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Abstract Transmissible gastroenteritis virus (TGEV) causes high mortality in young piglets (

AbstractBurn injuries can result in a significant inflammatory response, often leading to hypertrophic scarring (HTS). Local drug therapies e.g. corticoid injections are advised to treat HTS, although they are invasive, operator-dependent, extremely painful and do not permit extended drug release. Polymer-based microneedle (MN) arrays can offer a viable alternative to standard care, while allowing for direct, painless dermal drug delivery with tailorable drug release profile. In the current study, we synthesized photo-crosslinkable, acrylate-endcapped urethane-based poly(ε-caprolactone) (AUP-PCL) toward the fabrication of MNs. Physico-chemical characterization (1H-NMR, evaluation of swelling, gel fraction) of the developed polymer was performed and confirmed successful acrylation of PCL-diol. Subsequently, AUP-PCL, and commercially available PCL-based microneedle arrays were fabricated for comparative evaluation of the constructs. Hydrocortisone was chosen as model drug. To enhance the drug release efficiency of the MNs, Brij®35, a nonionic surfactant was exploited. The thermal properties of the MNs were evaluated via differential scanning calorimetry. Compression testing of the arrays confirmed that the MNs stay intact upon applying a load of 7 N, which correlates to the standard dermal insertion force of MNs. The drug release profile of the arrays was evaluated, suggesting that the developed PCL arrays can offer efficient drug delivery for up to two days, while the AUP-PCL arrays can provide a release up to three weeks. Finally, the insertion of MN arrays into skin samples was performed, followed by histological analysis demonstrating the AUP-PCL MNs outperforming the PCL arrays upon providing pyramidical-shaped perforations through the epidermal layer of the skin.

In order to recreate the complexity of human organs, the field of tissue engineering and regenerative medicine has been focusing on methods to build organs from the bottom up by assembling distinct small functional units consisting of a biomaterial and cells. This bottom-up engineering requires bioinks that can be assembled by 3D bioprinting and that permit fast vascularization of the construct to ensure survival of embedded cells. To this end, a small molecular weight alginate (SMWA) gel porogen is presented herein. Alginate is a biocompatible biomaterial, which can be easily converted into small porogen gels with the procedure reported in this article. The SMWA porogen is mixed with photo-crosslinkable hydrogels and leached from the hydrogel post-crosslinking to increase porosity and facilitate vascularization. As a proof of concept, this system is tested with the commonly used biomaterial Gelatin Methacryloyl (GelMA). The SMWA porogen-GelMA blend is proven to be bioprintable. Incubating the blend for 20 min in a low concentration phosphate buffered saline and sodium citrate solution significantly reduces the remaining porogen in the hydrogel . The intent to completely leach the porogen from the hydrogel was abandoned, as longer incubation times and higher concentrations of phosphate and citrate were detrimental to endothelial proliferation. Nonetheless, even with remnants of the porogen left in the hydrogel, the created porosity significantly improves viability, growth factor signaling, vasculogenesis, and angiogenesis in 3D bioprinted structures. This article concludes that the usage of the SMWA porogen can improve the assembly of microvasculature in 3D bioprinted structures. This technology can benefit the bottom-up assembly of large scaffolds with high cell density through 3D bioprinting by improving cell viability and allowing faster vascularization.

The current standard technique for vascular grafting in cerebral revascularization surgery employs the interposition of an autologous blood vessel. Technical complications have necessitated the development of a synthetic alternative, but classical biomaterials are not suited for small caliber vascular grafting due to the resulting neointimal hyperplasia and thrombosis. The electrospinning of polymers is a promising technique for the development of small vascular grafts. The in vivo performance and efficacy of electrospun polyurethane (ePU) grafts with an internal diameter of

