In order to increase the ability of oil-emulsion adjuvant to stimulate cellular immunity, chitosan hydrochloride with positive charge was selected to stabilize oil-in-water emulsion (CHE). In this paper, model antigen ovalbumin was selected to prepare vaccines with emulsion adjuvant, commercial adjuvant or no adjuvant. The emulsion was characterized by measuring the particle size, electric potential and antigen adsorption rate. BALB/c mice were immunized by intramuscular injection. Serum antibody levels, the numbers of IL-4-secreting cells in splenocytes, cytotoxic T lymphocyte (CTL) response, and the expression of central memory T cells were measured to evaluate the immunostimulatory effect. The results showed that chitosan hydrochloride can effectively stabilize the emulsion. The emulsion size is about 600 nm, and the antigen adsorption rate is more than 90%. After immunization, CHE could increase serum antibodies levels and increase IL-4 secretion. Expression of CTL surface activation molecules was also increased to stimulate CTL response further and to increase the CD44⁺CD62L⁺ in T cells proportion. CHE as adjuvant can stimulate humoral and cellular immunity more efficiently, and is expected to extend the duration of protection.
Animals ; Mice ; Chitosan ; Interleukin-4 ; Emulsions ; Immunization ; Adjuvants, Immunologic/pharmacology* ; Antigens ; Mice, Inbred BALB C

OBJECTIVE: This study tests the efficacy of Bletilla striata polysaccharide (BSP), carboxymethyl chitosan (CMC), baicalin (BA) and silver titanate (ST) in a wound dressings to fight infection, promote healing and provide superior biocompatibility. METHODS: The antibacterial activity of BA and ST was evaluated in vitro using the inhibition zone method. BA/ST/BSP/CMC porous sponge dressings were prepared and characterized. The biocompatibility of BA/ST/BSP/CMC was assessed using the cell counting kit-8 assay. The therapeutic effect of BA/ST/BSP/CMC was further investigated using the dorsal skin burn model in Sprague-Dawley rats. RESULTS: The wound dressing had good antibacterial activity against Escherichia coli and Staphylococcus aureus through BA and ST, while the combination of BSP and CMC played an important role in promoting wound healing. The BA/ST/BSP/CMC porous sponge dressings were prepared using a freeze-drying method with the concentrations of BA and ST at 20 and 0.83 mg/mL, respectively, and the optimal ratio of 5% BSP to 4% CMC was 1:3. The average porosity, water absorption and air permeability of BA/ST/BSP/CMC porous sponge dressings were measured to be 90.43%, 746.1% and 66.60%, respectively. After treatment for 3 and 7 days, the healing rates of the BA/ST/BSP/CMC group and BA/BSP/CMC group were significantly higher than those of the normal saline (NS) group and silver sulfadiazine (SSD) group (P < 0.05). Interleukin-1β expression in the BA/ST/BSP/CMC group at 1 and 3 days was significantly lower than that in the other three groups (P < 0.05). After being treated for 3 days, vascular endothelial growth factor expression in the BA/BSP/CMC group and BA/ST/BSP/CMC group was significantly higher than that in the NS group and SSD group (P < 0.05). Inspection of histological sections showed that the BA/ST/BSP/CMC group and BA/BSP/CMC group began to develop scabbing and peeling of damaged skin after 3 days of treatment, indicating accelerated healing relative to the NS group and SSD group. CONCLUSION The optimized concentration of BA/ST/BSP/CMC dressing was as follows: 6 mg BSP, 14.4 mg CMC, 0.5 mg ST and 12 mg BA. The BA/ST/BSP/CMC dressing, containing antibacterial constituents, was non-cytotoxic and effective in accelerating the healing of burn wounds, making it a promising candidate for wound healing. Please cite this article as: Gong YR, Zhang C, Xiang X, Wang ZB, Wang YQ, Su YH, Zhang HQ. Baicalin, silver titanate, Bletilla striata polysaccharide and carboxymethyl chitosan in a porous sponge dressing for burn wound healing. J Integr Med. 2023; 21(5): 487-495.
