ObjectiveTo investigate the feasibility of lung tissue flap repairing esophagus defect with an inner chitosan tube stentin in order to complete repairing and reconsruction of the esophagus defect.MethodsFifteen Japanese white rabbits were randomly divided into two groups， experiment group(n=10): esophagus defect was repaired with lung tissue flap having inner chitosan tube stent; control group(n=5): esophagus defect was repaired with lung tissue flap without inner chitosan tube stent; and then the gross and histological apearance in both groups were observed at 2, 4,8 weeks after operation, barium sulphate X-ray screen were observed at 10 weeks after operation.ResultsSix rabbits survived for over two weeks in experiment group, lung tissue flap healed with esophageal defect, squamous metaplasia were found on the surface of lung tissue flap in experiment group. At 10 weeks after operation, barium sulphate examination found that barium was fluent through the esophageal and no narrow or reversed peristalsis, the peristalsis was good in experiment group.Four rabbits survived for two weeks and the lung tissue flap healed with esophageal defect, fibrous tissue hyperplasy on the surface of the lung tissue flap in control group. At 10 weeks after operation, barium sulphate examination found that barium was fluent through the esophageal and slight narrow or reversed peristalsis, the peristalsis was not good in control group, otherwise.ConclusionIt is a feasible method to repair the esophageal defect with lung tissue flap with the inner chitosan stent.
To evaluate the effect of deacetylation degree (DDA) on the gelation behavior of thermosensitive chitosan-β glycerol phosphate disodium salt pentahydrate (CH-GP) system and to compare their rheological behaviors before and after gelation. Methods A series of thermosensitive CH-GP samples with different DDAs (70%, 85%, 90%, 97%)were prepared by dissolving CH with 0.1 mol/L HCl solution, 5 samples for every single DDA, and then all these CH-GP solution samples processed the frequency sweep test and temperature sweep test (10-70℃ , 1℃ /min) on AR 2000ex rheometer, with pH value of 7.02. Also, all the results of hydrogel samples were processed a frequency sweep test. Results With CH concentration of 2% (w/v) and pH value of 7.02 , the gelating temperature of CH-GP systems with different DDAs (85%, 90%, 97%) were (59.90 ± 0.08), (48.10 ± 0.08), (37.10 ± 0.11) ℃ , respectively. While the gelating temperature of CH-GP system with 70% DDA was over 70℃ . There were statistically significant differences in temperature and time of gelation among groups with different DDAs (P lt; 0.05). Furthermore, storage modulus of such system raised from dozens Pa to a magnitude of several kPa during gelation , while loss modulus kept almost steady. Conclusion Gelating temperature and mechanical property of the system could be measured objectively by rheological characterization. Thus during designing tissue engineered scaffolds for various purposes, it is helpful applying selected CH with optimal DDA to different target tissues.
ObjectiveTo investigate the mechanical properties of the novel compound calcium phosphate cement (CPC) biological material as well as the biological activity and osteogenesis effects of induced pluripotent stem cells (iPS) seeding on scaffold and compare their bone regeneration efficacy in cranial defects in rats.MethodsAc- cording to the different scaffold materials, the experiment was divided into 4 groups: pure CPC scaffold group (group A), CPC∶10%wt chitosan as 2∶1 ratio mixed scaffold group (group B), CPC∶10%wt chitosan∶whisker as 2∶1∶1 ratio mixed scaffold group (group C), and CPC∶10%wt chitosan∶whisker as 2∶1∶2 ratio mixed scaffold group (group D). Mechanical properties (bending strength, work-of-fracture, hardness, and modulus of elasticity) of each scaffold were detected. The scaffolds were cultured with fifth generation iPS-mesenchymal stem cells (MSCs), and the absorbance (A) values of each group were detected at 1, 3, 7, and 14 days by cell counting kit 8 (CCK-8) method; the alkaline phosphatase (ALP) activity, Live/Dead fluorescence staining and quantitative detection, ALP, Runx2, collagen typeⅠ, osteocalcin (OC), and bone morphogenetic protein 2 (BMP-2) gene expressions by RT-PCR were detected at 1, 7, and 14 days; and the alizarin red staining were detected at 1, 7, 14, and 21 days. Twenty-four 3-month-old male Sprague Dawley rats were used to establish the 8 mm-long skull bone defect model, and were randomly divided into 4 groups (n=6); 4 kinds of scaffold materials were implanted respectively. After 8 weeks, HE staining was used to observe the repair of bone defects and to detect the percentage of new bone volume and the density of neovascularization.ResultsThe bending strength, work-of-fracture, hardness, and modulus of elasticity in groups B, C, and D were significantly higher than those in group A, and in groups C, D than in group B, and in group D than in group C (P<0.05). CCK-8 assay showed that cell activity gradually increased with the increase of culture time, theA values in groups B, C, and D at 3, 7, 14 days were signifiantly