Objective To investigate the preparation of a chitosanencapsulated porus calcium polyphosphate (CPP) bioceramic so as to provide a feasible approach to repair of the bone defect. Methods The chitosan microspheres were produced by chemical procedures. The CPP bioceramic was made by the following steps: annealing, ball milling, admixing, and calcinating. The chemical method was used to encapsule the calcinated bioceramic by the porus chitosan film. The physicochemical property, biomechanical property, and toxicity of the chitosanencapsulated porus CPP bioceramic were analyzed. Results The uniform holes were observed in the CPP bioceramic under a microscope. The diameter of the hole was 100300 μm. The chitosan microballoons were amber in color. The particles were uniform with a diameter of 200-400 μm, with a poor compressive strength. They could be easily ground by hand. Themaximally tolerated dose of the CPP bioceramic leaching liquor given to the Jimpy mice of both sexes was gt;24g/kg on average. The compressive strength reached 200 MPa, and the interval porosity was about 60％-80％, which could completely meet with the compressive strength of the bone substitute. Conclusion The chitosancncapsulated porous CPP bioceramic can be used as a good porous bioceramic scaffold material, which has a good biomechanical property withno acute toxicity, and so may be used as an excellent material for the bone substitute.
Chitosan is a kind of biological material with good histocompatibility and gradual biodegradability in vivo. It has no toxicity or side-effect. For its gradual degradation, chitosan and adriamycin were mixed and formed drug delivery system (DDS). The release test of DDS and exudant of DDS in inhibiting OS-116 were examined in vitro. The results were as following: the DDS could release adriamycin in slow and stable way. The SO-116 inhidition rate of the exudant of the DDS on the 1st, 20th, 40th and 60th day was 58.11%, 36.48%, 24.32% and 21.62% respectively. It was concluded that the drug delivery system was a slow release system. It could maintain the concentration of adriamycin in a certain level. It was also suggested that the chitosan was a good carrier for slow release of chemotherapeutic drug in local therapy for postoperative treatment of bone tumor.
Objective To explore a way to make a new kind of chitosan-basedmicrosphere (MS), which can be used as a novel biodegradable haemostatic powder, and to confirm its haemostatic efficiency. MethodsChitosan(CTS), a haemostatic polysaccharide, was selected as a main material for the haemostatic powder; alginate (ALG), another haemostatic polysaccharide that has been found to be effective in promoting haemostasis in surgical procedures, was selected to be thecostar. The emulsification and the cross-link were chosen as a preparation process based on the interaction between the polysaccharides. The diameter of the prepared MS was determined by SPOS, and the surface of MS was observed under SEM. The swelling characteristics of MS in the simulative wound efflusion were investigated. In a splenic bleeding model in 6 rabbits, MS and Yunnanbaiyao were randomly used as a haemostatic agent, and the corresponding bleeding time was recorded. Results The MS prepared in the above-mentioned process was well proportioned and was similarly shaped. It became a kind of white powder after dehydration, and had a coralloid surface under SEM. The diameter of the MS was 4.05±2.55 μm, which was determined by SPOS. The swelling ratio of the MS was 280.139% within 5 min. The bleeding time was significantly decreased in the MStreated group (2.83±0.17 min) when compared with that in the control group (5.33±0.49 min)(P＜0.01). Conclusion The CTS/ALG-MS, which is made from haemostatic biomaterials (CTS, ALG) by emulsification and the cross-link processes, can be provided with favorable haemostatic efficiency. It can be used as a novel haemostaticpowder.However, its biodegrading rate and mode still remain to be further studied.
