角质形成细胞在纳米仿生功能支架上的快速贴壁及应用研究
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摘要
目的:构建“纳米银/壳聚糖膜”及“纳米金/壳聚糖膜”两种纳米仿生功能支架;研究仿生功能支架对治疗SD大鼠深II度切割伤的作用及其生物安全性;研究角质形成细胞在仿生功能支架上的贴壁率及生长情况。
方法:用自组装技术将纳米银、纳米金组装到壳聚糖膜上,得到“纳米银(金)/壳聚糖膜”仿生功能支架。众所周知,纳米银具有天然的抗菌性能,为此选用“纳米银/壳聚糖膜”仿生功能支架为主成分构造纳米银仿生敷料。用无菌、热原、皮肤刺激及全身急性毒性等试验,评价纳米银仿生敷料的生物安全性,再将其用于治疗SD大鼠深II度切割伤,通过肉眼观察、计算愈合率、组织病理切片等手段评价其治疗疗效,用火焰原子吸收分光光度法对经该敷料治疗的SD大鼠全血及肝、脑、肾等组织中的银含量进行测定,并与对照组(磺胺嘧啶银组,SD-Ag组)进行比较。
将构建的“纳米金/壳聚糖膜”仿生功能支架用于培养角质形成细胞,计算培养6h,12h,24h后的贴壁率,用免疫组织化学和透射电子显微镜等手段对培养的角质形成细胞进行表征、鉴定。
结果:基于“纳米银/壳聚糖膜”仿生功能支架的无菌、无热原、无刺激及无毒性等性能,将其用于治疗SD大鼠深II度切割伤,术后10d、13d愈合率分别为88.50±4.03%和98.98±6.09%,而对照I组(SD-Ag组)分别为67.78±3.86%和81.67±6.30%,对照II组(壳聚糖膜组)分别为73.70±3.25%和88.60±5.21%,且经纳米银仿生敷料治疗的SD大鼠感染少,且感染程度轻;对经纳米银仿生敷料及SD-Ag治疗后的SD大鼠全血及组织中的银进行测定,发现纳米银仿生敷料组各个时点全血银的含量明显比SD-Ag组低:纳米银仿生敷料组全血银含量最高是正常水平的7倍,而SD-Ag组高达26倍。第13d纳米银仿生敷料组SD大鼠全血银含量为0.11±0.06μg/g,基本恢复正常水平(与正常组银含量0.07±0.03μg/g相比,P>0.05),而此时SD-Ag组银含量是正常全血银的5倍(P<0.01)。深II度切割伤大鼠使用纳米银仿生敷料和SD-Ag后,各组织银含量都有不同程度的升高,SD-Ag组各组织银含量都比纳米银仿生敷料组高(P<0.05),其中肝银含量两组分别为11.48±0.32μg/g和1.64±0.12μg/g。在“纳米金/壳聚糖膜”仿生功能支架上培养新生SD大鼠的角质形成细胞,与壳聚糖膜组、空白培养板组相比,“纳米金/壳聚糖膜”仿生功能支架显著提高了角质形成细胞的贴壁率。24h时,角质形成细胞在“纳米金/壳聚糖膜”仿生功能支架上的贴壁率为58.72±3.40%,在空白培养板上仅为28.61±2.23%。12h、24h时,角质形成细胞在“纳米金/壳聚糖膜”仿生功能支架上的贴壁率是最高的。而6h时,壳聚糖膜上角质形成细胞的贴壁率(33.41±1.42%)最高;6h、12h、24h角质形成细胞在该支架上的贴壁率( 25.52±3.08% , 40.34±1.12% ,58.72±3.40%)变化比较明显,而在壳聚糖膜上(33.41±1.42%,35.52±2.69%,39.45±1.32%)、空白培养板上(18.02±6.22%,26.11±2.56%,28.61±2.23%)基本上没变化。
结论:纳米银仿生敷料无菌、无热原、无刺激、无毒性,具有促进创面愈合、缩短创面愈合周期及抗感染功能,且机体对纳米银的吸收比经典创面抗感染药SD-Ag少,减少了银中毒的可能性,提高了纳米材料的生物安全性,可用于治疗深II度创伤,在临床上具有良好的应用前景。
“纳米金/壳聚糖膜”仿生功能支架能显著提高角质形成细胞的贴壁率,并能促进其生长,且角质形成细胞保持原有的生物活性,为组织工程化皮肤的应用研究打下了基础。
Objective: To construct two nano-sized bionic-function scaffolds, i.e.“nano-silver/chitosan film”,“nano-gold/chitosan film”bionic function scaffold; and to investigate the efficacy, bio-safety , attached ration and growth of keratinocytes on bionic-function scaffold.
