(Photo-)crosslinkable gelatin derivatives for biofabrication applications

Over the recent decades gelatin has proven to be very suitable as an extracellular matrix mimic for bio-fabrication and tissue engineering applications. However, gelatin is prone to dissolution at typical cell culture conditions and is therefore often chemically modified to introduce (photo-)crosslinkable functionalities. These modifications allow to tune the material properties of gelatin, making it suitable for a wide range of biofabrication techniques both as a bioink and as a biomaterial ink (component). The present review provides a non-exhaustive overview of the different reported gelatin modification strategies to yield crosslinkable materials that can be used to form hydrogels suitable for biofabrication applications. The different crosslinking chemistries are discussed and classified according to their mechanism including chain-growth and step-growth polymerization. The step-growth polymerization mechanisms are further classified based on the specific chemistry including different (photo-)click chemistries and reversible systems. The benefits and drawbacks of each chemistry are also briefly discussed. Furthermore, focus is placed on different biofabrication strategies using either inkjet, deposition or light-based additive manufacturing techniques, and the applications of the obtained 3D constructs

Polymer Crosslinking: a new Strategy to Enhance Mechanical Properties and Structural Stability of Bioactive Glasses

The organic-inorganic hybrids fabricated by the sol-gel method are intrinsically bioactive materials with extensive applications in bone tissue engineering. The brittleness and limited water uptake capacity of these monoliths, however, restrict their applications for engineering the soft tissues and their interfaces with bone. To address these challenges, we developed a unique method in which polymer crosslinking was used to cease the over-condensation of a bioactive glass component and eradicate the formation of brittle structure. In this study, an organosilane-functionalized gelatin methacrylate was covalently bonded to a bioactive glass during the sol-gel process, and the condensation of silica networks was controlled by polymer-crosslinking. The physicochemical properties and mechanical strength of these hybrid hydrogels were then tuned by the incorporation of secondary crosslinking agents such as poly(ethylene glycol diacrylate). The resulting elastic hydrogels displayed tuneable compressive modulus in the range of 42 kPa to 530 kPa. The swelling behaviours of these hybrids and their structural integrities were also favourable for tissue engineering applications. Moreover, these hybrid hydrogels kept their structures for more than 28 days in simulated body fluid. The bioactivity of the constructs due to the presence of silica networks were confirmed by detecting nearly 2-fold increase in the alkaline phosphatase activity of the cultured bone progenitor cells on these hybrid hydrogels within 28 days of in vitro culture. Within the same period, in vivo studies on mice subcutaneous model showed that the hybrid hydrogels were highly biocompatible and well-tolerated. In summary, the bioactivity of the constructs, their tuneable physicochemical properties, the outstanding biocompatibility, and biodegradability of the hybrid hydrogels showed the high potential of the developed technique for fabrication of constructs for a variety of soft and hard tissue regeneration

Polymer Crosslinking: a new Strategy to Enhance Mechanical Properties and Structural Stability of Bioactive Glasses

The organic-inorganic hybrids fabricated by the sol-gel method are intrinsically bioactive materials with extensive applications in bone tissue engineering. The brittleness and limited water uptake capacity of these monoliths, however, restrict their applications for engineering the soft tissues and their interfaces with bone. To address these challenges, we developed a unique method in which polymer crosslinking was used to cease the over-condensation of a bioactive glass component and eradicate the formation of brittle structure. In this study, an organosilane-functionalized gelatin methacrylate was covalently bonded to a bioactive glass during the sol-gel process, and the condensation of silica networks was controlled by polymer-crosslinking. The physicochemical properties and mechanical strength of these hybrid hydrogels were then tuned by the incorporation of secondary crosslinking agents such as poly(ethylene glycol diacrylate). The resulting elastic hydrogels displayed tuneable compressive modulus in the range of 42 kPa to 530 kPa. The swelling behaviours of these hybrids and their structural integrities were also favourable for tissue engineering applications. Moreover, these hybrid hydrogels kept their structures for more than 28 days in simulated body fluid. The bioactivity of the constructs due to the presence of silica networks were confirmed by detecting nearly 2-fold increase in the alkaline phosphatase activity of the cultured bone progenitor cells on these hybrid hydrogels within 28 days of in vitro culture. Within the same period, in vivo studies on mice subcutaneous model showed that the hybrid hydrogels were highly biocompatible and well-tolerated. In summary, the bioactivity of the constructs, their tuneable physicochemical properties, the outstanding biocompatibility, and biodegradability of the hybrid hydrogels showed the high potential of the developed technique for fabrication of constructs for a variety of soft and hard tissue regeneration

