In vitro antimicrobial susceptibility testing methods: agar dilution to 3D tissue-engineered models.

In the field of orthopaedic surgery, bacterial invasion of implants and the resulting periprosthetic infections are a common and unresolved problem. Antimicrobial susceptibility testing methods help to define the optimal treatment and identify antimicrobial resistance. This review discusses proven gold-standard techniques and recently developed models for antimicrobial susceptibility testing, while also providing a future outlook. Conventional, gold-standard methods, such as broth microdilution, are still widely applied in clinical settings. Although recently developed methods based on microfluidics and microdroplets have shown advantages over conventional methods in terms of testing speed, safety and the potential to provide a deeper insight into resistance mechanisms, extensive validation is required to translate this research to clinical practice. Recent optical and mechanical methods are complex and expensive and, therefore, not immediately clinically applicable. Novel osteoblast infection and tissue models best resemble infections in vivo. However, the integration of biomaterials into these models remains challenging and they require a long tissue culture, making their rapid clinical implementation unlikely. A method applicable for both clinical and research environments is difficult to realise. With a continuous increase in antimicrobial resistance, there is an urgent need for methods that analyse recurrent infections to identify the optimal treatment approaches. Graphical abstract Timeline of published and partly applied antimicrobial susceptibility testing methods, listed according to their underlying mechanism, complexity and application in research or clinics.

Optimization of a tunable process for rapid production of calcium phosphate microparticles using a droplet-based microfluidic platform.

Calcium phosphate (CaP) biomaterials are amongst the most widely used synthetic bone graft substitutes, owing to their chemical similarities to the mineral part of bone matrix and off-the-shelf availability. However, their ability to regenerate bone in critical-sized bone defects has remained inferior to the gold standard autologous bone. Hence, there is a need for methods that can be employed to efficiently produce CaPs with different properties, enabling the screening and consequent fine-tuning of the properties of CaPs towards effective bone regeneration. To this end, we propose the use of droplet microfluidics for rapid production of a variety of CaP microparticles. Particularly, this study aims to optimize the steps of a droplet microfluidic-based production process, including droplet generation, in-droplet CaP synthesis, purification and sintering, in order to obtain a library of CaP microparticles with fine-tuned properties. The results showed that size-controlled, monodisperse water-in-oil microdroplets containing calcium- and phosphate-rich solutions can be produced using a flow-focusing droplet-generator microfluidic chip. We optimized synthesis protocols based on in-droplet mineralization to obtain a range of CaP microparticles without and with inorganic additives. This was achieved by adjusting synthesis parameters, such as precursor concentration, pH value, and aging time, and applying heat treatment. In addition, our results indicated that the synthesis and fabrication parameters of CaPs in this method can alter the microstructure and the degradation behavior of CaPs. Overall, the results highlight the potential of the droplet microfluidic platform for engineering CaP microparticle biomaterials with fine-tuned properties.

Decoupling the role of chemistry and microstructure in hMSCs response to an osteoinductive calcium phosphate ceramic.

Calcium phosphates (CaP) are widely used synthetic bone graft substitutes, having bioactivity that is regulated by a set of intertwined physico-chemical and structural properties. While some CaPs have shown to be as effective in regenerating large bone defects as autologous bone, there is still the need to understand the role of individual material properties in CaP performance. Here, we aimed to decouple the effects of chemical composition and surface-microstructure of a beta-tricalcium phosphate (TCP) ceramic, with proven osteoinductive potential, on human mesenchymal stromal cells (hMSCs) differentiation. To this end, we replicated the surface structure of the TCP ceramic into polylactic acid without inorganic additives, or containing the chemical constituents of the ceramic, i.e., a calcium salt, a phosphate salt, or TCP powder. The microstructure of the different materials was characterized by confocal laser profilometry. hMSCs were cultured on the materials, and the expression of a set of osteogenic genes was determined. The cell culture medium was collected and the levels of calcium and phosphate ions were quantified by inductively-coupled plasma mass spectrometry. The results revealed that none of the tested combinations of properties in polymer/composite replicas was as potent in supporting the osteogenic differentiation of hMSCs as the original ceramic. Nevertheless, we observed some effects of the surface structure in the absence of inorganics, as well as combined effects of surface structure and the added salts, in particular calcium, on osteogenic differentiation. The approach presented here can be used to study the role of independent properties in other CaP-based biomaterials.

