The Open Orthopaedics Journal




ISSN: 1874-3250 ― Volume 13, 2019

Osteocompatibility of Biofilm Inhibitors



Monica Rawson, Warren Haggard , Jessica A Jennings*
Biomedical Engineering, University of Memphis, 330 Engineering Technology Building, Memphis, TN 38152, USA

Abstract

The demand for infection prevention therapies has led to the discovery of several biofilm inhibitors. These inhibiting signals are released by bacteria, fungi, or marine organisms to signal biofilm dispersal or disruption in Gram-positive, Gram-negative, and fungal microorganisms. The purpose of this study was to test the biocompatibility of five different naturally-produced biofilm chemical dispersal and inhibition signals with osteoblast-like cells: D-amino acids (D-AA), lysostaphin (LS), farnesol, cis-2-decenoic acid (C2DA), and desformyl flustrabromine (dFBr). In this preliminary study, compatibility of these anti-biofilm agents with differentiating osteoblasts was examined over a 21 days period at levels above and below concentrations active against bacterial biofilm. Anti-biofilm compounds listed above were serially diluted in osteogenic media and added to cultures of MC3T3 cells. Cell viability and cytotoxicity, after exposure to each anti-biofilm agent, were measured using a DNA assay. Differentiation characteristics of osteoblasts were determined qualitatively by observing staining of mineral deposits and quantitatively with an alkaline phosphatase assay. D-AA, LS, and C2DA were all biocompatible within the reported biofilm inhibitory concentration ranges and supported osteoblast differentiation. Farnesol and dFBr induced cytotoxic responses within the reported biofilm inhibitory concentration range and low doses of dFBr were found to inhibit osteoblast differentiation. At high concentrations, such as those that may be present after local delivery, many of these biofilm inhibitors can have effects on cellular viability and osteoblast function. Concentrations at which negative effects on osteoblasts occur should serve as upper limits for delivery to orthopaedic trauma sites and guide development of these potential therapeutics for orthopaedics.

Keywords: Biocompatibility, biofilm, dispersal agents, infection, mineralization, osteoblast..


Article Information


Identifiers and Pagination:

Year: 2014
Volume: 8
First Page: 442
Last Page: 449
Publisher Id: TOORTHJ-8-442
DOI: 10.2174/1874325001408010442

Article History:

Received Date: 26/6/2014
Revision Received Date: 1/10/2014
Acceptance Date: 19/10/2014
Electronic publication date: 26 /11/2014
Collection year: 2014

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* Address correspondence to this author at the Biomedical Engineering, University of Memphis, 330 Engineering Technology Building, Memphis, TN 38152, USA; Tel: (901)678-3733; Fax: (901)678-5281; E-mail: jjnnings@memphis.edu




INTRODUCTION

Orthopaedic implants may be composed of a variety of different materials, such as metal alloys and polymers, which provide non-natural surfaces in the body and are a haven for biofilm formation [1Costerton JW, Montanaro L, Arciola CR. Biofilm in implant infections its production and regulation. Int J Artif Organs 2005; 28(11): 1062-8.]. Attachment of bacteria to medical device surfaces, such as orthopaedic implants, leads to the formation of a biofilm, which may cause life-threatening infections and healthcare cost burdens [2Campoccia D, Montanaro L, Arciola CR. The significance of infection related to orthopedic devices and issues of antibiotic resistance. Biomaterials 2006; 27(11): 2331-9.]. Orthopaedic infection is the second most common complication with large joint replacements and is very difficult to diagnose and treat [2Campoccia D, Montanaro L, Arciola CR. The significance of infection related to orthopedic devices and issues of antibiotic resistance. Biomaterials 2006; 27(11): 2331-9., 3Cahill JL, Shadbolt B, Scarvell JM, Smith PN. Quality of life after infection in total joint replacement. J Orthop Surg (Hong Kong) 2008; 16(1): 58-65.]. The consequences of infection include loss of implant function, damaged local tissues, and, in extreme cases, septic shock and death [4Cataldo MA, Petrosillo N, Cipriani M, Cauda R, Tacconelli E. Prosthetic joint infection recent developments in diagnosis and management. J Infect 2010; 61(6): 443-8.]. Treatment usually requires multiple surgeries and aggressive courses of systemic and local antibiotic therapies, with accompanying risks and side effects [3Cahill JL, Shadbolt B, Scarvell JM, Smith PN. Quality of life after infection in total joint replacement. J Orthop Surg (Hong Kong) 2008; 16(1): 58-65., 5Kahlmeter G, Dahlager JI. Aminoglycoside toxicity - a review of clinical studies published between 1975 and 1982 . J Antimicrob Chemother 1984; 13(Suppl A ): 9-22.].

