Perfused Platforms to Mimic Bone Microenvironment at the Macro/Milli/Microscale: Pros and Cons

As life expectancy increases, the population experiences progressive ageing. Ageing, in turn, is connected to an increase in bone-related diseases (i.e., osteoporosis and increased risk of fractures). Hence, the search for new approaches to study the occurrence of bone-related diseases and to develop new drugs for their prevention and treatment becomes more pressing. However, to date, a reliable in vitro model that can fully recapitulate the characteristics of bone tissue, either in physiological or altered conditions, is not available. Indeed, current methods for modelling normal and pathological bone are poor predictors of treatment outcomes in humans, as they fail to mimic the in vivo cellular microenvironment and tissue complexity. Bone, in fact, is a dynamic network including differently specialized cells and the extracellular matrix, constantly subjected to external and internal stimuli. To this regard, perfused vascularized models are a novel field of investigation that can offer a new technological approach to overcome the limitations of traditional cell culture methods. It allows the combination of perfusion, mechanical and biochemical stimuli, biological cues, biomaterials (mimicking the extracellular matrix of bone), and multiple cell types. This review will discuss macro, milli, and microscale perfused devices designed to model bone structure and microenvironment, focusing on the role of perfusion and encompassing different degrees of complexity. These devices are a very first, though promising, step for the development of 3D in vitro platforms for preclinical screening of novel anabolic or anti-catabolic therapeutic approaches to improve bone health

Dynamic Coculture of a Prevascularized Engineered Bone Construct

The generation of functional, vascularized tissues is a key challenge for the field of tissue engineering. Before clinical implantations of tissue engineered bone constructs can succeed, in vitro fabrication needs to address limitations in large-scale tissue development, including controlled osteogenesis and an inadequate vasculature network to prevent necrosis of large constructs. The tubular perfusion system (TPS) bioreactor is an effective culturing method to augment osteogenic differentiation and maintain viability of human mesenchymal stem cell (hMSC)-seeded scaffolds while they are developed in vitro. To further enhance this process, we developed a novel osteogenic growth factors delivery system for dynamically cultured hMSCs using microparticles encapsulated in three-dimensional alginate scaffolds. In light of this increased differentiation, we characterized the endogenous cytokine distribution throughout the TPS bioreactor. An advantageous effect in the ‘outlet’ portion of the uniaxial growth chamber was discovered due to the system’s downstream circulation and the unique modular aspect of the scaffolds. This unique trait allowed us to carefully tune the differentiation behavior of specific cell populations. We applied the knowledge gained from the growth profile of the TPS bioreactor to culture a high-volume bone composite in a 3D-printed femur mold. This resulted in a tissue engineered bone construct with a volume of 200cm3, a 20-fold increase over previously reported sizes. We demonstrated high viability of the cultured cells throughout the culture period as well as early signs of osteogenic differentiation. Taking one step closer toward a viable implant and minimize tissue necrosis after implantation, we designed a composite construct by coculturing endothelial cells (ECs) and differentiating hMSCs, encouraging prevascularization and anastomosis of the graft with the host vasculature. We discovered the necessity of cell to cell proximity between the two cell types as well as preference for the natural cell binding capabilities of hydrogels like collagen. Notably, the results suggested increased osteogenic and angiogenic potential of the encapsulated cells when dynamically cultured in the TPS bioreactor, suggesting a synergistic effect between coculture and applied shear stress. This work highlights the feasibility of fabricating a high-volume, prevascularized tissue engineered bone construct for the regeneration of a critical size defect

Finite element evaluations of the mechanical properties of polycaprolactone/hydroxyapatite scaffolds by direct ink writing: Effects of pore geometry

Osteochondral (OC) defects usually involve the damage of both the cartilage and its underneath subchondral bone. In recent years, tissue engineering (TE) has become the most promising method that combines scaffolds, growth factors, and cells for the repair of OC defects. An ideal OC scaffold should have a gradient structure to match the hierarchical mechanical properties of natural OC tissue. To satisfy such requirements, 3D printing, e.g., direct ink writing (DIW), has emerged as a technology for precise and customized scaffold fabrication with optimized structures and mechanical properties. In this study, finite element simulations were applied to investigate the effects of pore geometry on the mechanical properties of 3D printed scaffolds. Scaffold specimens with different lay-down angles, filament diameters, inter-filament spacing, and layer overlaps were simulated in compressive loading conditions. The results showed that Young's moduli of scaffolds decreased linearly with increasing scaffold porosity. The orthotropic characteristics increased as the lay-down angle decreased from 90° to 15°. Moreover, gradient transitions within a wide range of strain magnitudes were achieved in a single construct by assembling layers with different lay-down angles. The results provide quantitative relationships between pore geometry and mechanical properties of lattice scaffolds, and demonstrate that the hierarchical mechanical properties of natural OC tissue can be mimicked by tuning the porosity and local lay-down angles in 3D printed scaffolds

Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture

The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels

Osteoblastogenic differentiation of mesenchymal stem cells through nanoscale stimulation: the conception of a novel 3D osteogenic bioreactor

Throughout this body of work low amplitude high frequency (500 Hz – 5000 Hz) mechanical stimulation and its effect to induce osteogenesis on bone marrow derived MSCs has been investigated. Due to the nanolevel amplitudes of these high frequency vertical vibrations the term nanokicking appeared to be appropriate and was subsequently used throughout this thesis to refer to these high frequency sinusoidal stimulations provided by the bioreactor. In the first instance this work was performed in 2D and biological analyses to determine osteogenesis were carried out at a transcript (mRNA), protein and mineralisation level. Affirmative results for osteogensis were observed from genes and proteins (RUNX2, osteocalin, osteopontin) related to the osteoblast phenotype by qRT PCR, in cell western, and immunostaining. To determine the prescence of inorganic osseous minerals, more specific techniques such as Raman spectroscopy, micro computed tomography and histological stainings (Von Kossa/Alizarin Red) were further employed. The results observed remained in line with previously published material (Gentleman et al., 2009) drawing the conclusion that calcium phosphate (Ca10(PO4)6, through nanokicking,was formed in vitro. The natural progession of this research meant that a novel vibrational bioreactor was conceived and designed, through the use of Lean and Six Sigma principles (Andrew Thomas, 2004; Caldwell, 2006), in order to assess the potential of nanokicking in 3D. Here collagen was employed as a biomimetic scaffold and affirmative results for osteogenesis were observed. The bioreactor was unique in that long term (up to 46 days) sterile culture was achieved, it was easy to use and there was no requirement for osteogenic media, growth factors or complex chemistries (e.g. dexamethasone, rhBMP2) in order to induce osteogenesis. The cost of use and maintence was relatively cheap compared to available commercial bioreactors (Rauh et al., 2011b). It is envisaged that this technology may one day have real world use for ossesous tissue regeneration and care in a GMP and clinical setting, or for the preparation of autologous tissue for medical testing in the burgeoning field of personalised medicine

Mass Production of Mesenchymal Stem Cells — Impact of Bioreactor Design and Flow Conditions on Proliferation and Differentiation

The book serves as a good starting point for anyone interested in the application of tissue engineering. It offers a colorful mix of topics, which explain the obstacles and possible solutions for TE applications. The first part covers the use of adult stem cells and their applications. The following chapters offer an insight into the development of a tailored biomaterial for organ replacement and highlight the importance of cell-biomaterial interaction. In summary, this book offers insights into a wide variety of cells, biomaterials, interfaces and applications of the next generation biotechnology, which is tissue engineering