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.
Objective To study the grafting effect of tissue engineered artificial rat skin equivalent on full thickness wounds. Methods Full thickness wounds(Φ20mm) were made on the backs of twenty four nude mice which be divided in artificial skin(AS) group, chitosan membrane(CH) group and control group. All wounds were covered with AS, CH and petrolatum gauze , respectively. The wounds were observed daily by infrared ray scanning and histological examination on the 3rd , 7th, 14th, and 21st days. Results The wounds in AS group healed better than those in CH group and control group. The artificial skin achieved a good adherence to wound and there were some crescent regenerative blood vessel appeared in the AS group on the 3rd day of grafting. Then, the epidermal cells in artificial skin proliferated and differentiated to form a new epidermis consisting of stratum basal, stratum spinosum, stratum granulosum, stratum corneum almost like the natural skin. Dermis of the sd extracellular matrix secreted by fibroblasts; the chitosan lattice was degraded and replaced by the extracellular matrix. On the 14th day of grafting, the wounds healed. The color of artificial skin grafted was very similar to the natrual skin and the formed scar was very smaal. Conclusion A kind of new reconstructive tissue engineering artificial skin has good histocompatibility and can be transplanted into the full-thickness wounds.
Objective To develop a novel porous three-dimensional scaffold and to investigate its physico-chemical properties for tissue engineering cartilage.Methods Refined 88% deacetylation degree chitosan was prepared and dissolved in 0.2 mol/L acetate acid and fully mixed with highly purified porcine type Ⅱcollagen in 0.5 mol/L acetate acid solution in a ratio of 4 to 1 (wt/wt). Freeze-drying process was employed to fabricate the composite scaffold. The construct wascross-linked by use of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and Nhydroxysuccinimide (NHS). A mechanical tester was utilized to determine the tensilestrength change before and after cross-linking. The microstructure was observed via scanning electron microscopy (SEM). The lysozyme degradation was performedto evaluate the degradability of the scaffold in vitro. Results A bulk scaffold with desired configuration was obtained. The mechanical test showed that the crosslinking treatment could enhance the mechanical strength of the scaffold. The SEM results revealed that the two constituents evenly distributed in the scaffold and that the matrix was porous, sponge-like with interconnected pore sizing 100250 μm. In vitro lysozyme degradation indicated that crosslinked or uncross-linked composite scaffolds had faster degradation rate than the chitosan matrix. Conclusion Chitosan and typeⅡcollagen can be developed into a porous three-dimensional scaffold. The related physico-chemical tests suggest that the composite socaffold meets requirements for tissue engineered scaffold and may serve as an alternative cellcarrier for tissue engineering cartilage.
Abstract In order to study the influence of chitosan on bone infection, 27 New-Zealand rabbits were randomly divided into 3 groups. According to the Norden technique, the model of osteomyelitis of the tuberosity of tibia was produced experimentally by injection of staphylococcus aureus.Immediately following injection of the staphylococci, the chitosan gel, acetic acid solution and sterile distilled water were injected into the bones in the 3 groups respectively. The latter two groups were served as control. The severity of the infection was evaluated by clinical symptoms and signs, radiographicdata as well as the bone culture and bacterial counts. Compared the results from chitosan with the other two controls, it was found that the local injection ofchitosan could not reduce the incidence of bone infection, however, it could provide actual improvement when other data were concerned. The effects of chitosangel might be attributed to its bacteriostatic and immunological activity as well as its slow degradation in the body.
To observe the effect of chitosan/alginate (CTS/ALG) dressings on wound immersed in seawater. Methods Twenty-five healthy SD rats weighing 250-300 g were used to establ ish skin wound model through cutting 1.8 cm circle-shaped wound along spine bilaterally. The left side served as experimental group, and the right side as control group. The wounds were immersed in the prepared artificial seawater for 1 hour, then the experimental group was treated with CTS/ALG dressings, while the control group was treated with sterile gauze. Gross observation was performed andwound heal ing time was recorded. At 3, 5, 7, 10 and 12 days after operation, 2 cm × 2 cm skin tissues including the wounds were removed and underwent HE staining and immunohistochemistry staining using Envision method. Histological change of wound and expression of EGF receptor (EGFR) and bFGF were observed. Results In the experimental group, wound inflammatory response was sl ight and incrustation shrinked faster, while the incrustation in the control group shrinked slowly. The wound heal ing time of the experimental group and the control group was (11.68 ± 0.57) and (12.51 ± 0.54) days, respectively, suggesting there was a significant difference between two groups (P lt; 0.05). In the experimental group, granulation tissue prol iferation, cell infiltration, collagen tissue prol iferation, wound shrinkage and epithel ization appeared at 3 days after operation; regularly l ined collagen tissue, complete epithel ization and occurrence of skin appendages were observed at 10 days after operation; complete wound heal ing was noted at 12 days after operation; while in the control group, at the corresponding time point, late cell infiltration and epithel ization were observed and granulation tissue with ulcer was noted. Immunohistochemistry observation: high expression of bFGF in vascular endothel ial cells and interstitial fibroblasts and high expression of EGFR in vascular endothel ial cells were observed in the experimental group at 3 and 5 days after operation, and their expressions were low at 7, 10 and 12 days after operation; while in the control group, there were no or low expression of bFGF and EGFR at the same time point. Conclusion CTS/ALG dressings can promote the heal ing of wound immersed in seawater, but its mechanism needsfurther study.
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.
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.
【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.
【Abstract】 Objective To study the outcome of wound-heal ing hydrogel in treating chronic venous ulcer of lowerextremities so as to find a new therapy. Methods From April 2007 to September 2007, 60 patients with chronic venous ulcer of lower extremities were randomly assigned to wound-heal ing hydrogel group (group A, 30 cases) and control group (normal sal ine, group B, 30 cases). In group A, there were 24 males and 6 females, aging (57.3 ± 6.8) years; the disease course was (2.9 ± 0.7) years; and the ulcer area was (3.4 ± 0.6) cm2. In group B, there were 20 males and 10 females, aging (60.1 ± 7.4) years; the disease course was (3.3 ± 0.9) years; and the ulcer area was (3.1 ± 0.4) cm2. There were no differences in age, area of ulcer and course of disease between two groups (P gt; 0.05). The area of ulcer was measured every week after the treatment, and the effect of treatmentwas evaluated after 15 days. Results The ulcer area of 7 days and 14 days after treatment was (2.6 ± 0.7) and (1.1 ± 0.2) cm2 in group A, and (2.8 ± 0.6) and (2.3 ± 0.7) cm2 in group B, respectively; showing no statistically significant differences 7 days after treatment (P gt; 0.05), and showing statistically significant difference 14 days after treatment between two groups (P lt; 0.05).The average heal ing time was (12.0 ± 1.7) days in group A, and (31.0 ± 2.9) days in group B, respectively, showing statisticallysignificant difference (P lt; 0.01). The results were excellent, good, fair and poor in 16, 9, 4 and 1 of group A , and were in 3, 9, 14 and 4 of group B, respectively; showing statistically significant difference (P lt; 0.01). Conclusion Wound-heal ing hydrogel is effective in treating chronic venous ulcer of lower extremities.