中药复合材料在创伤修复中的应用
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摘要
本研究对丹参促进烧伤创面修复的研究进展和表皮细胞培养及其临床应用进行了系统的文献复习，回顾了丹参的主要化学成分及抗氧自由基损伤、保护内皮细胞、改善微循环、促进组织修复和再生、抑制过再生、免疫调节作用等功能；总结了培养的表皮细胞来源、表皮细胞的分离和培养方法及表皮细胞的临床应用，并对其将来的发展趋势进行了展望。同时也回顾了纳米技术在生物医学工程中的应用及纳米生物安全性问题。
目的：构建丹参纳米银壳聚糖膜复合材料。研究该复合材料对治疗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丹参纳米银壳聚糖膜复合材料上的贴壁、生长及增殖情况。方法：首先用纳米自组装技术将纳米银组装到壳聚糖膜上，再将丹参固定在组装有纳米银的壳聚糖膜上。用无菌试验、热原试验、原发性皮肤刺激试验、皮内刺激试验、急性全身毒性试验对丹参纳米银壳聚糖膜复合材料的生物安全性情况进行评价，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、组织病理切片手段评价该材料的治疗疗效，用火焰原子吸收分光光度法对经该材料治疗的SD大鼠全血及肝、脑、肾中的痕量银进行测定；将丹参纳米银壳聚糖膜复合材料用于培养角质形成细胞，计算培养6h，12h，24h后的贴壁率，用免疫组织化学的方法和透射电子显微镜等手段对培养的角质形成细胞进行鉴定。结果：通过无菌试验、热原试验、原发性皮肤刺激试验、皮内刺激试验、急性全身毒性试验发现丹参纳米银壳聚糖膜复合材料无菌、无热原、无皮肤刺激作用、无皮内刺激作用、无毒性；该材料用于治疗SD大鼠深Ⅱ度切割伤，术后10d、13d愈合率分别为90.65±4.03％和98.98±5.04％，比对照组显著提高了愈合率(P＜0.05)，且经丹参纳米银壳聚糖膜复合材料治疗的SD大鼠无感染；对经丹参纳米银壳聚糖膜复合材料及磺胺嘧啶银治疗后的SD大鼠全血及组织中的痕量银进行测定，发现丹参纳米银壳聚糖膜组各个时点全血银的含量明显比磺胺嘧啶银组低(P＜0.01)。第13d丹参纳米银壳聚糖膜组SD大鼠创面恢复时，全血银含量基本恢复正常水平(与正常组银含量相比，P＞0.05)，而此时磺胺嘧啶银组SD大鼠创面未恢复，其银含量是正常全血银的5倍(P＜0.01)。深Ⅱ度切割伤大鼠使用丹参纳米银壳聚糖膜复合材料和磺胺嘧啶银后，各组织银含量都有不同程度的升高，磺胺嘧啶银组各组织银含量都比丹参纳米银壳聚糖膜复合材料组高(P＜0.01)。在丹参纳米银壳聚糖膜复合材料上培养新生鼠的角质形成细胞，与壳聚糖膜组、空白培养板组相比，该材料显著提高了角质形成细胞的贴壁率。24h时，角质形成细胞在该材料上的贴壁率为65.55±3.01％，在空白培养板上仅为28.61±2.23％；12h、24h时，角质形成细胞在丹参纳米银壳聚糖膜复合材料上的贴壁率是最高的，而6h时，角质形成细胞在壳聚糖膜上的贴壁率最高。另外，在该复合材料上几乎没有成纤维细胞的生长。结论：丹参纳米银壳聚糖膜复合材料无菌、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磺胺嘧啶银要少得多，减少了银中毒的可能性，提高了纳米材料的生物安全性；该材料能显著提高角质形成细胞的贴壁率，并能促进其增殖，且增殖的角质形成细胞保持原有的生物活性，该材料在临床上具有良好的应用前景，将为研究应用组织工程化皮肤开阔美好的前景。
This dissertation reviewed research progress of salvia miltiorrhiza in promoting burn wound recovery and the culture and clinical application of epidermal cells and summarized chemical composition and anti-oxyradica injure, protecting endothelial cell, improving microcirculation, promoting tissue repair, inhibiting excessive regenerate and immunoregulation function of salvia miltiorrhiza. This paper also summarized resource of epidermal cells, separation of epidermal cells, cultural method of epidermal cells, clinical application ofepidermal cells and view its development. In addition, applications of nanotechnology in biomedical engineering and biological security of nanomaterials were reviewed.