Method:“Nano-silver(gold)/chitosan film”bionic-function scaffold were fabricated by self-assembly technology.“Nano-silver /chitosan film”bionic-function scaffold was used as nano-silver bionic wound dressing due to distinct antimicrobial properties of nano-silver. The sterility, pyrogen, skin irritation, subcutaneous, systemic acute toxicity tests were performed to evaluate the bio-safety of this nano-silver wound dressing. The efficacy of this wound dressing was tested in vivo experiments on deep partial-thickness wound created on Sprague Dawley rats (SD rats). Therapeutic effect was evaluated through macroscopic observation, calculating healing ration and pathological section. In addition, the concentration of silver in SD rats’blood and other tissues (liver, brain, kidney) was determined by flame atomic absorption spectrophotometry (FAAS). And the results were compared with that of silver sulfadiazine (SD-Ag) group. Keratinocytes were cultured on“nano-gold / chitosan film”bionic-function scaffold. 6h, 12h, 24h after inoculation, the cell attached rations were calculated respectively. The cultured cells were characterized and identified by immunohistochemistry and transmissive electron microscope (TEM).
Results: We found that this nano-silver wound dressing was sterility, no pyretogen. It had no irritation and systemic acute toxicity. 10 and 13 post scald day, the wound healing rates were 88.50±4.03% and 98.98±6.09% respectively in nano-silver dressing group. While the healing rates were 67.78±3.86% and 81.67±6.30% in silver sulfadiazine (SD-Ag) group (control group) and were 73.70±3.25%and 88.60±5.21% in chitosan film group (control group). In addition, there were few infected SD rats in nano-silver dressing group. The blood silver level in nano-silver dressing group was lower than that in SD-Ag group (p<0.01). The concentration of blood silver in nano-silver dressing group was 6 times higher than normal level, while in SD-Ag group was 25 times higher than normal level. On day 13 post-operation, the silver level in blood was 0.11±0.06μg/g, which returned normal level nearly (p>0.05, normal silver level was 0.07±0.03μg/g), in nano-silver dressing group and the silver level in SD-Ag group was 4 times higher than normal level. The concentration of tissue silver was higher than normal level in nano-silver dressing and SD-Ag group. But the silver level in tissues was higher than that in nano-silver dressing group (p<0.01). The concentration of liver silver was 11.48±0.32μg/g and 1.64±0.12μg/g in SD-Ag group and nano-silver dressing group, respectively.
In comparison to control groups,“nano-gold/chitosan film”bionic-function scaffold could significantly (P<0.01) increase the attached ration of keratinocytes and promote their growth. 24h after inoculation, the attached rations were 58.72±3.40% and 28.61±2.23% on the scaffold and cell culture plastic, respectively. After inoculation 12h, 24h, the attached rations on the scaffold were the highest. However, the attached ration (25.52±3.08%) after inoculation 6h on the scaffold was lower than that of on chitosan film (33.41±1.42%) (P<0.05). In addition, the attached rations (25.52±3.08%, 40.34±1.12%,58.72±3.40%) after inoculation 6h, 12h, 24h of keratinocytes cultured on the scaffold changed obviously. There were nearly no change on chitosan film (33.41±1.42%, 35.52±2.69%, 39.45±1.32%) and cell culture plastic (18.02±6.22%, 26.11±2.56%, 28.61±2.23%).
Conclusion: Nano-silver bionic wound dressing was of sterility, no- pyretogen, no irritation and systemic acute toxicity and can accelerate wound healing, and it can decrease the risk of silver poisoning. It could be used on deep partial-thickness wound with a prospect of wide-rage clinical application.