Mesoporous bioactive glass composition effects on degradation and bioactivity.

Mesoporous bioactive glasses (MBGs) are promising materials for regenerative medicine, due to their favorable properties including bioactivity and degradability. These key properties, but also their surface area, pore structure and pore volume are strongly dependent on synthesis parameters and glass stoichiometry. However, to date no systematic study on MBG properties covering a broad range of possible compositions exists. Here, 24 MBG compositions in the SiO2-CaO-P2O5 system were synthesized by varying SiO2 (60-90 mol %), CaO and P2O5 content (both 0 to 40 mol-%), while other synthesis parameters were kept constant. Mesopore characteristics, degradability and bioactivity were analysed. The results showed that, within the tested range of compositions, mesopore formation required a molar SiO2 content above 60% but was independent of CaO and P2O5 content. While mesopore size did not depend on glass stoichiometry, mesopore arrangement was influenced by the SiO2 content. Specific surface area and pore volume were slightly altered by the SiO2 content. All materials were degradable; however, degradation as well as bioactivity, i.e. the ability to form a CaP mineral on the surface, depended on stoichiometry. Major differences were found in early surface reactions in simulated body fluid: where some MBGs induced direct hydroxyapatite crystallization, high release of calcium in others resulted in calcite formation. In summary, degradation and bioactivity, both key parameters of MBGs, can be controlled by glass stoichiometry over a broad range while leaving the unique structural parameters of MBGs relatively unaffected. This allows targeted selection of material compositions for specific regenerative medicine applications.

3D porous Ti6Al4V-beta-tricalcium phosphate scaffolds directly fabricated by additive manufacturing.

3D Ti6Al4V-beta-tricalcium phosphate (TCP) hybrid scaffolds with interconnected porous network and controllable porosity and pore size were successfully produced by three-dimensional fiber deposition (3DF). The macrostructure of scaffolds was determined by the 3D design, whereas the micro- and submicron structure were derived from the Ti6Al4V powder sintering and the crystalline TCP powder, respectively. Ti6Al4V-TCP slurry was developed for 3DF by optimizing the TCP powder size, Ti6Al4V-to-TCP powder ratio and Ti6Al4V-TCP powder content. Moreover, the air pressure and fiber deposition rate were optimized. A maximum achievable ceramic content in the Ti6Al4V-TCP slurry that enables 3DF manufacturing was 10 wt%. The chemical analysis showed that limited contamination occurred during sintering. The compressive strength and Young's modulus of the scaffolds exhibited values between those of cancellous and cortical bone. The 3D Ti6Al4V-TCP scaffolds with 10 wt% TCP allowed deposition of a calcium phosphate layer on the surface in a simulated body fluid. Cumulative release of calcium and phosphate ions from the scaffolds was observed in a simulated physiological solution, in contrast to a cell culture medium. A pilot in vivo study, in which the scaffolds were implanted intramuscularly in dogs showed ectopic bone formation in the Ti6Al4V-TCP scaffolds with 10 wt% TCP, showing their osteoinductive potential. The porous 3D Ti6Al4V-TCP scaffolds developed here combine the mechanical properties of the metal with the bioactivity of the ceramic and are therefore likely to yield more effective strategies to control the implant-bone interface and thereby improve long-term clinical results in orthopaedics and craniomaxillofacial surgery. STATEMENT OF SIGNIFICANCE: In this work, 3D porous hybrid scaffolds made of a titanium alloy and a beta-tricalcium phosphate ceramic (Ti6Al4V-TCP) were developed using the direct additive manufacturing technique 3D fiber deposition. Upon optimization of the powders and slurry, scaffolds with up to 10 wt.% TCP with good mechanical properties and controllable porous structure at different length scales were successfully manufactured. A preliminary in vivo study in an intramuscular model demonstrated that the addition of TCP to the metal alloy improved its bioactivity. The combination of the two materials and the use of a direct additive manufacturing technique resulted in scaffolds that may lead to more effective strategies to control the implant-bone interface and thereby improve long-term clinical results in orthopaedics and craniomaxillofacial surgery.