Bacteria communicate within biofilm communities with chemical signals to trigger various events, such as detachment from a surface into the planktonic form when resources are scarce, a process known as quorum sensing [6Costerton W, Veeh R, Shirtliff M, Pasmore M, Post C, Ehrlich G. The application of biofilm science to the study and control of chronic bacterial infections. J Clin Invest 2003; 112(10): 1466-77., 7McDougald D, Rice SA, Barraud N, Steinberg PD, Kjelleberg S. Should we stay or should we go mechanisms and ecological consequences for biofilm dispersal. Nat Rev Microbiol 2012; 10(1): 39-50.]. The planktonic form of bacteria can be 10-1,000 times more susceptible to antibiotics than a colony protected by a biofilm [8Olson ME, Ceri H, Morck DW, Buret AG, Read RR. Biofilm bacteria formation and comparative susceptibility to antibiotics. Can J Vet Res 2002; 66(2): 86-92.]. Some of the different types of bacterial- and fungal-produced signals that disperse biofilms in Gram-positive, Gram-negative, and fungal microorganisms are D-amino acids (D-AA), lysostaphin (LS), farnesol, and cis-2-decenoic acid (C2DA) [9Davies DG, Marques CN. A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. J Bacteriol 2009; 191(5): 1393-403.-1Costerton JW, Montanaro L, Arciola CR. Biofilm in implant infections its production and regulation. Int J Artif Organs 2005; 28(11): 1062-8.]. In addition, Desformyl-flustrabromine (dFBr) is a marine-derived molecule that has been shown to have anti-biofilm activity against multiple bacterial strains [12Bunders CA, Minvielle MJ, Worthington RJ, Ortiz M, Cavanagh J, Melander C. Intercepting bacterial indole signaling with flustramine derivatives. J Am Chem Soc 2011; 133(50): 2016,0-3.]. Combining these biofilm dispersal and inhibition agents with antibiotics can greatly increase the susceptibility of bacterial biofilm to antibiotic therapy [13Jennings JA, Courtney HS, Haggard WO. Cis-2-decenoic acid inhibits, S. aureus growth and biofilm in vitro a pilot study. Clin Orthop Relat Res 2012; 470(10): 2663-70.].

While the antimicrobial effects of these biofilm disrupting agents have been characterized, little is known about their effects on osteoblast cell function. The presence of these chemical signals could slow down or halt the healing process depending on the toxic concentration released at the site [14Rathbone CR, Cross JD, Brown KV, Murray CK, Wenke JC. Effect of various concentrations of antibiotics on osteogenic cell viability and activity. J Orthop Res 2011; 29(7): 1070-4.]. Therefore, the purpose of this study is to test the biocompatibility of the aforementioned five different anti-biofilm agents with osteoblasts in vitro at levels above and below active concentrations through assessment of viability and differentiation over a 21 day time course compared to controls.

MATERIALS AND METHODS

Farnesol, D-AA (D-phenylalanine, D-proline, and D-tyrosine), dFBr, and LS were purchased from Sigma. C2DA was purchased from Grupo Nitrile.

