Chinese Journal of Tissue Engineering Research ›› 2024, Vol. 28 ›› Issue (15): 2364-2370.doi: 10.12307/2024.369
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Qi Fengying1, 2, Wang Lei2, Li Dongdong2, Yan Shaoduo2, Liu Kun1, 2, Zheng Yizhe1, 2, He Zixin1, 2, Yi Xiaoyang2, Wang Donggen2, Fu Qiuxia2, Liang Jun1
Received:
2023-04-04
Accepted:
2023-05-27
Online:
2024-05-28
Published:
2023-09-20
Contact:
Liang Jun, PhD, Professor, Department of Light Industry Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, China
Fu Qiuxia, PhD, Associate researcher, Institute of Health Services and Transfusion Medicine, Academy of Military Medical Sciences, Beijing 100850, China
About author:
Qi Fengying, Master candidate, Department of Light Industry Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, China; Institute of Health Services and Transfusion Medicine, Academy of Military Medical Sciences, Beijing 100850, China
CLC Number:
Qi Fengying, Wang Lei, Li Dongdong, Yan Shaoduo, Liu Kun, Zheng Yizhe, He Zixin, Yi Xiaoyang, Wang Donggen, Fu Qiuxia, Liang Jun. Preparation and characterization of a novel self-assembled polypeptide hydrogel sustainably releasing platelet-rich plasma growth factors[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(15): 2364-2370.
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2.1 富血小板血浆对多肽自组装的影响 富血小板血浆纯度达到98%以上,由凝血酶诱导的血小板聚集率达到100%,二磷酸腺苷诱导的血小板聚集率达到30%左右,聚集活性良好。 为了探究富血小板血浆对多肽自组装的影响,首先将贫血小板血浆与自组装多肽溶液混合,直接观察结果显示,血浆可激发多肽溶液自发组装形成多肽水凝胶;富血小板血浆与氯化钙/凝血酶溶液混合,使血浆中丰富的纤维蛋白原形成紧密排列聚合纤维蛋白网络,结果显示成功制备富血小板血浆水凝胶;将不同体积的多肽溶液与富血小板血浆混合均匀后立即添加氯化钙/凝血酶溶液,静置10 min,结果显示形成多肽-富血小板血浆水凝胶外观上与富血小板血浆水凝胶无差异。图1展示了质量分数0.3%多肽-富血小板血浆水凝胶的状态。"
2.3 自组装多肽的微观结构观察 自组装多肽的分析研究多集中于物性力学表征,包括利用透射电镜、扫描电镜等观察其表面形貌[31]。透射电镜观察多肽自组装情况,结果显示:组装前,多肽纳米纤维较短且无序堆积;添加人血清白蛋白后,多肽自组装,多肽纳米纤维明显变长,并且相互交联成束,形成致密的纤维网络结构,见图3。以上结果表明较低质量分数多肽溶液在人血清白蛋白激发下也可自组装,可能由于纳米纤维结合、缠绕的强度不够形成水凝胶。多肽溶液与人血清白蛋白溶液混合,多肽终质量分数为0.1%,干燥后喷金处理,通过扫描电镜进一步观察多肽组装后的微观结构,结果显示,组装前多肽呈无序堆积的片状结构;加入人血清白蛋白后,多肽自组装成相互交联且有序密集排列的多孔状结构,见图4。"
2.5 自组装多肽对富血小板血浆生长因子释放的影响 选择表皮生长因子与血管内皮生长因子为模型蛋白,探究多肽-富血小板血浆水凝胶对富血小板血浆生长因子释放的影响。线性拟合得到表皮生长因子的标准曲线方程为Y=0.008 641X+0.108 0,R2=0.981 7,表明表皮生长因子标准品在0-250 pg/mL范围内具有较好的线性关系。富血小板血浆水凝胶组在12 h时有突释现象,24 h后表皮生长因子的累积释放不再显著增加,释放达到平衡;质量分数0.1%,0.3%多肽-富血小板血浆水凝胶组12 h时的表皮生长因子累积释放量分别为(25.73±3.77),(22.26±4.94) pg,显著低于富血小板血浆水凝胶组的(60.45±3.24) pg(P均< 0.001)。并且质量分数0.1%多肽-富血小板血浆水凝胶组在48 h达到释放平衡,延长了表皮生长因子的释放时间,见图6A、B。 线性拟合得到血管内皮生长因子的标准曲线方程为Y=0.000 966 5X-0.051 82,R2=0.997 5,表明血管内皮生长因子标准品在0-2 000 pg/mL范围内具有较好的线性关系。释放24 h时,质量分数0.1%,0.3%多肽-富血小板血浆水凝胶组血管内皮生长因子累积释放量分别为(439.37±37.84),(327.88±34.96) pg,显著低于富血小板血浆水凝胶组的(752.26±67.11) pg(P均< 0.001),而且质量数0.1%多肽-富血小板血浆水凝胶组延长了血管内皮生长因子的释放时间,在48 h达到释放平衡,见图6C、D。以上结果表明,不同质量分数的自组装多肽(0.1%,0.3%)均可有效延长富血小板血浆中表皮生长因子与血管内皮生长因子的释放时间,对富血小板血浆生长因子具有一定的缓释效果。"
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