Injectable, self-healing mesoporous silica nanocomposite hydrogels with improved mechanical properties.

Self-healing hydrogels have emerged as promising biomaterials in regenerative medicine applications. However, an ongoing challenge is to create hydrogels that combine rapid self-healing with high mechanical strength to make them applicable to a wider range of organs/tissues. Incorporating nanoparticles within hydrogels is a popular strategy to improve the mechanical properties as well as to provide additional functionalities such as stimuli responsiveness or controlled drug delivery, further optimizing their use. In this context, mesoporous silica nanoparticles (MSNs) are promising candidates as they are bioactive, improve mechanical properties, and can controllably release various types of cargo. While commonly nanoparticles are added to hydrogels as filler component, in the current study we developed thiol surface-functionalized MSNs capable of acting as chemical crosslinkers with a known hydrophilic polymer, polyethylene glycol (PEG), through dynamic thiol-disulfide covalent interactions. Due to these dynamic exchange reactions, mechanically strong nanocomposites with a storage modulus of up to 32 ± 5 kPa compared to 1.3 ± 0.3 kPa for PEG hydrogels alone, with rapid self-healing capabilities, could be formed. When non-surface modified MSNs were used, the increase in storage modulus of the hydrogels was significantly lower (3.4 ± 0.7 kPa). In addition, the nanocomposites were shown to degrade slowly over 6 weeks upon exposure to glutathione while remaining intact at physiological conditions. Together, the data argue that creating nanocomposites using MSNs as dynamic crosslinkers is a promising strategy to confer mechanical strength and rapid self-healing capabilities to hydrogels. This approach offers new possibilities for creating multifunctional self-healing biomaterials for a wider range of applications in regenerative medicine.

3D alveolar in vitro model based on epithelialized biomimetically curved culture membranes.

There is increasing evidence that surface curvature at a near-cell-scale influences cell behaviour. Epithelial or endothelial cells lining small acinar or tubular body lumens, as those of the alveoli or blood vessels, experience such highly curved surfaces. In contrast, the most commonly used culture substrates for in vitro modelling of these human tissue barriers, ion track-etched membranes, offer only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically curved track-etched membranes, preserving the mainly spherical geometry of the cells' native microenvironment. The curved membranes were created by a combination of three-dimensional (3D) micro film (thermo)forming and ion track technology. We could successfully demonstrate the formation, the growth and a first characterization of confluent layers of lung epithelial cell lines and primary alveolar epithelial cells on membranes shaped into an array of hemispherical microwells. Besides their application in submerged culture, we could also demonstrate the compatibility of the bioinspired membranes for air-exposed culture. We observed a distinct cellular response to membrane curvature. Cells (or cell layers) on the curved membranes reveal significant differences compared to cells on flat membranes concerning membrane epithelialization, areal cell density of the formed epithelial layers, their cross-sectional morphology, and proliferation and apoptosis rates, and the same tight barrier function as on the flat membranes. The presented 3D membrane technology might pave the way for more predictive barrier in vitro models in future.

Comparative proteomic analysis of human mesenchymal stromal cell behavior on calcium phosphate ceramics with different osteoinductive potential.