Objective: To construct salvia miltiorrhiza /nano-silver/ chitosan film composite. Investigate the efficacy and bio-safety of salvia miltiorrhiza /nano-silver/ chitosan film composite and adherence, growth, proliferation of keratinocytes on this composite. Method: Using nanometer and self-assembly technology to construct salvia miltiorrhiza/nano-silver/ chitosan film composite. Sterility test, pyretogen test, primarily skin stimulus test, intradermally stimulation test, acute general toxicity test were performed to investigate the bio-safety of this composite. The efficacy of salvia miltiorrhiza/nano-silver/chitosan filmcomposite was tested in 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 were determined by flame atomic absorption spectrophotometry (FAAS); Keratinocytes were cultured on salvia miltiorrhiza/nano-silver/ chitosan film composite. 6h, 12h, 24h after inoculation, the cell attached rations were calculated respectively. The cultured cells identified and characterized by immunohistochemistry and transmissive electron microscope (TEM). Results: Salviamiltiorrhiza/nano-silver/chitosan film composite was sterility, no pyretogen, no stimulus to skin, no general toxicity. 10 and 13 post scald day, the wound healing rates were 90.65±4.03 % and 98.98±5.04 % respectively in salvia miltiorrhiza /nano-silver/ chitosan film composite group. This was higher than control groups (P＜0.05) and SD rats curing by this composite were free from infection. The silver level in blood was lower than SD-Ag group (P＜0.01). 13 post scald day, the blood silver content in salvia miltiorrhiza/nano-silver/chitosan film composite returned to normal level, while in SD-Ag group, the silver content was 5 times than normal level(P＜0.05). The concentration of tissue silver was higher than normal level in salvia miltiorrhiza/nano-silver/ chitosan film composite and SD-Ag group. But the silver level in tissues in SD-Ag group was much higher than composite group (P＜0.01). Salvia miltiorrhiza/nano-silver/chitosan film composite could significantly (P＜0.01) increase the attached ration of keratinocytes. 24h after inoculation, the attached ration of keratinocytes were 65.55±3.01% and 28.61±2.23 %on this composite and cell culture plastic, respectively. After inoculation 12h, 24h, the attached rations on this composite were the highest. However, the attached ration after inoculation 6h on this composite was lower than that on chitosan film. Meanwhile, there were not any fibroblasts growing on this composite. Conclusion: Salvia miltiorrhiza/nano-silver/chitosan film composite was sterility, no pyretogen, no stimulus to skin, no general toxicity. It can accelerate wound healing and decrease the risk of silver poisoning. In addition, it could significantly promote keratinocytes attached ration and effectively inhibited the growth of fibroblasts. The rapidly proliferating keratinocytes, which were cultured on this composite, maintained their bioactivity. Its good efficacy and bio-safety shows a prosperous future in clinical setting. This indicates that the composite was nontoxic and was a good candidate for wound dressing in skin tissue engineering.
目的：构建丹参纳米银壳聚糖膜复合材料。研究该复合材料对治疗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丹参纳米银壳聚糖膜复合材料上的贴壁、生长及增殖情况。方法：首先用纳米自组装技术将纳米银组装到壳聚糖膜上，再将丹参固定在组装有纳米银的壳聚糖膜上。用无菌试验、热原试验、原发性皮肤刺激试验、皮内刺激试验、急性全身毒性试验对丹参纳米银壳聚糖膜复合材料的生物安全性情况进行评价，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、组织病理切片手段评价该材料的治疗疗效，用火焰原子吸收分光光度法对经该材料治疗的SD大鼠全血及肝、脑、肾中的痕量银进行测定；将丹参纳米银壳聚糖膜复合材料用于培养角质形成细胞，计算培养6h，12h，24h后的贴壁率，用免疫组织化学的方法和透射电子显微镜等手段对培养的角质形成细胞进行鉴定。结果：通过无菌试验、热原试验、原发性皮肤刺激试验、皮内刺激试验、急性全身毒性试验发现丹参纳米银壳聚糖膜复合材料无菌、无热原、无皮肤刺激作用、无皮内刺激作用、无毒性；该材料用于治疗SD大鼠深Ⅱ度切割伤，术后10d、13d愈合率分别为90.65±4.03％和98.98±5.04％，比对照组显著提高了愈合率(P＜0.05)，且经丹参纳米银壳聚糖膜复合材料治疗的SD大鼠无感染；对经丹参纳米银壳聚糖膜复合材料及磺胺嘧啶银治疗后的SD大鼠全血及组织中的痕量银进行测定，发现丹参纳米银壳聚糖膜组各个时点全血银的含量明显比磺胺嘧啶银组低(P＜0.01)。第13d丹参纳米银壳聚糖膜组SD大鼠创面恢复时，全血银含量基本恢复正常水平(与正常组银含量相比，P＞0.05)，而此时磺胺嘧啶银组SD大鼠创面未恢复，其银含量是正常全血银的5倍(P＜0.01)。深Ⅱ度切割伤大鼠使用丹参纳米银壳聚糖膜复合材料和磺胺嘧啶银后，各组织银含量都有不同程度的升高，磺胺嘧啶银组各组织银含量都比丹参纳米银壳聚糖膜复合材料组高(P＜0.01)。在丹参纳米银壳聚糖膜复合材料上培养新生鼠的角质形成细胞，与壳聚糖膜组、空白培养板组相比，该材料显著提高了角质形成细胞的贴壁率。24h时，角质形成细胞在该材料上的贴壁率为65.55±3.01％，在空白培养板上仅为28.61±2.23％；12h、24h时，角质形成细胞在丹参纳米银壳聚糖膜复合材料上的贴壁率是最高的，而6h时，角质形成细胞在壳聚糖膜上的贴壁率最高。另外，在该复合材料上几乎没有成纤维细胞的生长。结论：丹参纳米银壳聚糖膜复合材料无菌、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磺胺嘧啶银要少得多，减少了银中毒的可能性，提高了纳米材料的生物安全性；该材料能显著提高角质形成细胞的贴壁率，并能促进其增殖，且增殖的角质形成细胞保持原有的生物活性，该材料在临床上具有良好的应用前景，将为研究应用组织工程化皮肤开阔美好的前景。
