Screening of Self-Assembling of Collagen IV Fragments into Stable Structures Potentially Useful in Regenerative Medicine.

Marcin Kolasa,Grzegorz Galita,Lukasz Pietrzak,Zbigniew Draczynski,Ireneusz Majsterek,Lukasz Szymanski,Joanna Wasko,Justyna Fraczyk,Agnieszka Krakowiak,Beata Kolesinska,Ewa Kucharska,Katarzyna Czerczak,Angelika Becht,Anna Gajda

doi:10.3390/ijms222413584

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.

Highlights

Regenerative medicine concerns the healing of tissue after an injury and restoring damaged tissues to their native function
The most interesting collagen peptides are generated from the primary collagen of the tissue interstitial matrix, type I collagen and the basement membrane, type IV collagen [49]
It was found that out of the pool of 37 fragments reconstructing the outer sphere of collagen IV (CO4A6_HUMAN Q14031 Collagen alpha6(IV) chain), nine fragments, due to self-assembling, form structures that mimic the structure of the triple helix of native collagens (CD studies)

Summary

Introduction

Regenerative medicine concerns the healing of tissue after an injury and restoring damaged tissues to their native function. The development of biomaterials useful in regenerative medicine has made it possible to obtain new medical materials that do not induce cellular system responses and stimulate tissue regeneration and restore their original functionality. They allow communication at the cellular level [1,2]. In the case of scaffolds based on natural materials, including collagens [13], the pore architecture can be designed to mimic the anisotropic nature of ECM in native tissues [14,15]. Isotropic scaffolds can be prepared with pores ranging in size from 90 to 300 mm This parameter influences the growth and mechanics of cells. Other components of the ECM are used as additives [20–23]

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