In recent years, synthetic calcium phosphate (CaP) ceramics have emerged as an alternative to bone grafts in the treatment of large critical-sized bone defects. To successfully substitute for bone grafts, materials must be osteoinductive, that is, they must induce osteogenic differentiation and subsequent bone formation in vivo. Although a set of osteoinductive CaP ceramics has been developed, the precise biological mechanism by which a material directs cells toward osteogenesis and the role of individual chemical and physical properties in this mechanism remain incompletely understood. Here, we used proteomics to compare serum protein adsorption to two CaP ceramics with different osteoinductive potential, namely an osteoinductive ß-tricalcium phosphate (TCP) and a non-osteoinductive hydroxyapatite (HA). Moreover, we analyzed the protein profiles of human mesenchymal stromal cells (hMSCs) cultured on these two ceramics. The serum protein adsorption experiments in the absence of cells highlighted the proteins that are highly abundant in the serum and/or have a high affinity to CaP. The extent of adsorption was suggested to be affected by the available surface area for binding and by the ion exchange dynamics on the surface. Several proteins were uniquely expressed by hMSCs on TCP and HA surfaces. Proteins identified as enriched on TCP were involved in processes related to wound healing, cell proliferation, and the production of extracellular matrix. On the other hand, proteins that were enriched on HA were involved in processes related to protein production, translation, localization, and secretion. In addition, we performed a separate proteomics analysis on TCP, HA, and two biphasic calcium phosphates with known osteoinductive potential and performed a clustering analysis on a combination of a set of proteins found to be enriched on osteoinductive materials with a set of proteins already known to be involved in osteogenesis. This yielded two protein networks potentially involved in the process of osteoinduction - one consisting of collagen fragments and collagen-related enzymes and a second consisting of endopeptidase inhibitors and regulatory proteins. The results of this study show that protein profiling can be a useful tool to help understand the effect of biomaterial properties on the interactions between a biomaterial and a biological system. Such understanding will contribute to the design and development of improved biomaterials for (bone) regenerative therapies.

Colorectal tumor-on-a-chip system: A 3D tool for precision onco-nanomedicine.

Awareness that traditional two-dimensional (2D) in vitro and nonrepresentative animal models may not completely emulate the 3D hierarchical complexity of tissues and organs is on the rise. Therefore, posterior translation into successful clinical application is compromised. To address this dearth, on-chip biomimetic microenvironments powered by microfluidic technologies are being developed to better capture the complexity of in vivo pathophysiology. Here, we describe a "tumor-on-a-chip" model for assessment of precision nanomedicine delivery on which we validate the efficacy of drug-loaded nanoparticles in a gradient fashion. The model validation was performed by viability studies integrated with live imaging to confirm the dose-response effect of cells exposed to the CMCht/PAMAM nanoparticle gradient. This platform also enables the analysis at the gene expression level, where a down-regulation of all the studied genes (MMP-1, Caspase-3, and Ki-67) was observed. This tumor-on-chip model represents an important development in the use of precision nanomedicine toward personalized treatment.

Development of a microfluidic platform integrating high-resolution microstructured biomaterials to study cell-material interactions.

Microfluidic screening platforms offer new possibilities for performing in vitro cell-based assays with higher throughput and in a setting that has the potential to closely mimic the physiological microenvironment. Integrating functional biomaterials into such platforms is a promising approach to obtain a deeper insight into the interactions occurring at the cell-material interface. The success of such an approach is, however, largely dependent on the ability to miniaturize the biomaterials as well as on the choice of the assay used to study the cell-material interactions. In this work, we developed a microfluidic device, the main component of which is made of a widely used biocompatible polymer, polylactic acid (PLA). This device enabled cell culture under different fluidic regimes, including perfusion and diffusion. Through a combination of photolithography, two-photon polymerization and hot embossing, it was possible to microstructure the surface of the cell culture chamber of the device with highly defined geometrical features. Furthermore, using pyramids with different heights and wall microtopographies as an example, adhesion, morphology and distribution of human MG63 osteosarcoma cells were studied. The results showed that both the height of the topographical features and the microstructural properties of their walls affected cell spreading and distribution. This proof-of-concept study shows that the platform developed here is a useful tool for studying interactions between cells and clinically relevant biomaterials under controlled fluidic regimes.

