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.
Objective To study the allograft effect of two kinds of tissue engineered oral mucosa lamina proprias on skin fullthickness wounds. Methods The cultured Wistar rat oral mucosa fibroblasts (OMF) were incorporated into collag en or chitosancollagen to construct the tissue engineered oral mucosa laminaproprias, and then the OMFs were labeled with BrdU. The fullthickness round skin defects were made with a round knife (diameter, 0.8 cm) on the backs of 36 Wistar rats (2125 weeks old), which were divided into 2 experimental groups: the fibroblastpopulated collagen lattices (FPCL) group (grafted by FPCLs) and the fibroblastpopulated chitosan collagen lattices (FPCCL) group (grafted by FPCCLs), and the control group (only covered with gauges). All the wounds were observed by the naked eyes or the light microscope, and were measured 4, 7, 14, and 21 days postoperatively. Results There were no infection during the wound healing period. At 7 days after the grafting, the wounds in the 3 groups were covered by scab and/or gauze; at 14 days, the gauze and scab on the wounds in the three groups were all replaced by the new epidermis naturally except one scab each in the FPCCL group and the control groups,which was replaced at 17 days.All the centers of the new epidermis were measurable as the pink red points. At 21 days, all the new skins were smooth without hairs, and their color was similar to the normal one. At 4, 7, and 14 days,there was an indication that the wound diameters became significantly smaller in the three groups; but after the 14th day, there was no significant indication of this kind. At 7 days, the wound diameter in the FPCL group was significantly smaller than that in the FPCCL group and the control group (Plt;0.01). Under the lightmicroscope, at 4 days postoperatively, the decayed tissue on the surfaces of the recipient wounds in the FPCL group and the FPCCL group was separated from the lower granular tissue in which there were many inflammatory cells, fibroblasts, and new vessels. There was a similar-phenomenon in the control group. Each skin wound in the three groups was only partly keratinocyted at 7 days postoperativel y. The recipient wounds were wholly keratinocyted with when rete ridges observed at 14 and 21 days, but in the control group the wounds were keratinocyted with no rete ridges. Fibers in the new dermis were thin. The OMFs with Brdu appeared in the granular tissue and new dermis at 4, 7, 14, and 21 days postoperatively, which could be illustr ated by the immunohistochemical staining. The positive OMFs and the granular tissue joined in the repair of the skin defe cts without any allergic reaction during the period of the wound healing. Conclusion The oral mucosa fibroblasts as the new seed cells can join i n the repair of the skin defects effectively and feasibly. The fibroblastpopul ated collagen lattices and the fibroblastpopulated chitosan collagen lat tices can repair skin defects effectively and feasibly, too. And the quality of the new skins was better in the two experimental groups than in the control group.
Objective To construct three kinds of collagen-chitosan porous scaffolds with enhanced biostability and to investigate the histocompatibility of the scaffolds in vivo. Methods Collagen-chitosan porous scaffolds were fabricated by freeze-drying method, cross-linked using dehydrothermal treatment and glutaraldehde, respectively. The morphology of the uncross-linked scaffold (scaffold1), dehydrothermally cross-linked scaffold (scaffold 2) and glutaraldehde crosslinked scaffold (scaffold 3) was studied by scanning electron microscopy. Threekinds of scaffolds were embedded subcutaneously on dorsal surface of 12 rabbit ears. The general and local responses were recorded daily. The biostability and histocompatibility of the scaffolds were observed by using HE staining after 3, 7, 14 and 28 days of operation. Results The scaffolds had three-dimensional porous structures with a porosity of more than 90%, and possessed pore sizes of 120±10 μm, 80±15 μm and 170±20 μm, respectively. All experimental rabbits survived with good general condition during the study. All skin incisions healed well without obvious reactive red or swelling. Histological study showed that scaffold 1 was degraded rapidly with obvious inflammation. The degradation of scaffold 2 was slower than that of scaffold 1 and the inflammation of scaffold 2 was also milder than that of scaffold 1. Scaffold 3 possessed slow degradation property with slight inflammatory reaction, and rapid tissue regeneration. Conclusion The collagenchitosan porous scaffolds have three-dimensional porous structures that are suitable for tissue regeneration. The biostability and histocompatiility of the scaffolds are enhanced after cross-linked. Glutaraldehde cross-linked is better than dehydrothermally cross-linked, which can facilitate dermal tissuereconstruction.
