Easy, Flexible and Standardizable Anti-Nascent Biofilm Activity Assay to Assess Implant Materials.

Medical implants have improved the quality of life of many patients. However, surgical intervention may eventually lead to implant microbial contamination. The aims of this research were to develop an easy, robust, quantitative assay to assess surface antimicrobial activities, especially the anti-nascent biofilm activity, and to identify control surfaces, allowing for international comparisons. Using new antimicrobial assays to assess the inhibition of nascent biofilm during persistent contact or after transient contact with bacteria, we show that the 5 cent Euro coin or other metal-based antibacterial coins can be used as positive controls, as more than 4 log reduction on bacterial survival was observed when using either S. aureus or P. aeruginosa as targets. The methods and controls described here could be useful to develop an easy, flexible and standardizable assay to assess relevant antimicrobial activities of new implant materials developed by industries and academics.

A Novel Bioreactor System Capable of Simulating the In Vivo Conditions of Synovial Joints.

Any significant in vitro evaluation of cartilage tissue engineering and cartilage repair strategies has to be performed under the harsh conditions encountered in vivo within synovial joints. To this end, we have developed a novel automated physiological robot reactor system (PRRS) that is capable of recapitulating complex physiological motions and load patterns within an environment similar to that found in the human knee. The PRRS consists of a mechanical stimulation unit (MSU) and an automatic sample changer (ASC) within an environment control box in which the humidity, temperature, and gas composition are tightly regulated. The MSU has three linear (orthogonal) axes and one rotational degree of freedom (around the z-axis). The ASC provides space for up to 24 samples, which can be allocated to individual stimulation patterns. Cell-seeded scaffolds and ex vivo tissue culture systems were established to demonstrate the applicability of the PRRS to the investigation of the effect of load and environmental conditions on engineering and maintenance of articular cartilage in vitro. The bioreactor is a flexible system that has the potential to be applied for culturing connective tissues other than cartilage, such as bone and intervertebral disc tissue, even though the mechanical and environmental parameters are very different.

In vitro methods for the evaluation of antimicrobial surface designs.

Bacterial adhesion and subsequent biofilm formation on biomedical implants and devices are a major cause of their failure. As systemic antibiotic treatment is often ineffective, there is an urgent need for antimicrobial biomaterials and coatings. The term "antimicrobial" can encompass different mechanisms of action (here termed "antimicrobial surface designs"), such as antimicrobial-releasing, contact-killing or non-adhesivity. Biomaterials equipped with antimicrobial surface designs based on different mechanisms of action require different in vitro evaluation methods. Available industrial standard evaluation tests do not address the specific mechanisms of different antimicrobial surface designs and have therefore been modified over the past years, adding to the myriad of methods available in the literature to evaluate antimicrobial surface designs. The aim of this review is to categorize fourteen presently available methods including industrial standard tests for the in vitro evaluation of antimicrobial surface designs according to their suitability with respect to their antimicrobial mechanism of action. There is no single method or industrial test that allows to distinguish antimicrobial designs according to all three mechanisms identified here. However, critical consideration of each method clearly relates the different methods to a specific mechanism of antimicrobial action. It is anticipated that use of the provided table with the fourteen methods will avoid the use of wrong methods for evaluating new antimicrobial designs and therewith facilitate translation of novel antimicrobial biomaterials and coatings to clinical use. The need for more and better updated industrial standard tests is emphasized. STATEMENT OF SIGNIFICANCE: European COST-action TD1305, IPROMEDAI aims to provide better understanding of mechanisms of antimicrobial surface designs of biomaterial implants and devices. Current industrial evaluation standard tests do not sufficiently account for different, advanced antimicrobial surface designs, yet are urgently needed to obtain convincing in vitro data for approval of animal experiments and clinical trials. This review aims to provide an innovative and clear guide to choose appropriate evaluation methods for three distinctly different mechanisms of antimicrobial design: (1) antimicrobial-releasing, (2) contact-killing and (3) non-adhesivity. Use of antimicrobial evaluation methods and definition of industrial standard tests, tailored toward the antimicrobial mechanism of the design, as identified here, fulfill a missing link in the translation of novel antimicrobial surface designs to clinical use.

Absorbable mineral nanocomposite for biomedical applications: Influence of homogenous fiber dispersity on mechanical properties.

