Antibacterial, anti-biofilm and angiogenic calcium sulfate-nano MgO composite bone void fillers for inhibiting Staphylococcus aureus infections

doi:10.1016/j.colcom.2020.100332

Colloid and Interface Science Communications

Volume 39, November 2020, 100332

https://doi.org/10.1016/j.colcom.2020.100332 Get rights and content

Highlights

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Addition of N, O-CMC as a binder had enhanced the compressive strength.
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Prepared beads showed sustained Tigecycline drug release.
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Prepared beads showed pro-angiogenic effect and were cytocompatible.
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The prepared beads showed antibacterial effect against S. aureus & MRSA.
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Prepared beads were also effective in inhibiting biofilm produced by S. aureus.

Abstract

Antibiotic loaded calcium sulfate (CS) beads are widely reported to be used as bone void fillers. Application of these beads has been found effective in the inhibition of bacterial infections and enhanced bone regeneration, though lacking the ability to enhance angiogenesis. Thus, realizing the importance of angiogenesis for complete bone regeneration, magnesium oxide nanoparticles (MgO Nps) of ≤50 nm and tigecycline (Tg) incorporated CS based nanocomposite (Tg-NC) (Tigecycline-Nanocomposite) beads were prepared. N, O-carboxymethyl chitosan solution was chosen as a binder in the preparation of CS-based beads for the enhanced stability. The prepared Tg-NC beads were characterized using SEM, EDS and FTIR. The Tg-NC beads were cytocompatible showing pro-angiogenic property. The sustained drug release for 10 days from Tg-NC beads was found to be very effective in the inhibition of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). Also, Tg-NC beads effectively inhibited biofilms exhibited by Staphylococcus aureus. Thus prepared Tg-NC beads can be used as bone void fillers for inhibiting infections and promoting angiogenesis.

Graphical abstract

Introduction

Antibiotic-impregnated bone cements use has been increasing in the treatment of arthroplasty related infections [1]. Poly-(methyl methacrylate) (PMMA) is one among the earlier reported antibiotic-impregnated polymer bone cement used in treating osteomyelitis. PMMA beads served as effective drug carriers, but a second revision surgery was required to remove the non-degradable beads by the end of the second week [2]. Therefore, the focus has shifted towards identifying suitable bone cement with better biodegradability and drug elution properties [3]. The use of bioceramic in such treatment procedures has been promising because of their advantages such as porosity, biodegradability and osteoconductivity [4]. CS is one such bioceramic whose importance has been realized in the treatment of periodontal, periprosthetic or implant-related infections, joint arthroplasty, pedal osteomyelitis etc. [5,6]. The studies have also demonstrated that the application of antibiotic-loaded CS bone fillers ensures sustained drug release at the infected site [7]. The CS also serves as an osteoconductive matrix to the surrounding pre-osteoblast cells and enhances bone formation [8]. Localized application of antibiotics loaded CS beads have shown to be effective in the inhibition of bacterial infection at the applied site [9,10]. Commercially available calcium sulfate-based beads have been reported as effective carriers of antibiotics in treating bacterial infections but lack the potential to enhance angiogenesis [11,12].

Realizing the importance of osteogenic and angiogenic coupling for the complete bone regeneration, this work was focused on developing CS beads incorporated with magnesium oxide nanoparticles (MgO NPs). The activity of magnesium ions in enhancing angiogenesis has been previously studied [13]. Osteogenic differentiation of mesenchymal stem cells (MSCs) in the presence of magnesium through the Notch signalling pathway has been reported [14]. The property of magnesium in enhancing both angiogenesis and osteogenesis was considered significant in the development of MgO NPs incorporated CS beads [15].

Ferguson et al. reported that the use of CS beads resulted in faster elution of antibiotics at the applied site since they tend to hydrolyse and dissolute quickly [3]. This insight suggested the use of a polymeric binder to attain sustained drug release. Chen et al., suggested that the incorporation of N, O-carboxymethyl chitosan (N,O-CMC) resulted in sustained localized delivery of proteins and also enhanced wound healing [16]. N,O-CMC (water soluble derivative of chitosan) is reported to be non-toxic with properties to improve the retention and bioavailability of drugs [17]. Thus, N,O-CMC was used as a binder in the preparation of the beads and its effect in mechanical properties of the prepared beads were also studied in the present work.

