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 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 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 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 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 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.
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 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 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 search the most suitable concentration of calcium in the medium for the basement membrane reconstruction in tissue engineering skin in vitro. Methods Composite chitosan tissue engineering skin was prepared according to previous studies. Four groups were included according to the concentrationof calcium (1.00, 1.45, 1.65 and 1.95 mmol/L respectively). After 7 days and 15 days of culture, the histological manifestation of basement membrane in tissue engineering skin was observed by hematoxylin amp; eosin staining and PAS staining, and collagen Ⅳ of basement membrane was detected immunohistochemicallyatthe dermalepidermal junction. Results This tissue engineering skin shared some histological features of normal skin, including a welldifferentiated stratifiedepidermis and a dense dermis. The epithelium in the group of 1.95 mmol/L calcium differentiated better than those in other groups. PAS staining showing a regularly red dying strap domain at the dermal-epidermal junction. Collagen Ⅳ was positively stained immunohistochemically at the dermalepidermal junction inthe tissue engineering skin. Conclusion The above results suggest that the medium with 1.95 mmol/L calcium should be suitable for the growth of composite chitosan tissue engineering skin and the reconstruction of basement membrane.