Screening of Self-Assembling of Collagen IV Fragments into Stable Structures Potentially Useful 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 ITGα1β1. 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.

Selection of collagen IV fragments forming the outer sphere of the native protein: Assessment of biological activity for regenerative medicine

“…However, it should be remembered that a large area of the protein structure is characterized by a low alignment value (pLDDT < 50), which may be due to inaccuracies related to the complexity of the structure resulting from the size of the protein. However, the results of modeling the collagen IV fragments selected in the dot‐blot test are consistent with the microscopic and CD data 121 …”

“…The peptides were obtained on a solid phase using DMT/NMM/TosO − as a coupling reagent 115 . Their susceptibility to the formation of porous peptide materials had been tested previously 121 …”

“…However, the results of modeling the collagen IV fragments selected in the dot-blot test are consistent with the microscopic and CD data. 121 The first screening was to examine the library of nonoverlapping fragments of collagen IV. In the next stage of the study, fragments showing a high ability to interact with antibodies (Group 1) and fragments with moderately strong interaction with antibodies (Group 2) were mapped to find the correct fragments capable of interacting with antibodies.…”

Study on the Possibility of Cross-Interactions of Selected Growth Factors with Specific and Non-Specific Antibodies

“…As a result of our research on the selection of protein fragments involved in the regeneration process of damaged tissues, we have shown [29] that for selection it is possible to use polyclonal antibodies specific to a protein, because finally the set of fragments forming the outer sphere of the protein is obtained. This is due to the fact that the amino acid sequence is important in the recognition of an antigen by antibodies, but a very important factor determining the interaction is the appropriate display of protein fragments, which allows the formation of an antigen-antibody complex.…”