Compounded topographical and physicochemical cueing by micro-engineered chitosan substrates on rat dorsal root ganglion neurons and human mesenchymal stem cells.

Given the intertwined physicochemical effects exerted in vivo by both natural and synthetic (e.g., biomaterial) interfaces on adhering cells, the evaluation of structure-function relationships governing cellular response to micro-engineered surfaces for applications in neuronal tissue engineering requires the use of in vitro testing platforms which consist of a clinically translatable material with tunable physiochemical properties. In this work, we micro-engineered chitosan substrates with arrays of parallel channels with variable width (20 and 60 µm). A citric acid (CA)-based crosslinking approach was used to provide an additional level of synergistic cueing on adhering cells by regulating the chitosan substrate's stiffness. Morphological and physicochemical characterization was conducted to unveil the structure-function relationships which govern the activity of rat dorsal root ganglion neurons (DRGs) and human mesenchymal stem cells (hMSCs), ultimately singling out the key role of microtopography, roughness and substrate's stiffness. While substrate's stiffness predominantly affected hMSC spreading, the modulation of the channels' design affected the neuronal architecture's complexity and guided the morphological transition of hMSCs. Finally, the combined analysis of tubulin expression and cell morphology allowed us to cast new light on the predominant role of the microtopography over substrate's stiffness in the process of hMSCs neurogenic differentiation.

The Implication of Spatial Statistics in Human Mesenchymal Stem Cell Response to Nanotubular Architectures.

INTRODUCTION: In recent years there has been ample interest in nanoscale modifications of synthetic biomaterials to understand fundamental aspects of cell-surface interactions towards improved biological outcomes. In this study, we aimed at closing in on the effects of nanotubular TiO2 surfaces with variable nanotopography on the response on human mesenchymal stem cells (hMSCs). Although the influence of TiO2 nanotubes on the cellular response, and in particular on hMSC activity, has already been addressed in the past, previous studies overlooked critical morphological, structural and physical aspects that go beyond the simple nanotube diameter, such as spatial statistics. METHODS: To bridge this gap, we implemented an extensive characterization of nanotubular surfaces generated by anodization of titanium with a focus on spatial structural variables including eccentricity, nearest neighbour distance (NND) and Voronoi entropy, and associated them to the hMSC response. In addition, we assessed the biological potential of a two-tiered honeycomb nanoarchitecture, which allowed the detection of combinatory effects that this hierarchical structure has on stem cells with respect to conventional nanotubular designs. We have combined experimental techniques, ranging from Scanning Electron (SEM) and Atomic Force (AFM) microscopy to Raman spectroscopy, with computational simulations to characterize and model nanotubular surfaces. We evaluated the cell response at 6 hrs, 1 and 2 days by fluorescence microscopy, as well as bone mineral deposition by Raman spectroscopy, demonstrating substrate-induced differential biological cueing at both the short- and long-term. RESULTS: Our work demonstrates that the nanotube diameter is not sufficient to comprehensively characterize nanotubular surfaces and equally important parameters, such as eccentricity and wall thickness, ought to be included since they all contribute to the overall spatial disorder which, in turn, dictates the overall bioactive potential. We have also demonstrated that nanotubular surfaces affect the quality of bone mineral deposited by differentiated stem cells. Lastly, we closed in on the integrated effects exerted by the superimposition of two dissimilar nanotubular arrays in the honeycomb architecture. DISCUSSION: This work delineates a novel approach for the characterization of TiO2 nanotubes which supports the incorporation of critical spatial structural aspects that have been overlooked in previous research. This is a crucial aspect to interpret cellular behaviour on nanotubular substrates. Consequently, we anticipate that this strategy will contribute to the unification of studies focused on the use of such powerful nanostructured surfaces not only for biomedical applications but also in other technology fields, such as catalysis.

Deterministic paracrine repair of injured myocardium using microfluidic-based cocooning of heart explant-derived cells.