MC3T3 mouse calvarial osteoblast cells (ATCC) were seeded at 1 × 104 cells/cm2 in 24 well plates in alpha-MEM containing 10% fetal bovine serum (FBS) with antibiotics, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. After overnight attachment, media was replaced with osteogenic media consisting of alpha-MEM with 10% FBS, 0.1 μM dexamethasone, 0.2 mM ascorbic acid 2-phosphate, 10 mM beta-glycerophosphate, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. Farnesol, C2DA, and an equal mixture of the three D-AAs were dissolved and diluted serially in 1.25% ethanol to improve solubility of these biofilm inhibitors with hydrophobic characteristics. LS and dFBr were solubilized and diluted in osteogenic media including antibiotics. Solutions of each chemical in alpha-MEM or alpha-MEM + ethanol were added to achieve the concentrations listed in Table 1 and a final ethanol concentration of 1.25% for those with added ethanol. Osteogenic media alone, osteogenic media + ethanol, and 10% FBS in alpha-MEM (non-osteogenic) were also evaluated as positive and negative controls. Media was refreshed every 3 days. At days 1, 3, 7, 14, and 21 cells in wells (n=4 per group per time point) were lysed with 25 mM Tris and 0.5% Triton X-100 and stored at -80°C until analysis. Cell number was estimated by DNA quantity using Quant-it™ PicoGreen (Invitrogen), and alkaline phosphatase (ALP) levels were determined through a colorimetric assay using p-nitrophenyl phosphate as a phosphatase substrate. In order to normalize ALP production in wells with varying cell quantity, ALP quantities measured in each well were divided by the DNA quantity from corresponding wells. Separate plates were fixed with 10% formalin and stained with alizarin red-S (MP Biomedicals) to visualize mineralization microscopically.

Table 1

Concentrations of each biofilm inhibitor evaluated for osteoblast biocompatibility.




Statistical analysis on DNA and ALP content was performed using ANOVA with Tukey post-hoc tests with n = 4 and significance level (α) of 0.05.

RESULTS

Visual Observations of the Chemical Dispersal Test Group Solutions

D-AA mixture formed white flakey precipitates at 500 μM and above, but the lower concentrations were soluble and did not precipitate. The farnesol and C2DA were very hydrophobic and formed micelles, but vigorous shaking created an even distribution of the solute prior to addition to cultures. The remaining agents tested, LS and dFBr, were polar and were therefore soluble and mixed evenly in solution.

Cell Viability

MC3T3 cells in osteogenic media had a positive growth rate until day 3, then cell viability remained steady over the following two weeks as cells became confluent (Fig. 1a).

Osteogenic media containing ethanol had statistically lower viability on day 3 in comparison to the two other control groups, but no significant differences in cell viability between control groups with or without osteogenic additives and ethanol were detected for any other time point. DNA quantity in cells treated with varying concentrations of lysostaphin remained consistent during the study for all concentrations tested (Fig. 1c), suggesting that it is not cytotoxic at concentrations equal to or less than 37.5 µg/mL (1.5 µM). For all evaluated anti-biofilm compounds, except for lysostaphin, there were dose-dependent decreases in viability during the initial 3 days shown in Fig. (1b-f). While cells exposed to C2DA did not exhibit significant decreases in viability by day 1, levels at 62.5 μM and above resulted in slower or inhibited cell growth at day 3 and beyond (Fig. 1e). D-AA initially decreased viability on the first day, but cells recovered and grew, though at decreased rates, at day 3 and beyond up to a concentration of 250 μM (Fig. 1b). Farnesol and dFBr had significant impacts on cell viability at levels over 48 μM (Fig. 1d) and 35 μM respectively (Fig. 1f).

Fig. (1)

DNA quantity for a) all the control groups and b-f) biofilm inhibitors to osteogenic controls on day 1 and 3 at varying concentrations. Data are represented as mean ± standard deviation; *indicates significant difference compared to osteogenic control.



