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.
ObjectiveTo investigate the effect of graphene oxide (GO)-carboxymethyl chitosan (CMC) hydrogel loaded with interleukin 4 (IL-4) and bone morphogenetic protein 2 (BMP-2) on macrophages M2 type differentiation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).MethodsGO solution was mixed with CMC, then the phosphate buffered saline (PBS), IL-4, BMP-2, or IL-4+BMP-2 were added to prepare different GO-CMC hydrogel scaffolds with or without different cytokines under crosslinking agents. The characteristics of pure GO-CMC hydrogel were characterized by gross observation, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FTIR), and the CMC hydrogel was used as control. The sustained release of GO-CMC hydrogels with different cytokines was also tested. Macrophages were isolated and cultured from female Sprague Dawley rats aged 4-5 weeks, and then cultured with GO-CMC hydrogels with and without different cytokines, respectively. CD206 immunofluorescence staining was used to detect the differentiation of macrophages after 24 hours. The 3rd generation of rats BMSCs were cultured with GO-CMC hydrogels with and without different cytokines respectively for osteogenic induction. The early osteogenesis was observed by alkaline phosphatase (ALP) staining after 10 days, and the late osteogenesis was observed by alizarin red staining after 21 days.ResultsGenerally, GO-CMC hydrogel was brown and translucent. SEM showed that the pore diameter and wall thickness of GO-CMC hydrogel were similar to that of CMC hydrogel, but the inner wall roughness increased. FTIR test showed that CMC polymerized to form hydrogel. In vitro, the sustained release experiments showed that the properties of GO-CMC hydrogels loaded with different cytokines were similar. CD206 immunofluorescence detection showed that GO-CMC hydrogels could induce macrophages differentiation into M2-type. ALP and alizarin red staining showed that GO-CMC hydrogels could induce BMSCs osteogenic differentiation, in which GO-CMC hydrogel loaded with IL-4+BMP-2 showed the most significant effect (P<0.05).ConclusionThe GO-CMC hydrogel loaded with IL-4 and BMP-2 can induce macrophages differentiation into M2-type and enhance the ability of BMSCs with osteogenic differentiation in vitro, which provide a new strategy for bone defect repair and immune regulation.
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 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.
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.
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.
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 explore the effect of chitosan (CS) hydrogel loaded with tendon-derived stem cells (TDSCs; hereinafter referred to as TDSCs/CS hydrogel) on tendon-to-bone healing after rotator cuff repair in rabbits. Methods TDSCs were isolated from the rotator cuff tissue of 3 adult New Zealand white rabbits by Henderson step-by-step enzymatic digestion method and identified by multidirectional differentiation and flow cytometry. The 3rd generation TDSCs were encapsulated in CS to construct TDSCs/CS hydrogel. The cell counting kit 8 (CCK-8) assay was used to detect the proliferation of TDSCs in the hydrogel after 1-5 days of culture in vitro, and cell compatibility of TDSCs/CS hydrogel was evaluated by using TDSCs alone as control. Another 36 adult New Zealand white rabbits were randomly divided into 3 groups (n=12): rotator cuff repair group (control group), rotator cuff repair+CS hydrogel injection group (CS group), and rotator cuff repair+TDSCs/CS hydrogel injection group (TDSCs/CS group). After establishing the rotator cuff repair models, the corresponding hydrogel was injected into the tendon-to-bone interface in the CS group and TDSCs/CS group, and no other treatment was performed in the control group. The general condition of the animals was observed after operation. At 4 and 8 weeks, real-time quantitative PCR (qPCR) was used to detect the relative expressions of tendon forming related genes (tenomodulin, scleraxis), chondrogenesis related genes (aggrecan, sex determining region Y-related high mobility group-box gene 9), and osteogenesis related genes (alkaline phosphatase, Runt-related transcription factor 2) at the tendon-to-bone interface. At 8 weeks, HE and Masson staining were used to observe the histological changes, and the biomechanical test was used to evaluate the ultimate load and the failure site of the repaired rotator cuff to evaluate the tendon-to-bone healing and biomechanical properties. Results CCK-8 assay showed that the CS hydrogel could promote the proliferation of TDSCs (P<0.05). qPCR results showed that the expressions of tendon-to-bone interface related genes were significantly higher in the TDSCs/CS group than in the CS group and control group at 4 and 8 weeks after operation (P<0.05). Moreover, the expressions of tendon-to-bone interface related genes at 8 weeks after operation were significantly higher than those at 4 weeks after operation in the TDSCs/CS group (P<0.05). Histological staining showed the clear cartilage tissue and dense and orderly collagen formation at the tendon-to-bone interface in the TDSCs/CS group. The results of semi-quantitative analysis showed that compared with the control group, the number of cells, the proportion of collagen fiber orientation, and the histological score in the TDSCs/CS group increased, the vascularity decreased, showing significant differences (P<0.05); compared with the CS group, the proportion of collagen fiber orientation and the histological score in the TDSCs/CS group significantly increased (P<0.05), while there was no significant difference in the number of cells and vascularity (P>0.05). All samples in biomechanical testing failed at the repair site during the testing process. The ultimate load of the TDSCs/CS group was significantly higher than that of the control group (P<0.05), but there was no significant difference compared to the CS group (P>0.05). Conclusion TDSCs/CS hydrogel can induce cartilage regeneration to promote rotator cuff tendon-to-bone healing.
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 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.