Surface micropatterning with zirconia and calcium phosphate ceramics by micromoulding in capillaries.

An increasing demand exists for biomaterials that are able to actively participate in the process of repair and regeneration of damaged or diseased organs and tissues. Patterning of surfaces of biomaterials with distinct chemical or physical cues is an attractive way to obtain spatial control over their interactions with the biological system. In the current study, micromoulding in capillaries method was used to pattern silicon substrates with bioinert yttria-stabilised zirconia or with bioactive calcium phosphate ceramics, both widely used biomaterials in orthopaedics and dentistry. Micrometer-scale patterns consisted of parallel lines with varying width and spacing. Both ceramics were successfully deposited on the substrate in a pattern defined by the mould. While the yttria-stabilised zirconia pattern was highly homogenous and smooth (Rq = 5.5 nm), the calcium phosphate pattern, consisting of dicalcium phosphate anhydrous before, and of ß-tricalcium phosphate after annealing, exhibited a less homogenous morphology and higher roughness (Rq = 893 nm). Both materials allowed attachment and proliferation of the MG-63 osteosarcoma cell line, independent of the pattern used. While a preferential orientation of cells in the direction of the pattern lines was observed for all patterns, this effect was more pronounced on the lines with a width of up to 20 µm on both yttria-stabilised zirconia and calcium phosphate ceramics, as compared to wider patterns. Furthermore, the cells retained an elongated morphology for a longer period of time on narrow patterns. Micromoulding in capillaries appeared to be a suitable method to pattern both types of ceramics, however further optimisation is needed to improve homogeneity and obtain better control over the chemical phase and crystalline structure of calcium phosphate patterns.

Evaluation of Cartilage Repair by Mesenchymal Stem Cells Seeded on a PEOT/PBT Scaffold in an Osteochondral Defect.

The main objective of this study was to evaluate the effectiveness of a mesenchymal stem cell (MSC)-seeded polyethylene-oxide-terephthalate/polybutylene-terephthalate (PEOT/PBT) scaffold for cartilage tissue repair in an osteochondral defect using a rabbit model. Material characterisation using scanning electron microscopy indicated that the scaffold had a 3D architecture characteristic of the additive manufacturing fabrication method, with a strut diameter of 296 ± 52 µm and a pore size of 512 ± 22 µm × 476 ± 25 µm × 180 ± 30 µm. In vitro optimisation revealed that the scaffold did not generate an adverse cell response, optimal cell loading conditions were achieved using 50 µg/ml fibronectin and a cell seeding density of 25 × 10(6) cells/ml and glycosaminoglycan (GAG) accumulation after 28 days culture in the presence of TGFß3 indicated positive chondrogenesis. Cell-seeded scaffolds were implanted in osteochondral defects for 12 weeks, with cell-free scaffolds and empty defects employed as controls. On examination of toluidine blue staining for chondrogenesis and GAG accumulation, both the empty defect and the cell-seeded scaffold appeared to promote repair. However, the empty defect and the cell-free scaffold stained positive for collagen type I or fibrocartilage, while the cell-seeded scaffold stained positive for collagen type II indicative of hyaline cartilage and was statistically better than the cell-free scaffold in the blinded histological evaluation. In summary, MSCs in combination with a 3D PEOT/PBT scaffold created a reparative environment for cartilage repair.

Surface modification of electrospun fibre meshes by oxygen plasma for bone regeneration.