Visual Observations of Cell Cultures

For focused analysis of mineralization response, the highest concentrations that supported viability were selected for reporting differentiation and mineralization markers (Table 2). In general, biocompatible concentrations of farnesol, D-AA, LS, and C2DA remained confluent and elongated similar to control groups, but dFBr was an exception. dFBr test samples changed morphology from the elongated fibroblast-like spindle shape form to a cuboidal form, similar to that of epithelial cells instead of osteoblast-like cells (Fig. 2h, i) [15Weinstein IB, Orenstein JM, Gebert R, Kaighn ME, Stadler UC. Growth and structural properties of epithelial cell cultures established from normal rat liver and chemically induced hepatomas. Cancer Res 1975; 35(1): 253-63.]. Significant staining for mineral deposits in osteogenic control groups became apparent by day 14 (Fig. 2b, c).

Table 2

Comparison of concentration evaluated, reported effective ranges and highest identified sub-toxic concentration.




Fig. (2)

Microscopic images (4X magnification) taken at day 21 of the control groups (a-c) and the highest sub-toxic concentrations of each test group (d-h) stained with Alizarin Red-S to show calcium deposits in dark red-brown. A 20X magnification of cells exposed to dFBr (i) shows the rounded morphology of these cells.



Cell Differentiation

ALP, an early indicator of osteogenic differentiation, peaked around day 3 for the osteogenic and non-osteogenic control groups (Fig. 3a). The osteogenic control group with ethanol had a decreased day 3 ALP production, but had higher ALP/DNA ratios than the other groups after day 3 and continued to positively increase throughout the 21day study. The D-AA test samples decreased in ALP/DNA production over time in comparison to the osteogenic control groups with ethanol (Fig. 3b). No significant differences from the osteogenic control group were detected in the evaluated concentrations of lysostaphin (Fig. 3c). Farnesol stimulated early significant increases in ALP/DNA on day 3 compared to both osteogenic control groups with and without ethanol, then continued to decrease in ALP production throughout the remainder of the 21 day study (Fig. 3d). In a similar fashion to farnesol, the C2DA test group ALP/DNA ratio peaked at day 3 at a significantly higher value than the osteogenic control groups, but steadily declined over the remainder of the 21 day time period (Fig. 3e). The farnesol and C2DA test groups displayed high variability, which may correspond to higher variability in the DNA levels. dFBr significantly lowered ALP production in the early stages of exposure to the cells, but peaked at levels similar to the osteogenic control by day 14 (Fig. 3f). With day 3 representing the peak in control ALP production, only farnesol, C2DA, non-osteogenic media, and osteogenic media containing ethanol sig-nificantly differed from the osteogenic positive control (Fig. 4).

Fig. (3)

Graphical representations ofALP/DNA ratio over time (mean ± standard deviation) for a) control and b-f) test groups. ◊indicates significant difference compared to non-osteogenic control, * indicates significant difference compared to osteogenic control, # indicates significant difference compared to osteogenic ethanol control.



Fig. (4)

Graphical representation comparing day 3 ALP/DNA production for control groups and sub-toxic concentrations of test groups. Data are represented as mean ± standard deviation; * indicates significant difference compared to osteogenic control, # indicates significant difference compared to osteogenic ethanol control.



DISCUSSION

The purpose of this study was to identify the biocompatible concentration range of five different biofilm-inhibitory molecules and to identify potential effects on osteoblast function prior to development of clinical applications for local delivery to inhibit orthopaedic infection. All the tested biocompatible concentration ranges were within reported biofilm inhibitory ranges, except farnesol and dFBr. While these anti-biofilm molecules have sub-toxic levels, some may influence osteogenic differentiation by altering ALP production.

