased the immediate µTBS.</p>

Inhibitory effect of chondroitinase ABC on ephrin A4 expression after spinal cord injury in rats / 生理学报

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.

Repair of subacute spinal cord crush injury by bone marrow stromal cell transplantation and chondroitinase ABC microinjection in adult rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the possibility of repairing spinal cord injury by bone marrow stromal cell (MSC) transplantation and microinjection of chondroitinase ABC (ChABC) in adult rats.</p><p><b>METHODS</b>MSCs 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.</p><p><b>RESULTS</b>At 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.</p><p><b>CONCLUSION</b>The strategy of MSC transplantation combined with ChABC can be effective for repairing spinal cord injury in adult rats.</p>

The effect of the removal of chondroitin sulfate on bond strength of dentin adhesives and collagen architecture / 대한치과보존학회지

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.

Degree of degeneration and Chondroitinase ABC treatment of human articular cartilage affect the adhesion of transplanted chondrocyte / 대한정형외과연구학회지

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.

Chondroitinase ABC Chemonucleolysis on Normal Rabbit's Lumbar Discs

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.

Chemonucleolysis using Chondroitinase ABC: An Expreimental Study / 대한정형외과학회잡지

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.