Plasma treatment is a method to modify the physicochemical properties of biomaterials, which consequently may affect interactions with cells. Based on the rationale that physical cues on the surface of culture substrates and implants, such as surface roughness, have proven to alter cell behaviour, we used electrospinning to fabricate fibrous three-dimensional scaffolds made of a poly (ethylene oxide terephthalate)/poly (butylene terephthalate) copolymer to mimic the physical microenvironment of extracellular matrix and applied radio-frequency oxygen plasma treatment to create nanoscale roughness. Scanning electron microscopy (SEM) analysis revealed a fibre diameter of 5.49 ± 0.96 µm for as-spun meshes. Atomic force microscopy (AFM) measurements determined an exponential increase of surface roughness with plasma treatment time. An increase in hydrophilicity after plasma treatment was observed, which was associated with higher oxygen content in plasma treated scaffolds compared to untreated ones. A more pronounced adsorption of bovine serum albumin occurred on scaffolds treated with plasma for 15 and 30 min compared to untreated fibres. Clinically relevant human mesenchymal stromal cells (hMSCs) were cultured on untreated, 15 and 30 min treated scaffolds. SEM analysis confirmed cell attachment and a pronounced spindle-like morphology on all scaffolds. No significant differences were observed between different scaffolds regarding the amount of DNA, metabolic activity and alkaline phosphatase (ALP) activity after 7 days of culture. The amount of ALP positive cells increased between 7 and 21 days of culture on both untreated and 30 min treated meshes. In addition, ALP staining of cells on plasma treated meshes appeared more pronounced than on untreated meshes after 21 days of culture. Quantitative polymerase chain reaction showed significant upregulation of bone sialoprotein and osteonectin expression on oxygen plasma treated fibres compared to untreated fibres in basic culture medium after 7 days of culture, while no differences were observed in the expression of other osteogenic markers. At 21 days, no osteocalcin protein could be detected by ELISA at any of the substrates. In conclusion, this study shows that oxygen plasma treatment can successfully be applied to modify the nanoscale surface properties of polymeric electrospun fibre meshes, which in turn may positively affect osteogenic differentiation of hMSCs.

Bioinorganics and biomaterials: bone repair.

The field of bioinorganics is well established in the development of a variety of therapies. However, their application to bone regeneration, specifically by way of localized delivery from functional implants, is in its infancy and is the topic of this review. The toxicity of inorganics is species, dose and duration specific. Little is known about how inorganic ions are effective therapeutically since their use is often the result of serendipity, observations from nutritional deficiency or excess and genetic disorders. Many researchers point to early work demonstrating a role for their element of interest as a micronutrient critical to or able to alter bone growth, often during skeletal development, as a basis for localized delivery. While one can appreciate how a deficiency can cause disruption of healing, it is difficult to explain how a locally delivered excess in a preclinical model or patient, which is presumably of normal nutritional status, can evoke more bone or faster healing. The review illustrates that inorganics can positively affect bone healing but various factors make literature comparisons difficult. Bioinorganics have the potential to have just as big an impact on bone regeneration as recombinant proteins without some of the safety concerns and high costs.

Influence of octacalcium phosphate coating on osteoinductive properties of biomaterials.

In this study, we investigated the influence of octacalcium phosphate (OCP) coating on osteoinductive behaviour of the biomaterials. Porous titanium alloy (Ti6Al4V), hydroxyapatite (HA), biphasic calcium phosphate (BCP) and polyethylene glyco terephtalate/polybuthylene terephtalate (PEGT-PBT) copolymer, all uncoated and coated with biomimetically produced OCP, were implanted in back muscles of 10 goats for 6 and 12 weeks. Uncoated Ti6Al4Vand HA did not show any bone formation after intramuscular implantation. All OCP coated implants, except PEGT-PBT, did induce bone in the soft tissue. The reason for the non-inductive behaviour of the copolymer is probably its softness, that makes it impossible to maintain its porous shape after implantation. Both uncoated and OCP coated BCP induced bone. However, the amount of animals in which the bone was induced was higher in the coated BCP implants in comparison to the uncoated ones. Osteoinductive potential of biomaterials is influenced by various material characteristics, such as chemical composition, crystallinity, macro- and microstructure. OCP coating has a positive effect on osteoinductivity of the biomaterials. The combination of the advantages of biomimetic coating method above traditional methods, and a good osteoinductivity of OCP coating that is produced by using this method, opens new possibilities for designing more advanced orthopaedic implants.