Preparation of pHEMA–CP composites with high interfacial adhesion via template-driven mineralization

Abstract

We report a template-driven nucleation and mineral growth process for the high-affinity integration of calcium phosphate (CP) with a poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogel scaffold. A mineralization technique was developed that exposes carboxylate groups on the surface of crosslinked pHEMA, promoting high-affinity nucleation and growth of calcium phosphate on the surface along with extensive calcification of the hydrogel interior. External factors such as the heating rate, the agitation of the mineral stock solution and the duration of the process that affect the outcome of the mineralization were investigated. This template-driven mineralization technique provides an efficient approach toward bonelike composites with high mineral–hydrogel interfacial adhesion strength.

Introduction

Current artificial materials used in the fabrication of orthopedic implants were originally developed for non-biological applications. Although they could provide an immediate solution for many patients, their long-term outcomes are not satisfactory. This is due to two major limitations: (1) these materials exhibit serious mechanical property mismatches with natural bone tissues;¹ and (2) these materials typically do not bear any functionalities that encourage the communication with their cellular environment, therefore limiting the potential for tissue attachment and in-growth.² The development of a new generation of bonelike composite materials with improved mechanical properties and enhanced biocompatibility over traditional implants calls for a biomimetic synthetic approach using natural bone as a guide.

Natural bone is a composite of collagen, a protein-based hydrogel template, and inorganic dahilite (carbonated apatite) crystals. The unusual combination of a hard inorganic material and an underlying elastic hydrogel network endows native bone with unique mechanical properties, such as low stiffness, resistance to tensile and compressive forces and high fracture toughness.³ Throughout the cavities of bone, there are bone cells and a myriad of soluble factors and extracellular matrix components that are constantly involved with the bone formation and remodeling process.⁴ For instance, it is suggested that acidic extracellular matrix proteins that are attached to the collagen scaffold play important templating and inhibitory roles during the mineralization process.5, 6 Presumably, the acidic groups serve as binding sites for calcium ions and align them in an orientation that matches the apatite crystal lattice.7, 8 It is the critical features like these that the biomimetic synthesis of artificial bone should emulate. We hypothesize that this can be realized by the generation of functional polymer scaffolds displaying surface ligands that mimic critical extracellular matrix components known to direct template-driven biomineralization, or to stimulate or assist cell adhesion, proliferation, migration and differentiation.9, 10, 11, 12, 13 Ideally, such 3-dimensional scaffolds should also possess proper physical properties, such as desired porosity and water retention ability, to allow both pre-implantation cell seeding and post-implantation tissue ingrowth.

Hydrogel polymers are particularly appealing candidates for the design of highly functional tissue engineering scaffolds.14, 15 The intrinsic elasticity and water retention ability of synthetic hydrogels resemble those of natural hydrogels, such as collagen matrices that are prevalent as structural scaffolds in various connective tissues including bone.¹⁴ The porosity of synthetic hydrogels may be controlled by various techniques including solvent casting/particulate leaching,16, 17 phase separation,¹⁸ gas foaming,¹⁹ solvent evaporation,²⁰ freeze drying,²¹ and blending with non-crosslinkable linear polymers²² to afford a range of mechanical properties. Another important feature of hydrogels is that they can be assembled in 3-dimensional form displaying multiple functional domains through copolymerization of different monomers. The polymerization chemistry is water compatible, allowing incorporation of polar ligands such as anionic peptides that mimic the acidic matrix proteins regulating mineral growth, and biological epitopes such as the tripeptide RGD23, 24, 25 that promote cellular adhesion.

Poly(2-hydroxyethyl methacrylate), or pHEMA, is one of the most well-studied synthetic hydrogel polymers.²⁶ With its high biocompatibility, pHEMA and its functionalized copolymers have become some of the most widely used synthetic hydrogels in tissue engineering. Applications include ophthalmic devices (e.g. contact lens),27, 28 cartilage replacements,²⁹ bonding agents in dental resins and bone cements,30, 31, 32 and various drug delivery vehicles.33, 34 One of the major challenges for its application as a 3-dimensional scaffold of artificial bonelike materials, however, is to realize a high-affinity integration of inorganic minerals with the pHEMA-based organic scaffold.

The most widely used existing approach of mineralizing organic polymer films with hydroxyapatite (HA) (Ca₁₀(PO₄)₆(OH)₂), a mineral closely related to carbonated apatite, the major inorganic component of natural bone, is the incubation of substrates with simulated body fluid (SBF).³⁵ This leads to slow growth of crystalline or amorphous biominerals that exhibit poor adhesion and lack a structural relationship with the substrate.35, 36 Recently, chemical treatment of biodegradable poly(lactide-co-glycolide) films with aqueous base has been shown to facilitate the growth of crystalline carbonate apatite on the surface.³⁷ Both the morphology and thickness of the resulting crystalline apatite layer, however, suggest that it is likely to suffer from inadequate interfacial adhesion (between the mineral and the polymer substrate) and poor mechanical properties. Other efforts aimed at improving the interface of HA with polymers include the use of silane coupling agents,38, 39 zirconyl salts,⁴⁰ polyacids41, 42 and isocyanates.43, 44 However, to this date, composite materials that integrate HA and organic scaffolds, especially pHEMA-based hydrogels, and demonstrate the level of integration of natural bone, have not yet been achieved.

Here we report the development of a rapid and effective mineralization method that leads to high affinity integration of HA with a pHEMA hydrogel scaffold. This work serves as an important first step towards the development of hydrogel-based biomimetic composite materials. External factors such as heating rate, agitation and the duration of the process that affect the outcome of the mineralization (e.g. the extent of mineralization, the strength of mineral adhesion at the gel–mineral interface and the mineral crystallinity) were investigated.