Thiol-gelatin-norbornene bioink for laser‐based high‐definition bioprinting

Two-photon polymerization (2PP) is a lithography-based 3D printing method allowing the fabrication of 3D structures with sub-micrometer resolution. This work focuses on the characterization of gelatin-norbornene (Gel-NB) bioinks which enables the embedding of cells via 2PP. The high reactivity of the thiol-ene system allows 2PP processing of cell-containing materials at remarkably high scanning speeds (1000 mm s(-1)) placing this technology in the domain of bioprinting. Atomic force microscopy results demonstrate that the indentation moduli of the produced hydrogel constructs can be adjusted in the 0.2-0.7 kPa range by controlling the 2PP processing parameters. Using this approach gradient 3D constructs are produced and the morphology of the embedded cells is observed in the course of 3 weeks. Furthermore, it is possible to tune the enzymatic degradation of the crosslinked bioink by varying the applied laser power. The 3D printed Gel-NB hydrogel constructs show exceptional biocompatibility, supported cell adhesion, and migration. Furthermore, cells maintain their proliferation capacity demonstrated by Ki-67 immunostaining. Moreover, the results demonstrate that direct embedding of cells provides uniform distribution and high cell loading independently of the pore size of the scaffold. The investigated photosensitive bioink enables high-definition bioprinting of well-defined constructs for long-term cell culture studies

Nanofibrous poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) scaffolds provide a functional microenvironment for cartilage repair

Articular cartilage defects, when repaired ineffectively, often lead to further deterioration of the tissue, secondary osteoarthritis and, ultimately, joint replacement. Unfortunately, current surgical procedures are unable to restore normal cartilage function. Tissue engineering of cartilage provides promising strategies for the regeneration of damaged articular cartilage. As yet, there are still significant challenges that need to be overcome to match the long-term mechanical stability and durability of native cartilage. Using electrospinning of different blends of biodegradable poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate), we produced polymer scaffolds and optimised their structure, stiffness, degradation rates and biocompatibility. Scaffolds with a poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) ratio of 1:0.25 exhibit randomly oriented fibres that closely mimic the collagen fibrillar meshwork of native cartilage and match the stiffness of native articular cartilage. Degradation of the scaffolds into products that could be easily removed from the body was indicated by changes in fibre structure, loss of molecular weight and a decrease in scaffold stiffness after one and four months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes revealed a hyaline-like cartilage matrix. The ability to fine tune the ultrastructure and mechanical properties using different blends of poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) allows to produce a cartilage repair kit for clinical use to reduce the risk of developing secondary osteoarthritis. We further suggest the development of a toolbox with tailor-made scaffolds for the repair of other tissues that require a ‘guiding’ structure to support the body’s self-healing process

Application of an acoustofluidic perfusion bioreactor for cartilage tissue engineering

Cartilage grafts generated using conventional static tissue engineering strategies are characterised by low cell viability, suboptimal hyaline cartilage formation and, critically, inferior mechanical competency, which limit their application for resurfacing articular cartilage defects. To address the limitations of conventional static cartilage bioengineering strategies and generate robust, scaffold-free neocartilage grafts of human articular chondrocytes, the present study utilised custom-built microfluidic perfusion bioreactors with integrated ultrasound standing wave traps. The system employed sweeping acoustic drive frequencies over the range of 890 to 910 kHz and continuous perfusion of the chondrogenic culture medium at a low-shear flow rate to promote the generation of three-dimensional agglomerates of human articular chondrocytes, and enhance cartilage formation by cells of the agglomerates via improved mechanical stimulation and mass transfer rates. Histological examination and assessment of micromechanical properties using indentation-type atomic force microscopy confirmed that the neocartilage grafts were analogous to native hyaline cartilage. Furthermore, in the ex vivo organ culture partial thickness cartilage defect model, implantation of the neocartilage grafts into defects for 16 weeks resulted in the formation of hyaline cartilage-like repair tissue that adhered to the host cartilage and contributed to significant improvements to the tissue architecture within the defects, compared to the empty defects. The study has demonstrated the first successful application of the acoustofluidic perfusion bioreactors to bioengineer scaffold-free neocartilage grafts of human articular chondrocytes that have the potential for subsequent use in second generation autologous chondrocyte implantation procedures for the repair of partial thickness cartilage defects