Electrospun micro- and nanosized fibers are frequently used as reinforcing elements in low temperature ceramic composites for biomedical applications. Electrospinning of fibers yield, however, not individual fibers, but rather fiber-mats that are difficult to separate. Most investigations have been performed on diced mats and highly nonhomogenous composites. We examined the influence of dispersed electrospun single micro- and nanometer fibers on the mechanical properties of calcium phosphate cement composites. Absorbable poly-l-lactic-acid was electrospun yielding fibers with diameters of 244 ± 78 nm, named nanofibers (NF), and 1.0 ± 0.3 µm, named microfibers (MF). These fibers were cut using a particle assisted ultrasonication process and dispersed with hydroxyapatite nanoparticles and composites of low (5%) and high (30%) NF/MF content were engineered. The homogeneity of the fiber distribution was investigated by analyzing fracture areas regarding the number of fibers and Voronoi area size distribution. Variation of fiber distribution was significantly lower in the NF group as compared to the MF group. For composites containing 5% NF (V/V), an eightfold increase in the compressive fracture strength, and for the 30% NF (V/V) a threefold increase compared was measured. The composite containing 5% NF was identified as optimal regarding fiber distribution and strength. Our new method of engineering these composites allows for high volume fractions of NF with low variation in fiber distribution to be incorporated into composites, and shows the importance of using single filaments as reinforcing agents. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 850-857, 2018.

Bovine Osteochondral Tissues: A Questionable Model to Evaluate Mechanical Loading In Vitro.

Articular cartilage exists within synovial joints to adsorb and distribute mechanical loads to the subchondral bone. Mechanical loading is one aspect of a wide range of microenvironmental stressors that contribute to the maintenance of articular cartilage. The aim of the current study was to characterize bovine osteochondral tissues and to assess their suitability to serve as a model for investigating the effects of mechanical loading on cartilage tissue in vitro using a custom-made reactor system. Osteochondral tissues were harvested from bovine knee joints and cultured up to 24 days in loaded and unloaded conditions. Notably, we found a considerable zone-specific heterogeneity between cartilage explants harvested from the same joint as evidenced by histology and gene expression levels. Results using the reactor system revealed that differences observed after mechanical loading varied within the range of the heterogeneity observed amongst the different cartilage explants. Thus, it may be difficult to obtain reliable and reproducible data in mechanical loading experiments from these tissues in vitro, especially in cases where small variations between the experimental groups are expected. This will likely lead to the reporting of false positives or negatives in studies investigating the effect of mechanical load on the function of cartilage tissue.

Transforming growth factor beta signaling is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes.

Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-ßs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-ß is essential for the formation of cartilage-like tissue and that alterations within the TGF-ß signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-ß1 was required to induce the formation of cartilage-like tissue. When TGF-ß signaling was blocked by inhibiting the TGF-ß receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-ß receptors 1 and 2 and TGF-ß2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-ß. We propose that a decrease in the expression of the chondrogenic growth factor TGF-ß2 and of the TGF-ß receptors in expanded chondrocytes accounts for a decrease in the activity of the TGF-ß signaling pathway and hence for the loss of the potential for autonomous cartilage-like tissue formation.

ASTM international workshop on standards and measurements for tissue engineering scaffolds.

The "Workshop on Standards & Measurements for Tissue Engineering Scaffolds" was held on May 21, 2013 in Indianapolis, IN, and was sponsored by the ASTM International (ASTM). The purpose of the workshop was to identify the highest priority items for future standards work for scaffolds used in the development and manufacture of tissue engineered medical products (TEMPs). Eighteen speakers and 78 attendees met to assess current scaffold standards and to prioritize needs for future standards. A key finding was that the ASTM TEMPs subcommittees (F04.41-46) have many active "guide" documents for educational purposes, but few standard "test methods" or "practices." Overwhelmingly, the most clearly identified need was standards for measuring the structure of scaffolds, followed by standards for biological characterization, including in vitro testing, animal models and cell-material interactions. The third most pressing need was to develop standards for assessing the mechanical properties of scaffolds. Additional needs included standards for assessing scaffold degradation, clinical outcomes with scaffolds, effects of sterilization on scaffolds, scaffold composition, and drug release from scaffolds. Discussions highlighted the need for additional scaffold reference materials and the need to use them for measurement traceability. Workshop participants emphasized the need to promote the use of standards in scaffold fabrication, characterization, and commercialization. Finally, participants noted that standards would be more broadly accepted if their impact in the TEMPs community could be quantified. Many scaffold standard needs have been identified and focus is turning to generating these standards to support the use of scaffolds in TEMPs.

Fabrication of biopolymer-based staple electrospun fibres for nanocomposite applications by particle-assisted low temperature ultrasonication.

