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.
This research was aimed to find the skin irritation and burns treatment effect of wound dressing with microspheres containing levofloxacin. We used reference GB/T16886.10-2005 to evaluate the dressing skin irritation. We prepared rabbit models divided into three groups. The control group was rapped with Vaseline gauze bandage, while the positive control group was rapped with the wounds of nano-silver paste bandage. The experimental sample group was rapped with wound dressing with microspheres containing levofloxacin. We measured the wound without healing area and the hydroxyproline content at the ends of 3 d, 6 d, 9 d, 14 d, 21 d, 28 d. and meanwhile performed histopathological examination. The experimental results showed that the dressing primary irritation index was 0. The nonhealing wound area of theexperimental sample group and positive control group at the ends of 6 d, 9 d, 14 d, 21 d were less than that of the control group (P<0.05). The nonhealing wound area of the experimental sample group at the ends of 9 d and 14 d was significantly lower than that of the positive control group (P<0.05). The hydroxyproline content of the experimental sample group at the ends of 6 d, 9 d and 14 d was significantly higher than that of the positive control group and blank control group (P<0.05). The pathology observed of the experimental sample group at 21 d were the earliest appendages. The wound dressing with microspheres containing levofloxacin has minimal skin irritation, effectively promote wound healing of burn.
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 evaluate the characterization, biocompatibil ity in vitro and in vivo, and antimicrobial activity of an injectable vancomycin-loaded borate glass/chitosan composite (VBC) so as to lay the foundation for its further cl inical application. Methods The sol id phase of VBC was constituted by borate glass and vancomycin, liquid phase was a mixture of chitosan, citric acid, and glucose with the proportion of 1 ∶ 10 ∶ 20. Solid phase and liquid phase was mixed withthe ratio of 2 ∶ 1. Vancomycin-loaded calcium sulfate (VCS) was produced by the same method using calcium sulfate instead of borate glass and sal ine instead of chitosan, as control. High performance liquid chromatography was applied to detect the release rate of antibiotics from VBC and VCS, and minimum inhibitory concentration (MIC) was tested by using an antibiotic tube dilution method. The structure of the VBC and VCS specimens before and 2, 4, 8, 16, and 40 days after immersion in D-Hank’s was examined by scanning electron microscopy, and the phase composition of VBC was analysed by X-ray diffraction after soaked for 40 days. Thirty-three healthy adult New Zealand white rabbits (weighing, 2.25-3.10 kg; male or female) were used to establ ish the osteomyel itis models according to Norden method. After 4 weeks, the models of osteomyel itis were successfully established in 28 rabbits, and they were randomly divided into 4 groups (groups A, B, C, and D). In group A (n=8), simple debridement was performed; in groups B and C (n=8), defect was treated by injecting VCS or VBC after debridement; and in group D (n=4), no treatment was given. The effectiveness of treatment was assessed using radiological and histological techniques after 2 months. Results The releases of vancomycin from VBC lasted for 30 days; the release rate of vancomycin reached 75% at the first 8 days, then could reached more than 90%. The releases of vancomycin from VCS lasted only for 16 days. The MIC of VBC and VCS were both 2 μg/mL. The VCS had a smooth glass crystal surface before immersion, however, it was almost degradated after 4 days. The fairly smooth surface of the VBC pellet became more porous and rougher with time, X-ray diffraction analysis of VBC soaked for 40 days indicated that the borate glass had gradually converted to hydroxyapatite. After 2 months, the best result of treatment was observed in group C, osteomyelitis symptoms disappeared. The X-ray scores of groups A, B, C, and D were 3.50 ± 0.63, 2.29 ± 0.39, 2.00 ± 0.41, and 4.25 ± 0.64, respectively; Smeltzer scores were 6.00 ± 0.89, 4.00 ± 0.82, 3.57 ± 0.98, and 7.25 ± 0.50, respectively. The scores were significantly higher in group D than in groups A, B, and C (P lt; 0.05), and in group A than in groups B and C (P lt; 0.05). The scores were higher in group B than in group C, but no significant difference was found (P gt; 0.05). Conclusion The VBC is effective in treating chronic osteomyelitis of rabbit by providing a sustained release of vancomycin, in addition to stimulating bone regeneration, so it may be a promising biomaterial for treating chronic osteomyelitis.
