OBJECTIVE: To repair esophageal defects with an artificial prosthesis composed of biodegradable materials and nonbiodegradable materials, which is gradually replaced by host tissue. METHODS: The artificial esophagus was a two-layer tube consisting of a chitosan-collagen sponge and an inner polyurethane stent with a diameter of 20 mm and a length of 50 mm. We used the artificial esophagus to replace 5 cm esophageal defects in group I (five dogs) and in group II (ten dogs), and nutritional support was given after operation. The inner polyurethane stent was removed after 2 weeks in group I and after 4 weeks in group II endoscopically and epithelization of the regenerated esophagus was observed by histologic examination and transmission electron microscope. RESULTS: In group I, the polyurethane stent was removed after 2 weeks, and partial regeneration of esophageal epithelial was observed; and constriction of the regenerated esophagus progressed and the dogs became unable to swallow after 4 weeks. In group II, the polyurethane stent was removed after 4 weeks, highly regenerated esophageal tissue successfully replaced the defect and complete epithelization of the regenerated esophagus was observed. After 12 weeks, complete regeneration of esophageal mucosa structures, including mucosal smooth muscle and mucosal glands and partial regeneration of esophageal muscle tissue were observed. CONCLUSION: Esophageal high-order structures can be regenerated and provided a temporary stent and support by polyurethane stent and an adequate three-dimensional structure for 4 weeks by collagen-chitosan sponge.
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 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 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.
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.
【Abstract】 Objective To increase the viscosity of chitosan/glycerol phosphate（C/GP）and to improve its preparation technique in order to develop the appl ication range of C/GP. Methods Chitosan was treated by high-pressure vapor steril ization in order to prepare high viscous C/GP（HV-C/GP）and prepare C/GP by standard methods. The rheologic changes of HV-C/GP and C/GP were detected dynamically by the Gemini rheometer. The initial solution viscosity, gelation temperature and gelation time were evaluated after the viscosity of the materials were increased. Two gelation materials were placed into continuous flow thermostated cells under the same condition and harvest them at predetermined time intervals, 1st, 2nd, 5th, 10th and 25th days, then they were dried, weighed and the mass loss rate was calculated. Ultrastructure of the freeze-dried samples was visual ized by the scanning electron microscope. Results The initial viscosity of C/GP was 1.81 Pas and that of HV-C/GP was 17.24 Pas. The latter one increased 10 times as well as the former one. The gelation temperature of C/GP was 37°C and that of HV-C/GP was 34°C. There was no remarkable difference in gelation time between them. The mass loss rate of HV-C/GP at first day was 72.5% and at 25th days was 90.8%, while that of C/GP was 55.4% and 78.2%. Porous network structure was observed by the scanning electron microscope in both of them. The pore diameter of C/GP was 50-100 μm and that of HV-C/GP was 30-50 μm, which was obviously smaller than the former. Conclusion The viscosity of HV-C/GP prepared by improved technique obviously increases and the thermosensitivity has no significant changes. The degradation time of HV-C/GP in vitro lengthens. The micrographs show that the HV-C/GP gels are porous and the pore diameter are smaller than C/GP.
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.
Objective To compare the effect and coverage of bacteriostasis of chitosan and sodium hyaluronate. Methods Each of the five bacteria, Proteus mirabilis, Escherichia coli, Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus, was cultivated for 33 tubes of broth culture. Leaving three tubes each group as control group, ploidy diluted concentration of high relative molecular weight chitosan, low relative molecular weight chitosan and sodium hyaluronate were added respectively in the broth culture. All the tubes were cultivated for 18 hours at 37 ℃ with homeothermia. Then the growth of bacteria was observed. ResultsThe minimal inhibitory concentrations (MIC) of high relative molecular weight chitosan were : Proteus mirabilis 0.031%, Escherichia coli 0.063%, Candida albicans 0.063%, Pseudomonas aerugionosa 0.063%, Staphylococcus aureus 0.063%; and the MIC of low relative molecular weight chitosan were: Proteus mirabilis 0.125%, Escherichia coli 0.025%, Candida albicans 0.25%, Pseudomonas aeruginosa 0.25%, Staphylococcus aureus 0.125%; bacteria grew well in each tube of sodium hyaluronate group and control group. Conclusion The above results show that sodium hyaluronate has no bacteriostasis, while chitosan has bacteriostasison broad spectrum and high relative molecular weight chitosan has ber effect.
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.
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.