Pore structure and dielectric behaviour of the 3D collagen-DAC scaffolds designed for nerve tissue repair

Highlights

• The methodology applied in this work may be instrumental for engineering scaffolds as biomimetic niches for neurally committed cells.

• The study demonstrated that the system of Col-DAC scaffolds had a mean pore size of approximately 32 μm and relatively high surface area to pore volume ratio.

• The magnitude of conductivity for modified Col, which is related to the decomposition of water, is approximately 40% lower than that recorded for the unmodified Col.

• The knowledge on the dielectric behaviour and microstructure of the Col-DAC scaffold may prove relevant to neural tissue engineering focused on the regeneration of the nervous system.

Abstract

The design and selection of a suitable scaffold with well-defined pores size distribution and dielectric properties are critical features for neural tissue engineering. In this study we use mercury porosimetry and the dielectric spectroscopy in the alpha-dispersion region of the electric field to determine the microarchitecture and activation energy of collagen (Col) modified by 2,3 dialdehyde cellulose (DAC). The scaffold was synthesized in three steps: (i) preparation of DAC by oxidation of cellulose, (ii) construction of a 3D Col sponge-shape or film, (iii) cross-linkage of the Col samples using DAC. The activation energy needed to break the bonds formed by water in the Col-DAC composite is approximately 2 times lower than that in the unmodified Col. In addition, the magnitude of conductivity for modified Col at 70 °C is approximately 40% lower than that recorded for the unmodified Col. The largest fraction, of which at least 70% of the total pore volume comprises the sponge, is occupied by pores ranging from 20 to 100 μm in size. The knowledge on the dielectric behaviour and microstructure of the Col-DAC scaffold may prove relevant to neural tissue engineering focused on the regeneration of the nervous system.

Introduction

Collagen-based scaffolds, among others, play a significant role in understanding and promoting the plasticity and repair of the central nervous system (CNS) [1]. Collagen (Col), mostly fibrillar type I, is a major component of the extracellular matrix (ECM) of many tissues and organs [2]. Our lab and others [3], [4], [5], [6] previously discussed more comprehensive data including conformational changes in helical structures and the thermomechanical and enzymatic stability of a few collagen scaffolds that were designed for use in the regeneration of the nervous system. However, the microarchitecture and electroconductive features of the scaffolds that optimize their interaction with stem/neural cells remains largely unexplored. In this work, we studied the pore structure and dielectric behaviour of Col modified by 2,3 dialdehyde cellulose (DAC) scaffolds to develop new materials with regenerative activity designed to repair tissue defects in the CNS.

The structure of type I Col, which is on the nanofiber scale of 50–500 nm, its degradability, high biocompatibility and relatively weak immunogenicity have long been known to play an important part in cell culture, and these properties make this bio-based polymer widely attractive for tissue engineering applications [7]. Advances in cellular and biomolecular delivery for tissue regeneration, with an emphasis on the CNS, has been described by Donaghue et al. [8]. Additionally, Col is an ideal scaffolding biopolymer due to the presence of Arg-Gly-Asp (RGD), peptides that render Col very cell-interactive. Col that contains the RGD attachment site and integrins that serve as their receptors constitute a major recognition system for cell adhesion [9], [10].

In contrast to native Col, the main disadvantage of reconstituted Col is its low mechanical strength, poor thermal stability and relatively rapid biodegradation. These disadvantages can be significantly improved by forming covalent bonds between Col polypeptide chains.

DAC was selected as the chemical crosslinking agent because it is biodegradable, biocompatible and toxicologically acceptable [11]. The study of anhydrous amorphous cellulose by dielectric spectroscopy [12] revealed the existence of the relaxation process corresponding to the motions of hydroxyl methyl and hydroxyl groups, which had an influence on the viscoelastic behaviour of this polymer. Additionally, recent results showed that the cellulose-derived conductive scaffold enhanced neural cell attachment and growth [13].

For neurodegenerative purpose, the Col scaffold must have certain dielectric properties and possess a stable micro-architecture that support cell attachment and growth. The pore size, pore distribution and their interconnectivity in scaffolds are extremely important factors that affect cell adhesion, migration, proliferation, differentiation and ECM biosynthesis [14]. It was shown [15] that nerve Col conduits, characterized by controlled porosity, have allowed the progressive substitution of non-neuronal tissue with close to normal neuronal tissue in vivo after nerve transfection in rats. The longitudinally oriented micro-architecture of 3D Col scaffolds may influence not only cell-substrate interactions, but also cell–cell interactions within the scaffold [16]. In addition, the porosity also facilitates the transport of nutrients and diffusion of metabolic waste. Without such regulations, the regenerated nerve system will not function properly.

Dielectric properties have a great influence on the design of engineered neural tissue-mimicking scaffolds. All physiological processes are accompanied by the flow of electric current as intra- and extracellular fluids and charges accumulate at the interface of structural elements, such as Col and water in the skin, tendon or bone [17], [18]. These conduction and polarization mechanisms in the tissues are possible because of their dielectric properties. Therefore, when engineering neural tissue, the dielectric properties of scaffolds should be similar or even better than those of the regenerating tissues and organs. Neurons are capable of using relatively weak electrochemical signals in the mV range to regulate cellular functions [19]. Electrical conductive scaffolds can help transmit these essential signals between neurons, which has a positive influence on the development of neural tissue [20]. The results of our previous studies [21], [22], performed by dielectric spectroscopy over a wide range temperatures, provided new information on molecular interactions in a bi-component system i.e., collagen-hyaluronic acid (Col-HA) and collagen-chondroitin sulphate (Col-CS) cross-linked by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC). For Col-HA or Col-CS, cross-linking is an effective way to not only decrease the biodegradation rate, but also optimize the dielectric properties of these systems. In fact, in the entire temperature range from 22° C to 230° C, the relative permittivity and the dielectric loss are much higher for Col-HA and Col-CS scaffolds than for unmodified Col because in modified samples occur a greater number of sites among which protons can jump and mobilize. More comprehensive data are published in our earlier article [22].

Another strategy for electrical communication in cell-substrate and cell–cell interactions was the construction of 3D electroactive polypyrrole-collagen (PPy-Col) fibrous scaffolds [23]. The optimization of the electrical stimulation process was needed to prevent the loss of human mesenchymal stem cell (hMSC) viability. The results showed a 3D electroactive fibrous scaffold could create the biomimetic microenvironment to facilitate the differentiation of inducible stem cells into neuronal lineage. Previously, dielectric techniques have also been used to study the structural characteristics of collagen-based biomaterials, such as collagen-DNA [24], collagen-polyvinyl alcohol [25], and collagen-guar gum or locust bean gum–collagen composites [26], for biomedical and biopharmaceutical applications.

Although the study of the influence of DAC on the mechanism of crosslinking and thermal stabilization of Col was carried out [27], [28], [29], the pore structure and dielectric properties of this type of biomaterial have not yet been addressed. In this work, we concentrated on the study of microstructural and dielectric properties of Col-DAC modified scaffolds (films, 3D sponges) using dielectric spectroscopy and measurements of the pores sizes and pores distributions.