The results of this preliminary in vitro study may be interpreted to predict possible in vivo outcomes of these biofilm-inhibitory agents delivered at locally active concentrations. One limitation of this study was the test sample size, n=4, with gapped time points. Samples were taken at time points to reflect early and later stages of differentiation, days 1, 3, 7, 14, and 21 [16Sowa H, Kaji H, Yamaguchi T, Sugimoto T, Chihara K. Smad3 promotes alkaline phosphatase activity and mineralization of osteoblastic MC3T3-E1 cells. J Bone Miner Res 2002; 17(7): 1190-9.]. Increasing the frequency of selected time points may provide more definitive characterization of cell growth and differentiation patterns after exposure to these anti-biofilm agents. The strategy used in this study was repetitive dosing, which applies more stress on the cell samples, as opposed to an approach that involves one initial dose at various concentrations. This strategy was chosen based on an ideal local delivery system, releasing continuous levels over a clinically-relevant time frame. Many local delivery systems display burst response followed by minimal release, which may result in different levels of recovery from the initial higher dose of biofilm-inhibitory chemicals [17Unnanuntana A, Bonsignore L, Shirtliff ME, Greenfield EM. The effects of farnesol on Staphylococcus aureus biofilms and osteoblasts.An in vitro study. J Bone Joint Surg Am 2009; 91(11): 2683-92.]. Other forms of quantitatively measuring differentiation can be used in future studies to further characterize differentiation state by measuring gene expression for osteoblast-specific proteins such as osteocalcin, matrix gla protein, osteopontin, collagen, and bone sialoprotein [18McCabe LR, Last TJ, Lynch M, Lian J, Stein J, Stein G. Expression of cell growth and bone phenotypic genes during the cell cycle of normal diploid osteoblasts and osteosarcoma cells. J Cell Biochem 1994; 56(2): 274-82., 19Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr Rev 1993; 14(4): 424-42.].

The D-AA test group was toxic at high concentrations, but supported cells at intermediate ranges over the course of 21 days with repetitive dosing. This toxic concentration level is much lower than previously reported toxic oral doses of D-AA [20Friedman M, Levin CE. Nutritional and medicinal aspects of D-amino acids. Amino Acids 2012; 42(5): 1553-82.]. D-Proline ingested orally has been shown to increase gastric inflammation, peptic ulcer formation, and kidney fibrosis and necrosis [20Friedman M, Levin CE. Nutritional and medicinal aspects of D-amino acids. Amino Acids 2012; 42(5): 1553-82.]. An equimolar mixture of D-AA was chosen for evaluation in this study based on results by Hochbaum et al. and Sanchez et al. showing optimal efficacy with a combination of various D-AA [10Hochbaum AI, Kolodkin-Gal I, Foulston L, Kolter R, Aizenberg J, Losick R. Inhibitory effects of D-amino acids on Staphylococcus aureus biofilm development. J Bacteriol 2011; 193(20): 5616-22., 21Sanchez CJ Jr, Prieto EM, Krueger CA , et al. Effects of local delivery of D-amino acids from biofilm-dispersive scaffolds on infection in contaminated rat segmental defects. Biomaterials 2013; 34(30): 7533-43.]. Sanchez et al.’s study also evaluated human dermal fibroblasts and osteoblasts exposed to D-AA for 24 hours, showing that cells exposed to 50mM (approximately 100 times the highest concentration evaluated in this study) of D-Methionine, D-Proline, and D-Tryptophan were more than 70% viable. It is important to note for comparison purposes that Sanchez et al.’s study was short term (24 hours), whereas this study was much longer (21 days). Time course, different cell lines, different D-AAs, and solubility enhancement of D-AA in media could contribute to the variation in viability results. The study by Sanchez et al. evaluated anti-biofilm efficacy of released D-AAs in vivo over 14 days, but did not report the quality of bone healing in the segmental defect. The interaction between D-AA and the host is a vital part of the healing process and should be evaluated further in vivo. Finally, the cytotoxicity of individual D-AA could be tested to determine whether a single component is more toxic than others.