Staphylococcus aureus, the common pathogen associated with most bone infections is of focus in this study [18]. Tg, a wide spectrum small molecule drug active against the inhibition of Staphylococcus aureus and methicillin-resistant strains of Staphylococcus aureus (MRSA) was chosen to be incorporated in CS beads along with MgO NPs [[19], [20]]. Therefore, this study was focused on developing and characterising CS, NC (nanocomposite) and Tg-NC beads. Also, in vitro antibacterial, anti-biofilm, cytocompatibility and angiogenic properties exhibited by the prepared beads were reported.

Section snippets

Materials

Calcium sulfate hemihydrate (CaSO₄.1/2H₂O) was purchased from Fisher Scientific, USA. Magnesium oxide nanopowder (≤ 50 nm) was purchased from Sigma Aldrich. Clinical strains of MRSA were obtained from Amrita Institute of Medical Sciences, Kochi, India. Mannitol salt agar (MSA), Luria Bertani broth (L.B.) and Tryptic soy broth (TSB) were purchased from Himedia, India. alamarBlue™, Fetal bovine serum (FBS), Iscove's modified Dulbecco's media (IMDM), Trypsin-EDTA, Large vessel endothelial

Preparation and characterization of CS, NC and Tg-NC beads

The obtained beads were hemispherical, 3 mm in diameter and weighed 26 ± 1.6 mg (n = 10). SEM images revealed the presence of randomly oriented crystallites throughout the CS and NC beads. Presence of MgO nanoparticles incorporated into the beads was confirmed using EDS. Respective atomic percentages of Mg, Ca, S, and O in NC beads estimated using EDS was found to be 12.17, 30.7, 3.83 and 37.30% (Fig. 2A & 2B) respectively. FTIR peaks corresponding to –OH stretching (3543–3242 cm⁻¹) and –OH

Conclusion

Herein, we have developed MgO NPs and Tg incorporated CS based beads with N, O-CMC as a binder. Tg-NC beads were capable of ensuring drug release for 10 days effectively inhibiting the growth of SA113 and clinical isolates of MRSA. SA113 biofilm formation was also reduced in the presence of Tg-NC beads. Importance of magnesium towards enhancing angiogenesis was also comprehended in this study. Capillary networking exhibited by HUVECs in the presence of Tg-NC beads proves its angiogenic nature.

Authors statement

Submission of an article implies that the work described has not been published previously, that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The authors are thankful for the support provided by Nanomission, Department of Science and Technology (DST), Government of India under “M.Tech Nanoscience and Technology” program. A. Sivashanmugam is grateful to the Council of Scientific and Industrial Research (CSIR), Government of India for the financial support through Senior Research Fellowship (SRF award no. 09/963 (0038) 2K17-EMR-I). The authors thank Dr. Raja Biswas for his guidance in anti-bacterial and biofilm inhibition studies.

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Antibacterial, anti-biofilm and angiogenic calcium sulfate-nano MgO composite bone void fillers for inhibiting Staphylococcus aureus infections

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Preparation and characterization of CS, NC and Tg-NC beads

Conclusion

Authors statement

Declaration of Competing Interest

Acknowledgement

J. Foot Ankle Surg.

Eur. J. Pharm. Biopharm.

J. Orthop. Res.

Biomaterials

Biomaterials

Biomaterials

FEMS Microbiol. Lett.

Int. J. Antimicrob. Agents

Diagn. Microbiol. Infect. Dis.

Biochim. Biophys. Acta - Gen. Subj.

Carbohydr. Polym.

Biochim. Biophys. Acta - Mol. Basis Dis.

Antibiotic prophylaxis in total hip replacement

Br. Med. J.

Ceramic biocomposites as biodegradable antibiotic carriers in the treatment of bone infections

J. Bone Jt. Infect.

Antibiotic-loaded synthetic calcium sulfate beads for prevention of bacterial colonization and biofilm formation in periprosthetic

Antimicrob. Agents Chemother.

Local delivery of antibiotics in the surgical treatment of bone infections

Tech. Orthop.

Local antibiotic therapy in osteomyelitis

Semin. Plast. Surg.

Adjuvant treatment of chronic osteomyelitis of the tibia following exogenous trauma using OSTEOSET®-T: a review of 21 patients in a regional trauma Centre

Strateg. Trauma Limb Reconstr.