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.
Objective To investigate the effect of carboxymethylated chitosan (CMCS) on the proliferation, cell cycle, and secretion of neurotrophic factors in cultured Schwann cells (SCs). Methods SCs were obtained from sciatic nerves of 20 Sprague Dawley rats (3-5 days old; male or female; weighing, 25-30 g) and cultured in vitro, SCs were identified and purified by immunofluorescence against S-100. The cell counting kit 8 (CCK-8) assay was used to determine the proliferation of SCs. The SCs were divided into 4 groups: 50 μg/mL CMCS (group B), 100 μg/mL CMCS (group C), 200 μg/mL CMCS (group D), and the same amount of PBS (group A) were added. The flow cytometry was used to analyze the cell cycle of SCs; the real-time quantitative PCR and Western blot analysis were used to detect the levels of never growth factor (NGF) and ciliary neurotrophic factor (CNTF) in cultured SCs induced by CMCS. Results The purity of cultured SCs was more than 90% by immunofluorescence against S-100; the CCK-8 results indicated that CMCS in concentrations of 10-1 000 μg/mL could promote the proliferation of SCs, especially in concentrations of 200 and 500 μg/mL (P lt; 0.01), but no significant difference was found between 200 and 500 μg/mL (P gt; 0.05). CMCS at a concentration of 200 μg/mL for 24 hours induced the highest proliferation, showing significant difference when compared with that at 0 hour (P lt; 0.01). The percentage of cells in phase S and the proliferation index were significantly higher in groups B, C, and D than in group A (P lt; 0.05), in groups C and D than in group B (P lt; 0.05); and there was no significant difference between group C and group D (P gt; 0.05). Real-time quantitative PCR and Western blot results showed that the levels of NGF and CNTF in groups B, C, and D were significantly higher than those in group A (P lt; 0.05), especially in group D. Conclusion CMCS can stimulate the proliferation, and induce the synthesis of neurotrophic factors in cultured SCs.
【Abstract】 Objective To investigate the anti-infection and bone repair effects of cationic l i posome-encapsulatedvancomycin combined with the nano-hydroxyapatite/chitosan/konjac glucomannan (n-HA/CS/KGM) composite scaffold invivo. Methods Fifty-one 6-month-old New Zealand white rabbits, weighing 1.5-3.0 kg, were selected to prepare chronicinfectious tibia bone defect model by using Staphylococcus aureus. After 4 weeks, 48 survival rabbits were randomly divided into 4 groups (n=12). After debridement, defect was treated with nothing in group A, with n-HA/CS/KGM composite scaffold in group B, with vancomycin and n-HA/CS/KGM composite scaffold in group C, and with cationic l i posome-encapsulated vancomycin and n-HA/CS/KGM composite scaffold in group D. After 8 weeks of treatment, general observation, X-ray, HE staining, the bacterial culture, and the measurement of the longest diameter of bone defect were done. Results At 4 weeks after modeling, 48 rabbits were diagnosed as having osteomyelitis, including periosteal new bone formation, destruction of bone, and soft tissue swell ing. The Norden score was 3.83 ± 0.52. At 8 weeks after treatment, sinus healed in groups C and D, but sinus was observed in groups A and B; the gross bone pathologieal scores of group D were significantly better than those of groups A and B (P lt; 0.05). Bone defects were repaired completely in group D, the results of the longest diameter of bone defects in group D was significantly better than those in the other 3 groups (P lt; 0.05). New bone formation was observed in groups C and D, but periosteal reactionand marrow low-density shadow were observed in groups A and B; Norden score in group D was significantly better than those in groups A, B, and C (P lt; 0.05). HE staining showed that there were a large number of trabecular bone formation and fibrosis, with no obvious signs of infection in groups C and D, but neutrophil accumulation was observed in groups A and B; Smeltzer scores in groups C and D were significantly better than those in groups A and B (P lt; 0.05). Bacteriological results showed higher negative rate in groups C and D than in groups A and B (P lt; 0.05). Conclusion Cationic l iposome-encapsulated vancomycin and n-HA/CS/KGM composite scaffold can be a good treatment for infectious bone defects in rabbits, providing a new strategy for the therapy of bone defects in chronic infection.
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 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 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 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.
Objective To compare the growth and extracellular matrix biosynthesis of nucleus pulposus cells (NPCs)and bone marrow mesenchymal stem cells (BMSCs) in thermo-sensitive chitosan hydrogel and to choose seed cells for injectable tissue engineered nucleus pulposus. Methods NPCs were isolated and cultured from 3-week-old New Zealand rabbits (male or female, weighing 150-200 g). BMSCs were isolated and cultured from bone marrow of 1-month-old New Zealand rabbits (male or female, weighing 1.0-1.5 kg). The thermo-sensitive chitosan hydrogel scaffold was made of chitosan, disodium β glycerophosphate, and hydroxyethyl cellulose. Then, NPCs at the 2nd passage or BMSCs at the 3rd passage were mixed with chitosan hydrogel to prepare NPCs or BMSCs-chitosan hydrogel complex as injectable tissue engineered nucleus pulposus. The viabil ities of NPCs and BMSCs in the chitosan hydrogel were observed 2 days after compound culture. The shapes and distributions of NPCs and BMSCs on the scaffold were observed by scanning electron microscope (SEM) 1 week after compound culture. The histology and immunohistochemistry examination were performed. The expressions of aggrecan and collagen type II mRNA were analyzed by RT-PCR 3 weeks after compound culture. Results The thermo-sensitive chitosan hydrogel was l iquid at room temperature and sol idified into gel at37 (after 15 minutes) due to crossl inking reaction. Acridine orange/propidium iodide staining showed that the viabil ity rates of NPCs and BMSCs in chitosan hydrogel were above 90%. The SEM observation demonstrated that the NPCs and BMSCs distributed in the reticulate scaffold, with extracellular matrix on their surfaces. The results of HE, safranin O histology and immunohistochemistry staining confirmed that the NPCs and BMSCs in chitosan hydrogel were capable of producing extracellular matrix. RT-PCR results showed that the expressions of collagen type II and aggrecan mRNA were 0.564 ± 0.071 and 0.725 ± 0.046 in NPCs culture with chitosan hydrogel, and 0.713 ± 0.058 and 0.852 ± 0.076 in BMSCs culture with chitosan hydrogel; showing significant difference (P lt; 0.05). Conclusion The thermo-sensitive chitosan hydrogel has good cellular compatibil ity. BMSCs culture with chitosan hydrogel maintains better cell shape, prol iferation, and extracellular matrix biosynthesis than NPCs.