There have been other studies involving fatty acid applications for antimicrobial effects against Gram-negative, Gram-positive, fungi, protozoan, and viruses [9Davies DG, Marques CN. A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. J Bacteriol 2009; 191(5): 1393-403., 13Jennings JA, Courtney HS, Haggard WO. Cis-2-decenoic acid inhibits, S. aureus growth and biofilm in vitro a pilot study. Clin Orthop Relat Res 2012; 470(10): 2663-70., 22Desbois AP, Smith VJ. Antibacterial free fatty acids activities, mechanisms of action and biotechnological potential. Appl Microbiol Biotechnol 2010; 85(6): 1629-42.]. Fatty acids have high potential as anti-biofilm therapeutics because many are nonspecific to particular types of microorganisms, although they have limitations of instability and a tendency to bind non-specifically to proteins [22Desbois AP, Smith VJ. Antibacterial free fatty acids activities, mechanisms of action and biotechnological potential. Appl Microbiol Biotechnol 2010; 85(6): 1629-42.]. Similar to a study by Jennings et al. of C2DA, up to 500 μg/mL (3 mM) supported fibroblast viability, but may have affected growth over the course of 48 hours [13Jennings JA, Courtney HS, Haggard WO. Cis-2-decenoic acid inhibits, S. aureus growth and biofilm in vitro a pilot study. Clin Orthop Relat Res 2012; 470(10): 2663-70.]. The present study resulted in cell death at concentrations of C2DA over 62.5 μM by day 3, which continued through the 21 day study. The difference in viability values may be due to the length of the test, 48 hours versus 21 days, or difference of cell lines, fibroblasts versus osteoblasts [13Jennings JA, Courtney HS, Haggard WO. Cis-2-decenoic acid inhibits, S. aureus growth and biofilm in vitro a pilot study. Clin Orthop Relat Res 2012; 470(10): 2663-70.]. However, the lower biocompatibility level this longer term study identified still has the potential to be effective against biofilm. Davies et al. have shown that bacterial colonies can be very sensitive to C2DA, some species in the nanomolar region [9Davies DG, Marques CN. A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. J Bacteriol 2009; 191(5): 1393-403.]. There have been several published studies of different cell reactions to the application of various fatty acids to the cultured cells, skin, and teeth in order to inhibit bacterial growth, as well as oral ingestion of fatty acids to control gut microbiota, showing dose-dependent toxicity of different fatty acids [23Masi LN, Portioli-Sanches EP, Lima-Salgado TM, Curi R. Toxicity of fatty acids on ECV-304 endothelial cells. Toxicol In Vitro 2011; 25(8): 2140-6., 24Kurihara H, Goto Y, Aida H, Hosokawa M, Takahashi K. Antibacterial activity against cariogenic bacteria and inhibition of insoluble glucan production by free fatty acids obtained from dried Gloiopeltis furcata. Fisheries Sci 1999; 65(1): 129-32.]. Cell type as well as fatty acid properties may play a role in viability, apoptosis, and necrosis, and effects of fatty acids on other cell types such as endothelial cells, lymphocytes, macrophages, and neutrophils should be studied further [23Masi LN, Portioli-Sanches EP, Lima-Salgado TM, Curi R. Toxicity of fatty acids on ECV-304 endothelial cells. Toxicol In Vitro 2011; 25(8): 2140-6.].

