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.
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.
ObjectiveTo investigate the possibility and effect of chitosan porous scaffolds combined with bone marrow mesenchymal stem cells (BMSCs) in repair of neurological deficit after traumatic brain injury (TBI) in rats.MethodsBMSCs were isolated, cultured, and passaged by the method of bone marrow adherent culture. The 3rd generation BMSCs were identified by the CD29 and CD45 surface antigens and marked by 5-bromo-2-deoxyuridine (BrdU). The chitosan porous scaffolds were produced by the method of freeze-drying. The BrdU-labelled BMSCs were co-cultured in vitro with chitosan porous scaffolds, and were observed by scanning electron microscopy. MTT assay was used to observe the cell growth within the scaffold. Fifty adult Sprague Dawley rats were randomly divided into 5 groups with 10 rats in each group. The rat TBI model was made in groups A, B, C, and D according to the principle of Feeney’s free fall combat injury. Orthotopic transplantation was carried out at 72 hours after TBI. Group A was the BMSCs and chitosan porous scaffolds transplantation group; group B was the BMSCs transplantation group; group C was the chitosan porous scaffolds transplantation group; group D was the complete medium transplantation group; and group E was only treated with scalp incision and skull window as sham-operation group. Before TBI and at 1, 7, 14, and 35 days after TBI, the modified neurological severity scores (mNSS) was used to measure the rats’ neurological function. The Morris water maze tests were used after TBI, including the positioning voyage test (the incubation period was detected at 31-35 days after TBI, once a day) and the space exploration test (the number of crossing detection platform was detected at 35 days after TBI). At 36 days after TBI, HE staining and immunohistochemistry double staining [BrdU and neurofilament triplet H (NF-H) immunohistochemistry double staining, and BrdU and glial fibrillary acidic protein (GFAP) immunohistochemistry double staining] were carried out to observe the transplanted BMSCs’ migration and differentiation in the damaged brain areas.ResultsFlow cytometry test showed that the positive rate of CD29 of the 3rd generation BMSCs was 98.49%, and the positive rate of CD45 was only 0.85%. After co-cultured with chitosan porous scaffolds in vitrofor 48 hours, BMSCs were spindle-shaped and secreted extracellular matrix to adhere in the scaffolds. MTT assay testing showed that chitosan porous scaffolds had no adverse effects on the BMSCs’ proliferation. At 35 days after TBI, the mNSS scores and the incubation period of positioning voyage test in group A were lower than those in groups B, C, and D, and the number of crossing detection platform of space exploration test in group A was higher than those in groups B, C, and D, all showing significant differences (P<0.05); but no significant difference was found between groups A and E in above indexes (P>0.05). HE staining showed that the chitosan porous scaffolds had partially degraded, and they integrated with brain tissue well in group A; the degree of repair in groups B, C, and D were worse than that of group A. Immunohistochemical double staining showed that the transplanted BMSCs could survive and differentiate into neurons and glial cells, some differentiated neural cells had relocated at the normal brain tissue; the degree of repair in groups B, C, and D were worse than that of group A.ConclusionThe transplantation of chitosan porous scaffolds combined with BMSCs can improve the neurological deficit of rats following TBI obviously, and also inhabit the glial scar’s formation in the brain damage zone, and can make BMSCs survive, proliferate, and differentiate into nerve cells in the brain damage zone.
Objective To verify the technics of inactivating/removing pathogens in medical chitosan derived from shrimp shell. Methods Possible pathogen species were included according to the raw material of shrimp shell used in production, then bacillus cereus, porcine parvovirus (PPV) and pseudorabies virus (PRV) were selected as indicator pathogens.Pathogen solution was prepared in accordance with Technical Standard for Disinfection. The processing procedure of medical chitosan was analyzed to determine whether the alkal ization of chitin and the filter steril ization of chitosan were capable of inactivating/removing pathogens and their efficiencies were tested. Results Bacillus cereus was removed by 8 184 cfu/ mL after alkal ization and 30 818 cfu/mL after filter steril ization. The average logarithm inactivation value (LIV) of PPV and PRV after alkal ization were equal to or above 4.76 logTCID50/0.1 mL and 6.67 logTCID50/0.1 mL, respectively, and their average LIV after filter steril ization were 2.25 logTCID50/0.1 mL and 3.04 logTCID50/0.1 mL. The alkal ization of chitin inactivated/removed indicator pathogens effectively, while the filter steril ization of chitosan removed bacterial effectually but could not inactivate viruses completely. Conclusion The alkal ization of chitin can be used as the technics of inactivating/removing pathogens during the preparation process of medical chitosan to guarantee the safety of the product.
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.
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.
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.
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 prepare collagen-chitosan /nano-hydroxyapatite-collagen-polylactic acid (Col-CS/ nHAC-PLA) biomimetic scaffold and to examine its biocompatibility so as to lay the foundation for its application on the treatment of osteochondral defect. Methods PLA was dissolved in dioxane for getting final concentration of 8%, and the nHAC power was added at a weight ratio of nHAC to PLA, 1 ∶ 1. The solution was poured into a mold and frozen. CS and Col were dissolved in 2% acetum for getting the final concentrations of 2% and 1% respectively, then compounded at a weight ratio of CS to Col, 20 ∶ 1. The solution was poured into the frozen mold containing nHAC-PLA, and then biomimetic osteochondral scaffold of Col-CS/nHAC-PLA was prepared by freeze-drying. Acute systemic toxicity test, intracutaneous stimulation test, pyrogen test, hemolysis test, cytotoxicity test, and bone implant test were performed to evaluate its biocompatibility. Results Col-CS/nHAC-PLA had no acute systemic toxicity. Primary irritation index was 0, indicating that Col-CS/nHAC-PLA had very slight skin irritation. In pyrogen test, the increasing temperature of each rabbit was less than 0.6℃, and the increasing temperature sum of 3 rabbits was less than 1.3℃, which was consistent with the evaluation criteria. Hemolytic rate of Col-CS/nHAC-PLA was 1.38% (far less than 5%). The toxicity grade of Col-CS/nHAC-PLA was classified as grade I. Bone implant test showed that Col-CS/nHAC-PLA had good biocompatibility with the surrounding tissue. Conclusion Col-CS/ nHAC-PLA scaffold has good biocompatibility, which can be used as an alternative osteochondral scaffold.
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.