Department of In Vitro Fertilization, Zeynep Kamil Gynecologic and Pediatric Teaching & Research Hospital, İstanbul, Turkey ; Department of OB/GYN, Yale School of Medicine, New Haven CT, USA.
Department of Histology&Embryology, İstanbul University Faculty of Medicine, İstanbul, Turkey.
Balkan Med J. 2015 Jan;32(1):69-78. doi: 10.5152/balkanmedj.2015.15102. Epub 2015 Jan 1.
Chitosan, a linear polysaccharide, has been recently used in biomedical applications. In vitro studies have demonstrated its effect on cellular growth and its stimulatory action on cellular layer formation.
The present study aims to compare the proliferative effects of chitosan in two forms, membranous and solution forms, on Swiss 3T3 mouse embryonic fibroblasts.
In vitro study.
Three experimental groups were formed: cells were cultured in a normal medium without chitosan (Control Group); cells were cultured either in a medium containing 2.0% chitosan in membranous form (Membrane Group) or chitosan solution at a concentration of 2.0% (Solution Group). Two different methods were used in the experiments: cells cultured on the medium containing chitosan in solution or membranous forms (method 1); and chitosan solution or membranous forms were added into the medium containing previously cultured cells (method 2).
Scanning electron microscopic investigations of the experimental groups revealed cells with well-defined cellular projections, intact cellular membranes and tight intercellular junctions. They were especially prominent in the membrane group of method 1 and in the membrane and solution groups of method 2. Mouse monoclonal anti-collagen 1 primary antibody was used to indicate collagen synthesis. Prominent collagen synthesis was detected in the membrane groups on the 10(th) day of culture for both methods. Bromodeoxyuridine (BrdU) and MTT assays were performed in order to assess cellular proliferation and viability, respectively. BrdU labelling tests indicated a higher proliferation index in the membrane group of method 1 on the 5(th) and 10(th) days. For the second method, the membranous form on the 10(th) day and solution form on the 5(th) day were the most effective groups in terms of cellular proliferation. MTT results reflected a high cellular viability in method 1 on the 5(th) day of treatment with the membranous form, whereas cellular viability was highest in the solution form of method 2 on the 5(th) day.
The membranous form of chitosan induced a significant proliferative effect and increased the ratio of cell-to-cell junctions of Swiss 3T3 mouse embryonic fibroblasts. Conveniently, the solution form also resulted in enhanced cell proliferation and viability compared to the control group. As the solution form is easy to prepare and apply to cells compared to the membrane form, the application of Chitosan directly to media appears to be a convenient alternative for tissue engineering approaches.
壳聚糖是一种线性多糖，最近已被应用于生物医学领域。体外研究表明，它对细胞生长具有影响，并能刺激细胞层的形成。
本研究旨在比较壳聚糖的两种形式，膜状和溶液形式，对瑞士 3T3 鼠胚胎成纤维细胞的增殖作用。
体外研究。
将实验分为三组：一组细胞在不含壳聚糖的正常培养基中培养（对照组）；一组细胞在含 2.0%壳聚糖的膜状形式培养基中培养（膜状组）；一组细胞在浓度为 2.0%的壳聚糖溶液培养基中培养（溶液组）。实验中使用了两种不同的方法：一组细胞在含壳聚糖溶液或膜状形式的培养基中培养（方法 1）；另一组细胞在含已培养细胞的培养基中加入壳聚糖溶液或膜状形式（方法 2）。
对实验组进行扫描电子显微镜观察，发现细胞具有清晰的细胞突起、完整的细胞膜和紧密的细胞连接。这些特征在方法 1 的膜状组和方法 2 的膜状和溶液组中尤为明显。使用鼠单克隆抗胶原蛋白 1 一抗来指示胶原蛋白的合成。在两种方法的第 10 天培养中，均在膜状组中检测到明显的胶原蛋白合成。溴脱氧尿苷（BrdU）和 MTT 测定分别用于评估细胞增殖和活力。BrdU 标记试验表明，方法 1 中膜状组在第 5 天和第 10 天的增殖指数较高。对于第二种方法，在第 10 天的膜状组和第 5 天的溶液组在细胞增殖方面效果最佳。MTT 结果反映了在方法 1 中，在第 5 天用膜状形式处理时细胞活力较高，而在方法 2 的第 5 天溶液形式时细胞活力最高。
壳聚糖的膜状形式诱导了显著的增殖效应，并增加了瑞士 3T3 鼠胚胎成纤维细胞的细胞间连接比例。方便的是，与对照组相比，溶液形式也导致细胞增殖和活力增加。由于与膜状形式相比，溶液形式更易于制备和应用于细胞，因此直接将壳聚糖应用于培养基似乎是组织工程方法的一种方便选择。
