Synthesis and characterization of hyaluronic acid–poly(ethylene glycol) hydrogels via Michael addition: An injectable biomaterial for cartilage repair

Abstract

Injectable hydrogels based on hyaluronic acid (HA) and poly(ethylene glycol) (PEG) were designed as biodegradable matrices for cartilage tissue engineering. Solutions of HA conjugates containing thiol functional groups (HA-SH) and PEG vinylsulfone (PEG-VS) macromers were cross-linked via Michael addition to form a three-dimensional network under physiological conditions. Gelation times varied from 14 min to less than 1 min, depending on the molecular weights of HA-SH and PEG-VS, degree of substitution (DS) of HA-SH and total polymer concentration. When the polymer concentration was increased from 2% to 6% (w/v) in the presence of 100 U ml⁻¹ hyaluronidase the degradation time increased from 3 to 15 days. Hydrogels with a homogeneous distribution of cells were obtained when chondrocytes were mixed with the precursor solutions. Culturing cell–hydrogel constructs prepared from HA185k-SH with a DS of 28 and cross-linked with PEG5k-4VS for 3 weeks in vitro revealed that the cells were viable and that cell division took place. Gel–cell matrices degraded in approximately 3 weeks, as shown by a significant decrease in dry gel mass. At day 21 glycosaminoglycans and collagen type II were found to have accumulated in hydrogels. These results indicate that these injectable hydrogels have a high potential for cartilage tissue engineering.

Introduction

Over the past decades hydrogels have been extensively studied as temporary scaffolds for cartilage regeneration [1], [2], [3]. Hydrogels are three-dimensional, hydrophilic, polymeric networks absorbing and retaining a large amount of water, with properties resembling those of native cartilage [4]. Moreover, hydrogels have a high permeability for nutrients and water-soluble metabolites, allowing cell survival and supporting cellular growth. The incorporation of chondrocytes or stem cells into hydrogels is a promising repair strategy for cartilage [5], [6], [7], [8].

In such a strategy preformed hydrogels with a permanent shape generally need to be surgically implanted into cartilage defects. However, good attachment and integration of preformed hydrogels in native cartilage is not easily realized because the defects usually have an irregular shape. Alternatively, injectable hydrogels, implanted via injection of a viscous polymer solution which subsequently gelates in situ at the defect site, offer an opportunity to circumvent this problem. These injectable hydrogels allow homogeneous encapsulation of chondrocytes and bioactive molecules during the gelation process, which may facilitate the formation of a new matrix.

One of the most studied methods to induce hydrogel formation at a cartilage defect site is photo-cross-linking of vinyl functionalized polymers, e.g. poly(ethylene glycol) (PEG) di(meth)acrylate [9], [10], [11], [12]. Elisseeff et al. pioneered injectable PEG-based hydrogels via transdermal photopolymerization for cartilage repair in vitro and in vivo and showed that neocartilage was formed after 7 weeks [11]. In another study, Bryant et al. fabricated biodegradable hydrogels based on PEG and polylactide (PLA) via photo-cross-linking and examined neocartilage formation in the hydrogels at different degradation rates [13], [14], [15]. One of the limitations of the photo-cross-linking method to prepare injectable hydrogels is the potential cytotoxicity of photo-initiators and UV light on cells [11], [16]. Also, the transmittance of UV light through the skin decreases with increasing skin thickness and no UV light penetrates human skin when the thickness is greater than 2 mm [11]. Thus, for hydrogel formation by photopolymerization in a cartilage defect UV light needs to be placed in close proximity to the defect.

Another approach to prepare injectable hydrogels as artificial cartilage matrices is use of the Michael-type addition reaction to cross-link precursor macromers. Cross-links are formed via addition reactions between nucleophiles (e.g. thiol groups) and electrophiles (e.g. vinyl/acrylate groups) [17]. Using this approach Park et al. reported on branched PEG–vinylsulfone macromers cross-linked with thiol-bearing, matrix metalloproteinase-sensitive peptides to form a three-dimensional network in situ under physiological conditions [18]. Gene expression experiments using real time polymerase chain reaction (RT-PCR) demonstrated the expression of type II collagen and aggrecan by chondrocytes. Recently, our group prepared injectable hydrogels based on vinylsulfone-conjugated dextrans and PEG thiols via the Michael addition reaction [19], [20]. Since cells do not adhere to dextran and PEG [21], [22], the hydrogels are expected not to facilitate the attachment of chondrocytes within the gels during the cartilage regeneration process.

Hyaluronic acid (HA), consisting of disaccharide units of glucuronic acid and N-acetylglucosamine [23], is a key glycosaminoglycan (GAG) component distributed throughout the extracellular matrix of cartilage and is a major constituent of synovial fluid. HA is a biodegradable polysaccharide which can be metabolized via enzymatic hydrolysis by hyaluronidase (HAase), an enzyme present in various mammalian tissues [24]. HA may interact with chondrocytes through surface receptors such as CD44, enabling modulation of cell activities such as migration, proliferation and differentiation, as well as matrix secretion [25], [26], [27]. Therefore, HA is considered a promising material for cell delivery in cartilage regeneration. Various reports on injectable HA-based hydrogels for cartilage repair have been published with photo-cross-linking as the gelation method [12], [28], [29], [30], [31], [32]. To the best of our knowledge only a few studies in cartilage tissue engineering have made use of HA-based hydrogels prepared by the Michael addition method. For example, Liu et al. reported on HA-based hydrogels prepared via Michael addition reactions between thiolated HA, thiolated gelatin and acrylated PEG for the repair of osteochondral defects in a rabbit model [33]. The results showed that defects filled with hydrogels seeded with mesenchymal stem cells (MSC) resulted in a firm, elastic, translucent cartilage with good integration with the surrounding cartilage.

In this study HA-based hydrogels were prepared via the Michael-type addition of thiolated HA (HA-SH) and 4-arm PEG-vinylsulfone (PEG-4VS). We focused our attention on factors important for successful application of an injectable hydrogel, such as gelation rate, enzymatic degradation profile and mechanical moduli. The degree of substitution of HA-SH and the molecular weight of HA and PEG, as well as the concentrations of the precursor solutions, were taken into account. Biocompatibility, biodegradability, cytotoxicity and cartilage formation were studied in vitro by cell culture experiments using bovine chondrocytes incorporated into the gels.