Application of an acoustofluidic perfusion bioreactor for cartilage tissue engineering

Cartilage grafts generated using conventional static tissue engineering strategies are characterised by low cell viability, suboptimal hyaline cartilage formation and, critically, inferior mechanical competency, which limit their application for resurfacing articular cartilage defects. To address the limitations of conventional static cartilage bioengineering strategies and generate robust, scaffold-free neocartilage grafts of human articular chondrocytes, the present study utilised custom-built microfluidic perfusion bioreactors with integrated ultrasound standing wave traps. The system employed sweeping acoustic drive frequencies over the range of 890 to 910 kHz and continuous perfusion of the chondrogenic culture medium at a low-shear flow rate to promote the generation of three-dimensional agglomerates of human articular chondrocytes, and enhance cartilage formation by cells of the agglomerates via improved mechanical stimulation and mass transfer rates. Histological examination and assessment of micromechanical properties using indentation-type atomic force microscopy confirmed that the neocartilage grafts were analogous to native hyaline cartilage. Furthermore, in the ex vivo organ culture partial thickness cartilage defect model, implantation of the neocartilage grafts into defects for 16 weeks resulted in the formation of hyaline cartilage-like repair tissue that adhered to the host cartilage and contributed to significant improvements to the tissue architecture within the defects, compared to the empty defects. The study has demonstrated the first successful application of the acoustofluidic perfusion bioreactors to bioengineer scaffold-free neocartilage grafts of human articular chondrocytes that have the potential for subsequent use in second generation autologous chondrocyte implantation procedures for the repair of partial thickness cartilage defects

Unpublished Mediterranean records of marine alien and cryptogenic species

Good datasets of geo-referenced records of alien species are a prerequisite for assessing the spatio-temporal dynamics of biological invasions, their invasive potential, and the magnitude of their impacts. However, with the exception of first records on a country level or wider regions, observations of species presence tend to remain unpublished, buried in scattered repositories or in the personal databases of experts. Through an initiative to collect, harmonize and make such unpublished data for marine alien and cryptogenic species in the Mediterranean Sea available, a large dataset comprising 5376 records was created. It includes records of 239 alien or cryptogenic taxa (192 Animalia, 24 Plantae, 23 Chromista) from 19 countries surrounding the Mediterranean Sea. In terms of records, the most reported Phyla in descending order were Chordata, Mollusca, Chlorophyta, Arthropoda, and Rhodophyta. The most recorded species was Caulerpa cylindracea, followed by Siganus luridus, Magallana sp. (cf. gigas or angulata) and Pterois miles. The dataset includes records from 1972 to 2020, with the highest number of records observed in 2018. Among the records of the dataset, Dictyota acutiloba is a first record for the Mediterranean Sea. Nine first country records are also included: the alga Caulerpa taxifolia var. distichophylla, the cube boxfish Ostracion cubicus, and the cleaner shrimp Urocaridella pulchella from Israel; the sponge Paraleucilla magna from Libya and Slovenia; the lumpfish Cyclopterus lumpus from Cyprus; the bryozoan Celleporaria vermiformis and the polychaetes Prionospio depauperata and Notomastus aberans from Malta

Nanostructure and mechanics of collagen fibrils from osteogenesis imperfecta mice and chronic asthma assessed with atomic force microscopy

