Biofabrication of multiscale bone extracellular matrix scaffolds for bone tissue engineering.

Interconnected porosity is critical to the design of regenerative scaffolds, as it permits cell migration, vascularisation and diffusion of nutrients and regulatory molecules inside the scaffold. 3D printing is a promising strategy to achieve this as it allows the control over scaffold pore size, porosity and interconnectivity. Thus, the aim of the present study was to integrate distinct biofabrication strategies to develop a multiscale porous scaffold that was not only mechanically functional at the time of implantation, but also facilitated rapid vascularisation and provided stem cells with appropriate cues to enable their differentiation into osteoblasts. To achieve this, polycaprolactone (PCL) was functionalised with decellularised bone extracellular matrix (ECM), to produce osteoinductive filaments for 3D printing. The addition of bone ECM to the PCL not only increased the mechanical properties of the resulting scaffold, but also increased cellular attachment and enhanced osteogenesis of mesenchymal stem cells (MSCs). In vivo, scaffold pore size determined the level of vascularisation, with a larger filament spacing supporting faster vessel in-growth and more new bone formation. By freeze-drying solubilised bone ECM within these 3D-printed scaffolds, it was possible to introduce a matrix network with microscale porosity that further enhanced cellular attachment in vitro and increased vessel infiltration and overall levels of new bone formation in vivo. To conclude, an "off-the-shelf" multiscale bone-ECM-derived scaffold was developed that was mechanically stable and, once implanted in vivo, will drive vascularisation and, ultimately, lead to bone regeneration.

Biophysical cues enhance myogenesis of human adipose derived stem/stromal cells.

Adipose-derived stem/stromal cell (ASC)-based tissue engineered muscle grafts could provide an effective alternative therapy to autografts - which are limited by their availability - for the regeneration of damaged muscle. However, the current myogenic potential of ASCs is limited by their low differentiation efficiency into myoblasts. The aim of this study was to enhance the myogenic response of human ASCs to biochemical cues by providing biophysical stimuli (11% cyclic uniaxial strain, 0.5 Hz, 1h/day) to mimic the cues present in the native muscle microenvironment. ASCs elongated and fused upon induction with myogenic induction medium alone. Yet, their myogenic characteristics were significantly enhanced with the addition of biophysical stimulation; the nuclei per cell increased approximately 4.5-fold by day 21 in dynamic compared to static conditions (23.3 ± 7.3 vs. 5.2 ± 1.6, respectively), they aligned at almost 45° to the direction of strain, and exhibited significantly higher expression of myogenic proteins (desmin, myoD and myosin heavy chain). These results demonstrate that mimicking the biophysical cues inherent to the native muscle microenvironment in monolayer ASC cultures significantly improves their differentiation along the myogenic lineage.

Spatial regulation of human mesenchymal stem cell differentiation in engineered osteochondral constructs: effects of pre-differentiation, soluble factors and medium perfusion.

OBJECTIVE: The objective of the study was to investigate the combined effects of three sets of regulatory factors: cell pre-differentiation, soluble factors and medium perfusion on spatial control of human mesenchymal stem cell (hMSC) differentiation into cells forming the cartilaginous and bone regions in engineered osteochondral constructs. DESIGN: Bone-marrow derived hMSCs were expanded in their undifferentiated state (UD) or pre-differentiated (PD) in monolayer culture, seeded into biphasic constructs by interfacing agarose gels and bone scaffolds and cultured for 5 weeks either statically (S) or in a bioreactor (BR) with perfusion of medium through the bone region. Each culture system was operated with medium containing either chondrogenic supplements (C) or a cocktail (Ck) of chondrogenic and osteogenic supplements. RESULTS: The formation of engineered cartilage in the gel region was most enhanced by using undifferentiated cells and chondrogenic medium, whereas the cartilaginous properties were negatively affected by using pre-differentiated cells or the combination of perfusion and cocktail medium. The formation of engineered bone in the porous scaffold region was most enhanced by using pre-differentiated cells, perfusion and cocktail medium. Perfusion also enhanced the integration of bone and cartilage regions. CONCLUSIONS: (1) Pre-differentiation of hMSCs before seeding on scaffold was beneficial for bone but not for cartilage formation. (2) The combination of medium perfusion and cocktail medium inhibited chondrogenesis of hMSCs. (3) Perfusion improved the cell and matrix distribution in the bone region and augmented the integration at the bone-cartilage interface. (4) Osteochondral grafts can be engineered by differentially regulating the culture conditions in the two regions of the scaffold seeded with hMSCs (hydrogel for cartilage, perfused porous scaffold for bone).