Journal of
Functional Biomaterials Article
Development and Evaluation of an Injectable Chitosan/β-Glycerophosphate Paste as a Local Antibiotic Delivery System for Trauma Care Logan Boles, Christopher Alexander, Leslie Pace, Warren Haggard, Joel Bumgardner and Jessica Jennings * Department of Biomedical Engineering, University of Memphis, Memphis, TN 38152, USA;
[email protected] (L.B.);
[email protected] (C.A.);
[email protected] (L.P.);
[email protected] (W.H.);
[email protected] (J.B.) * Correspondence:
[email protected]; Tel.: +1-205-310-3153 Received: 29 August 2018; Accepted: 9 October 2018; Published: 12 October 2018
Abstract: Complex open musculoskeletal wounds are a leading cause of morbidity worldwide, partially due to a high risk of bacterial contamination. Local delivery systems may be used as adjunctive therapies to prevent infection, but they may be nondegradable, possess inadequate wound coverage, or migrate from the wound site. To address this issue, a thermo-responsive, injectable chitosan paste was fabricated by incorporating beta-glycerophosphate. The efficacy of thermo-paste as an adjunctive infection prevention tool was evaluated in terms of cytocompatibility, degradation, antibacterial, injectability, and inflammation properties. In vitro studies demonstrated thermo-paste may be loaded with amikacin and vancomycin and release inhibitory levels for at least 3 days. Further, approximately 60% of thermo-paste was enzymatically degraded within 7 days in vitro. The viability of cells exposed to thermo-paste exceeded ISO 10993-5 standards with approximately 73% relative viability of a control chitosan sponge. The ejection force of thermo-paste, approximately 20 N, was lower than previously studied paste formulations and within relevant clinical ejection force ranges. An in vivo murine biocompatibility study demonstrated that thermo-paste induced minimal inflammation after implantation for 7 days, similar to previously developed chitosan pastes. Results from these preliminary preclinical studies indicate that thermo-paste shows promise for further development as an antibiotic delivery system for infection prevention. Keywords: chitosan; local antibiotic delivery; biofilm; complex musculoskeletal wounds; trauma care; injectable; in vivo evaluation; thermo-paste
1. Introduction High-energy trauma to the musculoskeletal system can cause complex open wounds characterized by lacerations, abrasions, and/or open fractures [1,2]. Complex musculoskeletal wounds, especially open wounds, are highly susceptible to bacterial contamination, with infection rates ranging from 20% to 50% [3,4]. Necrotic tissue surrounding the wound suffers from diminished perfusion, effectively restricting leukocytes and oral or IV administered antibiotics from accessing the site of injury. The wound provides a sheltered anaerobic environment that is favorable for bacterial proliferation [2]. Further, the complex morphology of severe musculoskeletal wounds creates a large surface area for bacteria to adhere, the initiating mechanism in biofilm formation, which can be resistant to antibiotics [5,6]. Biofilm-forming bacteria, such as Staphylococcus aureus and Pseudomonas aeruginosa, are commonly present in complex open musculoskeletal infections [7]. Some biofilm-containing infections can be eradicated with antibiotics administered systemically, but the dangerously high concentrations
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may produce nephrotoxicity or ototoxicity, among other ailments [8]. Local delivery systems used as adjunctive therapy to standard prophylactic antibiotics may be used to achieve high levels of antibiotic J. Funct. 2018, 9, xbiofilm-associated FOR PEER REVIEW 2 of 14 capable of Biomater. eliminating bacteria in local tissue without systemic concentrations reaching levels that produce toxic adverse effects [3,9]. dangerously high concentrations may produce nephrotoxicity or ototoxicity, among other ailments Local delivery of antibiotics from biomaterial systems supplements systemic therapy by increasing [8]. Local delivery systems used as adjunctive therapy to standard prophylactic antibiotics may be antibiotic concentrations at theoflocal site of contamination in complex wounds.bacteria Current available used to achieve high levels antibiotic capable of eliminating biofilm-associated in local local tissue delivery systems for complex musculoskeletal trauma applications including poly(methyl without systemic concentrations reaching levels that produce toxic adverse effects [3,9]. methacrylate) calcium sulfate (CaSO have significant shortcomings Local (PMMA), delivery of antibiotics from biomaterial systems supplements systemic therapy by[8,10]. 