Objective To investigate the effects of chitosan/polyvinyl alcohol (PVA) nerve conduits for repairing radial nerve defect in Macaques. Methods Twelve adult Macaques weighing 3.26-5.35 kg were made the models of radial nerve defect (2 cm in length) and were randomly divided into 3 groups according to nerve grafting, with 4 Macaques in each group. Chitosan/PVA nerve conduit, non-graft, and autografts were implanted in the defects in groups A, B, and C, respectively. And the right radial nerves were used as normal control. At 8 months postoperatively, the general observation,electrophysiological methods, and histological examination were performed. Results At 8 months postoperatively, theregenerated nerve bridged the radial nerve defect in group A, but no obvious adhesion was observed between the tube and the peripheral tissue. The regenerated nerve had not bridged the sciatic nerve defect in group B. The adhesions between the implanted nerve and the peri pheral tissue were significant in group C. Compound muscle action potentials (CMAP) were detected in group A and group C, and no CMAP in group B. Peak ampl itude showed a significantly higher value in normal control than in groups A and C (P lt; 0.05), but there was no significant difference between groups A and C (P gt; 0.05). Nerve conduction velocity and latency were better in normal control than in groups A and C, and in group C than in group A, all showing significant differences (Plt; 0.05). The density of myl inated fibers in groups A and C was significantly lower than that in normal control (P lt; 0.05), but there was no significant difference between groups A and C (P gt; 0.05). The diameter and the myel in sheath thickness of the myl inated fibers in normal control were significantly higher than those in groups A and C, and in group C than in group A, all showing significant differences (P lt; 0.05). Conclusion The chitosan/PVA nerve conduits can promote the peripheral nerve regeneration, and may promise alternative to nerve autograft for repairing peripheral nerve defects.
Objective To explore a green route for the fabrication of thermo-sensitive chitosan nerve conduits, improve the mechanical properties and decrease the degradation rate of the chitosan nerve conduits. Methods Taking advantage of the ionic specific effect of the thermo-sensitive chitosan, the strengthened chitosan nerve conduits were obtained by immersing the gel-casted conduits in salt solution for ion-induced phase transition, and rinsing, lyophilization, and 60Co sterilization afterwards. The nerve conduits after immersing in NaCl solutions for 0, 4, 12, 24, 36, 48, and 72 hours were obtained and characterized the general observation, diameters and mechanical properties. According to the above results, the optimal sample was chosen and characterized the microstructure, degradation properties, and cytocompatibility. The left sciatic nerve defect 15 mm in length was made in 20 male Sprague Dawley rats. The autologous nerves (control group, n=10) and the nerve conduits (experimental group, n=10) were used to repair the defects. At 8 weeks after operation, the compound muscle action potential (CMAP) was measured. The regenerated nerves were investigated by gross observation and toluidine blue staining. The gastrocnemius muscle was observed by HE staining. Results With the increased ionic phase transition time, the color of the conduit was gradually deepened and the diameter was gradually decreased, which showed no difference during 12 hours. The tensile strength of the nerve conduit was increased gradually. The ultimate tensile strength showed significant difference between the 48 hours and 12, 24, and 36 hours groups (P<0.05), and no significant difference between the 48 hours and 72 hours groups (P>0.05). As a result, the nerve conduit after ion-induced phase transition for 48 hours was chosen for further study. The scanning electron microscope (SEM) images showed that the nerve conduit had a uniform porous structure. The degradation rate of the the nerve conduit after ion-induced phase transition for 48 hours was significantly decreased as compared with that of the conduit without ion-induced phase transition. The nerve conduit could support the attachment and proliferation of rat Schwann cells on the inner surface. The animal experiments showed that at 8 weeks after operation, the CMAPs of the experimental and control groups were (3.5±0.9) and (4.3±1.1) m/V, respectively, which showed no significant difference between the two groups (P<0.05), and were significantly lower than that of the contralateral site ［(45.6±5.6 m/V), P>0.05］. The nerve conduit of the experimental group could repair the nerve defect. There was no significant difference between the experimental and control groups in terms of the histomorphology of the regenerated nerve fibers and the gastrocnemius muscle. Conclusion The green route for the fabrication of thermo-sensitive chitosan nerve conduits is free of any toxic reagents, and has simple steps, which is beneficial to the industrial transformation of the chitosan nerve conduit products. The prepared chitosan nerve conduit can be applied to rat peripheral nerve defect repair and nerve tissue engineering.
