Modulation of the Substitution Pattern of 5-Aryl-2-Aminoimidazoles Allows Fine-Tuning of Their Antibiofilm Activity Spectrum and Toxicity.

We previously synthesized several series of compounds, based on the 5-aryl-2-aminoimidazole scaffold, that showed activity preventing the formation of Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa biofilms. Here, we further studied the activity spectrum of a number of the most active N1- and 2N-substituted 5-aryl-2-aminoimidazoles against a broad panel of biofilms formed by monospecies and mixed species of bacteria and fungi. An N1-substituted compound showed very strong activity against the biofilms formed by Gram-negative and Gram-positive bacteria and the fungus Candida albicans but was previously shown to be toxic against various eukaryotic cell lines. In contrast, 2N-substituted compounds were nontoxic and active against biofilms formed by Gram-negative bacteria and C. albicans but had reduced activity against biofilms formed by Gram-positive bacteria. In an attempt to develop nontoxic compounds with potent activity against biofilms formed by Gram-positive bacteria for application in antibiofilm coatings for medical implants, we synthesized novel compounds with substituents at both the N1 and 2N positions and tested these compounds for antibiofilm activity and toxicity. Interestingly, most of these N1-,2N-disubstituted 5-aryl-2-aminoimidazoles showed very strong activity against biofilms formed by Gram-positive bacteria and C. albicans in various setups with biofilms formed by monospecies and mixed species but lost activity against biofilms formed by Gram-negative bacteria. In light of application of these compounds as anti-infective coatings on orthopedic implants, toxicity against two bone cell lines and the functionality of these cells were tested. The N1-,2N-disubstituted 5-aryl-2-aminoimidazoles in general did not affect the viability of bone cells and even induced calcium deposition. This indicates that modulating the substitution pattern on positions N1 and 2N of the 5-aryl-2-aminoimidazole scaffold allows fine-tuning of both the antibiofilm activity spectrum and toxicity.

Covalent immobilization of antimicrobial agents on titanium prevents Staphylococcus aureus and Candida albicans colonization and biofilm formation.

OBJECTIVES: Biofilm-associated implant infections represent a serious public health problem. Covalent immobilization of antimicrobial agents on titanium (Ti), thereby inhibiting biofilm formation of microbial pathogens, is a solution to this problem. METHODS: Vancomycin (VAN) and caspofungin (CAS) were covalently bound on Ti substrates using an improved processing technique adapted to large-scale coating of implants. Resistance of the VAN-coated Ti (VAN-Ti) and CAS-coated Ti (CAS-Ti) substrates against in vitro biofilm formation of the bacterium Staphylococcus aureus and the fungal pathogen Candida albicans was determined by plate counting and visualized by confocal laser scanning microscopy. The efficacy of the coated Ti substrates was also tested in vivo using an adapted biomaterial-associated murine infection model in which control-Ti, VAN-Ti or CAS-Ti substrates were implanted subcutaneously and subsequently challenged with the respective pathogens. The osseointegration potential of VAN-Ti and CAS-Ti was examined in vitro using human bone marrow-derived stromal cells, and for VAN-Ti also in a rat osseointegration model. RESULTS: In vitro biofilm formation of S. aureus and C. albicans on VAN-Ti and CAS-Ti substrates, respectively, was significantly reduced compared with biofilm formation on control-Ti. In vivo, we observed over 99.9% reduction in biofilm formation of S. aureus on VAN-Ti substrates and 89% reduction in biofilm formation of C. albicans on CAS-Ti substrates, compared with control-Ti substrates. The coated substrates supported osseointegration in vitro and in vivo. CONCLUSIONS: These data demonstrate the clinical potential of covalently bound VAN and CAS on Ti to reduce microbial biofilm formation without jeopardizing osseointegration.

Derivatives of the mouse cathelicidin-related antimicrobial peptide (CRAMP) inhibit fungal and bacterial biofilm formation.

