Objective To give a prel iminary experimental evidence and to prove chitosan and allogeneic morsel ized bone as potential bone substitutions in repairing rabbit radius segmental defect. Methods Chitosan and allogeneic morsel ized bone were mixed with various ratios (1 ∶ 5, 1 ∶ 10, 1 ∶ 25, 1 ∶ 50, and 1 ∶ 100). After preparation, the physicaland chemical properties of the composites were prel iminary detected; the composites at the ratios of 1 ∶ 50 and 1 ∶ 25 had good physical and chemical properties and were used for the animal experiment. The radius segmental defects of 15 mm in length were made in 50 adult New Zealand white rabbits (weighing 2.5-3.0 kg), then the animals were divided into 2 groups. In groups A and B, chitosan/allogeneic morsel ized bone composites were implanted at the ratio of 1 ∶ 50 and 1 ∶ 25, respectively. After 1, 2, 4, 8, and 12 weeks of operation, the gross, histological, immunohistochemical observations were performed. Before the rabbits were sacrified, X-ray films were taken; the serum calcium and alkal ine phosphatase (ALP) concentration were measured; and the biomechanical measurement was carried out at 12 weeks. Results The results of gross observation were essentially consistent with those of the X-ray films. The histological observation showed that the bone formation was earl ier in group A than in group B; the amount of new bone formation in group A was more than that in group B; and the bone forming area in group A was bigger than that in group B (P lt; 0.05) at 4 and 8 weeks after operation. The immunohistochemical staining showed that vascular endothel ial growth factor and insul in-l ike growth factor receptor II proteins expressed in the cytoplasm of 2 groups after 4 and 8 weeks, and the expression in group A was higher than that in group B (P lt; 0.05). There was no significant difference in the serum calcium concentration between 2 groups at each time point (P gt; 0.05). After 4 and 8 weeks, the ALP concentration in group A was significantly higher than that in group B (P lt; 0.05). After 12 weeks, the radius maximum bending loads of groups A and B were (299.75 ± 27.69) N and (278.54 ± 17.09) N, respectively, showing significant difference (t=4.045,P=0.002). Conclusion The composite of chitosan and allogeneic morsel ized bone has good osteogeneic activity and can beused as a bone tissue engineering scaffold, and the optimum ratio of chitosan to allogeneic morsel ized bone was 1 ∶ 50.
To develop a novel biodegradable collagen-chitosan-[poly(vinyl alcohol), PVA] composite artificial lacrimal canal iculus for treating tear overflowing (epiphora) caused by canal icular obstruction. Methods Homogeneously mixing solution composed of collagen, chitosan and PVA with different ratios was prepared. After several cycles of freezing/thawing process, the mixing solution was transferred into elastic hydrogel. Then the hydrogel was rinsed, punctured, dehydrated and trimmed, and three groups (T1, T2 and T3) of novel artificial lacrimal canal iculus were obtained. The appearance and diameter of all samples were observed under optical microscopy. The cross-section before and after drying as well as phase distribution of sample T2 was observed by SEM. The water absorption ratio and expanding ratio in PBS solution were calculated from three swell ing behavior curves. The degradabil ity of groups T1, T2 and T3 were prel iminary analyzed by degradation experiment in vitro. Results The micro-tubes with 0.5-0.7 mm in inner diameter, 0.9-1.5 mm in outer diameter and more than 20 mm in length were fabricated successfully through physical crossl inking without addition of toxic cross-l inker. SEM result showed that the sample had uniform phase distribution and smooth surface at dried state as well as interpenetrate network structure at hydrogel state. It was seen from the swell ing behavior curves that groups T1, T2 and T3 swelled rapidly within 10-30 minutes, and formed elastic composite hydrogel pipes. In addition, the expanding ratio of inner and outer diameter of the tube was 20%-30% and 100%-120% with swell ing, respectively. The equil ibrium water content of the hydrogel pipes increased with increase of collagen composition. Three groups of samples were immerged in PBS solution contained 2 mg/mL lysozyme at 37 for 1 month, their nozzle cracked, their wall became thin and more transparent. And also, there was small floc deposited on the tube surface. The samples were degraded into mash after they were soaked in PBS solution at 70 for 2 days. Conclusion The novel artificial lacrimal canal iculus with good mechanical property and high water absorption is in favor of operation, tear passing and anti-conglutination. It will be a potential candidate for treating the lacrimal passage occlusion.
【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 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.
