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 .
Chitosan is a kind of biological material with good histocompatibility and gradual biodegradability in vivo. It has no toxicity or side-effect. For its gradual degradation, chitosan and adriamycin were mixed and formed drug delivery system (DDS). The release test of DDS and exudant of DDS in inhibiting OS-116 were examined in vitro. The results were as following: the DDS could release adriamycin in slow and stable way. The SO-116 inhidition rate of the exudant of the DDS on the 1st, 20th, 40th and 60th day was 58.11%, 36.48%, 24.32% and 21.62% respectively. It was concluded that the drug delivery system was a slow release system. It could maintain the concentration of adriamycin in a certain level. It was also suggested that the chitosan was a good carrier for slow release of chemotherapeutic drug in local therapy for postoperative treatment of bone tumor.
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 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.
【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.
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 study hemostasis of a new chitosan hemostatic powder. Methods Twenty-four adult SD rats were made the models of l iver injury, male or female, and weighing 210-240 g. They were divided into three groups randomly (n=8) depending on different hemostatic powders. The incision of the l iver was treated with 300 mg Yunnan baiyao (group A1), chitosan hemostatic powder of pH6.5 (group B1) and pH7.5 (group C1), respectively. The bleeding time and bleeding amount were recorded. In vitro, with the modified Ree-White method, 2 mL artery blood from New Zealand whiterabbit was added into the 0.2 mL solution of Yunnan baiyao, chitosan hemostatic powder of pH6.5 and pH7.5 (concentration of 0.2 mg/mL), respectively. The blood coagulation time was recorded. The chitosan blood clots of group B2 and group C2 were observed with scanning electron microscopy (SEM). Results The bleeding time of group A1, group B1 and group C1 was (292 ± 31), (261 ± 23), and (224 ± 28) s, respectively, the bleeding amount was (1.63 ± 0.21), (1.47 ± 0.18), and (1.18 ± 0.17) g, respectively, showing statistically significant differences between groups B1, C1, and group A1 (P lt; 0.05), between group C1 and group B1 (P lt; 0.05). The blood clotting time of group A2, group B2, and group C2 was (653 ± 41), (255 ± 20), and (202 ± 11) s, respectively, showing statistically significant differences between groups B2, C2, and group A2 (P lt; 0.05), between group C2 and group B2 (P lt; 0.05). The SEM showed that the blood cells of group B2 and group C2 gathered around the chitosan. Conclusion Chitosan hemostatic powder of pH7.5 has good hemostasis.
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 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.
Objective To study repair of osteochondral defects by using composite of autologous BMSCs and chitosan/HAP (CS/HAP) bilayered scaffold in rabbits and its feasibil ity as osteochondral tissue engineering scaffolds. Methods CS/HAP bilayered scaffolds were produced with CS and HAP using a lyophil ization and sintering method. The pore size of the scaffold was observed by scanning electron microscopy (SEM). Anhydrous ethanol substitution method determined its porosity. BMSCs were isolated from bone marrow and cultured by general bone marrow methods. Both CD44 and CD45 on the BMSCs surface were detected with immunocytochemistry to identify BMSCs. Cell-scaffold complex was made with BMSCs as seed cells and CS/HAP bilayered scaffold as carrier by fibrin glue planting technique. The distribution ofBMSCs in CS/HAP scaffold was tested by SEM. The osteochondral defect (4 mm in diameter and 3 mm in height) model was made in the right knee joint of 36 Japanese white rabbits, which were randomly divided into 3 groups. Defects were repaired with CS/HAP and BMSCs composite ( group A, n=12) and with CS/HAP implants (group B, n=12); defects were not treated as a control (group C, n=12). Histological evaluation and gross observation were carried out at 6 weeks (n=6 in each group) and 12 weeks (n=6 in each group) postoperatively. Semi-quantitative histomorphological analysis was done to evaluate the repair cartilage tissue according to the modified Wakitani grading scale. Results CS/HAP bilayered scaffold possessed a porosity of 76.00% ± 5.01% and pore size of 200-400 μm (mean 300 μm ) in CS layer, and 72.00% ± 4.23% and 200-500 μm (mean 350 μm) in HAP layer, respectively. BMSCs formed colonies within 10-14 days. Immunocytochemistry results showed BMSCs had positive CD44 expression and negative CD45 expression. At 6 and 12 weeks after operation, gross and histological observation showed that the cartilage defects were fully filled with regenerated tissue, but bone defects were partially repaired in group A; the cartilage and bone defects were partially filled with regenerated tissue in group B and group C. The modified Wakitani grading scale were 5.17 ± 1.17 and 3.20 ± 0.75 in group A, 9.00 ± 0.63 and 6.00 ± 0.89 in group B, and 10.00 ± 0.89 and 9.60 ± 0.82 in group C at 6 weeks and 12 weeks postoperatively, respectively; showing significant differences between group A and groups B, C (P lt; 0.05). Conclusion The novel CS/HAP bilayered scaffold possesses porous structure and will possibly become a newbiomaterial of osteochondral tissue engineering.