We identified a 26-amino-acid truncated form of the 34-amino-acid cathelicidin-related antimicrobial peptide (CRAMP) in the islets of Langerhans of the murine pancreas. This peptide, P318, shares 67% identity with the LL-37 human antimicrobial peptide. As LL-37 displays antimicrobial and antibiofilm activity, we tested antifungal and antibiofilm activity of P318 against the fungal pathogen Candida albicans. P318 shows biofilm-specific activity as it inhibits C. albicans biofilm formation at 0.15 µM without affecting planktonic survival at that concentration. Next, we tested the C. albicans biofilm-inhibitory activity of a series of truncated and alanine-substituted derivatives of P318. Based on the biofilm-inhibitory activity of these derivatives and the length of the peptides, we decided to synthesize the shortened alanine-substituted peptide at position 10 (AS10; KLKKIAQKIKNFFQKLVP). AS10 inhibited C. albicans biofilm formation at 0.22 µM and acted synergistically with amphotericin B and caspofungin against mature biofilms. AS10 also inhibited biofilm formation of different bacteria as well as of fungi and bacteria in a mixed biofilm. In addition, AS10 does not affect the viability or functionality of different cell types involved in osseointegration of an implant, pointing to the potential of AS10 for further development as a lead peptide to coat implants.

Plant-derived decapeptide OSIP108 interferes with Candida albicans biofilm formation without affecting cell viability.

We previously identified a decapeptide from the model plant Arabidopsis thaliana, OSIP108, which is induced upon fungal pathogen infection. In this study, we demonstrated that OSIP108 interferes with biofilm formation of the fungal pathogen Candida albicans without affecting the viability or growth of C. albicans cells. OSIP108 displayed no cytotoxicity against various human cell lines. Furthermore, OSIP108 enhanced the activity of the antifungal agents amphotericin B and caspofungin in vitro and in vivo in a Caenorhabditis elegans-C. albicans biofilm infection model. These data point to the potential use of OSIP108 in combination therapy with conventional antifungal agents. In a first attempt to unravel its mode of action, we screened a library of 137 homozygous C. albicans mutants, affected in genes encoding cell wall proteins or transcription factors important for biofilm formation, for altered OSIP108 sensitivity. We identified 9 OSIP108-tolerant C. albicans mutants that were defective in either components important for cell wall integrity or the yeast-to-hypha transition. In line with these findings, we demonstrated that OSIP108 activates the C. albicans cell wall integrity pathway and that its antibiofilm activity can be blocked by compounds inhibiting the yeast-to-hypha transition. Furthermore, we found that OSIP108 is predominantly localized at the C. albicans cell surface. These data point to interference of OSIP108 with cell wall-related processes of C. albicans, resulting in impaired biofilm formation.

Identification and characterization of an anti-pseudomonal dichlorocarbazol derivative displaying anti-biofilm activity.

Pseudomonas aeruginosa strains resistant towards all currently available antibiotics are increasingly encountered, raising the need for new anti-pseudomonal drugs. We therefore conducted a medium-throughput screen of a small-molecule collection resulting in the identification of the N-alkylated 3,6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (MIC = 18.5 µg mL⁻¹). This compound, compound 1, is bacteriostatic towards a broad spectrum of Gram-positive and Gram-negative pathogens, including P. aeruginosa. Importantly, 1 also eradicates mature biofilms of P. aeruginosa. 1 displays no cytotoxicity against various human cell types, pointing to its potential for further development as a novel antibacterial drug.

Cell sources for nucleus pulposus regeneration.

PURPOSE: There is increasing interest in the development of cell therapy as a possible approach for the treatment of degenerative disc disease. To regenerate nucleus pulposus tissue, the cells must produce an appropriate proteoglycan-rich matrix, as this is essential for the functioning of the intervertebral disc. The natural environment within the disc is very challenging to implanted cells, particularly if they have been subcultured in normal laboratory conditions. The purpose of this work is to discuss parameters relevant to translating different proposed cell therapies of IVD into clinical use. RESULTS: Several sources of cells have been proposed, including nucleus pulposus cells, chondrocytes and mesenchymal stem cells derived from bone marrow or adipose tissue. There are some clinical trials and reports of attempts to regenerate nucleus pulposus utilising either autologous or allogenic cells. While the published results of clinical applications of these cell therapies do not indicate any safety issues, additional evidence will be needed to prove their long-term efficacy. CONCLUSION: This article discusses parameters relevant for successful translation of research on different cell sources into clinically applicable cell therapies: the influence of the intervertebral disc microenvironment on the cell phenotype, issues associated with cell culture and technical preparation of cell products, as well as discussing current regulatory requirements. There are advantages and disadvantages of each proposed cell type, but no strong evidence to favour any one particular cell source at the moment.