Abstract Background Long-term drug evaluation heavily relies upon rodent models. Drug discovery methods to reduce animal models in oncology may include three-dimensional (3D) cellular systems that take into account tumor microenvironment (TME) cell types and biomechanical properties. Methods In this study we reconstructed a 3D tumor using an elastic polymer (acrylate-endcapped urethane-based poly(ethylene glycol) (AUPPEG)) with clinical relevant stiffness. Single cell suspensions from low-grade serous ovarian cancer (LGSOC) patient-derived early passage cultures of cancer cells and cancer-associated fibroblasts (CAF) embedded in a collagen gel were introduced to the AUPPEG scaffold. After self-organization in to a 3D tumor, this model was evaluated by a long-term (> 40 days) exposure to a drug combination of MEK and HSP90 inhibitors. The drug-response results from this long-term in vitro model are compared with drug responses in an orthotopic LGSOC xenograft mouse model. Results The in vitro 3D scaffold LGSOC model mimics the growth ratio and spatial organization of the LGSOC. The AUPPEG scaffold approach allows to test new targeted treatments and monitor long-term drug responses. The results correlate with those of the orthotopic LGSOC xenograft mouse model. Conclusions The mechanically-tunable scaffolds colonized by a three-dimensional LGSOC allow long-term drug evaluation and can be considered as a valid alternative to reduce, replace and refine animal models in drug discovery. Graphical Abstract

Osteochondral defects are deep joint surface lesions that affect the articular cartilage and the underlying subchondral bone. In the current study, a tissue engineering approach encompassing individual cells encapsulated in a biocompatible hydrogel is explored in vitro and in vivo. Cell-laden hydrogels containing either human periosteum-derived progenitor cells (PDCs) or human induced pluripotent stem cell (iPSC)-derived chondrocytes encapsulated in gelatin methacryloyl (GelMA) were evaluated for their potential to regenerate the subchondral mineralized bone and the articular cartilage on the joint surface, respectively. PDCs are easily isolated and expanded progenitor cells that are capable of generating mineralized cartilage and bone tissue in vivo via endochondral ossification. iPSC-derived chondrocytes are an unlimited source of stable and highly metabolically active chondrocytes. Cell-laden hydrogel constructs were cultured for up to 28 days in a serum-free chemically defined chondrogenic medium. On day 1 and day 21 of the differentiation period, the cell-laden constructs were implanted subcutaneously in nude mice to evaluate ectopic tissue formation 4 weeks post-implantation. Taken together, the data suggest that iPSC-derived chondrocytes encapsulated in GelMA can generate hyaline cartilage-like tissue constructs with different levels of maturity, while using periosteum-derived cells in the same construct type generates mineralized tissue and cortical bone in vivo. Therefore, the aforementioned cell-laden hydrogels can be an important part of a multi-component strategy for the manufacturing of an osteochondral implant.

Poly(aspartic acid) (pAsp)-based fiber networks were developed via electrospinning and photo-crosslinking. Fiber networks hold the advantage that they exhibit a high surface-to-volume ratio, giving rise to a high water uptake and retention capacity, along with the ability to release moisture under desired circumstances (e.g. reduced relative humidity, mechanical pressure, etc.). Herein, polysuccinimide (PSI), the precursor of pAsp, was modified with 5-norbornene-2-methylamine to obtain crosslinkable norbornene-modified PSI (PSI-NB) with two different degrees of substitution, i.e. 19% and 46%. These derivatives were electrospun into thin uniform fibers after optimization of the processing parameters. The fiber sheets were crosslinked via a thiol-ene step-growth mechanism with three different thiol crosslinkers exploiting UV-A irradiation in the presence of TPO-L as photo-initiator. Using this strategy, fiber networks with diameters ranging between 1.27 ± 0.29 and 2.20 ± 1.05 µm were obtained, as visualized with scanning electron microscopy (SEM). Successful crosslinking was evidenced by a dissolution test in dimethylformamide and through X-ray photoelectron spectroscopy. Finally, the PSI-NB fiber networks were hydrolyzed to obtain pAsp-NB fiber networks by alkaline hydrolysis in a carbonate buffer solution, as confirmed by Fourier-transform infrared spectroscopy. The morphology of the fibers following hydrolysis was visualized by SEM and the average fiber diameters were calculated and compared to the diameters before hydrolysis, generally showing a diameter increase due to swelling of the fibers in aqueous solution. In conclusion, pAsp-based fiber networks were successfully developed and stabilized via photo-crosslinking.