Achieving Controlled Biomolecule-Biomaterial Conjugation

The conjugation of biomolecules can impart materials with the bioactivity necessary to modulate specific cell behaviors. While the biological roles of particular polypeptide, oligonucleotide, and glycan structures have been extensively reviewed, along with the influence of attachment on material structure and function, the key role played by the conjugation strategy in determining activity is often overlooked. In this review, we focus on the chemistry of biomolecule conjugation and provide a comprehensive overview of the key strategies for achieving controlled biomaterial functionalization. No universal method exists to provide optimal attachment, and here we will discuss both the relative advantages and disadvantages of each technique. In doing so, we highlight the importance of carefully considering the impact and suitability of a particular technique during biomaterial design

Microenvironments to regulate cellular behavior for neural development and regeneration

Strategies for regeneration after injury or during aging require the development of biomaterials able to reconstruct the essential components and properties of natural extracellular microenvironment. A particular feature in neural regeneration is the oriented disposition of neurons within nerves and cortex. In this thesis, through the spatiotemporal control of the availability of adhesive ligands at the surface of a biomaterial, these biomaterials allow directing the migration of neuron. This Thesis is structured in four parts. The first part presents microcontact printed patterns with adhesive compositions and geometries to allow directional migration and, uniquely, in vitro reconstruction of the somal translocation events occurring during cortical layering. In part 2 in situ directed neurites extension in defined directions is demonstrated using biomaterials functionalized with photo-activatable peptidomimetics of the laminin. In part 3 spatiotemporal and reversible regulations of actin dynamics in living cells is demonstrated using light-dosed delivery of Cytochalasin D. In the last part, the first demonstration of a light-regulated adhesive interaction between mammalian cells and a bacterial biointerface is provided.Strategien zur Regeneration nach Verletzungen oder während des Alterns benötigen die Entwicklung von Biomaterialien, die essentielle Komponenten und Eigenschaften der nativen extrazellulären Mikroumgebung rekonstruieren können. Eine besondere Eigenschaft in der Regeneration von Nervengewebe ist die elongierte Morphologie und gerichtete Disposition von Neuronen in Nerven und Kortex. Die räumlich-zeitliche Kontrolle der Verfügbarkeit von Zell-adhesiven Liganden auf der Oberfläche des Biomaterials, wie in dieser These beschrieben,erlaubt es hierbei die Migration von Neuronen steuern. Diese These ist in vier Abschnitte gegliedert. Der erste Teil präsentiert Mikrokontakt gedruckte Muster mit optimisierten, adhesiven Komponenten und Geometrien, um eine gerichtete Migration und in vitro erstmalig die Rekonstruktion der somalen Translokation zu gewährleisten, welche während der embryonalen Entwicklung des zerebralen Kortex stattfindet. In Teil 2 wird in situ der gerichtete Neuritenauswuchs in definierter Richtung gezeigt. Hierbei werden Biomaterialien verwendet, die mit Photo-aktivierbaren Peptidomimetika des Matrixproteins Laminin funktionalisiert sind. Im Teil 3 wird die räumlich-zeitliche und reversible Regulation der Dynamik des Actinzytoskellettes unter Zugabe von Licht-dosiertem Cytochalasin D gezeigt. Im letzten Teil, wird erstmalig die Licht-regulierte Interaktion zwischen Säugetierzellen und einer bakteriellen Biogrenzfläche demonstriert