higher than those in group A, and in groups C, D than in group B (P<0.05), but no significant difference was found between groups C and D (P>0.05). Live/Dead fluorescence staining showed that the proportion of living cells in groups B, C, and D at 7 and 14 days was significantly higher than that in group A (P<0.05), and in groups C, D at 7 days than in group B (P<0.05); but no significant difference was found between groups C and D (P>0.05). RT-PCR showed that the relative expressions of genes in groups B, C, and D at 7 and 14 days were significantly higher than those in group A, and in groups C, D than in group B (P<0.05); but no significant difference was found between groups C and D (P>0.05). Alizarin red staining showed that the red calcium deposition on the surface of scaffolds gradually deepened and thickened with the prolongation of culture time; theA values in groups B, C, and D at 14 and 21 days were significantly higher than those in group A (P<0.05), and in groups C and D than in group B (P<0.05), but no significant difference was found between groups C and D (P>0.05).In vivo repair experiments in animals showed that the new bone in each group was mainly filled with the space of scaffold material. Osteoblasts and neovascularization were surrounded by new bone tissue in the matrix, and osteoblasts were arranged on the new bone boundary. The new bone in groups B, C, and D increased significantly when compared with group A, and the new bone in groups C and D was significantly higher than that in group B. The percentage of new bone volume and the density of neovascularization in groups B, C, and D were significantly higher than those in group A, and in groups C and D than in group B (P<0.05); but no significant difference was found between groups C and D (P>0.05).ConclusionThe mechanical properties of the new reinforced composite scaffold made from composite chitosan, whisker, and CPC are obviously better than that of pure CPC scaffold material, which can meet the mechanical properties of cortical bone and cancellous bone. iPS-MSCs is attaching and proliferating on the new reinforced composite scaffold material, and the repair effect of bone tissue is good. It can meet the biological and osteogenic activity requirements of the implant materials in the bone defect repair.
ObjectiveTo prepare adipose-derived stem cells (ADSCs) and chitosan chloride (CSCl) gel complex to study the biocompatibility and the feasibility of repairing the wounds of deep partial thickness scald in rats. MethodsADSCs were prepared by enzymogen digestion and differential adherence method from the subcutaneous adipose tissue of SPF grade 6-week-old male Sprague Dawley (SD) rats. Temperature sensitive CSCl gel was prepared by mixing CSCl, β glycerol phosphate, and hydroxyethyl cellulose in 8∶2∶2.5 ratio. The proliferation of ADSCs was measured by cell counting kit 8 (CCK-8) assay and the survival of ADSCs was detected by the Live/Dead flurescent staining in vitro. A deep partial thickness burn animal model was made on the back of 72 SPF grade 6-week-old male SD rats by boiled water contact method and randomly divided into 3 groups (n=24). Group A was blank control group, group B was CSCl hydrogel group, group C was ADSCs/CSCl gel group. The wound closure rate at 3, 7, 14, 21 days was observed after operation. The number of inflammatory cells at 7 days and epidermal thickness at 21 days were observed by HE staining after operation. The angiogenesis at 7 days was evaluated by immunohistochemistry staining with CD31 expression. ResultsCSCl had a temperature sensitivity, at 4℃, the temperature-responsive hydrogel was liquid and became solid at 37℃. The CCK-8 assay and Live/Dead flurescent staining confirmed that ADSCs could grow and proliferate in the ADSCs/CSCl hydrogel complex. General observation showed the wound closure ratio in group C was superior to groups A and B after operation (P<0.05). HE staining showed that at 7 days after operation, the wound healing of the three groups entered fibrous proliferation stage. Collagen deposition and inflammatory cell infiltration were observed in the dermis of each group. The proportion of inflammatory cells in group C was significantly lower than that in groups A and B, and in group B than in group A (P<0.01). At 21 days after operation, the fibrous connective tissues of neoepithelium and dermis in groups B and C were arranged neatly, and fibroblasts and neocapillaries could be seen. In group A, neoepidermis could also be seen, but the fibrous connective tissues in dermis were arranged disorderly and sporadic capillaries could be seen. The thickness of neonatal epidermis in group C was significantly larger than that in groups A and B, and in group B than in group A (P<0.01). CD31 immunohistochemistry staining showed that the neovascularization could be seen in all groups. The number of neovascularization in group C was significantly higher than that in groups A and B, and in group B than in group A (P<0.05). ConclusionThe ADSCs/CSCl hydrogel complex has a good biocompatibility and possessed positive effects on promoting the deep partial thickness scald wound repairing in rats.