Objective To construct a ultraviolet-cross-linkable chitosan-carbon dots-morin (NMCM) hydrogel, observe whether it can repair cartilage injury by in vivo and in vitro experiments, and explore the related mechanism. Methods The chitosan was taken to prepare the ultraviolet (UV)-cross-linkable chitosan by combining methacrylic anhydride, and the carbon dots by combining acrylamide. The two solutions were mixed and added morin solution. After UV irradiation, the NMCM hydrogel was obtained, and its sustained release performance was tested. Chondrocytes were separated from normal and knee osteoarticular (KOA) cartilage tissue donated by patients with joint replacement and identified by toluidine blue staining. The 3rd generation KOA chondrocytes were co-cultured with the morin solutions with concentrations of 12.5, 25.0, 50.0 μmol/L and NMCM hydrogel loaded with morin of the same concentrations, respectively. The effects of morin and NMCM hydrogel on the proliferation of chondrocytes were detected by cell counting kit 8 (CCK-8). After co-cultured with NMCM hydrogel loaded with 50 μmol/L morin, the level of collagen type Ⅱ (COL-Ⅱ) of KOA chondrocytes was detected by immunofluorescence staining, and the level of reactive oxygen species (ROS) was detected by 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Twenty 4-week old Sprague Dawley rats were selected to construct a articular cartilage injury of right hind limb model, and were randomly divided into two groups (n=10). The cartilage injury of the experimental group was repaired with NMCM hydrogel loaded with 25 μmol/L morin, and the control group was not treated. At 4 weeks after operation, the repair of cartilage injury was observed by micro-CT and gross observation and scored by the International Cartilage Repair Association (ICRS) general scoring. The cartilage tissue and subchondral bone tissue were observed by Safranine-O-fast green staining and COL-Ⅱ immunohistochemistry staining and scored by ICRS histological scoring. The expressions of tumor necrosis factor α (TNF-α), nuclear factor κB (NK-κB), matrix metalloproteinase 13 (MMP-13), and COL-Ⅱ were detected by Western blot and real-time fluorescence quantitative PCR. Results NMCM hydrogels loaded with different concentrations of morin were successfully constructed. The drug release rate was fast in a short period of time, gradually slowed down after 24 hours, and the amount of drug release was close to 0 at 96 hours. At this time, the cumulative drug release rate reached 88%. Morin with a concentration ≤50 μmol/L had no toxic effect on chondrocytes, and the proliferation of chondrocytes improved under the intervention of NMCM hydrogel (P<0.05). NMCM hydrogel loaded with morin could increase the level of COL-Ⅱ in KOA chondrocytes (P<0.05) and reduce the level of ROS (P<0.05), but it did not reach the normal level (P<0.05). Animal experiments showed that in the experimental group, the articular surface was rough and the defects were visible at 4 weeks after operation, but the surrounding tissues were repaired and the joint space remained normal; in the control group, the articular surface was rougher, and no repair tissue was found for cartilage defects. Compared with the control group, the experimental group had more chondrocytes, increased COL-Ⅱ expression, and higher ICRS gross and histological scores (P<0.05); the relative expressions of MMP-13, NF-κB, and TNF-α protein and mRNA significantly decreased (P<0.05), and the relative expressions of COL-Ⅱ protein/COL-2a1 mRNA significantly increased (P<0.05). Conclusion NMCM hydrogel can promote chondrocytes proliferation, down regulate chondrocyte catabolism, resist oxidative stress, protect chondrocytes from cartilage injury, and promote cartilage repair.
