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.
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 ectopic bone formation of the chitosan/phosphonic chitosan sponge combined with human umbil ical cord mesenchymal stem cells (hUCMSCs) in vitro. Methods Phosphorous groups were introduced in chitosan molecules to prepare the phosphonic chitosan; 2% chitosan and phosphonic chitosan solutions were mixed at a volume ratio of 1 ∶ 1 and freeze-dried to build the complex sponge, and then was put in the simulated body fluid for biomimetic mineral ization in situ. The hUCMSCs were isolated by enzyme digestion method from human umbil ical cord and were cultured. The chitosan/phosphonic chitosan sponge was cultured with hUCMSCs at passage 3, and the cell-scaffoldcomposite was cultured in osteogenic medium. The growth and adhesion of the cells on the scaffolds were observed by l ight microscope and scanning electron microscope (SEM) at 1 and 2 weeks after culturing, respectively. The cell prol iferation was detected by MTT assay at 1, 2, 3, 4, 5, and 6 days, respectively. Bilateral back muscles defects were created on 40 New Zealand rabbits (3-4 months old, weighing 2.1-3.2 kg, male or female), which were divided into groups A, B, and C. In group A, cellscaffold composites were implanted into 40 right defects; in group B, the complex sponge was implanted into 20 left defects; and in group C, none was implanted into other 20 left defects. The gross and histological observations were made at 4 weeks postoperatively. Results The analysis results of phosphonic chitosan showed that the phosphorylation occurred mainly in the hydroxyl, and the proton type and chemical shifts intensity were conform to its chemical structure. The SEM results showed that the pores of the chitosan/phosphonic chitosan sponge were homogeneous, and the wall of the pore was thinner; the coating of calcium and phosphorus could be observed on the surface of the pore wall after mineral ized with crystal particles; the cells grew well on the surface of the chitosan/phosphonic chitosan sponge. The MTT assay showed that the chitosan/phosphonic chitosan sponge could not inhibit the prol iferation of hUCMSCs. The gross observation showed that the size and shape of the cell-scaffold composite remained intact and texture was toughened in group A, the size of the complex sponge gradually reducedin group B, and the muscle defects wound healed with a l ittle scar tissue in group C. The histological observation showed that part of the scaffold was absorbed and new blood vessels and new bone trabeculae formed in group A, the circular cavity and residual chitosan scaffolds were observed in group B, and the wound almost healed with a small amount of lymphocytes in group C. Conclusion The chitosan/phosphonic chitosan sponge has good biocompatibil ity, the tissue engineered bone by combining the hUCMSCs with chitosan/phosphonic chitosan sponge has the potential of the ectopic bone formation in rabbit.
Objective To prepare nano polypyrrole (PPy)/chitin composite membrane and observe their biocompatibility. Methods The nano PPy was synthesized by microemulsion polymerization, blended with chitosan and then formed membranes. The membranes were then modified by acetylation to get the experimental membranes (nano PPy/chitin composite membranes, group A). The chitosan membranes (group B) and chitin ones (group C) modified by acetylation acted as control. Scanning electron microscopy and FT-IR spectra were used to identify the nano PPy and the membranes of each group. And the conductivity of membranes of each group was measured. Schwann cells were co-cultured in vitro with each group membranes to observe the biocompatibility by inverted microscope observing, living cell staining, cell counting, and immunofluorescence staining. The lysozyme solution was used to evaluate the degradation of the membranes in vitro. Results The FT-IR spectra showed that the characteristic vibrational absorption peaks of C=C from nano PPy appeared at 1 543.4 cm–1 and 1 458.4 cm–1. Scanning electron microscopy observation revealed that the size of nano PPy particles was about 100-200 nm. The nano PPy particles were synthesized. It was successful to turn chitosan to chitin by the acetylation, which was investigated by FT-IR analysis of membranes in groups A and C. The characteristic peaks of the amide Ⅱ band around 1 562 cm–1 appeared after acetylated modification. Conductivity test showed that the conductivity of membranes in group A was about (1.259 2±0.005 7)×10–3 S/cm, while the conductivity of the membranes in groups B and C was not detected. The nano PPy particles uniformly distributed on the surface of membranes in group A were observed by scanning electron microscope; the membranes in control groups were smooth. As a result, the nano PPy/chitin composite membranes with electrical conductivity were obtained. The cultured Schwann cells were found to survive with good function by fluorescein diacetate live cell staining, soluble protein-100 immunofluorescence staining, and inverted microscope observing. The cell counting showed that the proliferation of Schwann cells after 2 days and 4 days of group A was more than that of the two control groups, and the differences were significant (P<0.05). It indicated that the nano PPy/chitin composite membranes had better ability of adhesion and proliferation than those of chitosan and chitin membranes. The degradation of membranesin vitro showed that the degradation rates of membranes in groups A and C were significantly higher than those in group B at all time points (P<0.05). In a word, the degradation performance of the membranes modified by acetylation was better than that of chitosan membranes under the same condition. Conclusion The nano PPy and chitosan can be blended and modified by acetylation successfully. Nano PPy/chitin composite membranes had electrical conductivity, degradability, and good biocompatibility in vitro.
