Experimental study on self-assembly of KLD-12 peptide hydrogel and 3-D culture of MSC encapsulated within hydrogel in vitro.

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.