Objective To investigate the effect of carboxymethylated chitosan (CMCS) on the proliferation, cell cycle, and secretion of neurotrophic factors in cultured Schwann cells (SCs). Methods SCs were obtained from sciatic nerves of 20 Sprague Dawley rats (3-5 days old; male or female; weighing, 25-30 g) and cultured in vitro, SCs were identified and purified by immunofluorescence against S-100. The cell counting kit 8 (CCK-8) assay was used to determine the proliferation of SCs. The SCs were divided into 4 groups: 50 μg/mL CMCS (group B), 100 μg/mL CMCS (group C), 200 μg/mL CMCS (group D), and the same amount of PBS (group A) were added. The flow cytometry was used to analyze the cell cycle of SCs; the real-time quantitative PCR and Western blot analysis were used to detect the levels of never growth factor (NGF) and ciliary neurotrophic factor (CNTF) in cultured SCs induced by CMCS. Results The purity of cultured SCs was more than 90% by immunofluorescence against S-100; the CCK-8 results indicated that CMCS in concentrations of 10-1 000 μg/mL could promote the proliferation of SCs, especially in concentrations of 200 and 500 μg/mL (P lt; 0.01), but no significant difference was found between 200 and 500 μg/mL (P gt; 0.05). CMCS at a concentration of 200 μg/mL for 24 hours induced the highest proliferation, showing significant difference when compared with that at 0 hour (P lt; 0.01). The percentage of cells in phase S and the proliferation index were significantly higher in groups B, C, and D than in group A (P lt; 0.05), in groups C and D than in group B (P lt; 0.05); and there was no significant difference between group C and group D (P gt; 0.05). Real-time quantitative PCR and Western blot results showed that the levels of NGF and CNTF in groups B, C, and D were significantly higher than those in group A (P lt; 0.05), especially in group D. Conclusion CMCS can stimulate the proliferation, and induce the synthesis of neurotrophic factors in cultured SCs.
Objective To improve the flexibil ity and hemostatic properties of chitosan (CS)/carboxymethyl chitosan (CMCS) hemostatic membrane by using glycerol and etamsylate to modify CS/CMCS hemostatic membrane. To investigate themechanical properties and hemostatic capabil ity of modified CS/CMCS hemostatic membrane. Methods The 2% CS solution, 2% CMCS solution, 10%, 15%, 20%, 25%, 30% glycerol with or without 0.5% etamsylate were used to prepare CS/CMCS hemostatic membrane with or without etamsylate by solution casting according to ratio of 16 ∶ 4 ∶ 5. The tensile properties were evaluated by tensile test according to GB 13022-1991. Twenty venous incisions and five arterial incisions hemorrhage of 1 cm × 1 cm in rabbit ears were treated by CS/CMCS hemostatic membrane modified by 15% (group A) and 25% (group B) of glycerol, and a combination of them and 0.5% etamsylate (groups C and D). The bleeding time and blood loss were recorded. Results The pH of yellow CS/ CMCS hemostatic membrane with thickness of 30-50 μm was 3-4. The incorporation glycerol into CS/CMCS hemostatic membrane resulted in decreasing in tensile strength (7.6%-60.2%) and modulus (97%-99%). However, elongation at break and water content increased 5.7-11.6 times and 13%-125% markedly. CS/CMCS hemostatic membrane adhered to wound rapidly, absorbed water from blood and became curly. The bleeding time and blood loss of venous incisions were (70 ± 3) seconds and (117.2 ± 10.8) mg, (120 ± 10) seconds and (121.2 ± 8.3) mg, (52 ± 4) seconds and (98.8 ± 5.5) mg, and (63 ± 3) seconds and (90.3 ± 7.1) mg in groups A, B, C, and D, respectively; showing significant differences (P lt; 0.05) between groups A, B and groups C, D. The bleeding time and blood loss of arterial incision were (123 ± 10) seconds and (453.3 ± 30.0) mg in group C. Conclusion CS/CMCS hemostatic membrane modified by glycerol and etamsylate can improve the flexibil ity, and shorten the bleeding time.