Comparing to results from Unnanuntana et al., concentrations of farnesol that are compatible with osteoblasts are more than six times lower in this study (48 μM compared to 300 μM) [17Unnanuntana A, Bonsignore L, Shirtliff ME, Greenfield EM. The effects of farnesol on Staphylococcus aureus biofilms and osteoblasts.An in vitro study. J Bone Joint Surg Am 2009; 91(11): 2683-92.]. The discrepancy between toxic concentrations found in this study and the study by Unnanuntana et al. could be related to the dosage strategy and the solubility procedure. The reversibility of farnesol effects was studied by exposing cultures to farnesol for two hours, suspended directly in media without additives to improve solubility, after which the farnesol was removed [17Unnanuntana A, Bonsignore L, Shirtliff ME, Greenfield EM. The effects of farnesol on Staphylococcus aureus biofilms and osteoblasts.An in vitro study. J Bone Joint Surg Am 2009; 91(11): 2683-92.]. Previous studies have indicated that the presence of fatty acids in serum can induce osteoblasts to undergo adipogenesis [25Diascro DD Jr, Vogel RL, Johnson TE , et al. High fatty acid content in rabbit serum is responsible for the differentiation of osteoblasts into adipocyte-like cells. J Bone Miner Res 1998; 13(1): 96-106.]. Masi et al. showed that endothelial cells incubated in fatty acids for 24 hours showed increased fatty deposits above 150 μM, over twice the biocompatible concentrations of farnesol or C2DA [23Masi LN, Portioli-Sanches EP, Lima-Salgado TM, Curi R. Toxicity of fatty acids on ECV-304 endothelial cells. Toxicol In Vitro 2011; 25(8): 2140-6.]. The farnesol, C2DA, and control groups maintained elongated spindle morphology (bone-like). In preliminary studies using oil-red O staining, there was no evidence of fat deposits in cells exposed to C2DA or farnesol compared to controls.

While no previous studies of dFBr in osteoblast culture have been published, it has been studied at concentrations much lower than those used in this study for its ability to interact with specific cell receptors as positive allosteric modulators for potential therapeutic application for various neurological and neuropsychiatric disorders [26German N, Kim JS, Jain A , et al. Deconstruction of the alpha4beta2 nicotinic acetylcholine receptor positive allosteric modulator desformylflustrabromine. J Med Chem 2011; 54(20): 7259-67.]. The chemical structure of dFBr can be modified to develop more selective agents for acetylcholine receptors and potentially biofilm inhibitors. Of the evaluated anti-biofilm agents, dFBr had the most significant effect on osteoblast differentiation activity and morphology. The morphologic change, lack of mineralization, and altered differentiation may have been a result of interaction with specific cell receptors, leading cells to differentiate into an undetermined lineage.

In a growth inhibitory context, LS is effective at concentrations as low as 0.004 µg/mL in some bacterial strains and disrupts S. aureus biofilm at concentrations as low as 0.8 µg/mL. Efficacy in eradicating biofilm in other staphylococcal strains, such as S. epidermidis, has been demonstrated at much higher concentrations of 200 µg/mL. This study only evaluated concentrations within the range effective against S. aureus. However, since the highest concentration of LS was not found to be toxic to osteoblasts in this study, the biocompatibility range could be further explored. This biofilm-disrupting agent may not be toxic within the reported anti-biofilm range because it is an endopeptidase protein that disrupts the cell wall of Staphylococcus aureus with specificity toward Staphylococcal microorganisms [11Wu JA, Kusuma C, Mond JJ, Kokai-Kun JF. Lysostaphin disrupts Staphylococcus aureus and Staphylococcus epidermidis biofilms on artificial surfaces. Antimicrob Agents Chemother 2003; 47(11): 3407-14., 27Walsh S, Shah A, Mond J. Improved pharmacokinetics and reduced antibody reactivity of lysostaphin conjugated to polyethylene glycol. Antimicrob Agents Chemother 2003; 47(2): 554-8.]. Therefore, LS may be non-toxic over a broad concentration range due to the presumably minimal disruption of mammalian cell membranes. While there have been no published studies evaluating LS effects on osteoblasts, there could be issues of immune reactivity when used in vivo in addition to short protein half-life and specificity toward Staphylococcal strains [27Walsh S, Shah A, Mond J. Improved pharmacokinetics and reduced antibody reactivity of lysostaphin conjugated to polyethylene glycol. Antimicrob Agents Chemother 2003; 47(2): 554-8.]. These limitations could be overcome by conjugation of these protein agents to polyethylene glycol (PEG), although the effect on biofilm inhibition of PEGylated LS is reported to be slightly decreased [27Walsh S, Shah A, Mond J. Improved pharmacokinetics and reduced antibody reactivity of lysostaphin conjugated to polyethylene glycol. Antimicrob Agents Chemother 2003; 47(2): 554-8.].