Objective To investigate the hemostasis of thermosensitive chitosan hemostatic film. Methods Fifty adult Sprague Dawley rats, male or female and weighing 190-210 g, were made the models of liver injury. The models were randomly divided into 5 groups (n=10) depending on different hemostatic materials. The incision of the liver was covered with the hemostatic materials of 2.0 cm × 1.0 cm × 0.5 cm in size: thermosensitive chitosan hemostatic film (group A), chitosan hemostatic film (group B), cellulose hemostatic cotton (group C), gelatin sponge (group D), and no treatment (group E), respectively. The bleeding time and bleeding amount were recorded. After 4 weeks, the incisions of the liver were observed with HE staining. Results Gross observation showed better hemostatic effect and faster hemostatic time in groups A, B, and C; group D had weaker hemostatic effect and slower hemostatic time; group E had no hemostatic effect. The bleeding time and bleeding amount of groups A, B, C, and D were significantly lower than those of group E (P lt; 0.05). The bleeding time and bleeding amount of groups A, B, and C were significantly lower than those of group D (P lt; 0.05), but no significant difference was found among groups A, B, and C (P gt; 0.05). The liver cells of group A had milder edema and ballooning degeneration than other 4 groups through histological observation. Conclusion The thermosensitive chitosan hemostatic film has good hemostasis effect on the liver incision of rats.
To observe the effect of chitosan/alginate (CTS/ALG) dressings on wound immersed in seawater. Methods Twenty-five healthy SD rats weighing 250-300 g were used to establ ish skin wound model through cutting 1.8 cm circle-shaped wound along spine bilaterally. The left side served as experimental group, and the right side as control group. The wounds were immersed in the prepared artificial seawater for 1 hour, then the experimental group was treated with CTS/ALG dressings, while the control group was treated with sterile gauze. Gross observation was performed andwound heal ing time was recorded. At 3, 5, 7, 10 and 12 days after operation, 2 cm × 2 cm skin tissues including the wounds were removed and underwent HE staining and immunohistochemistry staining using Envision method. Histological change of wound and expression of EGF receptor (EGFR) and bFGF were observed. Results In the experimental group, wound inflammatory response was sl ight and incrustation shrinked faster, while the incrustation in the control group shrinked slowly. The wound heal ing time of the experimental group and the control group was (11.68 ± 0.57) and (12.51 ± 0.54) days, respectively, suggesting there was a significant difference between two groups (P lt; 0.05). In the experimental group, granulation tissue prol iferation, cell infiltration, collagen tissue prol iferation, wound shrinkage and epithel ization appeared at 3 days after operation; regularly l ined collagen tissue, complete epithel ization and occurrence of skin appendages were observed at 10 days after operation; complete wound heal ing was noted at 12 days after operation; while in the control group, at the corresponding time point, late cell infiltration and epithel ization were observed and granulation tissue with ulcer was noted. Immunohistochemistry observation: high expression of bFGF in vascular endothel ial cells and interstitial fibroblasts and high expression of EGFR in vascular endothel ial cells were observed in the experimental group at 3 and 5 days after operation, and their expressions were low at 7, 10 and 12 days after operation; while in the control group, there were no or low expression of bFGF and EGFR at the same time point. Conclusion CTS/ALG dressings can promote the heal ing of wound immersed in seawater, but its mechanism needsfurther study.
To explore the effect of hydroxybutyl chitosan on the prevention of postoperative peritoneal adhesion in rats. Methods Ninety SD rats (half males and half females) weighing 250-280 g underwent laparotomy with subsequent cecal wall abrasion and peritoneal adhesion. Rats were randomized into 3 groups (n=30 per group): group A, injection of 2 mL hydroxybutyl chitosan solution (2%); group B, injection of 2 mL sodium hyaluronate solution(2%); group C, the abdomen of rat was exposed for 30 seconds and served as control group. The general condition of the rats was observed after operation. The rats were killed 2 and 4 weeks after operation, 15 rats per group at a time, to undergo gross and histologyobservation. The degree of adhesion was evaluated by double-bl ind method. The microstructure of injured electroscope cecal wall in groups A and C was observed with transmission electroscope 4 weeks after operation. Results All rats survived till the end of experiment. At 2 weeks after operation, the adhesion and the hyperplasia of fibrous connective tissue and collagen in groups A and B were sl ight while the adhesion in group C was serious with severe hyperplasia of fibrous connective tissue. According to the measurement classification by Nair histological grading, the difference between groups A and B and group C was significant (P lt; 0.05), while no significant difference was evident between group A and group B (P gt; 0.05). At 4 weeks after operation, the adhesion in group A was mild, and the hyperplasia of fibrous connective tissue and collagen were sl ight; the adhesion and the hyperplasia of fibrous connective tissue and collagen in group C were serious. The levels of group B were between group A and group C. The differences among three groups were significant (P lt; 0.05). Transmission electroscope showed inactive fibroblasts and loose thin collagen fibers in group A, and active fibroblasts and closely collagen fibers arranged in a disorderly manner in group C. Conclusion Hydroxybutyl chitosan can decrease the hyperplasia of fibrous connective tissue and inhibit the activity of fibroblasts significantly, and has a long-term role of preventing peritoneal adhesion.