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.
【Abstract】ObjectiveTo construct eukaryotic expression vector pSecTag2/HygroB-CD59 of human CD59 and transfect NIH3T3 cells after encapsulated by chitosan. MethodsThe human CD59 fragments were obtained by PCR form CD59-pGEM-T Easy Vector, cloned into the eukaryotic expression vector pSecTag2/HygroB, identified by restriction endonuclease’s digestion and DNA sequencing. After the particles of pSecTag2/HygroB-CD59 were encapsulated by chitosan, the NIH3T3 cells were transfected by chitosanCD59 nanoparticles and detected CD59 expression by immunohistochemistry stain. ResultsThe CD59 fragment was 312 bp. Its sequence was as same as CD59 cDNA in Genbank. After having been transfected by chitosan-CD59 nanoparticles in 24 hours, the 3T3 cells showed diffusely positive in the cytoplasms by anti-CD59 immunohistochemistry. ConclusionThe eukaryotic expression vector of human CD59 is constructed and transfected to NIH3T3 cells after encapsulated by chitosan. It will be very helpful for further study on transgenic livers.
Objective To construct a ultraviolet-cross-linkable chitosan-carbon dots-morin (NMCM) hydrogel, observe whether it can repair cartilage injury by in vivo and in vitro experiments, and explore the related mechanism. Methods The chitosan was taken to prepare the ultraviolet (UV)-cross-linkable chitosan by combining methacrylic anhydride, and the carbon dots by combining acrylamide. The two solutions were mixed and added morin solution. After UV irradiation, the NMCM hydrogel was obtained, and its sustained release performance was tested. Chondrocytes were separated from normal and knee osteoarticular (KOA) cartilage tissue donated by patients with joint replacement and identified by toluidine blue staining. The 3rd generation KOA chondrocytes were co-cultured with the morin solutions with concentrations of 12.5, 25.0, 50.0 µmol/L and NMCM hydrogel loaded with morin of the same concentrations, respectively. The effects of morin and NMCM hydrogel on the proliferation of chondrocytes were detected by cell counting kit 8 (CCK-8). After co-cultured with NMCM hydrogel loaded with 50 µmol/L morin, the level of collagen type Ⅱ (COL-Ⅱ) of KOA chondrocytes was detected by immunofluorescence staining, and the level of reactive oxygen species (ROS) was detected by 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Twenty 4-week old Sprague Dawley rats were selected to construct a articular cartilage injury of right hind limb model, and were randomly divided into two groups (n=10). The cartilage injury of the experimental group was repaired with NMCM hydrogel loaded with 25 µmol/L morin, and the control group was not treated. At 4 weeks after operation, the repair of cartilage injury was observed by micro-CT and gross observation and scored by the International Cartilage Repair Association (ICRS) general scoring. The cartilage tissue and subchondral bone tissue were observed by Safranine-O-fast green staining and COL-Ⅱ immunohistochemistry staining and scored by ICRS histological scoring. The expressions of tumor necrosis factor α (TNF-α), nuclear factor κB (NK-κB), matrix metalloproteinase 13 (MMP-13), and COL-Ⅱ were detected by Western blot and real-time fluorescence quantitative PCR. Results NMCM hydrogels loaded with different concentrations of morin were successfully constructed. The drug release rate was fast in a short period of time, gradually slowed down after 24 hours, and the amount of drug release was close to 0 at 96 hours. At this time, the cumulative drug release rate reached 88%. Morin with a concentration ≤50 µmol/L had no toxic effect on chondrocytes, and the proliferation of chondrocytes improved under the intervention of NMCM hydrogel (P<0.05). NMCM hydrogel loaded with morin could increase the level of COL-Ⅱ in KOA chondrocytes (P<0.05) and reduce the level of ROS (P<0.05), but it did not reach the normal level (P<0.05). Animal experiments showed that in the experimental group, the articular surface was rough and the defects were visible at 4 weeks after operation, but the surrounding tissues were repaired and the joint space remained normal; in the control group, the articular surface was rougher, and no repair tissue was found for cartilage defects. Compared with the control group, the experimental group had more chondrocytes, increased COL-Ⅱ expression, and higher ICRS gross and histological scores (P<0.05); the relative expressions of MMP-13, NF-κB, and TNF-α protein and mRNA significantly decreased (P<0.05), and the relative expressions of COL-Ⅱ protein/COL-2a1 mRNA significantly increased (P<0.05). Conclusion NMCM hydrogel can promote chondrocytes proliferation, down regulate chondrocyte catabolism, resist oxidative stress, protect chondrocytes from cartilage injury, and promote cartilage repair.