There is a high demand for in vitro intestinal models towards studying intestinal disfunctions, such as gastrointestinal diseases (Crohn's disease, irritable bowel syndrome, colorectal cancer, etc.) which are commonly diagnosed in modern society. In parallel, in vitro models are utilized for the evaluation of novel food supplements and pharmaceuticals in the intestinal micro-environment. Gelatin-based hydrogels can offer appropriate mechanical cues (down to G’ < 10 kPa) and cell interactivity to obtain physiologically relevant soft tissue models. Therefore, the current paper focused on the development of a novel, gelatin-methacryloyl-aminoethyl-methacrylate (gel-MA-AEMA)-based in vitro intestinal model, which also provides sufficient permeability towards nutrients and drugs. Moreover, the effect of the hydrogel morphology on the cell response was assessed by comparing the formation of a functional intestinal cell monolayer on flat hydrogel films versus 3D hydrogel scaffolds maintaining a close morphological resemblance with the intestinal architecture.A gel-MA-AEMA-based biomaterial ink was formulated and processed towards flat hydrogel films, while digital light processing was exploited to replicate the intestinal microarchitecture. The mechanical assessment of the 2D films confirmed physiologically relevant scaffold stiffness (G’ = 3.30±1.07 kPa). To evaluate the permeability of the hydrogels towards a medium size marker molecule (FITC-dextran 4 kg·mol−1), a static diffusion setup was exploited with custom-made adjustable inserts, which evidenced that both the 2D films and 3D constructs exhibited sufficient permeability. Finally, the combination of the hydrogels with a Caco-2/HT29-MTX co-culture evidenced hydrogel biocompatibility, enabling the formation of a functional cell monolayer after 21 days on the 2D hydrogel films, proven by transepithelial electrical resistance measurements and immunohistochemistry, while the 3D hydrogel constructs did not achieve confluency within 35 days.

A previously developed cellularized collagen-based vascular wall model showed promising results in mimicking the biological properties of a native vessel but lacked appropriate mechanical properties. In this work, we aim to improve this collagen-based model by reinforcing it using a tubular polymeric (reinforcement) scaffold. The polymeric reinforcements were fabricated exploiting commercial poly (ε-caprolactone) (PCL), a polymer already used to fabricate other FDA-approved and commercially available devices serving medical applications, through 1) solution electrospinning (SES), 2) 3D printing (3DP) and 3) melt electrowriting (MEW). The non-reinforced cellularized collagen-based model was used as a reference (COL). The effect of the scaffold’s architecture on the resulting mechanical and biological properties of the reinforced collagen-based model were evaluated. SEM imaging showed the differences in scaffolds’ architecture (fiber alignment, fiber diameter and pore size) at both the micro- and the macrolevel. The polymeric scaffold led to significantly improved mechanical properties for the reinforced collagen-based model (initial elastic moduli of 382.05 ± 132.01 kPa, 100.59 ± 31.15 kPa and 245.78 ± 33.54 kPa, respectively for SES, 3DP and MEW at day 7 of maturation) compared to the non-reinforced collagen-based model (16.63 ± 5.69 kPa). Moreover, on day 7, the developed collagen gels showed stresses (for strains between 20% and 55%) in the range of [5–15] kPa for COL, [80–350] kPa for SES, [20–70] kPa for 3DP and [100–190] kPa for MEW. In addition to the effect on the resulting mechanical properties, the polymeric tubes’ architecture influenced cell behavior, in terms of proliferation and attachment, along with collagen gel compaction and extracellular matrix protein expression. The MEW reinforcement resulted in a collagen gel compaction similar to the COL reference, whereas 3DP and SES led to thinner and longer collagen gels. Overall, it can be concluded that 1) the selected processing technique influences the scaffolds’ architecture, which in turn influences the resulting mechanical and biological properties, and 2) the incorporation of a polymeric reinforcement leads to mechanical properties closely matching those of native arteries.