Objective To study hemostasis of a new chitosan hemostatic powder. Methods Twenty-four adult SD rats were made the models of l iver injury, male or female, and weighing 210-240 g. They were divided into three groups randomly (n=8) depending on different hemostatic powders. The incision of the l iver was treated with 300 mg Yunnan baiyao (group A1), chitosan hemostatic powder of pH6.5 (group B1) and pH7.5 (group C1), respectively. The bleeding time and bleeding amount were recorded. In vitro, with the modified Ree-White method, 2 mL artery blood from New Zealand whiterabbit was added into the 0.2 mL solution of Yunnan baiyao, chitosan hemostatic powder of pH6.5 and pH7.5 (concentration of 0.2 mg/mL), respectively. The blood coagulation time was recorded. The chitosan blood clots of group B2 and group C2 were observed with scanning electron microscopy (SEM). Results The bleeding time of group A1, group B1 and group C1 was (292 ± 31), (261 ± 23), and (224 ± 28) s, respectively, the bleeding amount was (1.63 ± 0.21), (1.47 ± 0.18), and (1.18 ± 0.17) g, respectively, showing statistically significant differences between groups B1, C1, and group A1 (P lt; 0.05), between group C1 and group B1 (P lt; 0.05). The blood clotting time of group A2, group B2, and group C2 was (653 ± 41), (255 ± 20), and (202 ± 11) s, respectively, showing statistically significant differences between groups B2, C2, and group A2 (P lt; 0.05), between group C2 and group B2 (P lt; 0.05). The SEM showed that the blood cells of group B2 and group C2 gathered around the chitosan. Conclusion Chitosan hemostatic powder of pH7.5 has good hemostasis.
ObjectiveTo study the effect of intraarticular injection of crosslinked-chitosan in the treatment of knee osteoarthritis in rabbits.MethodsThirty-two New Zealand white rabbits were randomly divided into 4 groups (groups A, B, C, and D; 8 rabbits in each group). The knee osteoarthritis models were prepared by anterior cruciate ligament transection in the left hind in groups A, B, and C. At 4 weeks after operation, the rabbits were received intraarticular injection of 0.6 mL crosslinked-chitosan in group A, 0.3 mL chitosan (once per 2 weeks, for twice) in group B, and 0.3 mL saline (once per 2 weeks, for twice) in group C. The rabbits in group D were treated with sham operation in the left hind, and received intraarticular injection of 0.3 mL saline (once per 2 weeks, for twice). At 8 weeks, the macroscopic observation, histological examination (HE staining, Safranin-fast green double staining, and Mankin score), scanning electron microscopy (SEM) observation, and immunohistochemical staining of collagen type Ⅱ were performed.ResultsMacroscopic and SEM observations showed that the cartilage in group D was basically the same as normal and better than that in groups A and B, and the abrasion of cartilage in group C was the most serious. The histological observation results in groups A and B were slightly similar and better than those in group C, but not up to the structure of group D. The macroscopic score and Mankin score of groups B and C were significantly higher than those of group D (P<0.05), and there was no significant difference between group A and group B (P>0.05). Immunohistochemical staining results showed that the collagen type Ⅱ positive percentage of chondrocytes was significantly higher in group D than that in groups B and C, and no significant difference was found between group A and group B (P>0.05). ConclusionThe crosslinked-chitosan can significantly improve the osteoarthritis of the rabbit knee, delay the pathological changes of osteoarthritis, and decrease the frequency of injection.