Rats ; Animals ; Chitosan/pharmacology* ; Silver/pharmacology* ; Porosity ; Vascular Endothelial Growth Factor A/pharmacology* ; Rats, Sprague-Dawley ; Wound Healing ; Polysaccharides/pharmacology* ; Bandages ; Burns/drug therapy* ; Anti-Bacterial Agents/pharmacology* ; Silver Sulfadiazine/pharmacology*

The biofilms formed by pathogenic microorganisms seriously threaten human health and significantly enhance drug resistance, which urgently call for developing drugs specifically targeting on biofilms. Chitooligosaccharides extracted from shrimp and crab shells are natural alkaline oligosaccharides with excellent antibacterial effects. Nevertheless, their inhibition efficacy on biofilms still needs to be improved. Spirulina (SP) is a microalga with negatively charged surface, and its spiral structure facilitates colonization in the depth of the biofilm. Therefore, the complex of Spirulina and chitooligosaccharides may play a synergistic role in killing pathogens in the depth of biofilm. This research first screened chitooligosaccharides with significant bactericidal effects. Subsequently, Spirulina@Chitooligosaccharides (SP@COS complex was prepared by combining chitooligosaccharides with Spirulina through electrostatic adsorption. The binding of the complex was characterized by zeta potential, z-average size, and fluorescence labeling. Ultraviolet-visible spectroscopy (UV-Vis) showed the encapsulation efficiency and the drug loading efficiency reached up to 90% and 16%, respectively. The prepared SP@COS2 exhibited a profound synergistic inhibition effect on bacterial and fungal biofilms, which was mainly achieved by destroying the cell structure of the biofilm. These results demonstrate the potential of Spirulina-chitooligosaccharides complex as a biofilm inhibitor and provide a new idea for addressing the harm of pathogenic microorganisms.
Humans ; Spirulina ; Anti-Bacterial Agents/chemistry* ; Chitosan/pharmacology* ; Biofilms ; Chitin/pharmacology*

OBJECTIVE: To design and construct a graphene oxide (GO)/silver nitrate (Ag₃PO₄)/chitosan (CS) composite coating for rapidly killing bacteria and preventing postoperative infection in implant surgery. METHODS: GO/Ag₃PO₄ composites were prepared by ion exchange method, and CS and GO/Ag₃PO₄ composites were deposited on medical titanium (Ti) sheets successively. The morphology, physical image, photothermal and photocatalytic ability, antibacterial ability, and adhesion to the matrix of the materials were characterized. RESULTS: The GO/Ag₃PO₄ composites were successfully prepared by ion exchange method and the heterogeneous structure of GO/Ag₃PO₄ was proved by morphology phase test. The heterogeneous structure formed by Ag₃PO₄ and GO reduced the band gap from 1.79 eV to 1.39 eV which could be excited by 808 nm near-infrared light. The photothermal and photocatalytic experiments proved that the GO/Ag₃PO₄/CS coating had excellent photothermal and photodynamic properties. In vitro antibacterial experiments showed that the antibacterial rate of the GO/Ag₃PO₄/CS composite coating against Staphylococcus aureus reached 99.81% after 20 minutes irradiation with 808 nm near-infrared light. At the same time, the composite coating had excellent light stability, which could provide stable and sustained antibacterial effect. CONCLUSION GO/Ag₃PO₄/CS coating can be excited by 808 nm near infrared light to produce reactive oxygen species, which has excellent antibacterial activity under light.
Chitosan ; Silver Nitrate ; Titanium ; Anti-Bacterial Agents/pharmacology* ; Coloring Agents

Effective haemostatic materials can quickly control bleeding and achieve the purpose of saving patients' lives. In recent years, chitosan-based haemostatic materials have shown good haemostatic effects, but their application is limited because chitosan is almost insoluble in water. Carboxymethyl chitosan-based haemostatic materials can promote hemostasis by activating red blood cells and aggregating platelets. In addition, carboxymethyl chitosan can bind with Ca²⁺ to activate platelets and coagulation factors, and start endogenous coagulation pathways, which can adsorb fibrinogen in plasma to promote haemostasis. In this paper, the latest research progress of carboxymethyl chitosan-based haemostatic materials and their haemostatic mechanism were reviewed, in order to further strengthen the understanding of the haemostatic mechanism of carboxymethyl chitosan-based haemostatic materials, and provide new idea for the research and clinical application of carboxymethyl chitosan-based haemostatic materials.