Objective To introduce the application of polymer material, chitosan, in the cartilage tissue engineering. Methods The recent original articleson the application of chitosan in cartilage tissue engineering were extensivelyreviewed. The biocompatibility and biodegradation characters of chitosan and its application were analysed.Results Chitosan has a high degree of biocompatibility and a favorable chondrogenic characteristic. It can support the maintenance of the phenotypic morphology of chondrocytes besides being used as a scaffold for cell growth. Conclusion The perspect of the application of chitosan in cartilage tissue engineering is hopeful.
Objective To prepare chitosan microcarriers and to use it to cultivate rat primary hepatocytes. Methods The crosslinked chitosan microcarrier was prepared by the reaction of glutaraldehyde with chitosan. Various factors that influence the preparation were studied and the reaction conditions were optimized. Rat primary hepatocytes cultured on chitosan microcarrier were observed by using phase contrast microscope and scanning electron microscope. Results Chitosan microcarriers with good properties could be prepared by adjusting the concentration of chitosan solution and the quantity of glutaraldehyde. Rat hepatocytes cultured on chitosan microcarriers retained the spherical shape as they have in vivo. And albumin secretion can last over one week. The highest albumin secretion rate reached 26.7μg/24h/ml. Conclusion Chitosan microcarriers is a promising scaffold for hepatocyte attachment, which can be used in bioartificial liver support system.
Objective To investigate tissue engineered spinal cord which was constructed of bone marrow mesenchymal stem cells (BMSCs) seeded on the chitosan-alginate scaffolds bridging the both stumps of hemi-transection spinal cord injury (SCI) in rats to repair the acute SCI. Methods BMSCs were separated and cultured from adult male SD rat. Chitosan-alginate scaffold was produced via freeze drying, of which the structure was observed by scanning electron microscope (SEM) and the toxicity was determined through leaching l iquor test. Tissue engineered spinal cord was constructed by seeding second passage BMSCs on the chitosan-alginate scaffolds (1 × 106/mL) in vitro and its biocompatibil ity was observed under SEM at 1, 3, and 5 days. Moreover, 40 adult female SD rats were made SCI models by hemi-transecting at T9 level, and were randomly divided into 4 groups (each group, n=10). Tissue engineered spinal cord or chitosan-alginate scaffolds or BMSCs were implanted in groups A, B, and C, respectively. Group D was blank control whose spinal dura mater was sutured directly. After 1, 2, 4, and 6 weeks of surgery, the functional recovery of the hindl imbs was evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating score. Other indexes were tested by wheat germ agglutinin-horseradish peroxidase (WGA-HRP) retrograde tracing, HE staining and immunofluorescence staining after 6 weeks of surgery. Results Chitosan-alginate scaffold showed three-dimensional porous sponge structure under SEM. The cells adhered to and grew on the surface of scaffold, arranging in a directional manner after 3 days of co-culture. The cytotoxicity of chitosan-alginate scaffold was in grade 0-1. At 2, 4, and 6 weeks after operation, the BBB score was higher in group A than in other groups and was lower in group D than in other groups; showing significant differences (P lt; 0.05). At 4 and 6 weeks, the BBB score was higher in group B than in group C (P lt; 0.05). After 6 weeks of operation, WGA-HRP retrograde tracing indicated that there was no regenerated nerve fiber through the both stumps of SCI in each group. HE and immunofluorescence staining revealed that host spinal cord and tissue engineering spinal cord l inked much compactly, no scar tissue grew, and a large number of neurofilament 200 (NF-200) positive fibers and neuron specitic enolase (NSE) positive cells were detected in the lesioned area in group A. In group B, a small quantity of scar tissue intruded into non-degradative chitosan-alginate scaffold at the lesion area edge, and a few of NSE flourescence or NF-200 flourescence was observed at the junctional zone. The both stumps of SCI in group C or group D were filled with a large number of scar tissue, and NSE positive cells or NF-200 positive cells were not detected. Otherwise, there were obviously porosis at the SCI of group D. Conclusion The tissue engineered spinal cord constructed by multi-channel chitosan-alginate bioscaffolds and BMSCs would repair the acute SCI of rat. It would be widely appl ied as the matrix material in the future.