Objective To investigate the behavior of rat calvarial osteoblasts cultured on chitosan-gelatin/hydroxyapatite (CSGel/HA) composite scaffolds. Methods The rat calvarial osteoblasts (the 3rd passage) were seeded at a density of 1.01×106 cells/ml onto the CS-Gel/HA composite scaffolds having porosity 85.20%, 90.40% and 95.80%. Cell number was counted after cultured for 3 days,1 week, 2 weeks and 3 weeks. Cell proliferation, bone-like tissue formation, and mineralization were separately detected by HE, von Kossa histological stainingtechniques. Results The CS-Gel/HA composite scaffolds supported the attachmentof seeded rat calvarial osteoblasts. Cells proliferated faster in scaffold withhigher porosity 90.40% and 95.80% than scaffold with lower porosity 85.20%. The osteoblasts/scaffold constructs were feasible for mineral deposition, and bonelike tissue formation in 3 weeks. Conclusion This study suggests the feasibility of using CS-Gel/HA composite scaffolds for bone tissue engineering.
Objective To explore the in vitro osteogenesis of the chitosan-gelatin scaffold compounded with recombinant human bone morphogenetic protein 2 (rhBMP-2). Methods Recombinant human BMP-2 was compounded with chitosan-gelatin scaffolds by freezedrying. 2T3 mouse osteoblasts and C2C12 mouse myoblasts were cultured and seeded onto the complexes at thedensity of 2×104/ml respectively. The complexes were divided into two groups. Group A: 2T3 osteoblasts seeded, consisted of 14 rhBMP-2 modified complexes. Each time three scaffolds were taken on the 3rd, 7th, 14th, and 21st day of the culturing, then the expression of osteocalcin gene (as the marker of bone formation) in adherent cells was detected by semiquantitative RT-PCR with housekeeping gene β-tubulin as internalstandard. The other 2 rhBMP-2 modified complexes were stopped being cultured on 14th day after cell seeding, and the calcification of the complexes was detected by Alizarian Red S staining. Five scaffolds without rhBMP-2 modification as the control group A, they were stopped being cultured on 14th day after cell seeding. Of the 5 scaffolds, 3 were subjected tothe detection of osteocalcin gene expression and 2 were subjected to the detection of calcification. Group B: C2C12 myoblasts seeded, had equal composition andwas treated with the same as group A. Besides these 2 groups, another 2 rhBMP2 modified complexes with 2T3 osteoblasts seeding were cultured for 3 days and then scanned by electron microscope (SEM) as to detect the compatibility of the cell to the complex. ResultsSEM showed that cells attached closely to the complex and grew well. In group A, the expression level(1.28±0.17)of osteocalcin gene in cells on rhBMP-2 modified complexes was higher than that (0.56±0.09) of the control group A, being statistically -significantly different(P＜0.05) control. C2C12 myoblasts which did not express osteocalcin normally could also express osteocalcin after being stimulated by rhBMP-2 for at least 7 days. Alizarian Red S staining showed that there was more calcification on rhBMP-2 modified complexes in both groups. There were more calcification in the group compounded with rhBMP-2, when the groups were seeded with the same cells. Conclusion The complexmade of rhBMP-2 and chitosan-gelatin scaffolds has b osteogenesis ability in vitro.
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.