A Focal Adhesion Filament Cross-correlation Kit for fast, automated segmentation and correlation of focal adhesions and actin stress fibers in cells

This is the software described in our article 'A Focal Adhesion Filament Cross-correlation Kit for fast, automated segmentation and correlation of focal adhesions and actin stress fibers in cells' and the used datasets for image analysis and correlation.The zip file contains the microscopy images and segmentation and analysis. The software itself is the executable java (.jar) file 'GUIFocalAdhesionOnly.jar

Examining Uptake of Nanomaterials by Eukaryotic Cells with Digital Image Cytometry

Due to their small size and related interesting properties, artificial nanoma-terials are utilized for a great number of biological and medical applications. Cell entry routes, intracellular trafficking and processing of nanoparticles, which determine their fate, efficiency, and toxicity, are depending on various parameters of the specific nanomaterial, such as size, surface charge, surface chemistry and elasticity. Nanoparticle-cell interactions are typically elucidated by means of fluorescence microscopy. Cell functions can be observed by a multiplicity of commercially available probes. For the quantification of cell features from images (image cytometry), computer-based algorithms are favoured to avoid bias introduced by the subjective perception of the observer. By applying high throughput microscopy in combination with digital image cytometry the screening of high numbers of cells is made possible. With the large quantity of obtained data, cell populations can be identified and, in general, results that are statistically meaningful are obtained. In the first part of this work this method is applied in order to examine the cellular responses upon exposure to plasmonic poly(methacrylic acid)-coated gold nanoparticles (Au NPs) with respect to morphology and viability of human endothelial and epithelial cells (HUVECs and HeLa cells). Au NPs of 4-5 nm size were chosen which had been thoroughly characterized in terms of their physico-chemical parameters. These particles bear interesting properties for biomedical applications and, for several years, have been in the focus of research. In this work significant impacts on mitochondrial and lysosomal morphology upon exposure to the Au NPs are reported. The alteration of the structure of the cytoskeleton and a dramatically reduced proliferation are described. Interestingly, the smallest dose inducing the described cellular responses was of one or two magnitudes lower than those, where acute cytotoxicity and an increase in the production of reactive oxygen species (ROS) were observed. In the second part the process of endocytosis of polymer capsules is examined. These systems are seen as a promising tool for intracellular cargo delivery and release. After lipid raft-mediated phagocytosis, the capsules are transferred from the neutral extracellular medium to increasingly acidic intracellular vesicles. By embedding a pH-sensitive fluorescent dye into the cavity of the capsule the uptake process and the associated acidification can be monitored time-dependently. It is demonstrated that the kinetic of the acidification process strongly depends on the stiffness of the capsules. Soft particles with minor stiffness are transported faster into lysosomal structures than stiffer ones. Additionally, these sensor particles are used to confirm the importance of the V1G1-subunit of the vacuolar ATPase being responsible for vesicle acidification

Preparation and Properties of Isolated Z-disks

Z-disks form the boundaries of the sarcomeres, the basic contractile units of muscle cells. Within the Z-line thin filaments containing mainly actin interdigitate and are crosslinked by α-actinin. Ends of the giant proteins titin and nebulin are also anchored in the Z-disk. The Z-line was originally thought to have the purely mechanical function of transmitting contractile force along the myofibrils. However, more recently, the Z-disk has emerged as a highly dynamic structure involved in stress sensing and important signaling pathways that govern muscle homeostasis. In order to fully understand how the Z-disk functions a detailed description of its molecular organization is essential. Even though the structure the structure of the Z-disk has been studied by electron microscopy techniques its molecular organization is known only in outline to a resolution of about 5 nm, whereas at least 3 nm is required to begin distinguishing protein shapes and to accurately dock crystal structure. Reports describing the isolation of intact Z-disks from insect indirect flight muscle date from 30-40 years ago, but these preparations have not been subjected to modern electron microscopy techniques. We improved the existing methods for the isolation of the Z-disk from honeybee flight muscle and investigated its structure using cryo-electron tomography and subtomogram averaging. The preliminary data indicate that the resolution was improved when compared with past studies of plastic sectioned muscle. We have also investigated the protein composition of the preparations to monitor the components that are washed away during preparation. Methods for the isolation of intact Z-disks from vertebrate muscle are not available. We explored strategies for isolating Z-disks from skeletal and cardiac muscle. Even though such a preparation has not been achieved we present promising approaches that, with optimization, should enable isolation of Z-disks from vertebrate muscle