The distribution of photo-crosslinkable moieties onto a protein backbone can affect a biomaterial's crosslinking behavior, and therefore also its mechanical and biological properties. A profound insight in this respect is essential for biomaterials exploited in tissue engineering and regenerative medicine. In the present work, photo-crosslinkable moieties have been introduced on the primary amine groups of: (i) a recombinant collagen peptide (RCPhC1) with a known amino acid (AA) sequence, and (ii) bovine skin collagen (COL BS) with an unknown AA sequence. The degree of substitution (DS) was quantified with two conventional techniques: an ortho-phthalic dialdehyde (OPA) assay and 1H NMR spectroscopy. However, neither of both provides information on the exact type and location of the modified AAs. Therefore, for the first time, proteomic analysis was evaluated herein as a tool to identify functionalized AAs as well as the exact position of photo-crosslinkable moieties along the AA sequence, thereby enabling an in-depth, unprecedented characterization of functionalized photo-crosslinkable biopolymers. Moreover, our strategy enabled to visualize the spatial distribution of the modifications within the overall structure of the protein. Proteomics has proven to provide unprecedented insight in the distribution of photo-crosslinkable moieties along the protein backbone, undoubtedly contributing to superior functional biomaterial design to serve regenerative medicine.

The tissue engineering field is currently advancing towards minimally invasive procedures to reconstruct soft tissue defects. In this regard, injectable hydrogels are viewed as excellent scaffold candidates to support and promote the growth of encapsulated cells. Cross-linked gelatin methacryloyl (GelMA) gels have received substantial attention due to their extracellular matrix-mimicking properties. In particular, GelMA microgels were recently identified as interesting scaffold materials since the pores in between the microgel particles allow good cell movement and nutrient diffusion. The current work reports on a novel microgel preparation procedure in which a bulk GelMA hydrogel is ground into powder particles. These particles can be easily transformed into a microgel by swelling them in a suitable solvent. The rheological properties of the microgel are independent of the particle size and remain stable at body temperature, with only a minor reversible reduction in elastic modulus correlated to the unfolding of physical cross-links at elevated temperatures. Salts reduce the elastic modulus of the microgel network due to a deswelling of the particles, in addition to triple helix denaturation. The microgels are suited for clinical use, as proven by their excellent cytocompatibility. The latter is confirmed by the superior proliferation of encapsulated adipose tissue-derived stem cells in the microgel compared to the bulk hydrogel. Moreover, microgels made from the smallest particles are easily injected through a 20G needle, allowing a minimally invasive delivery. Hence, the current work reveals that powdered cross-linked GelMA is an excellent candidate to serve as an injectable hydrogel for adipose tissue engineering.

Abstract Multi-photon lithography (MPL) has proven to be a suitable tool to precisely control the microenvironment of cells in terms of the biochemical and biophysical properties of the hydrogel matrix. In this work, we present a novel method, based on multi-photon photografting of 4,4′-diazido-2,2′-stilbenedisulfonic acid (DSSA), and its capabilities to induce cell alignment, directional cell migration and endothelial sprouting in a gelatin-based hydrogel matrix. DSSA-photografting allows for the fabrication of complex patterns at a high-resolution and is a biocompatible, universally applicable and straightforward process that is comparably fast. We have demonstrated the preferential orientation of human adipose-derived stem cells (hASCs) in response to a photografted pattern. Co-culture spheroids of hASCs and human umbilical vein endothelial cells (HUVECs) have been utilized to study the directional migration of hASCs into the modified regions. Subsequently, we have highlighted the dependence of endothelial sprouting on the presence of hASCs and demonstrated the potential of photografting to control the direction of the sprouts. MPL-induced DSSA-photografting has been established as a promising method to selectively alter the microenvironment of cells.