Objective To investigate the effects of carboxymethylchitosan- carboxymethylcellulose (CMCH-CMC) film on the adhesion and heal ing of colonic anastomosis. Methods Sixty-four healthy adult male SD rats was randomly divided into control group and experimental group (n=32). The model of colonic anastomosis was made according to Buckenmaier’ smethod in all rats. The experimental group was treated by wrapping anastomosis with CMCH-CMC film (3 cm × 2 cm) and the control group was not treated. At 7 days and 14 days after operation, the adhesion formation of colonic anastomosis was observed, the tensile strength of the anstomosis was assessed and compared with 6 normal rats, and the hydroxyprol ine (HP) content of the anastomotsis was detected. Results There were 3 deaths in the experimental group and 2 deaths in the control group. The adhesive scores of the experimental group on the 7th and 14th postoperative day [(0.50 ± 0.16) points and (0.45 ± 0.14) points, (Plt; 0.05)] were significantly lower than those of the control group [(1.67 ± 0.15) points and (2.29 ± 0.18) points, (P lt; 0.05)], (Plt; 0.01). Tensile strength were more marked on the 14th postoperative day than on the 7th postoperative day in the control group (Plt; 0.05), but there was no significant difference between the 7th day and the 14th day in the experimental group. The tensile strength of thecontrol group and the experimental group on the 14th postoperative day [(178.36 ± 20.10) and (172.74 ± 22.18) mmHg] were respectively higher than those on the 7th postoperative day [(138.67 ± 16.65) and (130.81 ± 18.38) mmHg] (Plt; 0.01). The tensile strength of the control group and the experimental group on the 7th postoperative day were respectively significantly lower than that of the normal rats (P lt; 0.01). The level of HP in the anastomosis was significantly higher on the 7th postoperative day in the experimental group [(84.47 ± 11.87) μg/mg dried weight] than that of the control group [(55.47 ± 12.89) μg/mg dried weight), (Plt; 0.05)], but there was no significant difference between the experimental group and the control group on the 14th postoperative day [(146.07 ± 14.81) μg/mg dried weight, (137.14 ± 16.81) μg/mg dried weight, (P gt; 0.05)]. Conclusion The CMCH-CMC film can decrease adhesion the formation of colonic anastomosis, but does not interfere with the heal ing of colonic anastomosis.
Objective To investigate the feasibil ity of using thermo-sensitive chitosan hydrogen as a scaffold to construct tissue engineered injectable nucleus pulposus (NP). Methods Three-month-old neonatal New Zealand rabbits (male or female) weighing 150-200 g were selected to isolate and culture NP cells. The thermo-sensitive chitosan hydrogel scaffold wasmade of chitosan, disodium β-glycerophosphate and hydroxyethyl cellulose. Its physical properties and gross condition were observed. The tissue engineered NP was constructed by compounding the scaffold and rabbit NP cells. Then, the viabil ity of NP cells in the chitosan hydrogel was observed 2 days after compound culture and the growth condition of NP cells on the scaffold was observed by SEM 7 days after compound culture. NP cells went through histology and immunohistochemistry detection and their secretion of aggrecan and expression of Col II mRNA were analyzed by RT-PCR 21 days after compound culture. Results The thermo-sensitive chitosan hydrogel was l iquid at room temperature and sol idified into gel at 37 (15 minutes) due to crossl inking reaction. Acridine orange-propidiumiodide staining showed that the viabil ity rate of NP cells in chitosan hydrogel was above 90%. Scanning electron microscope observation demonstrated that the NP cells were distributed in the reticulate scaffold, with ECM on their surfaces. The results of HE, toluidine blue, safranin O and histology and immunohistochemistry staining confirmed that the NP cells in chitosan hydrogel were capable of producing ECM. RT-PCR results showed that the secretion of Col II and aggrecan mRNA in NP cells cultured three-dimensionally by chitosan hydrogen scaffold were 0.631 ± 0.064 and 0.832 ± 0.052, respectively,showing more strengths of producing matrix than that of monolayer culture (0.528 ± 0.039, 0.773 ± 0.046) with a significant difference (P lt; 0.05). Conclusion With good cellular compatibilities, the thermo-sensitive chitosan hydrogel makes it possible for NP cells to maintain their normal morphology and secretion after compound culture, and may be a potential NP cells carrier for tissue engineered NP.