We demonstrate the fabrication of staple polymer-based fibres by the ultrasound-assisted processing of electrospun meshes. Bioabsorbable Poly-L-Lactic Acid (PLLA) was electrospun from organic solvent mixtures, yielding continuous fibres with diameters in the range of 244±78 nm. Subsequently, the obtained fibres were sonicated at low temperatures in the presence of nanoparticles in order to obtain fibres with small aspect ratios. The influence of the dispersion medium, the sonication process parameters (temperature and time) and the dimensions of the particles used on the respective length distribution of the obtained nanofibres was investigated. Hexane was identified as an optimal dispersion medium for the system studied in this work. When a cooling bath temperature of 0°C was used, a slight increase in the obtained fibres' average length and distribution was observed as compared to cooling at -80°C (54±43 µm vs 44±31 µm). Moreover, in the presence of hydroxyapatite and hydrophilic and hydrophobic TiO2 nanoparticles in the dispersion medium longer fibres were obtained (44±31 µm, 63±47 µm, and 51±52 µm). Finally, the application of the obtained PLLA-fibre-hydroxyapatite (HA) nanoparticle precursors for the fabrication of a fibre-reinforced Brushite-based cement with high compressive strength is shown. This method of obtaining nanoscaled fibre-reinforced materials opens up a wide range of perspectives for the fabrication of composites for tissue engineering applications.

Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture.

For autologous chondrocyte transplantation, articular chondrocytes are harvested from cartilage tissue and expanded in vitro in monolayer culture. We aimed to characterize with a cellular resolution the synthesis of collagen type II (COL2) and collagen type I (COL1) during expansion in order to further understand why these cells lose the potential to form cartilage tissue when re-introduced into a microenvironment that supports chondrogenesis. During expansion for six passages, levels of transcripts encoding COL2 decreased to <0.1%, whereas transcript levels encoding COL1 increased 370-fold as compared to primary chondrocytes. Flow cytometry for intracellular proteins revealed that chondrocytes acquired a COL2/COL1-double positive phenotype during expansion, and the COL2 positive cells were able to enter the cell cycle. While the fraction of COL2 positive cells decreased from 70% to <2% in primary chondrocytes to passage six cells, the fraction of COL1 positive cells increased from <1% to >95%. In parallel to the decrease of the fraction of COL2 positive cells, the cells' potential to form cartilage-like tissue in pellet cultures steadily decreased. Intracellular staining for COL2 enables for characterization of chondrocyte lineage cells in more detail with a cellular resolution, and it may allow predicting the effectiveness of expanded chondrocytes to form cartilage-like tissue.

Tissue engineering - nanomaterials in the musculoskeletal system.

The musculoskeletal tissues bone, cartilage and ligament/tendon are highly structured nanocomposites consisting of nanofibres embedded in a matrix of different composition. Thus, it was a logical step that during the hype of nano in the last decade, nanotechnology and nanomaterials became a hot topic in the field of musculoskeletal repair. Especially the fact that using nanomaterials would encompass a biomimetic approach, thus copying nature, was promising. However, it became evident that using nanomaterials in the repair of musculoskeletal tissues had a longer history than initially thought and its way was paved with failures, which are important to remember when applying current ideas. This current opinion paper summarises some fundamental aspects of nanomaterials to be used for musculoskeletal application and discusses where this field might move to in the near future.

Evaluation of biocompatibility using in vitro methods: interpretation and limitations.

The in vitro biocompatibility of novel materials has to be proven before a material can be used as component of a medical device. This must be done in cell culture tests according to internationally recognized standard protocols. Subsequently, preclinical and clinical tests must be performed to verify the safety of the new material and device. The present chapter focuses on the first step, the in vitro testing according to ISO 10993-5, and critically discusses its limited significance. Alternative strategies and a brief overview of activities to improve the current in vitro tests are presented in the concluding section.

Chondrogenic pre-induction of human mesenchymal stem cells on beta-TCP: enhanced bone quality by endochondral heterotopic bone formation.

