Polyetheretherketone with citrate potentiated influx of copper boosts osteogenesis, angiogenesis, and bacteria-triggered antibacterial abilities

Highlights

• It was found that citrate can facilitate 2-fold influx of copper into cells.

• Polydopamine controlled the switch of implant’s bio-function through manipulating the release amount of copper and citrate.

• The synergistic antibacterial ability between copper and citrate killed 93 % bacteria.

• Copper and citrate can regulate different pathways of bone formation, thus enhanced osteodifferentiation of Ad-MSC in vitro and promoted new bone formation and osseointegration in vivo.

Abstract

A well designed coating for polyetheretherketone (PEEK) implants can provide enough support to overcome crucial medical challenges, which are insufficient osseointegration and high rate of infection. Herein, we utilize the co-deposition of polydopamine (PDA) and copper-citrate nanoclusters to construct a pH-responsive coating on porous PEEK for synergistic bone regeneration, vascular formation and anti-infection. Specifically, this PDA coating released high dose of copper and citrate at lower pH value, which increased intracellular copper content, boosted production of reactive oxygen species and severe damage of protein, leading to killing of 93 % planktonic bacterial and eradication of adherent bacteria. At pH of 7.4, the release of copper and citrate were in a slow and sustained behavior, synergistically enhanced vascular formation potential and osteodiffereration of Ad-MSC in vitro. After implanted in rabbit tibia for 6 and 12 weeks, the micro-CT evaluation and histological analysis consistently highlighted the ability of this PDA coating to increase new bone formation adjacent to coated PEEK implant and enhance bone-implant interfacial integration. These results were proven to be related to the synergistic effect that citrate facilitated a 2-fold influx of copper into cells, which not only enhanced the bacteria-killing ability but also encouraged bone regeneration of implants. This present work provides an effective method to control infections while promoting osseointegration simultaneously, which will show tremendous clinical application and can be a solution to current challenges facing orthopedics.

Introduction

Polyetheretherketone (PEEK) has attracted increasing attention as an orthopedic implant because of its excellent chemical resistance, natural radiolucency, magnetic resonance imaging compatibility, wear resistance, and most important, its similar mechanical properties to cortical bone [1]. However, its chemical and biological inertness, which usually associates with implant-associated infection and insufficient osseointegration, tends to limit its application. Therefore, it appears necessary to endow antibacterial and osteogenic ability to PEEK.

Many strategies have been applied to combatting with the implant-associated infection as it may lead to secondary surgery, which would increase not only patient suffering but also health costs. These strategies mainly include the coating of some antifouling polymers to repel bacteria and the immobilization/release of organic/inorganic bactericides to kill bacteria [[2], [3], [4], [5], [6]]. Nevertheless, the repelling coatings will inevitably become contaminated by bacteria and the bactericidal coatings are hindered by the rising problem of multidrug-resistant (MDR) bacteria. Therefore, it turns to be necessary to explore a new strategy to kill bacteria efficiently while prevent the development of MDR. Our previous study utilized the synergistic bactericidal effect of silver and gentamicin sulfate to eliminate bacteria and prevent the development of MDR [7]. However, the utilization of silver caused contradiction between antibacterial ability and biocompatibility as its antibacterial ability associates with cytotoxicity, which would lead to difficulties in balancing antibacterial and biocompatibility. Hence, the key is to find a strategy that can balance antibacterial and biocompatibility of implant, which means inhibiting bacteria while promoting osteodifferentiation of bone cells.

Copper has been reported to possess osteogenic and antimicrobial abilities, and it has been used in biomaterials to promote bone formation and prevent infection [[8], [9], [10]]. However, a high concentration of Cu²⁺ (256 μg/mL) is needed to kill bacteria while a relative low concentration of Cu²⁺ (0.1 mM/6.4 μg/mL) will be enough for osteogenesis [11,12]. Thus, we combined copper nanoclusters (CuNs) with citrate which also has been widely used in biomaterial to facilitate bone regeneration and kill bacteria [13]. In addition, citrate kill bacteria via traversing cell membranes and causing damage to particular enzymes [14], while copper kill bacteria through disrupting cell membranes, elevating production of reactive oxygen species (ROS), and binding to protein or DNA [15]. It is clear that copper and citrate share different pathways to kill bacteria and it is known that synergistic antibacterial effect can come from bactericides cocktail attacking bacteria from different fronts. In this way, bacteria can be killed at relative low concentration of Cu²⁺, which would be beneficial to the balance between biocompatibility and bactericidal activity. To achieve the switch of PEEK implant’s bio-function between osteogenesis and antibacterial, we planned to manipulate the release of Cu²⁺ under different situations through the utilization of pH-responsive controlled release property. It can help to achieve the low concentration and sustained release of copper-citrate nanoclusters under physiological conditions, which is expected to promote the bone formation; and achieve high concentration release of copper-citrate nanoclusters only if bacteria are present. To achieve this on-demand release of copper-citrate nanoclusters, polydopamine (PDA) was chosen. It is an important platform for surface functionality and stabilization agent in the fabrication of diverse organic-inorganic materials, most importantly it possesses excellent biocompatibility and pH responsive charge variation and degradation [16].

However, the two performances of osteogenesis and bacteria-killing may not be able to guarantee both rapid bone regeneration and sufficient early stability of implants. Impaired bone healing is also associated with a reduction of vascular supply, which would lead to compromised nutrient availability at the site of injury. It highlights the importance of functional angiogenic response to successful implantation [17]. Actually, active angiogenesis is a prerequisite step to achieve the successes of bone integration as blood vessels transport oxygen, nutrients and factors to the site of bone fracture [18]. Thus, it appears necessary to endow angiogenic ability to implants. The chosen copper has been proved to be able to promote angiogenesis by artificially mimicking hypoxia through stabilizing the structure of hypoxia-inducible factor (HIF-1α), in turn stimulating the secretion of VEGF [19], which plays an important role in the recruitment and differentiation of cells and in blood vessel formation [20,21]. In addition, citrate also has been reported to induce angiogenesis [22]. Hence, implants with copper-citrate nanoclusters are expected to facilitate angiogenesis.

The cross-talk between endothelial cells (ECs) and osteoblastic cells (OBs) also can affect bone repairing, as blood vessel growth and osteogenesis are coupled during bone remodeling [23]. Therefore, it would be important for us to understand the influences of implants on the crosstalk of ECs and OBs. For this purpose, a coculture system with adipose-derived mesenchymal stem cells (Ad-MSCs) and human umbilical vein endothelial cells (HUVECs) was designed, and effects of implants on the crosstalk of cells would be evaluated by their osteogenesis and angiogenesis performance.

In this study, copper-citrate nanoclusters were co-deposited with dopamine upon the porous surface of PEEK implants, aimed to promote implant’s osteogenesis, angiogenesis and antibacterial ability. In addition, the effects of implant on the communication between Ad-MSCs and HUVECs was investigated and the mechanism underlying the enhanced pH-responsive bacteria-killing effect was partially revealed.