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.
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.
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.
To explore the effect of hydroxybutyl chitosan on the prevention of postoperative peritoneal adhesion in rats. Methods Ninety SD rats (half males and half females) weighing 250-280 g underwent laparotomy with subsequent cecal wall abrasion and peritoneal adhesion. Rats were randomized into 3 groups (n=30 per group): group A, injection of 2 mL hydroxybutyl chitosan solution (2%); group B, injection of 2 mL sodium hyaluronate solution(2%); group C, the abdomen of rat was exposed for 30 seconds and served as control group. The general condition of the rats was observed after operation. The rats were killed 2 and 4 weeks after operation, 15 rats per group at a time, to undergo gross and histologyobservation. The degree of adhesion was evaluated by double-bl ind method. The microstructure of injured electroscope cecal wall in groups A and C was observed with transmission electroscope 4 weeks after operation. Results All rats survived till the end of experiment. At 2 weeks after operation, the adhesion and the hyperplasia of fibrous connective tissue and collagen in groups A and B were sl ight while the adhesion in group C was serious with severe hyperplasia of fibrous connective tissue. According to the measurement classification by Nair histological grading, the difference between groups A and B and group C was significant (P lt; 0.05), while no significant difference was evident between group A and group B (P gt; 0.05). At 4 weeks after operation, the adhesion in group A was mild, and the hyperplasia of fibrous connective tissue and collagen were sl ight; the adhesion and the hyperplasia of fibrous connective tissue and collagen in group C were serious. The levels of group B were between group A and group C. The differences among three groups were significant (P lt; 0.05). Transmission electroscope showed inactive fibroblasts and loose thin collagen fibers in group A, and active fibroblasts and closely collagen fibers arranged in a disorderly manner in group C. Conclusion Hydroxybutyl chitosan can decrease the hyperplasia of fibrous connective tissue and inhibit the activity of fibroblasts significantly, and has a long-term role of preventing peritoneal adhesion.
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.
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 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.
ObjectiveTo study the effect of intraarticular injection of crosslinked-chitosan in the treatment of knee osteoarthritis in rabbits.MethodsThirty-two New Zealand white rabbits were randomly divided into 4 groups (groups A, B, C, and D; 8 rabbits in each group). The knee osteoarthritis models were prepared by anterior cruciate ligament transection in the left hind in groups A, B, and C. At 4 weeks after operation, the rabbits were received intraarticular injection of 0.6 mL crosslinked-chitosan in group A, 0.3 mL chitosan (once per 2 weeks, for twice) in group B, and 0.3 mL saline (once per 2 weeks, for twice) in group C. The rabbits in group D were treated with sham operation in the left hind, and received intraarticular injection of 0.3 mL saline (once per 2 weeks, for twice). At 8 weeks, the macroscopic observation, histological examination (HE staining, Safranin-fast green double staining, and Mankin score), scanning electron microscopy (SEM) observation, and immunohistochemical staining of collagen type Ⅱ were performed.ResultsMacroscopic and SEM observations showed that the cartilage in group D was basically the same as normal and better than that in groups A and B, and the abrasion of cartilage in group C was the most serious. The histological observation results in groups A and B were slightly similar and better than those in group C, but not up to the structure of group D. The macroscopic score and Mankin score of groups B and C were significantly higher than those of group D (P<0.05), and there was no significant difference between group A and group B (P>0.05). Immunohistochemical staining results showed that the collagen type Ⅱ positive percentage of chondrocytes was significantly higher in group D than that in groups B and C, and no significant difference was found between group A and group B (P>0.05). ConclusionThe crosslinked-chitosan can significantly improve the osteoarthritis of the rabbit knee, delay the pathological changes of osteoarthritis, and decrease the frequency of injection.
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.