This dissertation reviewed research progress of salvia miltiorrhiza in promoting burn wound recovery and the culture and clinical application of epidermal cells and summarized chemical composition and anti-oxyradica injure, protecting endothelial cell, improving microcirculation, promoting tissue repair, inhibiting excessive regenerate and immunoregulation function of salvia miltiorrhiza. This paper also summarized resource of epidermal cells, separation of epidermal cells, cultural method of epidermal cells, clinical application ofepidermal cells and view its development. In addition, applications of nanotechnology in biomedical engineering and biological security of nanomaterials were reviewed.
Objective: To construct salvia miltiorrhiza /nano-silver/ chitosan film composite. Investigate the efficacy and bio-safety of salvia miltiorrhiza /nano-silver/ chitosan film composite and adherence, growth, proliferation of keratinocytes on this composite. Method: Using nanometer and self-assembly technology to construct salvia miltiorrhiza/nano-silver/ chitosan film composite. Sterility test, pyretogen test, primarily skin stimulus test, intradermally stimulation test, acute general toxicity test were performed to investigate the bio-safety of this composite. The efficacy of salvia miltiorrhiza/nano-silver/chitosan filmcomposite was tested in 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 were determined by flame atomic absorption spectrophotometry (FAAS); Keratinocytes were cultured on salvia miltiorrhiza/nano-silver/ chitosan film composite. 6h, 12h, 24h after inoculation, the cell attached rations were calculated respectively. The cultured cells identified and characterized by immunohistochemistry and transmissive electron microscope (TEM). Results: Salviamiltiorrhiza/nano-silver/chitosan film composite was sterility, no pyretogen, no stimulus to skin, no general toxicity. 10 and 13 post scald day, the wound healing rates were 90.65±4.03 % and 98.98±5.04 % respectively in salvia miltiorrhiza /nano-silver/ chitosan film composite group. This was higher than control groups (P＜0.05) and SD rats curing by this composite were free from infection. The silver level in blood was lower than SD-Ag group (P＜0.01). 13 post scald day, the blood silver content in salvia miltiorrhiza/nano-silver/chitosan film composite returned to normal level, while in SD-Ag group, the silver content was 5 times than normal level(P＜0.05). The concentration of tissue silver was higher than normal level in salvia miltiorrhiza/nano-silver/ chitosan film composite and SD-Ag group. But the silver level in tissues in SD-Ag group was much higher than composite group (P＜0.01). Salvia miltiorrhiza/nano-silver/chitosan film composite could significantly (P＜0.01) increase the attached ration of keratinocytes. 24h after inoculation, the attached ration of keratinocytes were 65.55±3.01% and 28.61±2.23 %on this composite and cell culture plastic, respectively. After inoculation 12h, 24h, the attached rations on this composite were the highest. However, the attached ration after inoculation 6h on this composite was lower than that on chitosan film. Meanwhile, there were not any fibroblasts growing on this composite. Conclusion: Salvia miltiorrhiza/nano-silver/chitosan film composite was sterility, no pyretogen, no stimulus to skin, no general toxicity. It can accelerate wound healing and decrease the risk of silver poisoning. In addition, it could significantly promote keratinocytes attached ration and effectively inhibited the growth of fibroblasts. The rapidly proliferating keratinocytes, which were cultured on this composite, maintained their bioactivity. Its good efficacy and bio-safety shows a prosperous future in clinical setting. This indicates that the composite was nontoxic and was a good candidate for wound dressing in skin tissue engineering.
引文
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