Nanofiber-Based Scaffold for Integrative Anterior Cruciate Ligament Reconstruction

The anterior cruciate ligament (ACL) is the most frequently injured ligament of the knee, with upwards of 100,000 ACL reconstructions performed annually. Current grafting techniques are limited by insufficient integration with subchondral bone and donor site morbidity issues related to graft harvest, potentially resulting in revision surgery and long-term joint pain. Therefore, significant demand exists for alternative grafting solutions that do not require additional surgery and can regenerate the native ACL-to-bone interface to promote biological fixation of the implanted ACL graft. To address this need, the ideal system must be able to withstand the functional demands of the native tissue by demonstrating physiologically equivalent mechanical properties, be comprised of compositionally varying phases in order to recapitulate the inherent heterogeneity of the native ligament to bone transition and be biodegradable such that it is gradually replaced by the regenerated tissue following implantation. It is hypothesized that a biomimetic, multi-phased scaffold comprised of optimized bone, interface and ligament regions coupled with controlled chemical and/or mechanical stimulation in vitro will guide phase-specific differentiation of mesenchymal stem cells (MSC) and result in a biologically integrated bone-ligament-bone complex in vivo. Mesenchymal stem cells are particularly attractive for this application as they can be routinely harvested from bone marrow, have been shown to respond to chemical, mechanical and structural cues, and are capable of differentiating towards the primary cell types (fibroblasts, osteoblasts and chondrocytes) found within ligament, bone and the ligament-to-bone interface. To this end, a nanofiber-based synthetic graft was designed with compositionally-varying phases to regenerate ligament, bone and interface tissues. The ligament phase was optimized in terms of nanofiber alignment, composition, mechanical stimulation and chemical stimulation. It was demonstrated that an aligned nanofiber substrate coupled with controlled mechanical stimulation was necessary to differentiate MSCs towards a fibroblastic phenotype. The bone phase was optimized in terms of ceramic content and it was shown that a threshold of mineral incorporation into nanofibers was necessary to differentiate MSCs towards an osteogenic phenotype. Lastly, a mechanoactive nanofiber collar was designed to induce interface formation. It was demonstrated that compressive stimulation applied via nanofiber collar contraction induced chondrogenic differentiation of MSCs. Subsequently, the three phases were incorporated to form a synthetic graft, for which graft architecture and cell seeding density were optimized. The resulting graft was cultured in vitro under the optimized parameters, demonstrating the formation of distinct and structurally continuous regions of bone, interface and ligament tissue. The graft was implanted in vivo where it was shown to be suitable for ACL reconstruction as it maintained knee stability and promoted ligament regeneration. In summary, this thesis focuses on the design of a biomimetic, nanofiber-based, integrated bone-ligament-bone construct, and elucidates chemical, mechanical and scaffold design-related parameters that can guide MSC differentiation towards desired tissue types. The impact of these studies extends beyond ligament reconstruction as they yield valuable scaffold design criteria, establish scaffold and culturing-related parameters to induce stem cell differentiation and can readily be applied to the formation of interfaces between soft-to-hard tissues as well as other complex tissues

Controlled Delivery of Angiogenic and Arteriogenic Growth Factors from Biodegradable Poly(ester amide) Electrospun Fibers for Therapeutic Angiogenesis