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.
ObjectiveTo investigate the in vivo degradation and histocompatibility of modified chitosan based on conductive composite nerve conduit, so as to provide a new scaffold material for the construction of tissue engineered nerve.MethodsThe nano polypyrrole (PPy) was synthesized by microemulsion polymerization, blended with chitosan, and then formed conduit by injecting the mixed solution into a customized conduit formation model. After freeze-drying and deacidification, the nano PPy/chitosan composite conduit (CP conduit) was prepared. Then the CP conduits with different acetyl degree were resulted undergoing varying acetylation for 30, 60, and 90 minutes (CAP1, CAP2, CAP3 conduits). Fourier infrared absorption spectrum and scanning electron microscopy (SEM) were used to identify the conduits. And the conductivity was measured by four-probe conductometer. The above conduits were implanted after the subcutaneous fascial tunnels were made symmetrically on both sides of the back of 30 female Sprague Dawley rats. At 2, 4, 6, 8, 10, and 12 weeks after operation, the morphology, the microstructure, and the degradation rate were observed and measured to assess the in vivo degradation of conduits. HE staining and anti-macrophage immunofluorescence staining were performed to observe the histocompatibility in vivo.ResultsThe characteristic peaks of the amide Ⅱ band around 1 562 cm−1 appeared after being acetylated, indicating that the acetylation modification of chitosan was successful. There was no significant difference in conductivity between conduits (P>0.05). SEM observation showed that the surfaces of the conduits in all groups were similar with relatively smooth surface and compact structure. After the conduits were implanted into the rats, with the extension of time, all conduits were collapsed, especially on the CAP3 conduit. All conduits had different degrees of mass loss, and the higher the degree of acetylation, the greater the mass change (P<0.05). SEM observation showed that there were more pores at 12 weeks after implantation, and the pores showed an increasing trend as the degree of acetylation increased. Histological observation showed that there were more macrophages and lymphocytes infiltration in each group at the early stage. With the extension of implantation time, lymphocytes decreased, fibroblasts increased, and collagen fibers proliferated significantly. ConclusionThe modified chitosan basedon conductive composite nerve conduit made of nano-PPy/chitosan composite with different acetylation degrees has good biocompatibility, conductivity, and biodegradability correlated with acetylation degree in vivo, which provide a new scaffold material for the construction of tissue engineered nerve.
Objective To study the effect of water soluble chitosan (WSC) on the apoptosis of peritoneal macrophage induced by lipopolysaccharides (LPS), and discuss the mechanism. Methods Peritoneal macrophages were divided to three groups: phosphate buffered saline (PBS) group, LPS group and LPS plus WSC group. At hour 24, apoptosis cell and active caspase-3 were detected by flow cytometry; nitric oxide (NO) was determined with Griess reagent. Results There were more apoptosis cells in the LPS group than the PBS group. The percentage of apoptosis cells was significantly decreased in the LPS plus WSC group than the LPS group. The expression of active caspase-3 and the secretion of NO were also inhibited by WSC after LPS intervention. Conclusion WSC inhibits apoptosis of peritoneal macrophage induced by LPS.
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.
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.
We in the present study observed the effect of N-fructose modified chitosan quaternary ammonium derivativeson on rat skin wound healing through animal experiments. Forty rats were randomly divided into eight groups (5 in each group). Four groups among the all 8 groups were the experimental groups, while the other 4 groups were the control groups. Next to the skin along the back of the spine, 1.50 cm×2.00 cm×0.16 cm full-thickness skin was cut to make an excision wound model for every rat. Those in the experimental groups were treated with the N-fructose-modified chitosan quaternary ammonium derivatives ointment dressing the wound, while those in the control groups with sterile medical vaseline processing. We dressed the wounds twice a day to observe the wound healing of all rats in different groups. We then observed the wound healing and wound pathology after 3, 7, 10, 15 days respectively in different groups. Results showed significant differences of the time of wound healing, area of wound healing and volume of wound healing between the experimental groups and control groups (P<0.05). It can be well concluded that N-fructose-modified chitosan quaternary ammonium derivatives does not harm the skin, but could promote skin healing, so that they could be suitable skin repair materials and ideal raw materials for medical dressing.