Humans ; Hemostatics ; Chitosan/pharmacology* ; Hemostasis ; Blood Coagulation/physiology* ; Hemorrhage

OBJECTIVE: To characterize the silver in chitosan antibacterial gel, and to establish a method for the determination of silver content in samples. METHODS: The silver in the samples was analysed by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and single particle inductively coupled plasma mass spectrometry (SP-ICP-MS). Microwave digestion was adopted to digest the chitosan antibacterial gel, and then the content of silver was determined by flame atomic absorption spectrometry. RESULTS: The analysises showed that the particle size of silver in chitosan antibacterial gel was about 150~ 200 nm. The silver showed good linearity in the concentration range of 25~250 μg/L (y=0.000 35x+0.001 7, r=0.999 9). The recovery rate (n=9) was 98.5%. CONCLUSIONS SEM, EDS and SP-ICP-MS can be used for the characterization of silver particles in chitosan antibacterial gel. Microwave digestion-flame atomic absorption spectrophotometry method is simple, practicable, high precision and high quantitative accuracy, which is suitable for the quantitative analysis of silver in chitosan antibacterial gel.
Anti-Bacterial Agents/pharmacology* ; Chitosan/chemistry* ; Microwaves ; Particle Size ; Silver

Objective: To explore the effect of P311 microspheres-loaded thermosensitive chitosan hydrogel on the wound healing of full-thickness skin defects in rats. Methods: The method of experimental study was adopted. The polyvinyl alcohol/sodium alginate microspheres (simple microspheres), P311 microspheres, and bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA) microspheres were prepared by water-in-oil emulsification, and then their morphology was observed under a light microscope/inverted fluorescence microscope. Chitosan solution was prepared, chitosan solution and β-glycerol phosphate disodium hydrate were mixed to prepare simple thermosensitive hydrogels, and thermosensitive hydrogels loaded with simple microspheres or P311 microspheres were prepared by adding corresponding substances in simple thermosensitive hydrogels. The morphological changes of the prepared four liquids in the state of tilt was observed at 37 ℃. After being freeze-dried, the micromorphology of the prepared four liquids was observed under a scanning electron microscope. Eighteen 3-4-week-old male Sprague-Dawley rats were divided into normal group without any treatment, dressing group, chitosan group, hydrogel alone group, simple microspheres-loaded hydrogel group, and P311 microspheres-loaded hydrogel group, which were inflicted with one full-thickness skin defect wound on both sides of the back spine and were dealt correspondingly, with 3 rats in each group. Rats with full-thickness skin defects in the five groups were collected, the wound healing was observed on post injury day (PID) 0 (immediately), 5, 10, and 15, and the wound healing rates on PID 5, 10, and 15 were calculated. The wound and wound margin tissue of rats with full-thickness skin defects in the five groups on PID 15 and normal skin tissue in the same site of rats in normal group were collected, hematoxylin and eosin staining was conducted to observe the histological changes, immunohistochemical staining was performed to observe the expressions of CD31 and vascular endothelial growth factor (VEGF), and Western blotting was conducted to detect the protein expressions of CD31 and VEGF. The number of samples was all three. Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, and Bonferroni correction. Results: Simple microspheres were spherical, with loose and porous surface. The surfaces of P311 microspheres and FITC-BSA microspheres were smooth without pores, and the FITC-BSA microspheres emitted uniform green fluorescence. The diameters of the three microspheres were basically consistent, being 33.1 to 37.7 μm. Compared with chitosan solution and simple thermosensitive hydrogel, the structures of the two microspheres-loaded hydrogels were more stable in the state of tilt at 37 ℃. The two microspheres-loaded hydrogels had denser network structures than those of chitosan solution and simple thermosensitive hydrogel, and in the cross section of which microspheres with a diameter of about 30 μm could be seen. Within PID 15, the wounds of rats in the five groups were healed to different degrees, and the wound healing of rats in P311 microspheres-loaded hydrogel group was the best. On PID 5, 10, and 15, the wound healing rates of rats in dressing group and chitosan group were (26.6±2.4)%, (38.5±3.1)%, (50.9±1.5)%, (47.6±2.0)%, (58.5±3.6)%, and (66.7±4.1)%, respectively, which were significantly lower than (59.3±4.8)%, (87.6±3.2)%, (97.2±1.0)% in P311 microspheres-loaded hydrogel group (P<0.05 or P<0.01). The wound healing rates of rats in hydrogel alone group on PID 10 and 15, and in simple microspheres-loaded hydrogel group on PID 15 were (76.0±3.3)%, (84.5±3.6)%, and (88.0±2.6)%, respectively, which were significantly lower than those in P311 microspheres-loaded hydrogel group (P<0.05). The epidermis, hair follicles, and sebaceous glands could be seen in the normal skin of rats in normal group, without positive expressions of CD31 or VEGF. The wounds of rats in P311 microspheres-loaded hydrogel group on PID 15 were almost completely epithelialized, with more blood vessels, hair follicles, sebaceous glands, and positive expressions of CD31 and VEGF in the wounds than those of rats with full-thickness skin defects in the other four groups, and more protein expressions of CD31 and VEGF than those of rats in the other five groups. Conclusions: The P311 microspheres-loaded thermosensitive chitosan hydrogel can release the encapsulated drug slowly, prolong the drug action time, and promote wound healing in rats with full-thickness skin defects by promoting wound angiogenesis and re-epithelialization.