Objective To investigate the effects of carboxymethylchitosan- carboxymethylcellulose (CMCH-CMC) film on the adhesion and heal ing of colonic anastomosis. Methods Sixty-four healthy adult male SD rats was randomly divided into control group and experimental group (n=32). The model of colonic anastomosis was made according to Buckenmaier’ smethod in all rats. The experimental group was treated by wrapping anastomosis with CMCH-CMC film (3 cm × 2 cm) and the control group was not treated. At 7 days and 14 days after operation, the adhesion formation of colonic anastomosis was observed, the tensile strength of the anstomosis was assessed and compared with 6 normal rats, and the hydroxyprol ine (HP) content of the anastomotsis was detected. Results There were 3 deaths in the experimental group and 2 deaths in the control group. The adhesive scores of the experimental group on the 7th and 14th postoperative day [(0.50 ± 0.16) points and (0.45 ± 0.14) points, (Plt; 0.05)] were significantly lower than those of the control group [(1.67 ± 0.15) points and (2.29 ± 0.18) points, (P lt; 0.05)], (Plt; 0.01). Tensile strength were more marked on the 14th postoperative day than on the 7th postoperative day in the control group (Plt; 0.05), but there was no significant difference between the 7th day and the 14th day in the experimental group. The tensile strength of thecontrol group and the experimental group on the 14th postoperative day [(178.36 ± 20.10) and (172.74 ± 22.18) mmHg] were respectively higher than those on the 7th postoperative day [(138.67 ± 16.65) and (130.81 ± 18.38) mmHg] (Plt; 0.01). The tensile strength of the control group and the experimental group on the 7th postoperative day were respectively significantly lower than that of the normal rats (P lt; 0.01). The level of HP in the anastomosis was significantly higher on the 7th postoperative day in the experimental group [(84.47 ± 11.87) μg/mg dried weight] than that of the control group [(55.47 ± 12.89) μg/mg dried weight), (Plt; 0.05)], but there was no significant difference between the experimental group and the control group on the 14th postoperative day [(146.07 ± 14.81) μg/mg dried weight, (137.14 ± 16.81) μg/mg dried weight, (P gt; 0.05)]. Conclusion The CMCH-CMC film can decrease adhesion the formation of colonic anastomosis, but does not interfere with the heal ing of colonic anastomosis.
Objective To prepare carboxymethyl-chitosan/hyaluronic acid/poly(vinyl alcohol) (CHP) blend membrane, evaluate its physicochemical properties and intraocular biocompatibil ity and to investigate its feasibil ity to be appl ied to glaucoma filtering surgery. Methods CHP blend membrane was prepared using solution casting method after blending carboxymethyl-chitosan, HA and poly(vinyl alcohol) in a proportion of 5 ∶ 4 ∶ 1 (M/M). Its water absorption rate, swell ing rate, permeabil ity, and mechanical properties were detected. Subconjunctival fibroblasts separated from subconjuncitival tissue of New Zealand white rabbits were cultured, and the cells at passage 4 were cultured on cell culture plate with or without the CHP blend membrane, serving as the experimental group and the control group, respectively. Effectof the CHP blend membrane on the subconjunctival fibroblasts was tested by MTT method 24, 48, and 72 hours after culture. Six New Zealand white rabbits were randomly divided into two groups (n=3 rabbits per group), and the CHP blend membrane and SK gel were implanted into the rabbits’ subconjunctival space and anterior chamber in the experimental group and the control group, respectively. Sl it lamp observation and binocular reaction record were conducted 1, 3, 5, 9, 11, 20, 30, 45, and 60 days after operation. Corneal tissue harvested from the experimental group was observed using scanning electron microscope 15 days after operation to study ophthalmic biocompatibil ity and biodegradabil ity. Results The water absorption rate and the swell ing rate of the CHP blend membrane was 83.8% ± 1.3% and 3.59 ± 0.50, respectively. The tensile strength of the dry and the wet CHP blend membrane was (20.59 ± 1.73) and (0.51 ± 0.13) MPa, respectively. The