3D scaffold with effective multidrug sequential release against bacteria biofilm

Abstract

Bone infection is a feared complication following surgery or trauma that remains as an extremely difficult disease to deal with. So far, the outcome of therapy could be improved with the design of 3D implants, which combine the merits of osseous regeneration and local multidrug therapy so as to avoid bacterial growth, drug resistance and the feared side effects. Herein, hierarchical 3D multidrug scaffolds based on nanocomposite bioceramic and polyvinyl alcohol (PVA) prepared by rapid prototyping with an external coating of gelatin-glutaraldehyde (Gel-Glu) have been fabricated. These 3D scaffolds contain three antimicrobial agents (rifampin, levofloxacin and vancomycin), which have been localized in different compartments of the scaffold to obtain different release kinetics and more effective combined therapy. Levofloxacin was loaded into the mesopores of nanocomposite bioceramic part, vancomycin was localized into PVA biopolymer part and rifampin was loaded in the external coating of Gel-Glu. The obtained results show an early and fast release of rifampin followed by sustained and prolonged release of vancomycin and levofloxacin, respectively, which are mainly governed by the progressive in vitro degradability rate of these scaffolds. This combined therapy is able to destroy Gram-positive and Gram-negative bacteria biofilms as well as inhibit the bacteria growth. In addition, these multifunctional scaffolds exhibit excellent bioactivity as well as good biocompatibility with complete cell colonization of preosteoblast in the entire surface, ensuring good bone regeneration. These findings suggest that these hierarchical 3D multidrug scaffolds are promising candidates as platforms for local bone infection therapy.

Statement of Significance

The present study is focused in finding an adequate therapeutic solution for the treatment of bone infection based on 3D multifunctional scaffolds, which combines the merits of osseous regeneration and local multidrug delivery. These 3D multidrug scaffolds, containing rifampin, levofloxacin and vancomycin, localized in different compartments to achieve different release kinetics. These 3D multidrug scaffolds displays an early and fast release of rifampin followed by sustained and prolonged release of vancomycin and levofloxacin, which are able to destroy Staphylococcus and Escherichia biofilms as well as inhibit bacteria growth in very short time periods. This new combined therapy approach involving the sequential delivery of antibiofilms with antibiotics constitutes an excellent and promising alternative for bone infection treatment.

Introduction

Bone infection is a potentially devastating complication with important clinical and socio-economic implications [1], [2], [3]. It is described as an inflammatory process that leads to bone destruction (osteolysis) usually caused by an underlying microbial infection, mainly by Staphylococcus aureus bacteria [4], [5]. Conventional treatments, involving systemic antibiotic administration [6], surgery and implant removal [7], [8], have important limitations and significant repercussions for the quality life of the patients such as, high side effects [9], prolonged hospital stays, additional surgical interventions [10], and even, high morbidity rate [11]. The main reason for the failure of these conventional treatments is the ability of bacteria to develop a biofilm [1], [2], [3], [4]. Biofilms are described as communities of microorganisms that grow attached to a surface or interphase and embedded in a self-produced extracellular matrix [12]. Biofilm development is one of the most common processes that bacteria accomplish in a cooperative manner. Inside the biofilm, bacteria grow protected from environmental stresses and resist antibiotics, disinfectants, phagocytosis and other components of the innate and adaptive immune and inflammatory defense system of the host giving as a consequence failure of the antibiotic treatment [13].

Currently, the nanotechnology field has emerged as a powerful tool to combat the infection process [14], [15], [16]. In this sense the development of novel multifunctional 3D scaffolds based on nanostructured materials to not only clear the infection but to also contribute to subsequent bone regeneration would be a very good alternative to conventional therapies [17], [18], [19], [20], [21], [22], [23]. The local antimicrobial administration will minimize side effects and risk of overdose, as well as to improve the bioavailability of the drug with the appropriate therapeutic concentration effectively reaching the target site [24]. Moreover, the possibility to achieve a combined therapy with different antimicrobial agents would be needed for a more efficient treatment of bone infection [25]. In this sense, an initial fast release of an antibiofilm drug to be able to destroy the biofilm capsule and subsequently more sustained and prolonged release of the different antibiotics would be desired [26].