Skeletal muscle tissue engineering (SMTE) aims at the in vitro generation of 3D skeletal muscle engineered constructs which mimic the native muscle structure and function. Although native skeletal muscle is a highly dynamic tissue, most research approaches still focus on static cell culture methods, while research on stimulation protocols indicates a positive effect, especially on myogenesis. A more mature muscle construct may be needed especially for the potential applications for regenerative medicine purposes, disease or drug disposition models. Most efforts towards dynamic cell or tissue culture methods have been geared towards mechanical or electrical stimulation or a combination of those. In the context of dynamic methods, pulsed electromagnetic field (PEMF) stimulation has been extensively used in bone tissue engineering, but the impact of PEMF on skeletal muscle development is poorly explored. Here, we evaluated the effects of PEMF stimulation on human skeletal muscle cells both in 2D and 3D experiments. First, PEMF was applied on 2D cultures of human myoblasts during differentiation. In 2D, enhanced myogenesis was observed, as evidenced by an increased myotube diameter and fusion index. Second, 2D results were translated towards 3D bioartificial muscles (BAMs). BAMs were subjected to PEMF for varying exposure times, where a 2-h daily stimulation was found to be effective in enhancing 3D myotube formation. Third, applying this protocol for the entire 16-days culture period was compared to a stimulation starting at day 8, once the myotubes were formed. The latter was found to result in significantly higher myotube diameter, fusion index, and increased myosin heavy chain 1 expression. This work shows the potential of electromagnetic stimulation for enhancing myotube formation both in 2D and 3D, warranting its further consideration in dynamic culturing techniques.

Digital microfluidics (DMF) holds great potential for the alleviation of laboratory procedures in assisted reproductive technologies (ARTs). The electrowetting on dielectric (EWOD) technology provides dynamic culture conditions in vitro that may better mimic the natural embryo microenvironment. Thus far, EWOD microdevices have been proposed for in vitro gamete and embryo handling in mice and for analyzing the human embryo secretome. This article presents the development of the first microfluidic chip utilizing EWOD technology designed for the manipulation of bovine embryos in vitro. The prototype sustains the cell cycles of embryos manipulated individually on the chips during in vitro culture (IVC). Challenges related to the chip fabrication as well as to its application during bovine embryo IVC in accordance with the adapted on-chip protocol are thoroughly discussed, and future directions for DMF in ARTs are indicated.

The authors would like to update the XPS spectrum in Figure 3c [...]

Overuse tendon injuries are a major cause of musculoskeletal morbidity in both human and equine athletes, due to the cumulative degenerative damage. These injuries present significant challenges as the healing process often results in the formation of inferior scar tissue. The poor success with conventional therapy supports the need to search for novel treatments to restore functionality and regenerate tissue as close to native tendon as possible. Mesenchymal stem cell (MSC)-based strategies represent promising therapeutic tools for tendon repair in both human and veterinary medicine. The translation of tissue engineering strategies from basic research findings, however, into clinical use has been hampered by the limited understanding of the multifaceted MSC mechanisms of action. In vitro models serve as important biological tools to study cell behavior, bypassing the confounding factors associated with in vivo experiments. Controllable and reproducible in vitro conditions should be provided to study the MSC healing mechanisms in tendon injuries. Unfortunately, no physiologically representative tendinopathy models exist to date. A major shortcoming of most currently available in vitro tendon models is the lack of extracellular tendon matrix and vascular supply. These models often make use of synthetic biomaterials, which do not reflect the natural tendon composition. Alternatively, decellularized tendon has been applied, but it is challenging to obtain reproducible results due to its variable composition, less efficient cell seeding approaches and lack of cell encapsulation and vascularization. The current review will overview pros and cons associated with the use of different biomaterials and technologies enabling scaffold production. In addition, the characteristics of the ideal, state-of-the-art tendinopathy model will be discussed. Briefly, a representative in vitro tendinopathy model should be vascularized and mimic the hierarchical structure of the tendon matrix with elongated cells being organized in a parallel fashion and subjected to uniaxial stretching. Incorporation of mechanical stimulation, preferably uniaxial stretching may be a key element in order to obtain appropriate matrix alignment and create a pathophysiological model. Together, a thorough discussion on the current status and future directions for tendon models will enhance fundamental MSC research, accelerating translation of MSC therapies for tendon injuries from bench to bedside.

To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 ± 2.80 μm, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting.