breaking elongation rate of the dry and the wet CHP blend membcane was 10.69% ± 1.16% and 53.15% ± 2.46%, respectively. The CHP blend membrane had good permeabil ity to NaCl and L-tyrosine. Absorbance (A) value of the experimental group 24, 48, and 72 hours after breeding was 0.207 ± 0.083, 0.174 ± 0.080, and 0.181 ± 0.048, respectively, while the A value of the control group was 0.284 ± 0.011, 0.272 ± 0.083, and 0.307 ± 0.056, respectively. Significant difference was evident between two groups (P lt; 0.05). In the experimental group, a small amount of floccus was exuded around the implanted membrane 1 day after operation; the floccus was absorbed on the third day, and there was no obvious inflammatory reaction occurring on the eleventh day. Most of the membrane degraded on the sixtieth day. Scanning electron microscope observation showed that the hexagonal morphology of the corneal endothel ial cells was intact, and no degradation particles adhered to the surface. In the control group, the implantation of SK gel into anterior chamber was unsuccessful because the SK gel was quite soft and easily broken. In the experimental group, mild hyperemia emerged around the implanted membrane 1 day after the subconjunctival implantation of the membrane, and it became normal on the ninth day. No corneal edema and inflammatory reaction of anterior chamber occurred till the sixtieth day. The results in the control group and the experiment group were similar. Conclusion Due to its good physicochemical properties and biocompatibil ity, the CHP blend membrane has potential appl ications in glaucomafiltering surgery.
ObjectiveTo investigate the protective effects of carboxymethylated chitosan (CMCS) on oxidative stress induced apoptosis of Schwann cells (SCs), and the expressions of brain derived neurotrophic factor (BDNF) and gl ial cell line derived neurotrophic factor (GDNF) in oxidative stress induced SCs. MethodsTwenty-four 3-5 days old Sprague Dawley rats (weighing 25-30 g, male or female) were involved in this study. The bilateral sciatic nerves of rats were harvested and SCs were isolated and cultured in vitro. The purity of SCs was identified by immunofluorescence staining of S-100. SCs were treated with different concentrations of hydrogen peroxide (H2O2, 0.01, 0.10, and 1.00 mmol/L) for 3, 6, 12, and 24 hours to establ ish the apoptotic model. The cell counting kit 8 (CCK-8) and flow cytometry analysis were used to detect the cell viabil ity and apoptosis induced by H2O2, and the optimal concentration and time for the apoptotic model of SCs were determined. The 2nd passage SCs were divided into 5 groups and were treated with PBS (control), with 1.00 mmol/L H2O2, with 1.00 mmol/L H2O2+50 μg/mL CMCS, with 1.00 mmol/L H2O2+100 μg/mL CMCS, and with 1.00 mmol/L H2O2+200 μg/mL CMCS, respectively. After cultured for 24 hours, the cell viabil ity was assessed by CCK-8, cell apoptosis was detected by flow cytometry analysis, the expressions of mRNA and protein of BDNF and GDNF were detected by real-time quantitative PCR and Western blot. ResultsThe immunofluorescence staining of S-100 indicated the positive rate was more than 95%. CCK-8 and flow cytometry results showed that H2O2 can inhibit the proliferation of SCs and induce the SCs apoptosis with dose dependent manner, the effect was the most significant at 1.00 mmol/L H2O2 for 24 hours; after addition of CMCS, SCs exhibited the increased proliferation and decreased apoptosis in a dose dependent manner. Real-time quantitative PCR and Western blot analysis showed that 1.00 mmol/L H2O2 can significantly inhibit BDNF and GDNF expression in SCs when compared with control group (P<0.05), 50-200 μg/mL CMCS can reverse the oxidative stress-induced BDNF and GDNF expression in SCs in a dose dependent manner, showing significant difference compared with control group and 1.00 mmol/L H2O2 induced group (P<0.05). There were significant differences among different CMCS treated groups (P<0.05). ConclusionCMCS has the protective stress on oxidative stress induced apoptosis of SCs, and may promote the BDNF and GDNF expressions of neurotrophic factors in oxidative stress induced SCs.