4 ), and collagen increasing antibiotic concentrations at properties, the local sitebut of contamination in complex wounds. PMMA possesses excellent mechanical it requires a secondary surgery Current for removal musculoskeletal applications including from available the body,local maydelivery serve assystems a nidusfor for complex secondary infection, andtrauma is incompatible with heat-labile poly(methyl methacrylate) (PMMA), calcium sulfate (CaSO 4), and collagen have significant antibiotics [11]. Ability to be resorbed by the body and providing calcium to facilitate fracture healing shortcomings [8,10]. PMMA possesses excellent mechanical properties, but it requires a secondary are assets of CaSO4 , but inadequate wound coverage and rapid resorption resulting in serous wound surgery for removal from the body, may serve as a nidus for secondary infection, and is incompatible drainage are undesirable characteristics [3,12]. Biodegradable collagen local delivery systems have with heat-labile antibiotics [11]. Ability to be resorbed by the body and providing calcium to facilitate excellent biocompatibility; however, the rapid release of antibiotics does not match its degradation fracture healing are assets of CaSO4, but inadequate wound coverage and rapid resorption resulting rate, and wound coverage does not clinicalcharacteristics needs [8]. [3,12]. Biodegradable collagen local in serous wound drainage are meet undesirable Chitosan, an abundant polysaccharide, has been investigated forrelease local antibiotic delivery due to delivery systems have excellent biocompatibility; however, the rapid of antibiotics does not its biocompatibility, biodegradation to non-toxic degradation products, and tailorability of degradation match its degradation rate, and wound coverage does not meet clinical needs [8]. abundant polysaccharide, been investigated for degree local antibiotic delivery due(DDA), to and drugChitosan, deliveryanrates through alteration has of molecular weight, of deacetylation its biocompatibility, biodegradation to non-toxic degradation products, and tailorability or cross-linking [13–16]. Lyophilized chitosan sponges were developed and are capable ofof local degradation and drug delivery rateshave through alteration degree of deacetylation therapeutic antibiotic release, but they limited abilityoftomolecular conformweight, to complex wound morphologies, (DDA), or cross-linking [13–16]. Lyophilized chitosan sponges were developed and are capable of as well as an unwanted initial burst release of loaded antibiotics [17–19]. To address the limitations of local therapeutic antibiotic release, but they have limited ability to conform to complex wound chitosan sponges, an injectable paste of finely ground, lyophilized chitosan hydrated with antibiotic morphologies, as well as an unwanted initial burst release of loaded antibiotics [17–19]. To address solution has been developed and has shown potential as an adjunctive infection-prevention tool [20,21]. the limitations of chitosan sponges, an injectable paste of finely ground, lyophilized chitosan The chitosan inhibitory of antibiotics for 3has days, degraded in aassimilar time frame, hydratedpaste with eluted antibiotic solution levels has been developed and shown potential an adjunctive and improved wound coverage. However, significant injection force made application the paste infection-prevention tool [20,21]. The chitosan paste eluted inhibitory levels of antibiotics forof 3 days, difficult, and migration from injection site couldwound reducecoverage. efficacy. However, Thus, there is a needinjection to improve degraded in a similar timethe frame, and improved significant force made application of the paste difficult, theprevent injectionmigration site couldof reduce the chitosan paste by reducing the force neededand formigration injection from and to the paste Thus, there is a need to improve the chitosan paste by reducing the force needed for injection from efficacy. the wound bed. and to prevent migration of(β-GP) the paste from a thepolyelectrolyte wound bed. Beta-glycerophosphate forms complex with chitosan that has low Beta-glycerophosphate (β-GP) forms a polyelectrolyte complex with chitosan that has low viscosity at low temperatures and gels at higher/body temperatures, as shown in Figure 1 [22,23]. viscosity at low temperatures and gels at higher/body temperatures, as shown in Figure 1 [22,23]. Taking advantage of this thermo-gelling characteristic, the addition of β-GP to chitosan paste may Taking advantage of this thermo-gelling characteristic, the addition of β-GP to chitosan paste may reduce the viscosity of theofpaste at room temperature to improve its injectability and wound coverage. reduce the viscosity the paste at room temperature to improve its injectability and wound Then,coverage. gelation Then, would occur at normal body temperature to form a thick paste that would not migrate gelation would occur at normal body temperature to form a thick paste that would from not themigrate woundfrom site the andwound wouldsite help maintain high local antibiotic concentrations at the local and would help maintain high local antibiotic concentrations at theinjury site tolocal prevent injuryinfection. site to prevent infection.
Figure 1. Schematic of proposed gelationof ofbeta-glycerophosphate beta-glycerophosphate (β-GP) and chitosan thermo-paste Figure 1. Schematic of proposed gelation (β-GP) and chitosan thermo-paste delivery system. delivery system.