A number of pathologies are characterized by long term functional impairment of tissues and organs in the human body. Some case pathologies are directly associated with gene mutations that alter the biosynthesis of collagen; the basic structural element and most vital protein of biological tissues in vertebrates.An example is osteogenesis imperfecta, a heritable bone disorder characterized by abnormally increased number of bone fractures. Other pathologies, however, develop over time as a result of tissue remodelling. In the case of asthma, the mechanical performance of the bronchial airways are now thought to be affliated with alterations in the quality and quantity of collagen fibrils. This thesis is based on the hypothesis that both the structure and biochemistry is altered in collagen{related pathologies, and as a result of these alterations the mechanical properties of collagen fibrils are impaired.The mechanical assessment of individual collagen fibrils (of larger than 100 nm in diameter) has been accomplished with a number of techniques, but atomic force microscopy (AFM) has been most widely used between several research groups and is the most promising technique at hand for this task. The main aim of this thesis was to assess the structure-mechanical function of collagen fibrils in osteogenesis imperfecta and asthma by employing AFM cantilever based-nanoindentation.Prior to conducting scientific studies, the AFM cantilever based-nanoindentation was successfully validated with conventional nanoindentation, a well established technique in thin film nanometrology. Collagen fibrils from the oim mouse model of osteogenesis imperfecta were mechanically characterized. This, as well as an in vitro study, using ribose, to artificially cross-link collagen fibrils, demonstrated that the indentation modulus was much dependent on the amount of non{enzymatic cross-links present in collagen fibrils. Further, it became clear that alterations in the structure can have an effect on the type of cross-link predominantly formed, as well as the hydration behaviour of collagen fibrils, and hence also on elasticity and in further instance likely on the ductility. Collagen fibrils from asthmatic donors showed close a trend towards a lower indentation modulus compared to collagen fibrils from healthy donors. An unparalleled biochemistry study could in part complement on this difference but further experimentation is required to draw conclusions with certainty.To date there are several avenues that could explain the nanomechanical changes seen in asthmatic collagen fibrils, yet the biochemical assays necessary to answer to these are, unfortunately, beyond the scope of this thesis. One possible change may be the amount of collagen types I and III, the abundant collagen types found in the submucosa. It is suggested that the fibril diameter of collagen type I is decreased with increasing the amount of collagen type III. The biochemistry study suggested a trend towards lower amount of type III collagen in asthmatics. In consequence of a possible decreased fibril diameter in asthmatics the density of the subepithelial layer in the airways could change and hence the mechanical properties at the tissue level. Beyond structural changes, there could also be changes in the immature to mature cross-links which would affect the fibril mechanics. This is supported by evidence of decreasing indentation modulus with increase of the immature to mature cross-linking ratio. Moreover, a strong effect of non-enzymatic glycation end products on collagen fibril mechanics was observed. The indentation modulus of collagen fibrils with artificially induced AGEs (advanced glycation end products) was significantly increased.This finding suggests that relative amount of glycosylated collagen fibrils in asthma could also be a contributing factor to the development of mechanical impairment of asthmatic airways. Nevertheless, findings within this thesis suggest that the biochemistry of collagen is directly associated with its mechanical performance. It has become clear during this thesis that the AFM serves as an important tool to characterize mechanically basic biological elements at the submicrometre scale. The technique used was highly effective towards associating the mechanics of individual collagen fibrils with pathology and biochemistry in model systems. Paralleled data are still needed to provide a holistic picture of a clear pathology state. Importantly, the technique is able to characterize the mechanics of individual collagen fibrils and the studies presented within this thesis have brought interesting results as well as further questions to be answered. With wider use of the technique it is anticipated that some of the issues raised within this thesis can also be discussed and answered by a larger research community

Load-bearing in cortical bone microstructure: selective stiffening and heterogeneous strain distribution at the lamellar level

An improved understanding of bone mechanics is vital in the development of evaluation strategies for patients at risk of bone fracture. The current evaluation approach based on bone mineral density (BMD) measurements lacks sensitivity, and it has become clear that as well as bone mass, bone quality should also be evaluated. The latter includes, among other parameters, the bone matrix material properties, which in turn depend on the hierarchical structural features that make up bone as well as their composition. Optimal load transfer, energy dissipation and toughening mechanisms have, to some extent, been uncovered in bone. Yet, the origin of these properties and their dependence upon the hierarchical structure and composition of bone are largely unknown. Here we investigate load transfer in the osteonal and sub-osteonal levels and the mechanical behaviour of osteonal lamellae and interlamellar areas during loading. Using cantilever-based nanoindentation, in situ microtensile testing during atomic force microscopy (AFM) and digital image correlation (DIC), we report evidence for a previously unknown mechanism. This mechanism transfers load and movement in a manner analogous to the engineered "elastomeric bearing pads" used in large engineering structures. µ-RAMAN microscopy investigations showed compositional differences between lamellae and interlamellar areas. The latter have lower collagen content but an increased concentration of noncollagenous proteins (NCPs). Hence, NC-enriched areas on the microscale might be similarly important for bone failure as ones on the nanoscale. Finally, we managed to capture stable crack propagation within the interlamellar areas in a time-lapsed fashion, proving their significant contribution towards fracture toughness