The increased production of ALP in control groups containing ethanol compared to osteogenic media without ethanol, in addition to all of the other test groups, was an unanticipated outcome. Models of chronic ethanol exposure in rats have shown inhibited bone development in vivo and increased proliferation and formation of bone nodules in stem cells isolated from ethanol-exposed rats, though no significant differences in ALP production were observed [28Rosa ML, Beloti MM, Prando N, Queiroz RH, de Oliveira PT, Rosa AL. Chronic ethanol intake inhibits in vitro osteogenesis induced by osteoblasts differentiated from stem cells. J Appl Toxicol 2008; 28(2): 205-11.]. Yeh et al. also reported that ethanol reduces osteogenesis in stromal cells isolated from humans with ethanol-induced osteonecrosis, showing that ethanol interferes with the signaling pathways that induced osteoblast formation [29Yeh CH, Chang JK, Wang YH, Ho ML, Wang GJ. Ethanol may suppress Wnt/beta-catenin signaling on human bone marrow stroma cells a preliminary study. Clin Orthop Relat Res 2008; May; 466(5): 1047-53.]. In the former studies, chronic alcohol consumption was studied and modeled by isolating cells from animals with ethanol exposure in addition to culture conditions with ethanol at a higher percentage than that used in this study for longer periods of time. An in vitro study by Farley et al. suggests that changes in cell function after ethanol exposure is due to ethanol increasing membrane fluidity [30Farley Jr, Fitzsimmons R, Taylor AK, Jorch UM, Lau KH. Direct effects of ethanol on bone resorption and formation in vitro. Arch Biochem Biophys 1985; 238(1): 305-14.]. Increased membrane permeability due to ethanol at low doses may have allowed for increased uptake of osteogenic factors. The increased ALP production in the C2DA and farnesol groups was also unexpected. Both of these biofilm inhibitors were delivered in ethanol but were much higher in ALP production than ethanol controls at early time points. This finding may have been an artifact of low cell number or may indicate that proliferative mechanisms have been affected, as several studies have demonstrated an inverse relationship between the sequential processes of proliferation and differentiation [31Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture an in vitro model of osteoblast development. J Bone Miner Res 1992; 7(6): 683-92., 32Ishaug SL, Yaszemski MJ, Bizios R, Mikos AG. Osteoblast function on synthetic biodegradable polymers. J Biomed Mater Res 1994; 28(12): 1445-53.]. As proliferative mechanisms decreased, ALP production may have begun at an earlier time point in these groups. However, the mineralization staining patterns do not suggest that this led to increased deposition of mineral or differentiation into bone.

In conclusion, in vitro studies of potential biofilm inhibiting/dispersing agents are a vital step in evaluating possible solutions to orthopaedic implant related infections. Several of the tested anti-biofilm agents were biocompatible within its reported effective ranges; however, others were proven cytotoxic within the reported anti-biofilm concentrations. dFBr and farnesol were not biocompatible within the effective anti-biofilm ranges and would require more testing before therapeutic use. High concentrations of naturally produced biofilm inhibitors, such as those that may be present after local delivery, may have effects on cellular viability and osteoblast function. In vivo studies based on dose response limits identified in these studies are needed to fully characterize the effects of these biofilm inhibitors prior to clinical use.

CONFLICT OF INTEREST

The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

ACKNOWLEDGEMENTS

Funding for portions of this research was provided by Department of Defense, Peer Reviewed Orthopaedic Research Program Award # OR090687, and departmental funds at the University of Memphis. The authors also wish to acknowledge Harry Courtney, PhD, from University of Tennessee Health Science Center and Mark Smeltzer, PhD, from University Arkansas for Medical Sciences for advice and assistance in experimental research.

REFERENCES

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[4] Cataldo MA, Petrosillo N, Cipriani M, Cauda R, Tacconelli E. Prosthetic joint infection recent developments in diagnosis and management. J Infect 2010; 61(6): 443-8.
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