Objectives

We have observed clinical cases where bone is formed in the overlaying muscle covering surgically created bone defects treated with a hydroxyapatite/calcium sulphate biomaterial. Our objective was to investigate the osteoinductive potential of the biomaterial and to determine if growth factors secreted from local bone cells induce osteoblastic differentiation of muscle cells.

Bone tissue engineering is a promising strategy to treat the huge number of bone fractures caused by progressive population ageing and diseases i.e., osteoporosis. The bioactive and biomimetic materials design modulating cell behaviour can support healthy bone tissue regeneration. In this frame, type I collagen and hydroxyapatite (HA) have been often combined to produce biomimetic scaffolds. In addition, mesoporous bioactive glasses (MBGs) are known for their ability to promote the deposition of HA nanocrystals and their potential to incorporate and release therapeutic ions. Furthermore, the use of 3D printing technologies enables the effective design of scaffolds reproducing the natural bone architecture. This study aims to design biomimetic and bioactive 3D printed scaffolds that mimic healthy bone tissue natural features in terms of chemical composition, topography and biochemical cues. Optimised collagenous hybrid systems will be processed by means of extrusion 3D printing technologies to obtain high resolution bone-like structures. Protocols of human co-cultures of osteoblasts and osteoclasts will be developed and used to test the 3D scaffolds. Type I collagen has been combined with rod-like nano-HA and strontium containing MBGs (micro- and nano-sized particles) in order to obtain hybrid systems resembling the composition of native bone tissue. A comprehensive rheological study has been performed to investigate the potential use of the hybrid systems as biomaterial inks. Mesh-like structures have been obtained by means of extrusion-based technologies exploiting the freeform reversible embedding of suspended hydrogels (FRESH) approach. Different crosslinking methods have been tested to improve final constructs mechanical properties. Both crosslinked and non-crosslinked biomaterials were cultured with human osteoblasts and osteoclasts to assay the hybrid matrix biocompatibility as well as its influence on cell behaviour. Homogeneous hybrid systems have been successfully developed and characterised, proving their suitability as biomaterial inks for 3D printing technologies. Mesh-like structures have been extruded in a thermo-reversible gelatine slurry, exploiting the sol-gel transition of the systems under physiological conditions. Covalent bonds between collagen molecules have been promoted by genipin treatment, leading to a significant increase in matrix strength and stability. The collagen methacrylation and the further UV-crosslinking are under investigation as alternative promising method to reinforce the 3D structure during the printing process. Biological tests showed the potential of the developed systems especially for genipin treated samples, with a significant adhesion of primary cells. Collagenous hybrid systems proved their suitability for bioactive 3D printed structures design for bone tissue engineering. The multiple stimuli provided by the scaffold composition and structure will be investigated on both direct and indirect human osteoblasts and osteoclasts co-culture, according to the developed protocols

Cells directly probe and respond to the physicomechanical properties of their extracellular environment, a dynamic process which has been shown to play a key role in regulating both cellular adhesive processes and differential function. Recent studies indicate that stem cells show lineage-specific differentiation when cultured on substrates approximating the stiffness profiles of specific tissues. Although tissues are associated with ranging Young's modulus values for bulk rigidity, at the sub-cellular level, and particularly at the micro- and nanoscales, tissues are comprised of heterogeneous distributions of rigidity. Lithographic processes have been widely explored in cell biology for the generation of analytical substrates to probe cellular physicomechanical responses. In this work, we show for the first time that that direct-write e-beam exposure can significantly alter the rigidity of elastomeric PDMS substrates and develop a new class of two-dimensional elastomeric substrates with controlled patterned rigidity ranging from the micron to the nanoscale. The mechano-response of human mesenchymal stem cells to e-beam patterned substrates was subsequently probed in vitro and significant modulation of focal adhesion formation and osteochondral lineage commitment was observed as a function of both feature diameter and rigidity, establishing the groundwork for a new generation of biomimetic material interfaces

Aims: The aim of the research is the functionalization of biosurfaces by anchoring on them biomolecules involved in the process of osteointegration (cellular adhesion, proliferation, differentiation, migration, matrix mineralization). Alkaline phosphatase (ALP) was used as model protein, because it is involved in the mineralization processes. The functionalized surfaces are biomimetic, because they show the biological signals triggering new tissue generation. A rapid osseointegration are the final goal and a good response and fast healing of bad quality bones is one of the main issues. The devices of interest for the research are dental or orthopaedic implants and substitutes of small bones. Methods: Bioactive glasses of various compositions were employed as substrates. Bioactive glasses, when in contact with biological fluids, stimulate the precipitation of a hydroxyapatite layer on their surfaces, which in turn promotes effective osteointegration of the implant. Since bioactive glasses are prone to hydroxylation, they could be successfully functionalized and grafted by biomolecules. So the biomimetic materials considered will be bioactive both from a physicochemical (osteoconduction and apatite precipitation) and from a biochemical (osteoinduction) point of view. The research was focused first of all on the methods for developing active sites on the substrates. In the case of bioactive glasses the surface must be cleaned of any contaminants and the reactive hydroxyls activated. Results: The immobilization of ALP was performed both with and without spacer molecules and a comparison among the different techniques will be presented. XPS was used for the analysis of the immobilized enzyme on titanium and bioglasses and specific signals for its identification were set. After the addition of the specific substrate, the ALP activity was evaluated by UV-VIS spectroscopy. Conclusions: ALP was successfully grafted on the surface of bioactive glasses with and without the use of an intermediate layer of spacer molecules. The presence of ALP was determined on all the samples, as well as its enzymatic activity. Further analyses are necessary to evaluate the opportunity of using a spacer molecule. Cell adhesion and proliferation tests are in progress

The ability to generate replacement human tissues on demand is a major clinical need. Indeed the paucity of techniques in reconstructive surgery and trauma emphasize the urgent requirement for alternative strategies for the formation of new tissues and organs. The idea of biomimesis is to abstract good design principles and optimizations from nature and incorporate them in the construction of synthetic materials and structures. Direct appropriation of natural inorganic skeletons is also biomimetic since their unique properties inform us on ways to generate functional, optimized scaffolds. A number of well characterized natural skeletons were investigated as potential scaffolds for tissue regeneration using mesenchymal stem cell populations. Marine sponges, sea urchin skeletons and nacre were found to possess unique functional properties that supported human cell attachment, growth and proliferation and provided organic/ inorganic extracellular matrix analogues for guided tissue regeneration. A good understanding of the processses involved in biomineralisation and the emergence of complex inorganic forms has inspired synthetic strategies for the formation of biological analogues (organised inorganic materials with biological form). We have developed two functional examples of biological structures generated using biomimetic materials chemistry with applications for human tissue regeneration. Mineralised biopoly-saccharide microcapsules provided enclosed micro-environments with an appropriate physical structure and physiological milieu, for the support of the initial stages of tissue regeneration combined with a capacity to deliver human cells, plasmid DNA and controlled release of biological factors such as cytokines. Calcium carbonate porous microspheres analogous to microscopic coccolithophore shells provided a template for tissue formation and a mechanism for the delivery of DNA and functional biological factors. These biomi-metic structures have considerable potential as scaffolds for skeletal repair and regeneration, particularly when combined with inductive and stimulatory biological factors (cytokines, morphogens, signal molecules) and plasmid DNA carrying with them chemical cues that modulate and direct permanent tissue formation complimentary with the host