Sun Jianhua, Zheng Qixin
Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
J Huazhong Univ Sci Technolog Med Sci. 2009 Aug;29(4):512-6. doi: 10.1007/s11596-009-0424-6. Epub 2009 Aug 7.
To synthesize KLD-12 peptide with sequence of AcN-KLDLKLDLKLDL-CNH(2) and trigger its self-assembly in vitro, to encapsulate rabbit MSCs within peptide hydrogel for 3-D culture and to evaluate the feasibility of using it as injectable scaffold for tissue engineering of IVD. KLD-12 peptide was purified and tested with high performance liquid chromatography (HPLC) and mass spectroscopy (MS). KLD-12 peptide solutions with concentrations of 5 g/L, 2.5 g/L and 1 g/L were triggered to self-assembly with 1xPBS in vitro, and the self-assembled peptide hydrogel was morphologically observed. Atomic force microscope (AFM) was employed to examine the inner structure of self-assembled peptide hydrogel. Mesenchymal stem cells (MSCs) were encapsulated within peptide hydrogel for 3-D culture for 2 weeks. Calcein-AM/PI fluorescence staining was used to detect living and dead cells. Cell viability was observed to evaluate the bioactivity of MSCs in KLD-12 peptide hydrogel. The results of HPLC and MS showed that the relative molecular mass of KLD-12 peptide was 1467.83, with a purity quotient of 95.36%. KLD-12 peptide at 5 g/L could self-assemble to produce a hydrogel, which was structurally integral and homogeneous and was able to provide sufficient cohesion to retain the shape of hydrogel. AFM demonstrated that the self-assembly of KLD-12 peptide hydrogel was successful and the assembled material was composed of a kind of nano-fiber with a diameter of 30-40 nm and a length of hundreds of nm. Calcein-AM/PI fluorescence staining revealed that MSCs in KLD-12 peptide hydrogel grew well. Cell activity detection exhibited that the A value increased over the culture time. It is concluded that KLD-12 peptide was synthesized successfully and was able to self-assemble to produce nano-fiber hydrogel in vitro. MSCs in KLD-12 peptide hydrogel grew well and proliferated with the culture time. KLD-12 peptide hydrogel can serve as an excellent injectable material of biological scaffolds in tissue engineering of IVD.
合成序列为AcN-KLDLKLDLKLDL-CNH₂的KLD-12肽，并在体外触发其自组装，将兔间充质干细胞封装在肽水凝胶中进行三维培养，评估其作为椎间盘组织工程可注射支架的可行性。对KLD-12肽进行纯化，并用高效液相色谱（HPLC）和质谱（MS）进行检测。将浓度为5 g/L、2.5 g/L和1 g/L的KLD-12肽溶液与1×PBS在体外触发自组装，对自组装的肽水凝胶进行形态学观察。采用原子力显微镜（AFM）检查自组装肽水凝胶的内部结构。将间充质干细胞（MSCs）封装在肽水凝胶中进行三维培养2周。采用钙黄绿素-AM/PI荧光染色检测活细胞和死细胞。观察细胞活力以评估MSCs在KLD-12肽水凝胶中的生物活性。HPLC和MS结果显示，KLD-12肽的相对分子质量为1467.83，纯度为95.36%。5 g/L的KLD-12肽能够自组装形成水凝胶，其结构完整且均匀，能够提供足够的凝聚力以保持水凝胶的形状。AFM表明KLD-12肽水凝胶的自组装成功，组装材料由一种直径为30-40 nm、长度为数百纳米的纳米纤维组成。钙黄绿素-AM/PI荧光染色显示，KLD-12肽水凝胶中的MSCs生长良好。细胞活性检测表明，随着培养时间的延长，A值增加。结论是，KLD-12肽成功合成，能够在体外自组装形成纳米纤维水凝胶。KLD-12肽水凝胶中的MSCs生长良好，并随培养时间增殖。KLD-12肽水凝胶可作为椎间盘组织工程中一种优良的可注射生物支架材料。