Objective To evaluate the effect of implantation of the complex of high viscous chitosan/glycerol phosphate with demineral ized bone matrix (HV-C/GP-DBM) in repairing cartilage defects of rabbits. Methods HV-C/ GPDBM was prepared by compounding HV-C/GP and DBM by 2:1 (W/W). Twenty-four 34-week-old New Zealand white adult rabbits, weighing 3.5-4.5 kg, were included. A bit with the diameter of 3.5 mm was used to drill 3-cm-deep holes in both sides of femoral condyle to make cartilage defects. The complex of HV-C/GP-DBM was then injected into the right holes as the experimental group and the left ones serve as the control group. The rabbits were killed at 4, 8 and 16 weeks after theoperation, respectively. The obtained specimens were observed macroscopically, microscopically and histologically. According to the International Cartilage Repair Society Histological Scoring (ICRS), the effect of cartilage repair was assessed at 16 weeks postoperatively. Results At 4-8 weeks postoperatively, in the experimental group, the defects were filled with hyal ine cartilage-l ike tissues; the majority of chitosan degradated; and the DBM particles were partly absorbed. However, in the control group, there were small quantities of discontinuous fibrous tissues and maldistributed chondrocytes at the border and the bottom of the defects. At 16 weeks postoperatively, 6 joints in the experimental group had smooth surface, and the defects were basically repaired by hyal ine cartilage-l ike tissues. The newly-formed tissues integrated well with the surrounding area. Under the cartilage, the new bone formation was still active and some DBM particles could be seen. However, the defects in the control group were repaired by fibrous tissues. The result of histological scoring of the specimens at 16 weeks showed that a total of 6 aspects including formation of chondrocytes and integration with the surrounding cartilages were superior in the experimental group to those in the control group, and there were significant differences between the two groups (P lt; 0.05). Conclusion The biodegradable and injectable complex of HV-C/GP-DBM with good histocompatibil ity and non-toxic side effects can repair cartilage defects and is a promising biomaterial for cartilage defect repair.
ObjectiveGelatin methacryloyl (GelMA)/hyaluronic acid methacryloyl (HAMA)/chitosan oligosaccharide (COS) hydrogel was used to construct islet biomimetic microenvironment, and to explore the improvement effect of GelMA/HAMA/COS on islet activity and function under hypoxia. Methods Islets cultured on the tissue culture plate was set as the control group, on the GelMA/HAMA/COS hydrogel with COS concentrations of 0, 1, 5, 10, and 20 mg/mL respectively as the experimental groups. Scanning electron microscopy was used to observe the microscopic morphology, rheometer test to evaluate the gel-forming properties, contact angle to detect the hydrophilicity, and the biocompatibility was evaluated by the scaffold extract to L929 cells [using cell counting kit 8 (CCK-8) assay]. The islets were extracted from the pancreas of 8-week-old Sprague Dawley rats and the islet purity and function were identified by dithizone staining and glucose-stimulated insulin secretion (GSIS) assays, respectively. Islets were cultured under hypoxia (1%O2) for 24, 48, and 72 hours, respectively. Calcein-acetyl methyl/propidium iodide (Calcein-AM/PI) staining was used to evaluate the effect of hypoxia on islet viability. Islets were cultured in GelMA/HAMA/COS hydrogels with different COS concentrations for 48 hours, and the reactive oxygen species kits were used to evaluate the antagonism of COS against islet reactive oxygen species production under normoxia (20%O2) and hypoxia (1%O2) conditions. Calcein-AM/PI staining was used to evaluate the effect of COS on islet activity under hypoxia (1%O2) conditions. Islets were cultured in tissue culture plates (group A), GelMA/HAMA hydrogels (group B), and GelMA/HAMA/COS hydrogels (group C) for 48 hours, respectively. Immunofluorescence and GSIS assays were used to evaluate the effect of COS on islet activity under hypoxia (1%O2) conditions, respectively. Results GelMA/HAMA/COS hydrogel had a porous structure, the rheometer test showed that it had good gel-forming properties, and the contact angle test showed good hydrophilicity. CCK-8 assay showed that the hydrogel in each group had good biocompatibility. The isolated rat islets were almost round, with high islet purity and insulin secretion ability. Islets were treated with hypoxia for 24, 48, and 72 hours, Calcein-AM/PI staining showed that the number of dead cells gradually increased with time, which were significantly higher than those in the non-hypoxia-treated group (P<0.001). Reactive oxygen staining showed that GelMA/HAMA/COS hydrogels with different COS concentrations could antagonize the production of reactive oxygen under normal oxygen and hypoxia conditions, and this ability was positively correlated with COS concentration. Calcein-AM/PI staining indicated that GelMA/HAMA/COS hydrogels with different COS concentrations could improve islet viability under hypoxia conditions, and cell viability was positively correlated with COS concentration. Immunofluorescence staining showed that GelMA/HAMA/COS hydrogel could promote the expression of islet function-related genes under hypoxia conditions. GSIS assay results showed that the insulin secretion of islets in hypoxia condition of group C was significantly higher than that of groups B and C (P<0.05). Conclusion GelMA/HAMA/COS hydrogel has good biocompatibility, promotes islet survival and function by inhibiting reactive oxygen species, and is an ideal carrier for building islet biomimetic microenvironment for islet culture and transplantation.