Objective To extend its application in the field of bone repair by adding oxygen-carboxymethylated chitosan (O-CMC) and gentamicin for modification of the calcium sulfate cement (CSC). Methods The O-CMC/CSC was prepared by adding O-CMC with different concentrations (0.1wt%, 0.3wt%, 0.5wt%, 0.7wt%, and 1.0wt%) in the CSC liquid phase. The effect of O-CMC on the CSC was evaluated by testing the injectability, compressive strength, degradation rate, pH value, cytotoxicity and osteogenesis. After the optimal concentration of O-CMC was determined, gentamicin with different concentrations (0.5wt%, 1.5wt%, and 2.5wt%) was added in the O-CMC/CSC, and then the compressive strength and antibacterial properties were investigated. Results After adding O-CMC in the CSC liquid phase, the injection time of O-CMC/CSC was increased to more than 5 minutes; it significantly prolonged with increased concentration of O-CMC (P<0.05). The compressive strength of the modified bone cement was in the range of 11-18 MPa and it was the highest when the concentration of O-CMC was 0.5wt% (P<0.05). The degradation rate of O-CMC/CSC was not influenced obviously by O-CMC (P>0.05). The pH value was in the range of 7.2-7.4 and Ca2+ concentration was in the range of 6-8 mmol/L.In vitro mineralization experiment indicated that the induced mineralization ability of O-CMC/CSC was much higher than that of pure CSC. The 0.5wt% O-CMC/CSC had the best performance; the compressive strength of the composite bone cement was above 5 MPa after gentamicin was added, which had antibacterial effect. Conclusion O-CMC is able to effectively improve the injection, compressive strength, and osteogenic activity of CSC; in addition, antibacterial properties is obtained in the CSC after adding gentamicin.
ObjectiveTo investigate the effect of graphene oxide (GO)-carboxymethyl chitosan (CMC) hydrogel loaded with interleukin 4 (IL-4) and bone morphogenetic protein 2 (BMP-2) on macrophages M2 type differentiation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).MethodsGO solution was mixed with CMC, then the phosphate buffered saline (PBS), IL-4, BMP-2, or IL-4+BMP-2 were added to prepare different GO-CMC hydrogel scaffolds with or without different cytokines under crosslinking agents. The characteristics of pure GO-CMC hydrogel were characterized by gross observation, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FTIR), and the CMC hydrogel was used as control. The sustained release of GO-CMC hydrogels with different cytokines was also tested. Macrophages were isolated and cultured from female Sprague Dawley rats aged 4-5 weeks, and then cultured with GO-CMC hydrogels with and without different cytokines, respectively. CD206 immunofluorescence staining was used to detect the differentiation of macrophages after 24 hours. The 3rd generation of rats BMSCs were cultured with GO-CMC hydrogels with and without different cytokines respectively for osteogenic induction. The early osteogenesis was observed by alkaline phosphatase (ALP) staining after 10 days, and the late osteogenesis was observed by alizarin red staining after 21 days.ResultsGenerally, GO-CMC hydrogel was brown and translucent. SEM showed that the pore diameter and wall thickness of GO-CMC hydrogel were similar to that of CMC hydrogel, but the inner wall roughness increased. FTIR test showed that CMC polymerized to form hydrogel. In vitro, the sustained release experiments showed that the properties of GO-CMC hydrogels loaded with different cytokines were similar. CD206 immunofluorescence detection showed that GO-CMC hydrogels could induce macrophages differentiation into M2-type. ALP and alizarin red staining showed that GO-CMC hydrogels could induce BMSCs osteogenic differentiation, in which GO-CMC hydrogel loaded with IL-4+BMP-2 showed the most significant effect (P<0.05).ConclusionThe GO-CMC hydrogel loaded with IL-4 and BMP-2 can induce macrophages differentiation into M2-type and enhance the ability of BMSCs with osteogenic differentiation in vitro, which provide a new strategy for bone defect repair and immune regulation.