ABSTRACT
New scaffold materials composed of biodegradable components are of great interest in regenerative medicine. These materials should be: stable, nontoxic, and biodegrade slowly and steadily, allowing the stable release of biodegradable and biologically active substances. We analyzed peptide-polysaccharide conjugates derived from peptides containing RGD motif (H-RGDS-OH (1), H-GRGDS-NH2 (2), and cyclo(RGDfC) (3)) and polysaccharides as scaffolds to select the most appropriate biomaterials for application in regenerative medicine. Based on the results of MTT and Ki-67 assays, we can state that the conjugates containing calcium alginate and the ternary nonwoven material were the most supportive of muscle tissue regeneration. Scanning electron microscopy imaging and light microscopy studies with hematoxylin-eosin staining showed that C2C12 cells were able to interact with the tested peptide-polysaccharide conjugates. The release factor (Q) varied depending on both the peptide and the structure of the polysaccharide matrix. LDH, Alamarblue®, Ki-67, and cell cycle assays indicated that peptides 1 and 2 were characterized by the best biological properties. Conjugates containing chitosan and the ternary polysaccharide nonwoven with peptide 1 exhibited very high antibacterial activity against Staphylococcus aureus and Klebsiella pneumoniae. Overall, the results of the study suggested that polysaccharide conjugates with peptides 1 and 2 can be potentially used in regenerative medicine.
ABSTRACT
The aim of the research was to check whether it is possible to use fragments of type IV collagen to obtain, as a result of self-assembling, stable spatial structures that could be used to prepare new materials useful in regenerative medicine. Collagen IV fragments were obtained by using DMT/NMM/TosO- as a coupling reagent. The ability to self-organize and form stable spatial structures was tested by the CD method and microscopic techniques. Biological studies covered: resazurin assay (cytotoxicity assessment) on BJ, BJ-5TA and C2C12 cell lines; an alkaline version of the comet assay (genotoxicity), Biolegend Legendplex human inflammation panel 1 assay (SC cell lines, assessment of the inflammation activity) and MTT test to determine the cytotoxicity of the porous materials based on collagen IV fragments. It was found that out of the pool of 37 fragments (peptides 1-33 and 2.1-2.4) reconstructing the outer sphere of collagen IV, nine fragments (peptides: 2, 4, 5, 6, 14, 15, 25, 26 and 30), as a result of self-assembling, form structures mimicking the structure of the triple helix of native collagens. The stability of spatial structures formed as a result of self-organization at temperatures of 4 °C, 20 °C, and 40 °C was found. The application of the MST method allowed us to determine the Kd of binding of selected fragments of collagen IV to ITGα1ß1. The stability of the spatial structures of selected peptides made it possible to obtain porous materials based on their equimolar mixture. The formation of the porous materials was found for cross-linked structures and the material stabilized only by weak interactions. All tested peptides are non-cytotoxic against all tested cell lines. Selected peptides also showed no genotoxicity and no induction of immune system responses. Research on the use of porous materials based on fragments of type IV collagen, able to form stable spatial structures as scaffolds useful in regenerative medicine, will be continued.