Therapeutic angiogenesis relies on the delivery of exogenous growth factors to stimulate neovessel formation. However, systemic administration of angiogenic factors results in rapid clearance from the site of interest due to their short biological half-life. In this work, we are reporting controlled delivery of a ‘cocktail’ of growth factors, an angiogenic factor −fibroblast growth factor-2 (FGF2), and an arteriogenic factor −fibroblast growth factor-9 (FGF9), from biodegradable poly(ester amide) (PEA) electrospun fibers towards targeting neovascular formation and maturation. FGF2 and FGF9 were dual loaded into PEA fibers using a mixed blend and emulsion electrospinning technique. Matrigel tube formation and Boyden chamber assays were used to evaluate neovessel formation in vitro. Chick chorioallantoic membrane (CAM) model coupled with power Doppler ultrasound imaging, and ischemic hindlimb mouse model were employed to assess the in vivo angiogenic capacity of the delivery system. Co-released FGF2 and FGF9 from dual loaded PEA fibers enhanced endothelial cell tube formation, directed-migration of smooth muscle cells towards PDGF-BB, and tube stabilization in vitro. The 3D power Doppler volumes of the CAM displayed enhanced localized angiogenesis underneath the fibrous mats with enhanced blood perfusion and flow. Histological analysis of the ischemic tibialis anterior muscle revealed increased percentage of mural cell-covered microvessels in mice treated with FGF9-loaded PEA fibers than those treated with unloaded fibers. This supports the premise that controlled delivery of fibroblast growth factors from biodegradable PEA electrospun fibers can provide means for minimally invasive revascularization of ischemic tissues as a novel approach for treatment of ischemic vascular disease

3D-printed Synthetic Polymer Templates for Bone Tissue Engineering : Bulk Modifications and Osteoconduction Assessment