The combination of natural and synthetic polymers to form hybrid hydrogels offers the potential of fabricating new materials that possess a combination of properties resulting from both types of polymer classes. Within this work, two alkene-functionalized poly(2-alkyl/aryl–2-oxazoline) (PAOx) copolymers and one gelatin derivative, thiolated gelatin (gel-SH), are synthesized as precursors for hybrid hydrogels through a photo-induced radical thiol-ene crosslinking process. In-situ photo-rheology revealed an increased mechanical stability for hydrogels that possess an excess amount of PAOx precursor. A final qualitative investigation of the thermo-responsive properties of a P(EtOx270–norbornenOx30):gel-SH (2:1) hydrogel film revealed a cloud point temperature (Tcp) in the same range as the Tcp of the P(EtOx270–norbornenOx30) polymer precursor, which is around 30 °C. This promising result demonstrates that thermo-responsive hybrid poly(2-oxazoline)-gelatin hydrogels could be prepared with predictable Tcps and that further investigation into this appealing feature might be of interest. Ultimately, this work shows a proof-of-concept of using PAOx as potential hybrid hydrogel precursor in combination with cell-interactive gelatin derivatives to potentially improve the mechanical stability of the final scaffolds and introduce additional features such as thermo-responsiveness for the purpose of drug delivery.

Cardiovascular diseases (CVDs) account for the 31% of total death per year, making them the first cause of death in the world. Atherosclerosis is at the root of the most life-threatening CVDs. Vascular bypass/replacement surgery is the primary therapy for patients with atherosclerosis. The use of polymeric grafts for this application is still burdened by high-rate failure, mostly caused by thrombosis and neointima hyperplasia at the implantation site. As a solution for these problems, the fast re-establishment of a functional endothelial cell (EC) layer has been proposed, representing a strategy of crucial importance to reduce these adverse outcomes. Implant modifications using molecules and growth factors with the aim of speeding up the re-endothelialization process has been proposed over the last years. Collagen, by virtue of several favorable properties, has been widely studied for its application in vascular graft enrichment, mainly as a coating for vascular graft luminal surface and as a drug delivery system for the release of pro-endothelialization factors. Collagen coatings provide receptor–ligand binding sites for ECs on the graft surface and, at the same time, act as biological sealants, effectively reducing graft porosity. The development of collagen-based drug delivery systems, in which small-molecule and protein-based drugs are immobilized within a collagen scaffold in order to control their release for biomedical applications, has been widely explored. These systems help in protecting the biological activity of the loaded molecules while slowing their diffusion from collagen scaffolds, providing optimal effects on the targeted vascular cells. Moreover, collagen-based vascular tissue engineering substitutes, despite not showing yet optimal mechanical properties for their use in the therapy, have shown a high potential as physiologically relevant models for the study of cardiovascular therapeutic drugs and diseases. In this review, the current state of the art about the use of collagen-based strategies, mainly as a coating material for the functionalization of vascular graft luminal surface, as a drug delivery system for the release of pro-endothelialization factors, and as physiologically relevant in vitro vascular models, and the future trend in this field of research will be presented and discussed.

We investigate the possibilities to realize light extraction from single crystal diamond (SCD) nanopillars. This was achieved by dedicated 519 nm laser-induced spin-state initiation of negatively charged nitrogen vacancies (NV−). We focus on the naturally-generated by chemical vapor deposition (CVD) growth of NV−. Applied diamond was neither implanted with 14N+, nor was the CVD synthesized SCD annealed. To investigate the possibility of light extraction by the utilization of NV−’s bright photoluminescence at room temperature and ambient conditions with the waveguiding effect, we have performed a top-down nanofabrication of SCD by electron beam lithography (EBL) and dry inductively-coupled plasma/reactive ion etching (ICP-RIE) to generate light focusing nanopillars. In addition, we have fluorinated the diamond’s surface by dedicated 0 V SF6 ICP plasma. Light extraction and spin manipulations were performed with photoluminescence (PL) spectroscopy and optically detected magnetic resonance (ODMR) at room temperature. We have observed a remarkable effect based on the selective 0 V SF6 plasma etching and surprisingly, in contrast to literature findings, deactivation of NV− centers. We discuss the possible deactivation mechanism in detail.