An clinical and pharmacokinetic study for a drug delivery system (DDS) of gentamycin-loaded chitosan bar were carried out with the purpose to evaluate its efficacy and giving further data for its clinical applications. Eighteen cases of chronic osteomyelitis were treated by surgical necrectomy with implantation of gentamycin-load chitosan bar in the prepared bone cavity. After operation, the concentration of gentamycin in serum and wound drainage fluid were examined at different times and blood urea nitrogen (BUN) and serum creatinine (Cr) as well. The clinical results were evaluated by the conditions of wound healing and clinical and roentgenographic manifestations. The results showed that the serum gentamycin concentration reached its peak level (0.86 microgram/ml) at 24 hours after operation and lasted for 4 days. No increase in the concentrations of BUN and Cr were observed after implantation. The gentamycin concentration in wound drainage fluid was several hundred times higher than the minimum inhibitory concentration (MIC) for staphylococcus aureus. All of the 18 cases were followed up for 24.8 months (in an range of 6-34 months) 16 patients received initial cure and without any recurrence. So, it could be concluded that the gentamycin-loaded chitosan DDS was a simple and effective method for the treatment of chronic osteomylitis without the necessity to carry out a second operation to remove the drug carrier, and it was sound to popularize its clinical application.
Objective To investigate the effects of chitosan on the cell cycle of the human fibroblasts and on the Ki-67 antigen expression in vitro and to investigate the mechanism of chitosan preventing the postoperative tissue adhesion. Methods The cultured fibroblasts were treated for 48 hours with 0，0.01，0.1，1.0，10.0 mg/ml of chitosan, respectively;then, the cell cycle of the fibroblasts was measured by the flow cytometry. The cultured fibroblasts were treated for 24 hours with the chitiosan at the above concentrations; then, the Ki-67 antigen in the cell nucleus was detected with the immunohistochemical staining toobserve its expression. Results The growth of the fibroblastswas obviously suppressed by chitosan, especially in the cell morphology. When the concentrations of chitosan were 1.0 mg/ml and 10.0 mg/ml, the percentages of the fibroblasts in the proliferation stage were 32.3%±5.2% and 14.7%±2.9%, respectively,which were significantly smaller than the percentage of the fibroblasts when the concentration of chitosan was 0 mg/ml (the control group) (41.9%±5.8%， P＜0.05). When the concentrations were 0.01 mg/ml and 0.1 mg/ml, the percentages of the fibroblasts in the proliferation stage were 39.0%±6.0% and 35.5%±3.4%, respectively, which were smaller than that of the control, but not significantly different from that of the control (P＞0.05). When the concentrations of chitosan were 0.1 mg/ml,1.0 mg/ml and 10.0 mg/ml, the percentages of the fibroblasts that had the positiveKi-67 antigen were 37.3%±3.4%, 30.5%±6.2% and 17.8%±3.0%，respectively, which were significantly smaller than that of the control (57.6%±8.9%， P＜0.05). When the concentration was 0.01 mg/ml, the percentage of the fibroblasts that had the positive Ki-67 antigen was 54.1%±8.0%, which was smaller than that of the control, but not significantly different from that of the control (P＞0.05). ConclusionChitosan can inhibit the proliferation of the fibroblasts and increase the percentage of the fibroblasts in the quiescent stage, which can be considered as one of the mechanisms that chitosan can prevent the postoperative tissueadhesion.
【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 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.
ObjectiveTo study the long-term prevention effect of self-developed chitosan electrospun membrane on cerebrospinal fluid leakage. MethodsTwenty-five healthy adult New Zealand rabbits were selected to prepare the bilateral dural defect (0.8 cm×0.8 cm in size) via midline incision of head.Defect of the right was repaired with chitosan electrospun membrane as the experimental group; defect of the left was not repaired as the control group.At 2-16 weeks after operation,one rabbit was sacrificed for the general observation of inflammatory response surrounding bone window and absorption of chitosan electrospun membrane; at 3 and 6 weeks after operation,5 rabbits were sacrificed for sampling to observe histological change and collagen expression by HE and Masson staining,and to measure the expressions of epidermal growth factor receptor (EGFR) and basic fibroblast growth factor (bFGF) by immunohistochemical staining. ResultsNo inflammatory reaction of swelling,exudation,and sppuration appeared in the skin and subcutaneous tissue after operation in 2 groups.There was no adhesion around the chitosan electrospun membrane,and new fiber membrane formed under the chitosan electrospun membrane in the experimental group; no cerebrospinal fluid leakage happened; the chitosan electrospun membrane was gradually degraded with time,and was completely absorbed at 16 weeks.There was uneven scar around the dural detect in control group.Histological observation showed less inflammatory cell infiltration in the experimental group,showing significant difference in the number of inflammatory cells compared with control group at 3,6 weeks (P<0.05); capillary,granulation tissue and collagen fiber massively proliferated; collagen fiber arranged in line,and there was a clear borderline between chitosan electrospun membrane and adjacent collagen fiber.The immunohistochemical staining showed that there were high expressions of bFGF and EGFR in the experimental group,and low expressions of bFGF and EGFR in the control group. ConclusionChitosan electrospun membrane for dural defect of rabbit can effectively reconstruct the dura,and it has exact long-term prevention effect on cerebrospinal fluid leakage.