Evaluation of the toxicity of 5-aryl-2-aminoimidazole-based biofilm inhibitors against eukaryotic cell lines, bone cells and the nematode Caenorhabditis elegans.

Previously, we have synthesized several series of compounds based on the 5-aryl-2-aminoimidazole scaffold, which showed a preventive activity against microbial biofilms. We here studied the cytotoxicity of the most active compounds of each series. First, the cytostatic activity was investigated against a number of tumor cell lines (L1210, CEM and HeLa). A subset of monosubstituted 5-aryl-2-aminoimidazoles showed a moderate safety window, with therapeutic indices (TIs) ranging between 3 and 20. Whereas introduction of a (cyclo-)alkyl chain at the N1-position strongly reduced the TI, introduction of a (cyclo-)alkyl chain or a triazole moiety at the 2N-position increased the TI up to 370. Since a promising application of preventive anti-biofilm agents is their use in anti-biofilm coatings for orthopedic implants, their effects on cell viability and functional behavior of human osteoblasts and bone marrow derived mesenchymal stem cells were tested. The 2N-substituted 5-aryl-2-aminoimidazoles consistently showed the lowest toxicity and allowed survival of the bone cells for up to 4 weeks. Moreover they did not negatively affect the osteogenic differentiation potential of the bone cells. Finally, we examined the effect of the compounds on the survival of Caenorhabditis elegans, which confirmed the higher safety window of 2N-substituted 5-aryl-2-aminoimidazoles.

Engineering anatomically shaped human bone grafts.

The ability to engineer anatomically correct pieces of viable and functional human bone would have tremendous potential for bone reconstructions after congenital defects, cancer resections, and trauma. We report that clinically sized, anatomically shaped, viable human bone grafts can be engineered by using human mesenchymal stem cells (hMSCs) and a "biomimetic" scaffold-bioreactor system. We selected the temporomandibular joint (TMJ) condylar bone as our tissue model, because of its clinical importance and the challenges associated with its complex shape. Anatomically shaped scaffolds were generated from fully decellularized trabecular bone by using digitized clinical images, seeded with hMSCs, and cultured with interstitial flow of culture medium. A bioreactor with a chamber in the exact shape of a human TMJ was designed for controllable perfusion throughout the engineered construct. By 5 weeks of cultivation, tissue growth was evidenced by the formation of confluent layers of lamellar bone (by scanning electron microscopy), markedly increased volume of mineralized matrix (by quantitative microcomputer tomography), and the formation of osteoids (histologically). Within bone grafts of this size and complexity cells were fully viable at a physiologic density, likely an important factor of graft function. Moreover, the density and architecture of bone matrix correlated with the intensity and pattern of the interstitial flow, as determined in experimental and modeling studies. This approach has potential to overcome a critical hurdle-in vitro cultivation of viable bone grafts of complex geometries-to provide patient-specific bone grafts for craniofacial and orthopedic reconstructions.

Optimizing the medium perfusion rate in bone tissue engineering bioreactors.

There is a critical need to increase the size of bone grafts that can be cultured in vitro for use in regenerative medicine. Perfusion bioreactors have been used to improve the nutrient and gas transfer capabilities and reduce the size limitations inherent to static culture, as well as to modulate cellular responses by hydrodynamic shear. Our aim was to understand the effects of medium flow velocity on cellular phenotype and the formation of bone-like tissues in three-dimensional engineered constructs. We utilized custom-designed perfusion bioreactors to culture bone constructs for 5 weeks using a wide range of superficial flow velocities (80, 400, 800, 1,200, and 1,800 µm/s), corresponding to estimated initial shear stresses ranging from 0.6 to 20 mPa. Increasing the flow velocity significantly affected cell morphology, cell-cell interactions, matrix production and composition, and the expression of osteogenic genes. Within the range studied, the flow velocities ranging from 400 to 800 µm/s yielded the best overall osteogenic responses. Using mathematical models, we determined that even at the lowest flow velocity (80 µm/s) the oxygen provided was sufficient to maintain viability of the cells within the construct. Yet it was clear that this flow velocity did not adequately support the development of bone-like tissue. The complexity of the cellular responses found at different flow velocities underscores the need to use a range of evaluation parameters to determine the quality of engineered bone.

An antibiofilm coating of 5-aryl-2-aminoimidazole covalently attached to a titanium surface.