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study is toisdevelop an adjunctive infection prevention therapytherapy for trauma The goal goalofofthe thecurrent current study to develop an adjunctive infection prevention for care. It care. is hypothesized that by modifying the chitosan paste to include β-glycerophosphate, a trauma It is hypothesized that by modifying the chitosan paste to include β-glycerophosphate, thermo-responsive paste will be formed that is easily injectable, provides effective wound coverage, a thermo-responsive paste will be formed that is easily injectable, provides effective wound coverage, point-of-care loading loading with with antibiotic antibiotic solutions, solutions, locally delivers therapeutic levels of is capable of point-of-care antibiotics, and degrades over the course of fracture healing. Specific objectives of this study were to determine the the cytocompatibility, antibiotic characteristics, determine cytocompatibility, antibiotic release release characteristics, enzymatic enzymatic degradation,degradation, injectability, injectability, and ininflammatory vivo tissue inflammatory response of the thermo-paste. and in vivo tissue response of the thermo-paste. 2. Results 2.1. 2.1. Cytocompatibility Cytocompatibility The The cytocompatibility cytocompatibility of of thermo-paste thermo-paste met met the the cytocompatibility cytocompatibility standard standard outlined outlined in in ISO ISO 10993-5 [24]. No statistically significant differences in relative viability were observed between 10993-5 [24]. No statistically significant differences in relative viability were observed between the the groups groups at at the the 0.05 0.05 level level of of significance significance (Figure (Figure 2). 2).
Figure 2. Relative viability of NIH3T3 cells after 72 h of indirect exposure to chitosan samples (n = 3); presented as as mean mean ± ± standard data is presented standard deviation deviation (STD). (STD).
2.2. Antibiotic Elution 2.2. Antibiotic Elution and and Activity Activity The vancomycin and amikacin at infection-inhibiting levels for atfor least The thermo-paste thermo-pastereleased releasedboth both vancomycin and amikacin at infection-inhibiting levels at 72 h (Figures 3a and3a 4a), and this was confirmed by zoneby of inhibition (ZOI) analysis 1, Figures 5 least 72 h (Figures and 4a), and this was confirmed zone of inhibition (ZOI) (Table analysis (Table 1, and 6). The vancomycin release kinetics of thermo-paste are comparable to poly(ethylene glycol) (PEG) Figures 5 and 6). The vancomycin release kinetics of thermo-paste are comparable to poly(ethylene paste a cumulative of 86.2release ± 14.3% vs. 91.9 ± 18.3% and± detectable for 5levels days glycol)with (PEG) paste with release a cumulative of 86.2 ± 14.3% vs. 91.9 18.3% and levels detectable (Figure 3b).(Figure Activity3b). against S. aureus wasS.sustained for sustained 72 h basedfor on72 ZOI for 5 days Activity against aureus was h analysis. based on Thermo-paste ZOI analysis. cumulatively released 46.0 ± 12.9% of amikacin vs. 27.4 ± 4.1% of PEG paste and of maintained active Thermo-paste cumulatively released 46.0 ± 12.9% of amikacin vs. 27.4 ± 4.1% PEG paste and concentrations against P. aeruginosa for 4 days (Figure 4b). Thermo-paste samples released amikacin maintained active concentrations against P. aeruginosa for 4 days (Figure 4b). Thermo-paste samples at concentrations than PEG paste onthan DayPEG 1 and were all highest groups among on Dayall 2. released amikacinhigher at concentrations higher paste onhighest Day 1 among and were Detectable levels of antibiotics were not observed on Days 6 or 7 for any group. Extended elution groups on Day 2. Detectable levels of antibiotics were not observed on Days 6 or 7 for any group. profiles were observed for thermo-paste compared to chitosancompared sponge for antibiotics. Extended elution profiles were observed for thermo-paste toboth chitosan sponge for both
antibiotics.
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(a)
(b) Figure 3. (a) (a) Concentrations Concentrationsof ofvancomycin vancomycininineluate eluatesamples, samples,(b) (b) cumulative vancomycin released cumulative vancomycin released (n (n = 3). Data presented as mean ± STD. Minimum inhibitory concentration (MIC) values reported = 3). Data presented as mean ± STD. Minimum inhibitory concentration (MIC) values reported are are lab lab generated values. * represents a significant difference compared to PEG paste < 0.05). generated values. * represents a significant difference compared to PEG paste (p
