Strong binding of bioactive BMP-2 to nanocrystalline diamond by physisorption

Abstract

Nano-crystalline diamond (NCD)-coated surfaces were efficiently functionalized with bone morphogenetic protein-2 (BMP-2) by means of physisorption. Due to their randomly oriented texture, NCD-coated surfaces appear to bind complex molecules firmly. Applying various highly sensitive analytical methods, the interaction was found extremely stable. The strength of the experimentally measured adherence between BMP-2 and NCD was further corroborated by theoretical calculations. Oxygen treatment rendered NCD hydrophilic by the appearance of surface oxygen containing groups. This particular NCD surface exhibited even higher binding energies towards BMP-2 than the hydrophobic surface, and this surface was also favoured by cultured cells. Most importantly in this context, bound BMP-2 was found fully active. When cultured on BMP-2-treated NCD, osteosarcoma cells strongly up-regulated alkaline phosphatase, a specific marker for osteogenic differentiation. Hence, this simple method will allow generating highly versatile surfaces with complex biomimetic coatings, essentials for novel medical devices and implants as well as for innovative scaffolds in tissue engineering.

Introduction

Bio-nano-technological advancements put forward new materials and strategies for tissue engineering, thus supporting innovative strategies, by which now combinations of biocompatible, yet inert material can be employed together with living cells or bioactive proteins in order to sustain regeneration and repair of damaged, diseased and aged tissues [1], [2]. For this purpose, novel materials such as synthetic polymers, carbon nanotubes, fullerene or nanocrystalline diamond (NCD) have been applied [3]. Pertinent requirements are biocompatibility, chemical stability and inertness, properties, which are perfectly fulfilled by NCD-coated surfaces. Moreover, the micro-architecture of surfaces, which were found optimal for an improved healing process [4], [5], remains highly preserved when coated with NCD.

As a result of surface termination of the NCD surface by saturating the dangling bonds either with hydrogen or oxygen the reactivity of the surface to adsorbed intermediates can be influenced [6]. Functional groups such as carbonyl, carboxyl, hydroxyl, ether, ester etc. as well as H provide surfaces representing special chemical reactivities, varying physical properties or exhibiting hydrophilic or hydrophobic attitude. This variability of the surface was found essential for the functionalization with bioactive molecules. When attaching cytokines or growth factors in particular bone morphogenetic protein-2 (BMP-2) to these surfaces, one gains a stable biomimetic coating which e.g. results in improved osseointegration [7], [8]. BMP-2 and other therapeutical drugs have been applied either adsorbed to biocompatible material or by means of “slow releasing systems.” Within the organism, the carrier material will degrade and the bioactive substances are being released [9]. Inevitably, this may also lead to unwanted, systemic effects, which can only be avoided when the drug is firmly hold in place. To achieve a controlled local effect, enzymes have been covalently bound on NCD while maintaining biological activity. Obviously, this involves complex chemistry [9], [10]. To date, stable binding of complex, bioactive molecules by physisorption, comparable to or even stronger than covalent binding, has not been described. Physisorption of proteins to a surface is commonly believed to result in a weak, unspecific binding [11]. Furthermore, no distinct specifications with regard to protein binding onto NCD are available nor have quality or quantity of binding been thoroughly determined.