In situ-forming injectable hydrogels for regenerative medicine

Abstract

Regenerative medicine involves interdisciplinary biomimetic approaches for cell therapy and tissue regeneration, employing the triad of cells, signals, and/or scaffolds. Remarkably, the field of therapeutic cells has evolved from the use of embryonic and adult stem cells to the use of induced pluripotent stem cells. For application of these cells in regenerative medicine, cell fate needs to be carefully controlled via external signals, such as the physical properties of an artificial extracellular matrix (ECM) and biologically active molecules in the form of small molecules, peptides, and proteins. It is therefore crucial to develop biomimetic scaffolds, reflecting the nanoenvironment of three-dimensional (3D) ECM in the body. Here, we describe in situ-forming injectable hydrogel systems, prepared using a variety of chemical crosslinkers and/or physical interactions, for application in regenerative medicine. Selective and fast chemical reactions under physiological conditions are prerequisites for in situ formation of injectable hydrogels. These hydrogels are attractive for regenerative medicine because of their ease of administration, facile encapsulation of cells and biomolecules without severe toxic effects, minimally invasive treatment, and possibly enhanced patient compliance. Recently, the Michael addition reaction between thiol and vinyl groups, the click reaction between bis(yne) molecules and multiarm azides, and the Schiff base reaction have been investigated for generation of injectable hydrogels, due to the high selectivity and biocompatibility of these reactions. Noncovalent physical interactions have also been proposed as crosslinking mechanisms for in situ forming injectable hydrogels. Hydrophobic interactions, ionic interactions, stereocomplex formation, complementary pair formation, and host–guest interactions drive the formation of 3D polymeric networks. In particular, supramolecular hydrogels have been developed using the host–guest chemistry of cyclodextrin (CD) and cucurbituril (CB), which allows highly selective, simple, and biocompatible crosslinking. Molecular recognition and complex formation of supramolecules, without the need for additional additives, have been successfully applied to the 3D network formation of polymer chains. Finally, we review the current state of the art of injectable hydrogel systems for application in regenerative medicine, including cell therapy and tissue regeneration.

Introduction

Regenerative medicine is the term for biomedical approaches to improve, restore, or replace biological functions of damaged tissues and organs, using cells, physico-chemical factors, and/or engineered scaffolds [1]. These translational approaches include cell therapies, immunomodulation therapy via infused cells, and tissue regeneration or engineering using artificial organs and tissues [1], [2]. In recent years, engineered scaffolds have been investigated widely as an artificial extracellular matrix (ECM) to provide structural support and growth factors for the spatiotemporal control of encapsulated cells [3]. The ECM naturally consists of an interlocking mesh of fibrous proteins and glycosaminoglycans (GAGs). GAGs are carbohydrate polymers, such as heparan sulfate, chondroitin sulfate, and keratan sulfate, which are usually attached to ECM proteins to form proteoglycans. Hyaluronic acid (HA) is an exceptional non-proteoglycan polysaccharide. Collagens are the most abundant protein in the ECM, while elastin, laminin, and fibronectin are also important components of the ECM. Cell-to-ECM adhesion is regulated by cellular adhesion molecules (CAMs) of integrins that bind cells to fibronectin and laminin in the ECM [4], [5]. The cell fate is regulated by external signals, such as cell–cell interactions, the physical properties of the ECM, and growth factors [6], [7].

As an artificial ECM, synthetic scaffolds should possess appropriate mechanical properties, porous structures that allow free diffusion of nutrients and wastes, and degradability that matches the rate of cellular growth, enabling spatiotemporal control of encapsulated cells. They should be easily fabricated in a way that minimizes cell damage and cytotoxic byproducts. A variety of synthetic scaffolds have been developed for tissue engineering, including hydrogels [8], [9], porous nanostructures [10], [11], [12], and nanofibers [13], [14]. In particular, hydrogels, three-dimensional (3D) networks of crosslinked polymer chains, have been widely investigated as promising artificial ECMs for in vitro and in vivo tissue engineering applications [15], [16], [17]. As schematically shown in Fig. 1, in situ-forming cytocompatible hydrogels can be prepared using non-toxic chemical crosslinkers, enzymes for biological crosslinking, physical interactions such as hydrophobic interaction and ionic interaction, and supramolecular chemistry. These injectable hydrogels have many advantages for various biomedical applications, including their ease of administration, simple cell encapsulation, the minimally invasive treatment, and the possibly enhanced patient compliance [18], [19], [20], [21], [22]. In addition, injectable hydrogels can easily take on a complex shape and can adhere to the surrounding tissues during hydrogel formation. However, to our knowledge, there are few in situ-forming hydrogels available for long-term cell encapsulation, due to the absence of chemical crosslinking systems that are completely biocompatible, the narrow range of physiologically acceptable triggering stimuli for physical crosslinking, and the low stability of physically crosslinked hydrogels in the body [15], [23]. Furthermore, it is not easy to provide adequate growth factors to cells encapsulated in hydrogels for their proliferation and differentiation. To achieve this, cellular adhesion moieties and/or growth factors should be introduced into the hydrogel in a spatiotemporally controlled manner (Fig. 1). The systematic investigation on the fate of cells encapsulated in the hydrogels over time is one of the most important but currently unmet biomedical requirements for the successful application of hydrogels to regenerative medicine.

In this review, we describe various injectable hydrogel systems used in regenerative medicine, including cell therapy and tissue regeneration. First, we review injectable hydrogels prepared using non-toxic chemical crosslinkers, employing the Michael addition reaction between thiol and vinyl groups, the click reaction between bis(yne) molecules and multiarm azides, and the Schiff base reaction. Second, we review injectable hydrogels prepared using enzymes for biological crosslinking, such as hydrogen peroxidase for the crosslinking of an HA–tyramine conjugate. Third, we review injectable hydrogels prepared using physical interactions, such as hydrophobic interactions and ionic interactions, and external stimuli-responsive hydrogel systems, of which the formation is triggered by temperature and/or pH changes. Fourth, we discuss supramolecular hydrogels prepared using host–guest chemistry involving cyclodextrin (CD) and cucurbituril (CB). Finally, we describe several examples of in situ-forming injectable hydrogels applied in cell therapy and tissue regeneration, with a view to further clinical development.