Study of collagen organization in cell-laden hydrogels and animal tissue samples for effective tissue engineering scaffolds

The interaction of biomaterials with biological systems is a complex process, that is triggered in response to implants and wounds. It is essential to understand the phases of wound healing response, particularly the interactions of immune cells such as macrophages and fibroblasts, with the local extracellular matrix which can influence implant acceptance or the restoration of the damaged wound site. Materials properties such as compressive modulus, surface geometry, functionalization, and topology can be tuned to modulate the inflammatory and fibrotic responses to wounds and implants. Naturally derived materials, such as alginate, are widely used biomaterials owing to their biocompatibility and the diverse crosslinking strategies that can be used for fabrication. Soft alginate gels can be synthesized after methacrylation to be relatively stable under physiological conditions, while retaining pH sensitivity, which can be useful in the treatment of chronic wounds. Studying the collagen response to NIH/3T3 fibroblasts encapsulated in these soft hydrogels can develop wound healing strategies to promote faster wound healing. The transition of collagen organization from aligned to isotropic states in the dually crosslinked stiffer methacrylated alginate (ALGMA) hydrogels shows promise towards the development of topical gels for wound care. Modifying the surface properties using arginine-like derivatives is effective in modulating the fibroblast response to implanted glass beads in SKH1-E mice. Collagen response to modified glass beads using SHG microscopy was evaluated using several factors such as collagen amount, secretion of collagen III, and organization of collagen. The albizziin modification showed both isotropic collagen organization as well similar collagen type III as unwounded skin. Furthermore, statistical analysis uncovered correlations between SHG derived parameters and the materials properties of the chemical modifiers. Collagen type III was correlated with the surface tension of the modifier, and an empirical equation was derived relating materials parameters with the observed collagen measurements. The effectiveness of diverse wound care strategies on shallow and deep wounds on porcine subjects was conducted using SHG microscopy. Treatment duration, as well as scaffold preparation were instrumental in reducing a scarring response and accelerating wound closure rates. By combining the understanding of wound healing in diverse tissue environments, with environmentally responsive wound dressings, it is possible to improve the quality of life for millions of patients across the world

Optically manipulated control over micron-scale signalling dynamics for directing cellular differentiation and migration

Cellular microenvironments are an important area of study, and their implications with regard to development, tissue function, and disease, mean that they have particular relevance in tissue engineering. The development of tissue engineered therapeutics is underpinned by the understanding of how the cells exist in their natural environment. A fundamental lack of insight into the signalling mechanisms within microenvironments, due to in part a lack of appropriate technologies, has meant that the therapeutic potential of tissue engineering is limited. To this end, the development of a micropatterning technology that enables control over solute signalling dynamics on the micron scale has been investigated. A bespoke holographic optical tweezers (HOTs) system was used to precisely position cells and controlled release vehicles into three-dimensional arrangements that resemble basic cellular micro-architectures. Via optical manipulation, release vehicles could be patterned to create solute release patterns to mimic signalling events in vitro. A proof of concept was established to demonstrate fluorophore release from microparticles positioned with high precision, into previously unobtainable micron-scale patterns. Such developments required optimisation of the system and protocols, for use with cell and microparticle manipulation and, creating a tool-set suitable for address unsolved biological questions. Biological investigations were completed to demonstrate how the HOTs can be used to control zonal cell differentiation and migration. These processes are paramount to cell microenvironment function, and this study has shown that the HOTs patterning setup is capable of achieving such signalling models in vitro. Herein is presented compelling evidence that optically manipulated release sources can achieve new levels of precision over signalling dynamics, over the length scales suitable for even the smallest cell microenvironments. It is hoped that through the better in vitro modelling of such cellular microenvironments and other signalling events, investigators will be able to elucidate new mechanisms through which cells proliferate and function