Hipster microcarriers: Exploring geometrical and topographical cues of non-spherical microcarriers in biomedical applications

Structure and organisation are key aspects of the native tissue environment, which ultimately condition cell fate via a myriad of processes, including the activation of mechanotransduction pathways. By modulating the...

Biofabrication of development-inspired scaffolds for regeneration of the annulus fibrosus macro- and microarchitecture

Annulus fibrosus (AF) tissue engineering is a promising strategy for repairing the degenerated intervertebral disc (IVD) and a research area that could benefit from improved tissue models to drive translation. AF tissue is composed of concentric layers of aligned collagen bundles arranged in an angle-ply pattern, an architecture which is challenging to recapitulate with current scaffold design strategies. In response to this need, we developed a strategy to print 3D scaffolds that induce cell and tissue organization into oriented patterns mimicking the AF. Polycaprolactone (PCL) was printed in an angle-ply macroarchitecture possessing microscale aligned topographical cues. The topography was achieved by extrusion through custom-designed printer nozzles which were either round or possessing circumferential sinusoidal peaks. Whereas the round nozzle produced extruded filaments with a slight uniaxial texture, patterned nozzles with peak heights of 60 or 120 μm produced grooves, 10.87 ± 3.09 μm or 17.77 ± 4.91 μm wide, respectively. Bone marrow derived mesenchymal stem cells (BM-MSCs) cultured on the scaffolds for four weeks exhibited similar degrees of alignment within ± 10 ° of the printing direction and upregulation of outer AF markers (COL1, COL12, SFRP, MKX, MCAM, SCX and TAGLN), with no statistically significant differences as a function of topography. Interestingly, the grooves generated by the patterned nozzles induced longitudinal end-to-end alignment of cells, capturing the arrangement of cells during fibrillogenesis. In contrast, topography produced from the round nozzle induced a continuous web of elongated cells without end-to-end alignment. Extracellular collagen I, decorin and fibromodulin were detected in patterns closely following cellular organization. Taken together, we present a single-step biofabrication strategy to induce anisotropic cellular alignments in x-, y-, and z-space, with potential application as an in vitro model for studying AF tissue morphogenesis and growth.

Investigating the Regulation of Neural Differentiation and Injury in PC12 Cells Using Microstructure Topographic Cues

In this study, we designed and manufactured a series of different microstructure topographical cues for inducing neuronal differentiation of cells in vitro, with different topography, sizes, and structural complexities. We cultured PC12 cells in these microstructure cues and then induced neural differentiation using nerve growth factor (NGF). The pheochromocytoma cell line PC12 is a validated neuronal cell model that is widely used to study neuronal differentiation. Relevant markers of neural differentiation and cytoskeletal F-actin were characterized. Cellular immunofluorescence detection and axon length analysis showed that the differentiation of PC12 cells was significantly different under different isotropic and anisotropic topographic cues. The expression differences of the growth cone marker growth-associated protein 43 (GAP-43) and sympathetic nerve marker tyrosine hydroxylase (TH) genes were also studied in different topographic cues. Our results revealed that the physical environment has an important influence on the differentiation of neuronal cells, and 3D constraints could be used to guide axon extension. In addition, the neurotoxin 6-hydroxydopamine (6-OHDA) was used to detect the differentiation and injury of PC12 cells under different topographic cues. Finally, we discussed the feasibility of combining the topographic cues and the microfluidic chip for neural differentiation research.

The Influence of the Surface Topographical Cues of Biomaterials on Nerve Cells in Peripheral Nerve Regeneration: A Review

The surface topographies of artificial implants including surface roughness, surface groove size and orientation, and surface pore size and distribution have a great influence on the adhesion, migration, proliferation, and differentiation of nerve cells in the nerve regeneration process. Optimizing the surface topographies of biomaterials can be a key strategy for achieving excellent cell performance in various applications such as nerve tissue engineering. In this review, we offer a comprehensive summary of the surface topographies of nerve implants and their effects on nerve cell behavior. This review also emphasizes the latest work progress of the layered structure of the natural extracellular matrix that can be imitated by the material surface topology. Finally, the future development of surface topographies on nerve regeneration was prospectively remarked.

Physical and mechanical cues affecting biomaterial-mediated plasmid DNA delivery: insights into non-viral delivery systems