Objective
To investigate the effects of nucleus localization signal linked nucleic kinase substrate short peptide (NNS) conjugated chitosan (CS) (NNSCS) mediated the transfection of microRNA-140 (miR-140) in rabbit articular chondrocytes in vitro.
Methods
Recombinant plasmid GV268-miR-140 and empty plasmid GV268 were combined with NNSCS to form NNSCS/pDNA complexes, respectively. Chondrocytes were isolated and cultured through trypsin and collagenase digestion from articular cartilage of newborn New Zealand white rabbits. The second generation chondrocytes were divided into 3 intervention groups: normal cell control group (group A), NNSCS/GV268 empty plasmid transfection group (group B), and NNSCS/GV268-miR-140 transfection group (group C). NNSCS/GV268 and NNSCS/GV268-miR- 140 complexes were transiently transfected into cells of groups B and C. After transfection, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expressions of exogenous miR-140; Annexin Ⅴ-FITC/PI double staining and MTT assay were used to detect the effect of exogenous miR-140 on apoptosis and proliferation of transfected chondrocytes; the expressions of Sox9, Aggrecan, and histone deacetylase 4 (Hdac4) were detected by RT-qPCR.
Results
RT-qPCR showed that the expression of miR-140 in group C was significantly higher than that in groups A and B (P<0.05). Compared with groups A and B, the apoptosis rate in group C was decreased and the proliferation activity was improved, Sox9 and Aggrecan gene expressions were significantly up-regulated, and Hdac4 gene expression was significantly down-regulated (P<0.05). There was no significant difference in above indexes between groups A and B (P>0.05).
Conclusion
Exogenous gene can be carried into the chondrocytes by NNSCS and expressed efficiently, the high expression of miR-140 can improve the biological activity of chondrocytes cultured in vitro, which provides important experimental basis for the treatment of cartilage damage diseases.
Marine-derived biopolymers are excellent raw materials for biomedical products due to their abundant resources, good biocompatibility, low cost and other unique functions. Marine-derived biomaterials become a major branch of biomedical industry and possess promising development prospects since the industry is in line with the trend of " green industry and low-carbon economy”. Chitosan and alginates are the most commonly commercialized marine-derived biomaterials and have exhibited great potential in biomedical applications such as wound dressing, dental materials, antibacterial treatment, drug delivery and tissue engineering. This review focuses on the properties and applications of chitosan and alginates in biomedicine.
ObjectiveTo explore a method of three-dimensional (3D) printing technology for preparation of personalized rat brain tissue cavity scaffolds so as to lay the foundation for the repair of traumatic brain injury (TBI) with tissue engineered customized cavity scaffolds.
MethodsFive male Sprague Dawley rats[weighing (300±10) g] were induced to TBI models by electric controlled cortical impactor. Mimics software was used to reconstruct the surface profile of the damaged cavity based on the MRI data, computer aided design to construct the internal structure. Then collagen-chitosan composite was prepared for 3D bioprinter of bionic brain cavity scaffold.
ResultsMRI scans showed the changes of brain tissue injury in the injured side, and the position of the cavity was limited to the right side of the rat brain cortex. The 3D model of personalized cavity containing the internal structure was successfully constructed, and cavity scaffolds were prepared by 3D printing technology. The external contour of cavity scaffolds was similar to that of the injured zone in the rat TBI; the inner positive crossing structure arranged in order, and the pore connectivity was good.
ConclusionCombined with 3D reconstruction based on MRI data, the appearance of cavity scaffolds by 3D printing technology is similar to that of injured cavity of rat brain tissue, and internal positive cross structure can simulate the topological structure of the extracellular matrix, and printing materials are collagen-chitosan complexes having good biocompatibility, so it will provide a new method for customized cavity scaffolds to repair brain tissue cavity after TBI.