Human fibroblasts and human epidermal keratinocytes were used for culture. Chitosan solution were added in the culture solution(DMEM). After 72 hours, the fibroblasts showed rapid growth in the control culture without Chitosan, But the numbers of human fibroblasts from growth was decreased as the concentration of Chitosan was increasing. On the contrary the human epidermal keratinocytes growed more rapidly in the culture with Chitosan than in the culture without Chitosan. The results showed that Chitosan inhibited the growwth of human fibroblast and stimulated the growth of human epidermal keratinocyte .
OBJECTIVE: To prepare chitosan-gelatin/hydroxyapatite (CS-Gel/HA) composite scaffolds, and to investigate the influence of components and preparing conditions to their micromorphology. METHODS: The CS-Gel/HA composite scaffolds were prepared by phase-separation method. Micromorphology and porosity were detected by using scanning electron microscope and liquid displacement method respectively. RESULTS: Porous CS-Gel/HA composite scaffolds could be prepared by phase-separation method, and their density and porosity could be controlled by adjusting components and quenching temperature. CONCLUSION: The study suggests the feasibility of using CS-Gel/HA composite scaffolds for the transplantation of autogenous osteoblasts to regenerate bone tissue.
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.
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 constitute a new collagen gel artificial skin by using ch ito san as one of the components. Methods Human fo resk in fibroblasts were incorporated into thechitosan-collagen-GAGs to constitute dermal equivalent(DE). The growth of fibroblasts incorporated in gels and several factors which influenced the contraction of the gel were observed. The influence of different chitosan contents on the growth of fibroblast and keratinocyte and on the antibacterial effect were studied. Keratinocytes separated from normal children foresk in were seeded on the matured DE to reconstruct artificial skin, which was immersed at the early stage of culture, then lifted to an air-liquid interface. The structure of the DE and artificial skin were analysed by histology and scanning electron microscope. Results The contraction rate of the DE was proportional to the number of fibroblasts, and the final size of the DE was inversely proportional to the concent ration of collagen protein. Fibroblasts incorporated into the gel showed the exponential growth from the 2nd day to the 9th day. Chitosan-collagen-GAGs had no inhibition effect on the growth of fibroblasts, but promoted the growth of eratinocytes. Staphylococcus aureus was inh ibited even more as chitosan content increased. Scanning electron micro scopy indicated that the DE had abundant porous fabrication. Artificial skin shared some histological features of normal skin, which consisted of a good strat ifiedepiderm is and a dense dermis. Conclusion Chitosan-Collagen-GAGs collagen gelart ificial skin is a new collagen gel living artificial skin which has certain antibacterial ability and stratified epiderm is and dense dermis structure like normal skin.
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.
Objective To review and analyze the properties, products, and appl ications of chitosan so as to explore the key molecular structure parameters which can affect the properties and appl ications significantly, and to reveal the relationship between molecular structures and properties so as to provide reference for further development of chitosan industryand scientific research. Methods Based on the collection and analysis of related l iterature, patents and medical productsderived from chitosan, as well as the author’s experiences in research and development, evaluation and standardization of chitosan, the paper was prepared to bring more attentions into the correlativity between structure and properties of chitosan. Results Potential risks in cl inical appl ication of chitosan-based preparations were seriously proposed in addition to a scientific review and analysis on relationships between chitosan structure and properties, as well as the present situations of developments and appl ications of chitosan. Conclusion The molecular structure is the crucial factor that can bring not only positive but also passive effects to the properties and appl ications of chitosan, especially for highly purified chitosan, molecular weight, and deacetylation degree are the most important parameters that should be focused more attention on.