Recently, a nanocomposite bioceramic (MGHA), formed by particles of nanocrystalline apatite embedded into amorphous mesoporous bioactive glass in the SiO₂–P₂O₅–CaO system, has been reported. Due to the synergy of the features of its two components, including (i) ordered mesoporous arrangement with pores of 8 nm, (ii) high surface area and pore volume, (iii) high bioactivity, (iv) presence of nanocrystalline apatite particles homogeneously distributed, and (v) improved in vitro biocompatibility, this nanocomposite material is an excellent candidate for bone tissue engineering and local drug delivery [27], [28], [29], [30]. Concerning the fabrication processes to obtain scaffolds, 3D plotting techniques (also called direct writing or printing) have been widely developed to prepare porous scaffolds in recent years [31]. In this sense, recently, hierarchical meso-macro 3D porous scaffolds have been fabricated by a combination of a single-step sol–gel route in the presence of a surfactant as the mesostructure directing agent and a biomacromolecular polymer (methylcellulose) as the macrostructure template followed by rapid prototyping technique with a high potential in bone tissue engineering [32], [33]. However, this methodology for preparing scaffolds containing different antimicrobial agent is inconvenient, because of the need for methylcellulose and the additional sintering procedure.

Currently, the fabrication of composite scaffolds based on bioceramic-polymer mixture has allowed improvement of their properties due to the synergy of the features of their components being widely used for different applications [34], [35]. Therefore, the incorporation of biocompatible polymer have enhanced the mechanical properties of scaffolds by using room temperature in the process, which offers the possibility to incorporate drugs for the subsequently controlled release [36]. In this case, polyvinyl alcohol (PVA) is selected because it is generally biocompatible, degradable and water soluble so toxic solvents do not need to be used in the preparation. In addition, PVA can be cross-linked to improve its crystallinity and to control its dissolution by a simple heat treatment at low temperature [37]. A previous study has shown that mixing mesoporous glasses powders with an aqueous PVA solution to form an injectable paste is very efficient to fabricate 3D scaffolds. In addition, several studies have shown that gelatin-glutaraldehyde (Gel-Glu) coatings onto the 3D scaffolds could improve both, its mechanical properties as well as the early and fast release of a drug depending on the cross-linking degree of the gelatin [38], [39], [40].

The present study is focused in finding an adequate therapeutic solution for the treatment of bone infection by the design of hierarchical 3D multidrug scaffolds based on nanocomposite bioceramic, highly bioactive and biocompatible, with PVA polymer. These structures must be able to incorporate different drugs in various compartments for combined therapy, which allows to eradicate the bacterial biofilm and thus, to completely eliminate the bone infection. For antimicrobial therapy, currently there are many antimicrobial agents and combinations [1], [7]. It is important to assure the maxima antimicrobial efficacy by sustained and prolonged administration in the time. Levofloxacin (LEV) is successfully used in clinical record in the treatment of bone infection due to ability to penetrate into trabecular and cortical bone, minimizing the risk of resistance selection [41], [42]. Moreover, LEV exhibits a sustained release in mesoporous matrices due to the strong interaction with the silanol groups [30]. On the other hand, vancomycin (VAN) is widely used during the prophylaxis and postoperative surgery for prevention and treatment of bone infection [7]. VAN is described as a tricyclic glycopeptide antibiotic commonly used for treatment of severe infections caused by Gram-positive bacteria and especially indicated for methicillin-resistant S. aureus (MRSA), penicillin-resistant pneumococci, or patients allergic to penicillins and cephalosporins [43], [44], [45]. Moreover, due to its hydrophilic character exhibits sustained and prolonged release in polymeric systems [46], [47]. Finally, rifampin (RIF) is an antibiofilm antibiotic, which is able to attack and destroy the Staphylococci in biofilm. RIF must always combined with another antibiotic because bacteria can develop resistance very rapidly when it is used as a monotherapy [48]. In this sense, this drug has been reported to present a synergy when it is administrated with other compounds such as levofloxacin and vancomycin [49].

Herein, the present study proposes a 3D multifunctional scaffold as a novel drug delivery system for treatment of bone infection and bone regeneration. This 3D system will be constituted by a mixture of nanocomposite MGHA and PVA containing different antimicrobial agents in different compartment to achieve different release kinetics. The 3D multifunctional scaffolds will be fabricated by rapid prototyping technique using a paste formed by aqueous mixture of calcined MGHA powder and PVA. Previous to scaffolds fabrication, both LEV and VAN will be incorporated into the mesopore structure and polymer matrix, respectively. Finally, this 3D scaffolds will be coated by a Gel-Glu layer containing RIF to obtain an early and fast release of this antibiofilm agent. In vitro degradability assays in simulated body fluid and biocompatibility assays in presence of preosteoblast have been performed in order to study the bone regeneration capability of these scaffolds. Moreover, antimicrobial tests to study the effectiveness on 3D multifunctional scaffolds against Staphylococcus and Escherichia biofilms have been also reported. Fig. 1 displays the schematic design of multidrug 3D scaffold as well as the processing of fabrication.