New techniques to heal bone defects include the combination of bone substitute materials with mesenchymal stem cells (MSC). To find solutions not hampered by low material resorbability or high donor variability of human MSC, the potency of such composites is usually evaluated by heterotopic bone formation assays in immunocompromised animals. The aim of this study was to investigate whether resorbable phase-pure beta-tricalcium-phosphate (beta-TCP) could support heterotopic bone formation by MSC comparable to partially resorbable hydroxyapatite/tricalcium-phosphate (HA/TCP). Furthermore, in light of disappointing results with osteogenic in vitro priming of MSC, we tested whether chondrogenic pre-induction of constructs may allow for enhanced bone formation by triggering the endochondral pathway. beta-TCP granules of three different sizes and HA/TCP were seeded with MSC and transplanted subcutaneously into immunocompromised mice either immediately or after a chondrogenic pre-induction for 6 weeks. After 8 weeks, explants were analysed by histology. beta-TCP seeded with unprimed MSC revealed intramembranous bone formation without haematopoietic marrow with 3.8-fold more bone formed with granules smaller than 0.7 mm than with 0.7-1.4mm particles (p< or =0.018). Chondrogenic pre-induction of beta-TCP/MSC composites resulted in collagen type II and proteoglycan-rich cartilage-like tissue which, after transplantation, underwent endochondral ossification, yielding ectopic bone produced by human cells while haematopoietic marrow was derived from the mouse. Transdifferentiation of MSC-derived chondrocytes to osteoblasts or direct osteogenesis of cartilage-resident MSC is postulated to explain the human origin of new bone. In conclusion, beta-TCP was significantly more osteo-permissive (p=0.004) than HA/TCP for human MSC, and chondrogenic priming of beta-TCP/MSC represented a superior approach capable of supporting full bone formation, including marrow organization.

Covalent immobilization of antibacterial furanones via photochemical activation of perfluorophenylazide.

N-(3-trimethoxysilylpropyl)-4-azido-2,3,5,6-tetrafluorobenzamide (PFPA-silane) was used as a photoactive cross-linker to immobilize antibacterial furanone molecules on silicon oxide surfaces. This immobilization strategy is useful, especially for substrates and molecules that lack reactive functional groups. To this end, cleaned wafers were initially incubated in solutions of different concentrations of PFPA-silane to form a monolayer presenting azido groups on the surface. The functionalized surfaces were then treated with a furanone solution followed by illumination with UV light and extensive rinsing with ethanol to remove noncovalently adhered molecules. In the presented study, we demonstrate the ability to control the surface density of the immobilized furanone molecules by adjusting the concentration of PFPA-silane solution used for surface functionalization using complementary surface analytical techniques. The fluorine in PFPA-silane and the bromine in furanone molecules were convenient markers for the XPS study. The ellipsometric layer thickness of the immobilized furanone molecules on the surface decreased with decreasing PFPA-silane concentration, which correlated with a decline of water contact angle as a sign of film collapse. The intensity of characteristic azide vibration in the MTR IR spectra was monitored as a function of PFPA-silane concentration, and the peak disappeared completely after furanone application followed by UV irradiation. As a complementary technique to XPS, TOF-SIMS provided valuable information on the chemical and molecular structure of the modified surfaces and spatial distribution of the immobilized furanone molecules. Finally, this report presents a convenient, reproducible, and robust strategy to design antibacterial coating based on furanone compounds for applications in human health care.

Furanone at subinhibitory concentrations enhances staphylococcal biofilm formation by luxS repression.

Brominated furanones from marine algae inhibit multicellular behaviors of gram-negative bacteria such as biofilm formation and quorum sensing (QS) without affecting their growth. The interaction of furanone with QS in gram-positive bacteria is unknown. Staphylococci have two QS systems, agr and luxS, which lower biofilm formation by two different pathways, RNAIII upregulation and bacterial detachment, and polysaccharide intercellular adhesin (PIA) reduction, respectively. We synthesized natural furanone compound 2 [(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone] from Delisea pulchra and three analogues to investigate their effect on biofilm formation in gram-positive bacteria. Compound 2, but not the analogues, enhanced the biofilms of Staphylococcus epidermidis 1457 and 047 and of S. aureus Newman at concentrations between 1.25 and 20 microM. We show the growth inhibition of S. epidermidis and S. aureus by free furanone and demonstrate bactericidal activity. An induction of biofilm occurred at concentrations of 10 to 20% of the MIC and correlated with an increase in PIA. The biofilm effect was agr independent. It was due to interference with luxS, as shown by reduced luxS expression in the presence of compound 2 and independence of the strong biofilm formation in a luxS mutant upon furanone addition. Poly(l-lysine)-grafted/poly(ethylene glycol)-grafted furanone was ineffective on biofilm and not bactericidal, indicating the necessity for free furanone. Free furanone was similarly toxic for murine fibroblasts as for staphylococci, excluding a therapeutic application of this compound. In summary, we observed a biofilm enhancement by furanone in staphylococci at subinhibitory concentrations, which was manifested by an increase in PIA and dependent on luxS.