ObjectiveTo investigate the effect of electrospun chitosan/polylactic acid (ch/PLA) nerve conduit for repairing peripheral nerve defect in rats. MethodsNerve conducts loaded with ch/PLA was made by the way of electrospun. The mechanical property, hydrophility, biocompatibility were tested, and the scanning electron microscope was used to observe the ultrastructure. The same experiments were also performed on pure PLA nerve conducts as a comparison. Then, 54 Sprague Dawley rats were divided into 3 groups randomly, 18 rats in each group. Firstly, the 10 mm defects in the right sciatic nerves were made in the rats and were respectively repaired with ch/PLA (group A), autografts (group B), and no implant (group C). At 4, 8, and 12 weeks after operation, general observations, sciatic functional index (SFI), electrophysiological evaluation, wet weight of gastrocnemius and soleus muscles, histological examination, immunohistological analysis, and transmission electron microscopy were performed to evaluate the effects. ResultsCompared with pure PLA nerve conducts, the addition of chitosan could improve the mechanical property, hydrophility, biocompatibility, and ultrastructure of the nerve conducts. At 4 weeks postoperatively, the regenerated nerve bridged the nerve defect in group A. The SFI improved gradually in both group A and group B, showing no significant difference (P>0.05). Compound muscle action potentials and nerve conduction velocity could be detected in both group A and group B at 8 and 12 weeks after operation, and significant improvements were shown in both groups (P<0.05). The wet weight and myocyte cross section of gastrocnemius and soleus muscles showed no significant difference between group A and group B (P>0.05), but there was significant difference when compared with group C (P<0.05) at 12 weeks postoperatively. Immunohistological analysis revealed that S-100 positive Schwann cells migrated in both group A and group B, and axon also regenerated by immunohistological staining for growth associated protein 43 and neurofilaments 160. Transmission electron microscopy showed no significant difference in the diameter of nerve fiber between group A and group B (P>0.05), but the thickness of myelin sheath in group A was significantly larger than that in group B (P<0.05). ConclusionThe electrospun ch/PLA nerve conduits can effectively promote the peripheral nerve regeneration, and may promise an alternative to nerve autograft for repairing peripheral nerve defect.
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.
Objective To prepare carboxymethyl-chitosan/hyaluronic acid/poly(vinyl alcohol) (CHP) blend membrane, evaluate its physicochemical properties and intraocular biocompatibil ity and to investigate its feasibil ity to be appl ied to glaucoma filtering surgery. Methods CHP blend membrane was prepared using solution casting method after blending carboxymethyl-chitosan, HA and poly(vinyl alcohol) in a proportion of 5 ∶ 4 ∶ 1 (M/M). Its water absorption rate, swell ing rate, permeabil ity, and mechanical properties were detected. Subconjunctival fibroblasts separated from subconjuncitival tissue of New Zealand white rabbits were cultured, and the cells at passage 4 were cultured on cell culture plate with or without the CHP blend membrane, serving as the experimental group and the control group, respectively. Effectof the CHP blend membrane on the subconjunctival fibroblasts was tested by MTT method 24, 48, and 72 hours after culture. Six New Zealand white rabbits were randomly divided into two groups (n=3 rabbits per group), and the CHP blend membrane and SK gel were implanted into the rabbits’ subconjunctival space and anterior chamber in the experimental group and the control group, respectively. Sl it lamp observation and binocular reaction record were conducted 1, 3, 5, 9, 11, 20, 30, 45, and 60 days after operation. Corneal tissue harvested from the experimental group was observed using scanning electron microscope 15 days after operation to study ophthalmic biocompatibil ity and biodegradabil ity. Results The water absorption rate and the swell ing rate of the CHP blend membrane was 83.8% ± 1.3% and 3.59 ± 0.50, respectively. The tensile strength of the dry and the wet CHP blend membrane was (20.59 ± 1.73) and (0.51 ± 0.13) MPa, respectively. The breaking elongation rate of the dry and the wet CHP blend membcane was 10.69% ± 1.16% and 53.15% ± 2.46%, respectively. The CHP blend membrane had good permeabil ity to NaCl and L-tyrosine. Absorbance (A) value of the experimental group 24, 48, and 72 hours after breeding was 0.207 ± 0.083, 0.174 ± 0.080, and 0.181 ± 0.048, respectively, while the A value of the control group was 0.284 ± 0.011, 0.272 ± 0.083, and 0.307 ± 0.056, respectively. Significant difference was evident between two groups (P lt; 0.05). In the experimental group, a small amount of floccus was exuded around the implanted membrane 1 day after operation; the floccus was absorbed on the third day, and there was no obvious inflammatory reaction occurring on the eleventh day. Most of the membrane degraded on the sixtieth day. Scanning electron microscope observation showed that the hexagonal morphology of the corneal endothel ial cells was intact, and no degradation particles adhered to the surface. In the control group, the implantation of SK gel into anterior chamber was unsuccessful because the SK gel was quite soft and easily broken. In the experimental group, mild hyperemia emerged around the implanted membrane 1 day after the subconjunctival implantation of the membrane, and it became normal on the ninth day. No corneal edema and inflammatory reaction of anterior chamber occurred till the sixtieth day. The results in the control group and the experiment group were similar. Conclusion Due to its good physicochemical properties and biocompatibil ity, the CHP blend membrane has potential appl ications in glaucomafiltering surgery.