Objective To study the release properties of basic fibroblast growth factor (bFGF) chitosan microspheres prepared by cross-linking-emulsion method using chitosan as a carrier material so as to lay a foundation for further study. Methods Using 0.6% sodium tripolyphosphate solution as a crosslinking agent and 1.5% solution of chitosan as a carrier material, bFGF chitosan microspheres were prepared by cross-linking-emulsion method. Laser particle size analyzer and Zeta electric potential analyzer were used to measure the particle diameter distribution, scanning electronic microscope to observe the morphology, and ELISA to determine the drug loading, the encapsulation rate, and the drug release properties. Results The particle size of bFGF chitosan microspheres ranged 20.312-24.152 μm. The microspheres were round with a smooth surface and uniform distribution, and it had no apparent porosity. The drug loading and encapsulation rate of microspheres were (7.57 ± 0.34) mg/g and 95.14% ± 1.58%, respectively. The bFGF chitosan microspheres could continuously release bFGF for 24 days; the bFGF level increased gradually with time and reached (820.45 ± 21.34) ng/mL at 24 days; and the microspheres had a burst effect, and the burst rate was 18.08%, and the accumulative release rate of the microspheres reached 82.05% during 24 days. Conclusion It is easy-to-operate to prepare the bFGF chitosan microspheres with the cross-linking-emulsion method. The bFGF chitosan microspheres have smooth surface, uniform distribution, and no apparent porosity.
ObjectiveTo explore the feasibility of chitosan/allogeneic bone powder composite porous scaffold as scaffold material of bone tissue engineering in repairing bone defect. MethodsThe composite porous scaffolds were prepared with chitosan and decalcified allogeneic bone powder at a ratio of 1∶5 by vacuum freeze-drying technique. Chitosan scaffold served as control. Ethanol alternative method was used to measure its porosity, and scanning electron microscopy (SEM) to measure pore size. The hole of 3.5 mm in diameter was made on the bilateral femoral condyles of 40 adult Sprague Dawley rats. The composite porous scaffolds and chitosan scaffolds were implanted into the hole of the left femoral condyle (experimental group) and the hole of the right femoral condyle (control group), respectively. At 2, 4, 8, and 12 weeks after implantation, the tissues were harvested for gross observation, histological observation, and immunohistochemical staining. ResultsThe composite porous scaffold prepared by vacuum freeze-drying technique had yellowish color, and brittle and easily broken texture; pore size was mostly 200-300μm; and the porosity was 76.8%±1.1%, showing no significant difference when compared with the porosity of pure chitosan scaffold (78.4%±1.4%) (t=-2.10, P=0.09). The gross observation and histological observation showed that the defect area was filled with new bone with time, and new bone of the experimental group was significantly more than that of the control group. At 4, 8, and 12 weeks after implantation, the bone forming area of the experimental group was significantly larger than that of the control group (P < 0.05). The immunohistochemical staining results showed that osteoprotegerin (OPG) positive expression was found in the experimental group at different time points, and the positive expression level was significantly higher than that in the control group (P < 0.05). ConclusionChitosan/allogeneic bone powder composite porous scaffold has suitable porosity and good osteogenic activity, so it is a good material for repairing bone defect, and its bone forming volume and bone formation rate are better than those of pure chitosan scaffold.