Syntetiske polymerbiomaterialer er enkle å bearbeide, biologisk inerte og brukes derfor i en rekke biomedisinske applikasjoner. Forskning har i lang tid fokusert på å øke biologiske aktivitet til slike materialers, og å tilpasse egenskapene til ulike bruksområder. Tredimensjonal (3D)-printing er velegnet til framstille biomaterialmaler med stor presisjon etter bestemte designparametre. Målet med denne avhandlingen var å undersøke 3D-printede syntetiske polymermaler for bruk til dyrkning og regenerasjon av beinvev (BTE). Undersøkelsene bestod av tre faser: Først ble det utført en systematisk litteraturundersøkelse for å analysere relevante faktorer ved bruk av 3D-printede, nedbrytbare maler og virkningen deres på beinregenerering i kraniale beindefekter hos ulike dyrearter (Studie I). En meta-analyse ble utført for å sammenligne nydannelse av bein for hver materialtype (polymerer, keramer eller kompositter). Man fant at effekten på beinregenereasjon var høyest hos kompositter bestående av polymerer og biokeramer, men også materialstrukturen gitt av 3D-printing. Parallelt ble det utført en studie på funksjonalisering av 3D-printede polykaprolakton (PCL) maler med gelatin (GL) som ble testet in vitro (Studie II). Til tross for at økt mengde GL (ved 8 og 16%) forbedret osteogen differensieringen av stamceller (fra rotter) ble malene ikke videreført på grunn av materialets lave strekkfasthet. I neste fase, ble poly(lactide-co-trimethylenecarbonate) (PLATMC) sammenlignet med PCL, og man fant at PLATMC hadde gunstigere både nedbrytnings- og mekaniske egenskaper enn PCL (studie III). I tillegg viste PLATMC seg bedre egnet for å fremme mineralisering av ekstracellulær matriks (ECM) fra humane stamceller in vitro. I en subkutan implantasjonsmodell i kanin (varighet 8 uker) var vertsresponsen på PLATMC mild, med innvekst av løst bindevevs og høy infiltrasjon av celler, der PCL bar preg av tett fibrøs vevsinnkapsling. Videre, når begge malene ble implantert i skallebensdefekter i kaniner, viste PLATMC-malene størst innvekst av bein. Det ble også funnet nydannelse av bein direkte på materialoverflaten, noe som hittil ikke beskrevet for syntetiske polymer. I tredje fasen valgte man å modifisere PLATMC ved å kombinere polymeren med hydroksapatitt (HA), et mineral og en viktig komponent i beinmasse. 3D-printede blandinger med ulike andeler HA (10, 30 og 50 %) ble sammenlignet med umodifisert PLATMC og testet for fysiske og biologiske egenskaper (Studie IV). Man fant at tilsatt HA reduserte strekkfastheten sammenlignet med ren PLATMC. HA10 viste noe redusert nedbrytningshastighet og lave nivåer av frigitt kalsium, mens de høye nedbrytningsprofilene til HA30 og HA50 ble tidlig ledsaget av omfattende frigivelse av kalsium. Ved bruk av stamceller (fra menneske) (in vitro), fant man for HA10 høyere mineralisering av ECM etter 14 og 21 dager enn for PLATMC alene, mens HA30 og HA50 ikke fremmet mineralisering i like stor grad. I tillegg viste HA30 og HA50 markant mindre beininnvekst når de ble implantert i skallebeinsdefekter i kaniner. Oppsummert fant man at umodifisert 3D-printet PLATMC fremmet mineralisering av ECM både in vitro og in vivo, men at man ved å tilsette HA i for store mengder, gjennom frigivelse av kalsium, forstyrrer denne prosessen i tillegg til å redusere materialets strekkfasthet. Resultatene fra disse studiene samlet støtter bruken av 3D-printede PLATMC-maler for beinregenerering.Synthetic polymer biomaterials are used in numerous biomedical applications providing biological inertness and ease of processing and shaping. Current research is directed towards boosting their biological activity, customized per application. 3D-printing is a promising technique for producing biomaterial templates with the required design parameters. The aim of the thesis was therefore to investigate the fabrication of osteoconductive 3D-printed synthetic polymer-based templates for bone tissue engineering (BTE). The investigation comprised three phases: In phase I, a literature survey was conducted, to review factors of relevance in applying potentially-degradable 3D-printed templates and their influence on bone regeneration in the calvarial bone defect (CBD) model, across various animal species (Study I). A meta-analysis was undertaken to compare the yield of new bone for each type of template material (polymer, ceramic or composites/blends). The highest impact on new bone formation was associated with the blended polymers and bioceramics, and the interconnected porosity generated by the 3D-printing. In parallel, an experimental study was undertaken on the functionalization of 3D-printed polycaprolactone (PCL) templates with gelatin (GL) due to its good biodegradation and biocompatibilty. Their physical and osteoconductive properties were tested in vitro (Study II). The biochemical compatibility contributed by GL (at 8 and 16%) improved the osteogenic differentiation of the seeded rat-BMSCs. However, this led to quite low tensile resistance and PCL/GL templates were therefore not studied in further in vivo trials. In phase II, poly(lactide-co-trimethylene carbonate) (PLATMC) was compared to PCL, and revealed that PLATMC had better degradation and mechanical properties than PCL (Study III), with prominent osteoconductivity and mineralized extracellular matrix (ECM) deposition (in vitro). In a subcutaneous implantation model in rabbits (8 weeks), the host response to PLATMC was mild, with loose connective tissue interface and high cellular invasion. In contrast, PCL was characterized by dense fibrous tissue encapsulation. When both templates were implanted in CBD in rabbits, PLATMC templates showed greater amount of new bone formation together with obvious contact osteogenesis presented on its surface, which was unique and unreported for a synthetic polymer before. In phase III, PLATMC was blended with hydroxyapatite (HA), in several ratios: 10 % HA (HA10), 30 % (HA30) and 50 % (HA50). Printability, physical, mechanical, and biological properties were compared (Study IV). The disclosed tensile properties of all 3D-printed HA blends were reduced, compared to PLATMC. HA10 showed reduced degradation and mild Ca release rate, while the high degradation profile of HA30 and HA50 was accompanied by massive early Ca release rates. On the biological aspect in vitro, using human-BMSCs seeded up to 28 days, HA10 disclosed higher mineralized ECM secretion at 14 and 21 days than PLATMC, while the osteoconductivity of HA30 and HA50 were markedly reduced and exhibited no advantages over pristine PLATMC templates. Moreover, HA30 and HA50, exhibited marked less osteoconductivity and reduced bone ingrowth when implanted in CBD. Thus high Ca release were correlated to reduced bone ingrowth and reduced osteoconduction, and the rate of Ca release should be considered in characterizing new HA-based templates. In summary, 3D-printed PLATMC showed promising osteoconductive activity, stimulating abundant mineralized ECM secretion in vitro, and demonstrated contact osteogenesis in vivo. However, the addition of HA reduced its tensile properties and high Ca release rates exhibited less osteoconductive properties than PLATMC. The results of these studies support the application of 3D-printed PLATMC templates for BTE.Doktorgradsavhandlin

Synthesis, Applications and Biological Impact of Nanocellulose

This book aims to highlight recent advances in the synthesis of nanocellulose and surface modifications for the design of functional nanocellulose, as well as its applications and potential biological impact. It features two review articles and four original research articles which targets a broad readership of chemists, materials scientists, biochemists, nanotechnologists and others with an interest in nanocellulose research