“Nano-gold/chitosan film”bionic-function scaffold could significantly (P<0.01) increase the attached ration of keratinocytes and promote their growth. The rapidly proliferating keratinocytes, which were cultured on this scaffold, maintained their bioactivity. It was a good candidate for wound dressing in skin tissue engineering.
方法:用自组装技术将纳米银、纳米金组装到壳聚糖膜上,得到“纳米银(金)/壳聚糖膜”仿生功能支架。众所周知,纳米银具有天然的抗菌性能,为此选用“纳米银/壳聚糖膜”仿生功能支架为主成分构造纳米银仿生敷料。用无菌、热原、皮肤刺激及全身急性毒性等试验,评价纳米银仿生敷料的生物安全性,再将其用于治疗SD大鼠深II度切割伤,通过肉眼观察、计算愈合率、组织病理切片等手段评价其治疗疗效,用火焰原子吸收分光光度法对经该敷料治疗的SD大鼠全血及肝、脑、肾等组织中的银含量进行测定,并与对照组(磺胺嘧啶银组,SD-Ag组)进行比较。
将构建的“纳米金/壳聚糖膜”仿生功能支架用于培养角质形成细胞,计算培养6h,12h,24h后的贴壁率,用免疫组织化学和透射电子显微镜等手段对培养的角质形成细胞进行表征、鉴定。
结果:基于“纳米银/壳聚糖膜”仿生功能支架的无菌、无热原、无刺激及无毒性等性能,将其用于治疗SD大鼠深II度切割伤,术后10d、13d愈合率分别为88.50±4.03%和98.98±6.09%,而对照I组(SD-Ag组)分别为67.78±3.86%和81.67±6.30%,对照II组(壳聚糖膜组)分别为73.70±3.25%和88.60±5.21%,且经纳米银仿生敷料治疗的SD大鼠感染少,且感染程度轻;对经纳米银仿生敷料及SD-Ag治疗后的SD大鼠全血及组织中的银进行测定,发现纳米银仿生敷料组各个时点全血银的含量明显比SD-Ag组低:纳米银仿生敷料组全血银含量最高是正常水平的7倍,而SD-Ag组高达26倍。第13d纳米银仿生敷料组SD大鼠全血银含量为0.11±0.06μg/g,基本恢复正常水平(与正常组银含量0.07±0.03μg/g相比,P>0.05),而此时SD-Ag组银含量是正常全血银的5倍(P<0.01)。深II度切割伤大鼠使用纳米银仿生敷料和SD-Ag后,各组织银含量都有不同程度的升高,SD-Ag组各组织银含量都比纳米银仿生敷料组高(P<0.05),其中肝银含量两组分别为11.48±0.32μg/g和1.64±0.12μg/g。在“纳米金/壳聚糖膜”仿生功能支架上培养新生SD大鼠的角质形成细胞,与壳聚糖膜组、空白培养板组相比,“纳米金/壳聚糖膜”仿生功能支架显著提高了角质形成细胞的贴壁率。24h时,角质形成细胞在“纳米金/壳聚糖膜”仿生功能支架上的贴壁率为58.72±3.40%,在空白培养板上仅为28.61±2.23%。12h、24h时,角质形成细胞在“纳米金/壳聚糖膜”仿生功能支架上的贴壁率是最高的。而6h时,壳聚糖膜上角质形成细胞的贴壁率(33.41±1.42%)最高;6h、12h、24h角质形成细胞在该支架上的贴壁率( 25.52±3.08% , 40.34±1.12% ,58.72±3.40%)变化比较明显,而在壳聚糖膜上(33.41±1.42%,35.52±2.69%,39.45±1.32%)、空白培养板上(18.02±6.22%,26.11±2.56%,28.61±2.23%)基本上没变化。
结论:纳米银仿生敷料无菌、无热原、无刺激、无毒性,具有促进创面愈合、缩短创面愈合周期及抗感染功能,且机体对纳米银的吸收比经典创面抗感染药SD-Ag少,减少了银中毒的可能性,提高了纳米材料的生物安全性,可用于治疗深II度创伤,在临床上具有良好的应用前景。
“纳米金/壳聚糖膜”仿生功能支架能显著提高角质形成细胞的贴壁率,并能促进其生长,且角质形成细胞保持原有的生物活性,为组织工程化皮肤的应用研究打下了基础。
Objective: To construct two nano-sized bionic-function scaffolds, i.e.“nano-silver/chitosan film”,“nano-gold/chitosan film”bionic function scaffold; and to investigate the efficacy, bio-safety , attached ration and growth of keratinocytes on bionic-function scaffold.
