Effect of nanoparticles carrying chondroitin sulfate ABC on the migration of schwann cells in a magnetic field / 中国组织工程研究

BACKGROUND: The clinical effect of spinal cord injury is usually unfavorable due to the lack of axon regeneration and the formation of glial scar. Schwann cells, as the support cells for nerve regeneration, have poor migration ability in the central nervous system with abundant astrocytes, which limit its effect on axon regeneration. OBJECTIVE: To explore the effect on the migration of Schwann cells containing superparamagnetic nanoparticles loaded with chondroitinase ABC (ChABC) in the region of astrocytes in the external magnetic field. METHODS: Schwann cells and astrocytes were extracted from sciatic nerves and brachial plexus and cerebral cortex of Sprague-Dawley rats of postnatal day 1 to 3. Cell purity was identified by immunofluorescence staining. The toxicity of superparamagnetic nanoparticles (PEI-SPIONs) to Schwann cells was analyzed by live/dead cell staining. Schwann cells were transfected with PEI-SPIONS in an external magnetic field of 1.4Td for 2 days. The optimal transfection concentration of PEI-SPIONS used was 2 mg/L and the optimal mass ratio of PEI-SPIONS to ChABC was 1:4. Cell migration test was used to evaluate the migration ability of not-treated Schwann cells, PEI-SPIONs/ ChABC transfected Schwann cells, and PEI-SPIONs/ChABC transfected Schwann cells in an external magnetic field. RESULTS AND CONCLUSION: The purity of Schwann cells and astrocytes reached to (91.7±1.2)% and (93.3±2.2)%, respectively. Compared with the Schwann cells group, the number of PEI-SPIONs/ChABC-transfected Schwann cells that entered the region of astrocytes was significantly increased (P < 0.05). Under the external magnetic field, the number of PEI-SPIONs/ChABC-transfected Schwann cells that entered the region of astrocytes and the cell migration distance were significantly increased as compared with the Schwann cells group (P < 0.005). In summary, PEI-SPIONs/ChABC transfection can enhance the ability of Schwann cells to break the glial scar, and increase the fusion of astrocytes. Under the guidance of external magnetic field, the migration ability of Schwann cells is significantly elevated. This method may be a new strategy to promote nerve regeneration after spinal cord injury.

Curative effect of porous silk fibroin scaffolds combined with chondroitinase ABC in treatment of spinal cord injury / 中华创伤杂志

Objective To investigate the effect of porous silk fibroin scaffolds (PSFSs) combined with chondroitinase ABC (ChABC)for treatment of rats with spinal cord injury (SCI).Methods After exposed to T9 spinal cord transection injury,96 SD rats were divided into control group,PSFSs group,ChABC group,and PSFSs plus ChABC group according to random number table.BBB scoring system was used to evaluate hindlimb motor function in rats.Immunohistochemistry and Western blot analysis were performed to detect expression levels of neurofilament-200 (NF-200),glial fibrillary acidic protein (GFAP),and growth associated protein-43 (GAP-43) of the injured spinal cord.Immuno-fluorescence staining was carried out to evaluate regeneration of nerve fiber.Results BBB score improved in PSFSs group (8.1 ± 0.8),ChABC group (9.0 ± 1.1),and PSFSs plus ChABC group (13.7 ± 1.3) compared with control group 4 weeks after injury (5.3 ±0.7,P ＜0.05).Immunohistochemistry showed higher integral absorbance (IA) values of NF-200 and GAP-43 in those treatment groups,but smaller GFAP-positive area was observed compared with control group (P ＜ 0.05).Immuno-fluorescence staining indicated more GAP-43 growth at injury sites in PSFSs plus ChABC group in contrast with other 3 groups.Western blotting showed levels of NF-200,GFAP,and GAP-43 differed among groups (P ＜ 0.05).Conclusion PSFSs combined with chondroitinase ABC transplantation can enhance axonal regeneration,inhibit glial scar proliferation and hence promote motor function recovery.

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.