Rats ; Male ; Animals ; Hydrogels ; Vascular Endothelial Growth Factor A ; Chitosan/pharmacology* ; Serum Albumin, Bovine/pharmacology* ; Microspheres ; Polyvinyl Alcohol/pharmacology* ; Hematoxylin/pharmacology* ; Eosine Yellowish-(YS)/pharmacology* ; Rats, Sprague-Dawley ; Wound Healing ; Skin/injuries* ; Skin Abnormalities ; Soft Tissue Injuries ; Water/pharmacology* ; Alginates/pharmacology*

Objective: To explore the effects and mechanism of water-soluble chitosan hydrogel on infected full-thickness skin defect wounds in diabetic mice. Methods: The experimental research method was adopted. The control hydrogel composed of polyvinyl alcohol and gelatin, and the water-soluble chitosan hydrogel composed of the aforementioned two materials and water-soluble chitosan were prepared by the cyclic freeze-thaw method. The fluidity of the two dressings in test tube before and after the first freeze-thawing was generally observed, and the difference in appearance of the final state of two dressings in 12-well plates were compared. According to random number table (the same grouping method below), the cell strains of L929 and HaCaT were both divided into water-soluble chitosan hydrogel group and control hydrogel group, respectively. After adding corresponding dressings and culturing for 24 h, the cell proliferation activity was measured using cell counting kit 8. Rabbit blood erythrocyte suspensions were divided into normal saline group, polyethylene glycol octyl phenyl ether (Triton X-100) group, water-soluble chitosan hydrogel group, and control hydrogel group, which were treated accordingly and incubated for 1 hour, and then the hemolysis degree of erythrocyte was detected by a microplate reader. Twenty-four female db/db mice aged 11-14 weeks were selected, and full-thickness skin defect wounds on their backs were inflicted and inoculated with the methicillin-resistant Staphylococcus aureus (MRSA), 72 h later, the mice were divided into blank control group, sulfadiazine silver hydrogel group, control hydrogel group, and water-soluble chitosan hydrogel group, which were treated accordingly. On post injury day (PID) 0 (immediately), 7, 14, and 21, the healing of the wound was observed. On PID 14 and 21, the wound healing rate was calculated. On PID 14, MRSA concentration in wounds was determined. On PID 21, the wounds were histologically analyzed by hematoxylin and eosin staining; the expression of CD31 in the wounds was detected by immunofluorescence method, and its positive percentage was calculated. Raw264.7 cells were taken and divided into interleukin-4 (IL-4) group, blank control group, control hydrogel group, and water-soluble chitosan hydrogel group, which were treated accordingly. At 48 h of culture, the percentages of CD206 positive cells were detected by flow cytometry. The number of samples was all 3. Data were statistically analyzed with independent sample t test, one-way analysis of variance, analysis of variance for repeated measurement, least significant difference test, and Dunnett T3 test. Results: Two dressings in test tube had certain fluidity before freeze-thawing and formed semi-solid gels after freeze-thawing for once. The final forms of two dressings in 12-well plates were basically stable and translucent sheets, with little difference in transparency. At 24 h of culture, the cell proliferation activities of L929 and HaCaT in water-soluble chitosan hydrogel group were significantly higher than those in control hydrogel group (with t values of 6.37 and 7.50, respectively, P<0.01). At 1 h of incubation, the hemolysis degree of erythrocyte in water-soluble chitosan hydrogel group was significantly lower than that in Triton X-100 group (P<0.01), but similar to that in normal saline group and control hydrogel group (P>0.05). On PID 0, the traumatic conditions of mice in the 4 groups were similar. On PID 7, more yellowish