Method:“Nano-silver(gold)/chitosan film”bionic-function scaffold were fabricated by self-assembly technology.“Nano-silver /chitosan film”bionic-function scaffold was used as nano-silver bionic wound dressing due to distinct antimicrobial properties of nano-silver. The sterility, pyrogen, skin irritation, subcutaneous, systemic acute toxicity tests were performed to evaluate the bio-safety of this nano-silver wound dressing. The efficacy of this wound dressing was tested in vivo experiments on deep partial-thickness wound created on Sprague Dawley rats (SD rats). Therapeutic effect was evaluated through macroscopic observation, calculating healing ration and pathological section. In addition, the concentration of silver in SD rats’blood and other tissues (liver, brain, kidney) was determined by flame atomic absorption spectrophotometry (FAAS). And the results were compared with that of silver sulfadiazine (SD-Ag) group. Keratinocytes were cultured on“nano-gold / chitosan film”bionic-function scaffold. 6h, 12h, 24h after inoculation, the cell attached rations were calculated respectively. The cultured cells were characterized and identified by immunohistochemistry and transmissive electron microscope (TEM).
Results: We found that this nano-silver wound dressing was sterility, no pyretogen. It had no irritation and systemic acute toxicity. 10 and 13 post scald day, the wound healing rates were 88.50±4.03% and 98.98±6.09% respectively in nano-silver dressing group. While the healing rates were 67.78±3.86% and 81.67±6.30% in silver sulfadiazine (SD-Ag) group (control group) and were 73.70±3.25%and 88.60±5.21% in chitosan film group (control group). In addition, there were few infected SD rats in nano-silver dressing group. The blood silver level in nano-silver dressing group was lower than that in SD-Ag group (p<0.01). The concentration of blood silver in nano-silver dressing group was 6 times higher than normal level, while in SD-Ag group was 25 times higher than normal level. On day 13 post-operation, the silver level in blood was 0.11±0.06μg/g, which returned normal level nearly (p>0.05, normal silver level was 0.07±0.03μg/g), in nano-silver dressing group and the silver level in SD-Ag group was 4 times higher than normal level. The concentration of tissue silver was higher than normal level in nano-silver dressing and SD-Ag group. But the silver level in tissues was higher than that in nano-silver dressing group (p<0.01). The concentration of liver silver was 11.48±0.32μg/g and 1.64±0.12μg/g in SD-Ag group and nano-silver dressing group, respectively.
In comparison to control groups,“nano-gold/chitosan film”bionic-function scaffold could significantly (P<0.01) increase the attached ration of keratinocytes and promote their growth. 24h after inoculation, the attached rations were 58.72±3.40% and 28.61±2.23% on the scaffold and cell culture plastic, respectively. After inoculation 12h, 24h, the attached rations on the scaffold were the highest. However, the attached ration (25.52±3.08%) after inoculation 6h on the scaffold was lower than that of on chitosan film (33.41±1.42%) (P<0.05). In addition, the attached rations (25.52±3.08%, 40.34±1.12%,58.72±3.40%) after inoculation 6h, 12h, 24h of keratinocytes cultured on the scaffold changed obviously. There were nearly no change on chitosan film (33.41±1.42%, 35.52±2.69%, 39.45±1.32%) and cell culture plastic (18.02±6.22%, 26.11±2.56%, 28.61±2.23%).
Conclusion: Nano-silver bionic wound dressing was of sterility, no- pyretogen, no irritation and systemic acute toxicity and can accelerate wound healing, and it can decrease the risk of silver poisoning. It could be used on deep partial-thickness wound with a prospect of wide-rage clinical application.
“Nano-gold/chitosan film”bionic-function scaffold could significantly (P<0.01) increase the attached ration of keratinocytes and promote their growth. The rapidly proliferating keratinocytes, which were cultured on this scaffold, maintained their bioactivity. It was a good candidate for wound dressing in skin tissue engineering.
引文
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