Biofilms, especially those formed by Staphylococcus aureus, play a key role in the development of orthopedic implant infections. Eradication of these infections is challenging due to the elevated tolerance of biofilm cells against antimicrobial agents. In this study, we developed an antibiofilm coating consisting of 5-(4-bromophenyl)-N-cyclopentyl-1-octyl-1H-imidazol-2-amine, designated as LC0024, covalently bound to a titanium implant surface (LC0024-Ti). We showed in vitro that the LC0024-Ti surface reduces biofilm formation of S. aureus in a specific manner without reducing the planktonic cells above the biofilm, as evaluated by plate counting and fluorescence microscopy. The advantage of compounds that only inhibit biofilm formation without affecting the viability of the planktonic cells, is that reduced development of bacterial resistance is expected. To determine the antibiofilm activity of LC0024-Ti surfaces in vivo, a biomaterial-associated murine infection model was used. The results indicated a significant reduction in S. aureus biofilm formation (up to 96%) on the LC0024-Ti substrates compared to pristine titanium controls. Additionally, we found that the LC0024-Ti substrates did not affect the attachment and proliferation of human cells involved in osseointegration and bone repair. In summary, our results emphasize the clinical potential of covalent coatings of LC0024 on titanium implant surfaces to reduce the risk of orthopedic implant infections. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1908-1919, 2019.

Adipose-Derived Stem Cells Respond to Increased Osmolarities.

Cell therapies present a feasible option for the treatment of degenerated cartilaginous and intervertebral disc (IVD) tissues. Microenvironments of these tissues are specific and often differ from the microenvironment of cells that, could be potentially used for therapy, e.g. human adipose-derived stem cells (hASC). To ensure safe and efficient implantation of hASC, it is important to evaluate how microenvironmental conditions at the site of implantation affect the implanted cells. This study has demonstrated that cartilaginous tissue-specific osmolarities ranging from 400-600 mOsm/L affected hASC in a dose- and time-dependent fashion in comparison to 300 mOsm/L. Increased osmolarities resulted in transient (nuclear DNA and actin reorganisation) and non-transient, long-term morphological changes (vesicle formation, increase in cell area, and culture morphology), as well as reduced proliferation in monolayer cultures. Increased osmolarities diminished acid proteoglycan production and compactness of chondrogenically induced pellet cultures, indicating decreased chondrogenic potential. Viability of hASC was strongly dependent on the type of culture, with hASC in monolayer culture being more tolerant to increased osmolarity compared to hASC in suspension, alginate-agarose hydrogel, and pellet cultures, thus emphasizing the importance of choosing relevant in vitro conditions according to the specifics of clinical application.

Antibacterial activity of a new broad-spectrum antibiotic covalently bound to titanium surfaces.

Biofilm-associated infections, particularly those caused by Staphylococcus aureus, are a major cause of implant failure. Covalent coupling of broad-spectrum antimicrobials to implants is a promising approach to reduce the risk of infections. In this study, we developed titanium substrates on which the recently discovered antibacterial agent SPI031, a N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol, was covalently linked (SPI031-Ti). We found that SPI031-Ti substrates prevent biofilm formation of S. aureus and Pseudomonas aeruginosa in vitro, as quantified by plate counting and fluorescence microscopy. To test the effectiveness of SPI031-Ti substrates in vivo, we used an adapted in vivo biomaterial-associated infection model in mice in which SPI031-Ti substrates were implanted subcutaneously and subsequently inoculated with S. aureus. Using this model, we found a significant reduction in biofilm formation (up to 98%) on SPI031-Ti substrates compared to control substrates. Finally, we demonstrated that the functionalization of the titanium surfaces with SPI031 did not influence the adhesion and proliferation of human cells important for osseointegration and bone repair. In conclusion, these data demonstrate the clinical potential of SPI031 to be used as an antibacterial coating for implants, thereby reducing the incidence of implant-associated infections. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2191-2198, 2016.

Covalent Binding of Heparin to Functionalized PET Materials for Improved Haemocompatibility.