exudates were observed inside the wound in blank control group and control hydrogel group, while a small amount of exudates were observed in the wound in sulfadiazine silver hydrogel group and water-soluble chitosan hydrogel group. On PID 14, the wounds in blank control group and control hydrogel group were dry and crusted without obvious epithelial coverage; in sulfadiazine silver hydrogel group, the scabs fell off and purulent exudate was visible on the wound; in water-soluble chitosan hydrogel group, the base of wound was light red and obvious epithelial coverage could be observed on the wound. On PID 14, the wound healing rate in water-soluble chitosan hydrogel group was significantly higher than that in the other 3 groups (all P<0.01). On PID 21, the wound in water-soluble chitosan hydrogel group was completely closed, while the wounds in the other 3 groups were not completely healed; the wound healing rate in water-soluble chitosan hydrogel group was significantly higher than that in the other 3 groups (all P<0.01). On PID 14, the concentration of MRSA in the wound in water-soluble chitosan hydrogel group was significantly lower than that in blank control group (P<0.01), but similar to that in control hydrogel group and sulfadiazine silver hydrogel group (P>0.05). On PID 21, the new epidermis was severely damaged in blank control group; the epidermis on the wound in control hydrogel group also had a large area of defect; complete new epidermis had not yet being formed on the wound in sulfadiazine silver hydrogel group; the wound in water-soluble chitosan hydrogel group was not only completely covered by the new epidermis, the basal cells of the new epidermis were also regularly aligned. On PID 21, the percentage of CD31 positivity in the wound in water-soluble chitosan hydrogel group was (2.19±0.35)%, which was significantly higher than (0.18±0.05)% in blank control group, (0.23±0.06)% in control hydrogel group, and (0.62±0.25)% in sulfadiazine silver hydrogel group, all P<0.01. At 48 h of culture, the percentage of CD206 positive Raw264.7 cells in water-soluble chitosan hydrogel group was lower than that in IL-4 group (P>0.01) but significantly higher than that in blank control group and control hydrogel group (P<0.05 or P<0.01). Conclusions: The water-soluble chitosan hydrogel has good biosafety and can induce higher level of macrophage M2 polarization than control hydrogel without water-soluble chitosan, so it can enhance the repair effect of MRSA-infected full-thickness skin defect wounds in diabetic mice and promote rapid wound healing.
Mice ; Female ; Animals ; Rabbits ; Interleukin-4 ; Hydrogels/pharmacology* ; Wound Healing ; Chitosan/pharmacology* ; Diabetes Mellitus, Experimental ; Water ; Methicillin-Resistant Staphylococcus aureus ; Gelatin ; Polyvinyl Alcohol ; Hemolysis ; Saline Solution ; Eosine Yellowish-(YS) ; Hematoxylin ; Octoxynol ; Silver ; Phenyl Ethers ; Sulfadiazine

OBJECTIVE: To compare a new thermosensitive recombinant human amelogenin (rhAm) carrier and traditional propylene glycol alginate (PGA) carrier for their characteristics, antibacterial activity, and biocompatibility with human periodontal membrane fibroblasts. METHODS: PGA-rhAm was prepared by mixing 3.3% PGA and rhAm, and CS-βGP-rhAm was prepared by mixing 2% chitosan (CS) with rhAm and then with 60% β-sodium glycerophosphate solution (βGP) as the crosslinking agent. The biophysical properties of the prepared carriers were characterized, and their antibacterial activity was assessed by observing Staphylococcus aureus growth. The biocompatibility of the carriers was evaluated in human periodontal membrane fibroblasts (hPDLFs) using CCK8 assay and scratch test, and mRNA and protein expressions of osteogenic genes of the cells incubated with the carriers were detected using RT-qPCR and Western blotting; osteogenic differentiation of the