Background Whilst traditional strategies to increase transfection efficiency of non-viral systems aimed at modifying the vector or the polyplexes/lipoplexes, biomaterial-mediated gene delivery has recently sparked increased interest. This review aims at discussing biomaterial properties and unravelling underlying mechanisms of action, for biomaterial-mediated gene delivery. DNA internalisation and cytoplasmic transport are initially discussed. DNA immobilisation, encapsulation and surface-mediated gene delivery (SMD), the role of extracellular matrix (ECM) and topographical cues, biomaterial stiffness and mechanical stimulation are finally outlined. Main text Endocytic pathways and mechanisms to escape the lysosomal network are highly variable. They depend on cell and DNA complex types but can be diverted using appropriate biomaterials. 3D scaffolds are generally fabricated via DNA immobilisation or encapsulation. Degradation rate and interaction with the vector affect temporal patterns of DNA release and transgene expression. In SMD, DNA is instead coated on 2D surfaces. SMD allows the incorporation of topographical cues, which, by inducing cytoskeletal re-arrangements, modulate DNA endocytosis. Incorporation of ECM mimetics allows cell type-specific transfection, whereas in spite of discordances in terms of optimal loading regimens, it is recognised that mechanical loading facilitates gene transfection. Finally, stiffer 2D substrates enhance DNA internalisation, whereas in 3D scaffolds, the role of stiffness is still dubious. Conclusion Although it is recognised that biomaterials allow the creation of tailored non-viral gene delivery systems, there still are many outstanding questions. A better characterisation of endocytic pathways would allow the diversion of cell adhesion processes and cytoskeletal dynamics, in order to increase cellular transfection. Further research on optimal biomaterial mechanical properties, cell ligand density and loading regimens is limited by the fact that such parameters influence a plethora of other different processes (e.g. cellular adhesion, spreading, migration, infiltration, and proliferation, DNA diffusion and release) which may in turn modulate gene delivery. Only a better understanding of these processes may allow the creation of novel robust engineered systems, potentially opening up a whole new area of biomaterial-guided gene delivery for non-viral systems.

Self-agglomerated collagen patterns govern cell behaviour

Reciprocity between cells and their surrounding extracellular matrix is one of the main drivers for cellular function and, in turn, matrix maintenance and remodelling. Unravelling how cells respond to their environment is key in understanding mechanisms of health and disease. In all these examples, matrix anisotropy is an important element, since it can alter the cell shape and fate. In this work, the objective is to develop and exploit easy-to-produce platforms that can be used to study the cellular response to natural proteins assembled into diverse topographical cues. We demonstrate a robust and simple approach to form collagen substrates with different topographies by evaporating droplets of a collagen solution. Upon evaporation of the collagen solution, a stain of collagen is left behind, composed of three regions with a distinct pattern: an isotropic region, a concentric ring pattern, and a radially oriented region. The formation and size of these regions can be controlled by the evaporation rate of the droplet and initial collagen concentration. The patterns form topographical cues inducing a pattern-specific cell (tenocyte) morphology, density, and proliferation. Rapid and cost-effective production of different self-agglomerated collagen topographies and their interfaces enables further study of the cell shape-phenotype relationship in vitro. Substrate topography and in analogy tissue architecture remains a cue that can and will be used to steer and understand cell function in vitro, which in turn can be applied in vivo, e.g. in optimizing tissue engineering applications.

Planar organization of airway epithelial cell morphology using hydrogel grooves during ciliogenesis fails to induce ciliary alignment

Topographical cues are known to influence cell organization both in native tissues and in vitro. In the trachea, the matrix beneath the epithelial lining is composed of collagen fibres that...

Bioengineered in vitro skeletal muscles as new tools for muscular dystrophies preclinical studies

Muscular dystrophies are a group of highly disabling disorders that share degenerative muscle weakness and wasting as common symptoms. To date, there is not an effective cure for these diseases. In the last years, bioengineered tissues have emerged as powerful tools for preclinical studies. In this review, we summarize the recent technological advances in skeletal muscle tissue engineering. We identify several ground-breaking techniques to fabricate in vitro bioartificial muscles. Accumulating evidence shows that scaffold-based tissue engineering provides topographical cues that enhance the viability and maturation of skeletal muscle. Functional bioartificial muscles have been developed using human myoblasts. These tissues accurately responded to electrical and biological stimulation. Moreover, advanced drug screening tools can be fabricated integrating these tissues in electrical stimulation platforms. However, more work introducing patient-derived cells and integrating these tissues in microdevices is needed to promote the clinical translation of bioengineered skeletal muscle as preclinical tools for muscular dystrophies.

Combined effect of shear stress and laser-patterned topography on Schwann cell outgrowth: synergistic or antagonistic?

Shear stress can act either synergistically or antagonistically with topographical cues in specific cell responses such as orientation and elongation.

Magnetically-driven 2D cells organization on superparamagnetic micromagnets fabricated by laser direct writing

We demonstrate a proof of concept for magnetically-driven 2D cells organization on superparamagnetic micromagnets fabricated by laser direct writing via two photon polymerization (LDW via TPP) of a photopolymerizable superparamagnetic composite. The composite consisted of a commercially available, biocompatible photopolymer (Ormocore) mixed with 4 mg/mL superparamagnetic nanoparticles (MNPs). The micromagnets were designed in the shape of squares with 70 µm lateral dimension. To minimize the role of topographical cues on the cellular attachment, we fabricated 2D microarrays similar with a chessboard: the superparamagnetic micromagnets alternated with non-magnetic areas of identical shape and lateral size as the micromagnets, made from Ormocore by LDW via TPP. The height difference between the superparamagnetic and non-magnetic areas was of ~ 6 µm. In the absence of a static magnetic field, MNPs-free fibroblasts attached uniformly on the entire 2D microarray, with no preference for the superparamagnetic or non-magnetic areas. Under a static magnetic field of 1.3 T, the fibroblasts attached exclusively on the superparamagnetic micromagnets, resulting a precise 2D cell organization on the chessboard-like microarray. The described method has significant potential for fabricating biocompatible micromagnets with well-defined geometries for building skin grafts adapted for optimum tissue integration, starting from single cell manipulation up to the engineering of whole tissues.