Glyoxal crosslinking of cell-seeded chitosan/collagen hydrogels for bone regeneration

Abstract

Chitosan and collagen are natural biomaterials that have been used extensively in tissue engineering, both separately and as composite materials. Most methods to fabricate chitosan/collagen composites use freeze drying and chemical crosslinking to create stable porous scaffolds, which subsequently can be seeded with cells. In this study, we directly embedded human bone marrow stem cells (hBMSC) in chitosan/collagen materials by initiating gelation using β-glycerophosphate at physiological temperature and pH. We further examined the use of glyoxal, a dialdehyde with relatively low toxicity, to crosslink these materials and characterized the resulting changes in matrix and cell properties. The cytocompatibility of glyoxal and the crosslinked gels were investigated in terms of hBMSC metabolic activity, viability, proliferation and osteogenic differentiation. These studies revealed that glyoxal was cytocompatible at concentrations below about 1 mM for periods of exposure up to 15 h, though the degree of cell spreading and proliferation were dependent on matrix composition. Glyoxal-crosslinked matrices were stiffer and compacted less than uncrosslinked controls. It was further demonstrated that hBMSC can attach and proliferate in three-dimensional matrices composed of 50/50 chitosan/collagen, and that these materials supported osteogenic differentiation in response to stimulation. Such glyoxal-crosslinked chitosan/collagen composite materials may find utility as cell delivery vehicles for enhancing the repair of bone defects.

Introduction

Chitosan and collagen type I composite materials have been proposed for many regenerative applications in tissues as diverse as bone [1], ligament [2], skin [3] and blood vessels [4]. Both of these materials are derived from natural sources and have key features that make them attractive in regenerative medicine, including compatibility with implantation and the ability to be degraded over time. Type I collagen is a main structural protein in many tissues and contains a variety of bioactive sites that promote cell attachment [5], [6] and regulate cell differentiation [7], [8]. Chitosan is a polysaccharide derived from the exoskeleton of crustaceans, which has been commercialized in wound-healing applications because of its tissue adhesive and antibacterial properties [9]. Composites of these materials have been explored because they offer the opportunity to integrate the benefits of each component while avoiding the drawbacks. For example, such composites offer good cell attachment because of the collagen component, while also being stable and resistant to rapid remodeling because of the chitosan component. In bone tissue engineering, chitosan/collagen composites have been shown to enhance the mechanical stiffness of scaffolds and to induce osteogenic differentiation of bone marrow stem cells, relative to either pure component [10].

A common method to create chitosan/collagen composite scaffolds involves a freeze-drying process that results in a sponge-like network with interconnected pores [10], [11]. Chitosan/collagen sponges often require an additional crosslinking step to stabilize hydrogels and enhance their mechanical properties and prevent degradation. Glutaraldehyde is one of the most widely used reagents to crosslink both chitosan and collagen due to its high effectiveness in creating irreversible crosslinking bonds [12], [13]. In most cases, cells cannot be incorporated into such materials during fabrication because of the high cytotoxicity of glutaraldehyde, and therefore must be seeded onto the exterior of scaffolds post-fabrication. More recently, injectable formulations of chitosan/collagen materials have been developed using β-glycerophosphate (β-GP) to initiate gelation [14], [15]. In this case the β-GP helps to create physical crosslinks in the polymers, but eventually leaks out of the gel. Although the mechanism is not entirely clear, it is thought that β-GP plays two important roles in chitosan/collagen composite gel formation. First, it acts as a proton receiver from the positively charged chitosan at elevated temperature and thereby induces chitosan gelation [16]. Second, β-GP is a weak base and therefore can neutralize weakly acidic collagen type I solutions and initiate self-assembly of a fibrous network that mimics native collagen conformation. In a previous study, we showed that β-GP-initiated chitosan/collagen composites could be created at physiological pH and temperature, and that human bone marrow stem cells (hBMSC) remained viable and continued to proliferate when embedded in such materials at the time of fabrication [14].

Composite chitosan/collagen gels created using β-GP-initiated gelation are however mechanically weak, because they are stabilized only by electrostatic bonds, as opposed to covalent or ionic bonds [16]. It has been reported that the small aldehyde glyoxal can be used in combination with β-GP to create stable and biocompatible chitosan hydrogels for cartilage regeneration [17]. This study showed that a human epithelial cell line (HEK293) could be embedded in three-dimensional (3-D) gels and tolerated up to 0.15 mM glyoxal while retaining their viability and ability to proliferate in culture. Calf chondrocytes were also subsequently encapsulated in such gels for in vivo evaluation. Although glyoxal is an aldehyde and has been implicated in contact dermatitis [18], it exhibits generally lower cytotoxicity when compared to glutaraldehyde [19]. In the current study, we have investigated the use of glyoxal to crosslink and therefore stabilize chitosan/collagen composite materials gelled with β-GP and seeded with hBMSC. Our study includes examination of the effects of glyoxal on both the cellular and matrix components of these materials. In addition, we have assessed the osteogenic potential of hBMSC when cultured in 3-D glyoxal-crosslinked chitosan/collagen hydrogels and stimulated by osteogenic supplements. We expect that such crosslinked chitosan/collagen materials will find utility in bone tissue engineering because they provide a stable osteoconductive matrix that also promotes cell attachment.