cells was detected using alkaline phosphatase staining. RESULTS: PGA-rhAm had a viscosity value of 3.262±0.055 Pa.s. CS-βGP-rhAm had a solidification capacity of 6 min at 37 ℃ with a pH value close to that of the oral cavity and a swelling rate of about 90%. CS-β GP-rhAm maintained sustained release of rhAm for over 2 weeks with a self-degradation time over 3 weeks. CS-βGPrhAm more effectively inhibited the growth of S. aureus than rhAm-loaded PGA. While PGA did not obviously affect the proliferation of hPDLFs, both CS-βGP and CS-βGP-rhAm significantly promoted the cell proliferation(P < 0.001). Scratch test showed that after rhAm loading, both CS-βGP and PGA promoted cell migration (P < 0.01). CS-βGP-rhAm significantly enhanced the mRNA expressions of RUNX2 and OCN mRNA level and the protein expressions of Ki67, RUNX2, collagen I, and β-catenin (P < 0.05); PGA-rhAm only enhanced RUNX2 (P < 0.05) and OCN (P < 0.01) mRNA expressions without significant effects on the protein expressions. Alkaline phosphatase staining results showed that CS-βGP, but not PGA, promoted osteogenic differentiation of hPDLFs. CONCLUSION CS-βGP carrier is capable of sustained release of rhAm, inhibiting the growth of S. aureus, and improving the biological activity of hPDLFs without affecting the bioactivity of rhAm after drug loading.
Alginates ; Alkaline Phosphatase ; Amelogenin ; Anti-Bacterial Agents/pharmacology* ; Cell Differentiation ; Cells, Cultured ; Chitosan/pharmacology* ; Collagen ; Core Binding Factor Alpha 1 Subunit ; Delayed-Action Preparations ; Glycerophosphates ; Humans ; Ki-67 Antigen ; Osteogenesis ; Periodontal Ligament ; RNA, Messenger ; Staphylococcus aureus ; beta Catenin

OBJECTIVE: A dynamic gel loaded with lyophilized platelet-rich plasma-chitosan/difunctionalized polyethylene glycol (LPRP-CP) was prepared to investigate its hemostatic antibacterial and promoting wound healing of scald wounds through in vitro and in vivo experiments. METHODS: In this study, normal gauze/blank tablet (Ctrl), LPRP-CP, Chitosan HUCHUANG Powder(Chito P)and ChitoGauze XP PRO group (Chito G group) were set. The hemostatic effect and promoting healing effect of the four groups of materials were evaluated by establishing rabbit ear artery hemorrhage model and superficial Ⅱ° scalded model of skin on the back. The hemostatic time and bleeding amount were calculated and the gross and histological results of scald healing were observed. The antibacterial effect of the four groups of materials was evaluated by antibacterial test in vitro. RESULTS: In the rabbit ear arterial hemorrhage model, the hemostasis of all materials was successful. The hemostatic time of Ctrl, Chito P, LPRP-CP and Chito G groups was 213.33±38.30, 118.33±24.01, 115.00±8.37 and 111.67±11.69 s, respectively. The blood loss was 1233.83±992.27, 346.67±176.00, 193.33±121.47 and 147.50±80.66 mg, respectively. Compared with Ctrl, the hemostasis time of LPRP-CP, Chito P and Chito G group was significantly shorter (P<0.001), and the amount of blood loss of LPRP-CP and Chito G group was decreased (P<0.05). Compared with LPRP-CP, there were no significant differences in hemostatic time and blood loss between Chito P and Chito G group (P>0.05). In the model of superficial Ⅱ° scalded on the back of rabbit, the wound healing rate of LPRP-CP was faster than that of the other three groups at the same time, and the healing effect was perfect. In the antibacterial test in vitro, only LPRP-CP had better anti-S. aureus effect, and all groups had no anti-E. coli effect. CONCLUSION LPRP-CP is an excellent hemostatic material for superficial wounds, and has certain antibacterial and wound healing effects, which has a wide academic value and research prospects.
Animals ; Anti-Bacterial Agents/pharmacology* ; Chitosan/pharmacology* ; Hemorrhage ; Hemostasis ; Hemostatics ; Humans ; Platelet-Rich Plasma ; Rabbits