To mitigate autogenous shrinkage in cementitious materials and simultaneously preserve the material’s mechanical performance, superabsorbent polymers and nanosilica are included in the mixture design. The use of the specific additives influences both the hydration process and the hardened microstructure, while autogenous healing of cracks can be stimulated. These three stages are monitored by means of non-destructive testing, showing the sensitivity of elastic waves to the occurring phenomena. Whereas the action of the superabsorbent polymers was evidenced by acoustic emission, the use of ultrasound revealed the differences in the developed microstructure and the self-healing of cracks by a comparison with more commonly performed mechanical tests. The ability of NDT to determine these various features renders it a promising measuring method for future characterization of innovative cementitious materials.

To progress the fields of tissue engineering (TE) and regenerative medicine, development of quantitative methods for non-invasive three dimensional characterization of engineered constructs (i.e. cells/tissue combined with scaffolds) becomes essential. In this study, we have defined the most optimal staining conditions for contrast-enhanced nanofocus computed tomography for three dimensional visualization and quantitative analysis of in vitro engineered neo-tissue (i.e. extracellular matrix containing cells) in perfusion bioreactor-developed Ti6Al4V constructs. A fractional factorial 'design of experiments' approach was used to elucidate the influence of the staining time and concentration of two contrast agents (Hexabrix and phosphotungstic acid) and the neo-tissue volume on the image contrast and dataset quality. Additionally, the neo-tissue shrinkage that was induced by phosphotungstic acid staining was quantified to determine the operating window within which this contrast agent can be accurately applied. For Hexabrix the staining concentration was the main parameter influencing image contrast and dataset quality. Using phosphotungstic acid the staining concentration had a significant influence on the image contrast while both staining concentration and neo-tissue volume had an influence on the dataset quality. The use of high concentrations of phosphotungstic acid did however introduce significant shrinkage of the neo-tissue indicating that, despite sub-optimal image contrast, low concentrations of this staining agent should be used to enable quantitative analysis. To conclude, design of experiments allowed us to define the most optimal staining conditions for contrast-enhanced nanofocus computed tomography to be used as a routine screening tool of neo-tissue formation in Ti6Al4V constructs, transforming it into a robust three dimensional quality control methodology.

Pseudorabies virus (PRV) initially replicates in the porcine upper respiratory tract. It easily invades the mucosae and submucosae for subsequent spread throughout the body via blood vessels and nervous system. In this context, PRV developed ingenious processes to overcome different barriers such as epithelial cells and the basement membrane. Another important but often overlooked barrier is the substantial mucus layer which coats the mucosae. However, little is known about how PRV particles interact with porcine respiratory mucus. We therefore measured the barrier properties of porcine tracheal respiratory mucus, and investigated the mobility of nanoparticles including PRV in this mucus. We developed an in vitro model utilizing single particle tracking microscopy. Firstly, the mucus pore size was evaluated with polyethylene glycol coupled (PEGylated) nanoparticles and atomic force microscope. Secondly, the mobility of PRV in porcine tracheal respiratory mucus was examined and compared with that of negative, positive and PEGylated nanoparticles. The pore size of porcine tracheal respiratory mucus ranged from 80 to 1500 nm, with an average diameter of 455±240 nm. PRV (zeta potential: -31.8±1.5 mV) experienced a severe obstruction in porcine tracheal respiratory mucus, diffusing 59-fold more slowly than in water. Similarly, the highly negatively (-49.8±0.6 mV) and positively (36.7±1.1 mV) charged nanoparticles were significantly trapped. In contrast, the nearly neutral, hydrophilic PEGylated nanoparticles (-9.6±0.8 mV) diffused rapidly, with the majority of particles moving 50-fold faster than PRV. The mobility of the particles measured was found to be related but not correlated to their surface charge. Furthermore, PEGylated PRV (-13.8±0.9 mV) was observed to diffuse 13-fold faster than native PRV. These findings clearly show that the mobility of PRV was significantly hindered in porcine tracheal respiratory mucus, and that the obstruction of PRV was due to complex mucoadhesive interactions including charge interactions rather than size exclusion.