The hemocompatibility of vascular grafts made from poly(ethylene terephthalate) (PET) is insufficient due to the rapid adhesion and activation of blood platelets that occur upon incubation with whole blood. PET polymer was treated with NHx radicals created by passing ammonia through gaseous plasma formed by a microwave discharge, which allowed for functionalization with amino groups. X-ray photoelectron spectroscopy characterization using derivatization with 4-chlorobenzaldehyde indicated that approximately 4% of the -NH2 groups were associated with the PET surface after treatment with the gaseous radicals. The functionalized polymers were coated with an ultra-thin layer of heparin and incubated with fresh blood. The free-hemoglobin technique, which is based on the haemolysis of erythrocytes, indicated improved hemocompatibility, which was confirmed by imaging the samples using confocal optical microscopy. A significant decrease in number of adhered platelets was observed on such samples. Proliferation of both human umbilical vein endothelial cells and human microvascular endothelial cells was enhanced on treated polymers, especially after a few hours of cell seeding. Thus, the technique represents a promising substitute for wet-chemical modification of PET materials prior to coating with heparin.

Bone grafts engineered from human adipose-derived stem cells in perfusion bioreactor culture.

We report engineering of half-centimeter-sized bone constructs created in vitro using human adipose-derived stem cells (hASCs), decellularized bone scaffolds, and perfusion bioreactors. The hASCs are easily accessible, can be used in an autologous fashion, are rapidly expanded in culture, and are capable of osteogenic differentiation. hASCs from four donors were characterized for their osteogenic capacity, and one representative cell population was used for tissue engineering experiments. Culture-expanded hASCs were seeded on fully decellularized native bone scaffolds (4 mm diameter x 4 mm thick), providing the necessary structural and mechanical environment for osteogenic differentiation, and cultured in bioreactors with medium perfusion. The interstitial flow velocity was set to a level necessary to maintain cell viability and function throughout the construct volume (400 microm/s), via enhanced mass transport. After 5 weeks of cultivation, the addition of osteogenic supplements (dexamethasone, sodium-beta-glycerophosphate, and ascorbic acid-2-phosphate) to culture medium significantly increased the construct cellularity and the amounts of bone matrix components (collagen, bone sialoprotein, and bone osteopontin). Medium perfusion markedly improved the distribution of cells and bone matrix in engineered constructs. In summary, a combination of hASCs, decellularized bone scaffold, perfusion culture, and osteogenic supplements resulted in the formation of compact and viable bone tissue constructs.

Hypoxia and stem cell-based engineering of mesenchymal tissues.

Stem cells have the ability for prolonged self-renewal and differentiation into mature cells of various lineages, which makes them important cell sources for tissue engineering applications. Their remarkable ability to replenish and differentiate in vivo is regulated by both intrinsic and extrinsic cellular mechanisms. The anatomical location where the stem cells reside, known as the "stem cell niche or microenvironment," provides signals conducive to the maintenance of definitive stem cell properties. Physiological condition including oxygen tension is an important component of the stem cell microenvironment and has been shown to play a role in regulating both embryonic and adult stem cells. This review focuses on oxygen as a signaling molecule and the way it regulates the stem cells' development into mesenchymal tissues in vitro. The physiological relevance of low oxygen tension as an environmental parameter that uniquely benefits stem cells' expansion and maintenance is described along with recent findings on the regulatory effects of oxygen on embryonic stem cells and adult mesenchymal stem cells. The relevance to tissue engineering is discussed in the context of the need to specifically regulate the oxygen content in the cellular microenvironment in order to optimize in vitro tissue development.

Tissue engineered bone grafts: biological requirements, tissue culture and clinical relevance.

The tremendous need for bone tissue in numerous clinical situations and the limited availability of suitable bone grafts are driving the development of tissue engineering approaches to bone repair. In order to engineer viable bone grafts, one needs to understand the mechanisms of native bone development and fracture healing, as these processes should ideally guide the selection of optimal conditions for tissue culture and implantation. Engineered bone grafts have been shown to have capacity for osteogenesis, osteoconduction, osteoinduction and osteointegration - functional connection between the host bone and the graft. Cells from various anatomical sources in conjunction with scaffolds and osteogenic factors have been shown to form bone tissue in vitro. The use of bioreactor systems to culture cells on scaffolds before implantation further improved the quality of the resulting bone grafts. Animal studies confirmed the capability of engineered grafts to form bone and integrate with the host tissues. However, the vascularization of bone remains one of the hurdles that need to be overcome if clinically sized, fully viable bone grafts are to be engineered and implanted. We discuss here the biological guidelines for tissue engineering of bone, the bioreactor cultivation of human mesenchymal stem cells on three-dimensional scaffolds, and the need for vascularization and functional integration of bone grafts following implantation.