While encapsulation of cells within protective nanoporous gel cocoons increases cell retention and pro-survival integrin signaling, the influence of cocoon size and intra-capsular cell-cell interactions on therapeutic repair are unknown. Here, we employ a microfluidic platform to dissect the impact of cocoon size and intracapsular cell number on the regenerative potential of transplanted heart explant-derived cells. Deterministic increases in cocoon size boosted the proportion of multicellular aggregates within cocoons, reduced vascular clearance of transplanted cells and enhanced stimulation of endogenous repair. The latter being attributable to cell-cell stimulation of cytokine and extracellular vesicle production while also broadening of the miRNA cargo within extracellular vesicles. Thus, by tuning cocoon size and cell occupancy, the paracrine signature and retention of transplanted cells can be enhanced to promote paracrine stimulation of endogenous tissue repair.

Collagen-Based Microcapsules As Therapeutic Materials for Stem Cell Therapies in Infarcted Myocardium.

As cell therapies emerged, it was quickly realized that pro-regenerative cells directly injected into injured tissue struggled within the inflammatory microenvironment. By using microencapsulation, i.e., encapsulating cells within polymeric biomaterials, they are henceforth protected from the harmful extracellular cues, while still being able to receive oxygen and nutrients and release secreted factors. Previous work showed that stem cells encapsulated within a biologically inert material (agarose) were able to significantly improve the function of the infarcted mouse heart. With the aim of using more bioresponsive microcapsules, we sought to develop an enzymatically degradable, type I collagen-based microcapsule for the intramyocardial delivery of bone marrow-derived mesenchymal stromal cells in a murine model of myocardial infarction.

Elucidating structure-function relationships governing the interfacial response of human mesenchymal stem cells to polydopamine coatings.

Deposition of mussel-inspired polydopamine (PDA) has rapidly emerged as a simple yet effective strategy to functionalize the surface of biomaterials. The experimental simplicity of the deposition process, combined with native bioactivity and bioadhesive properties, make PDA an attractive solution for biomedical applications, ranging from functional biomaterials for tissue engineering to antibacterial surfaces. Unveiling the interplay among deposition parameters, physicochemical properties of the resulting structures and their functions, is a fundamental aspect to unlock a more sophisticated knowledge of PDA biofunctionalization and its role in controlling key biological events, such as stem cell response. Although the mechanism for the bioinductive capacity of PDA is not fully understood, surface topography, chemistry and adhesive properties are believed to play a critical role, either individually or in combination. This work addresses the differential roles of such surface properties on PDA bioactivity. We achieved novel insights on the physicochemical makeup of two PDA coatings obtained by varying one critical parameter (i.e., solution agitation) during the deposition. Successively, we focused on the effects on human mesenchymal stem cells (hMSCs) in both normal and serum-free culturing conditions. This study reveals both the serum-dependent and independent cueing involved in bioactive induction caused by PDA.

Evaluation of the direct effects of poly(dopamine) on the in vitro response of human osteoblastic cells.

Functional polymeric coatings have rapidly become one of the most efficient strategies to endow biomaterials with enhanced bioactive properties. Among the bio-inspired polymers used for biomedical applications, mussel-derived poly(dopamine) (PDA) has increasingly attracted considerable interest because of its unique characteristics. In this work, we carried out detailed physicochemical characterization of a PDA film deposited on nanoporous titanium. In particular, we employed spectroscopic techniques (Raman and ATR-FTIR) and Digital Pulsed Force Mode Atomic Force microscopy (DPFM-AFM) to probe the chemical makeup and the nanomechanical properties of PDA-coated surfaces. In addition, we investigated protein adsorption by ATR-FTIR and quantified it with ten different serum proteins by Liquid Chromatography Mass spectroscopy (LC-MS), aiming at elucidating their potential contribution to the subsequent cell colonization. Successively, we assessed the response of MG-63 human osteoblastic cells to PDA-coated titanium both the multiple- and single-cell levels. Results for this study demonstrate that, compared to bare and nanoporous titanium, the PDA coating positively influences the adhesion and proliferation of MG-63 cells. In addition, we focus on how the three different substrates influence cell morphology (i.e. aspect ratio and form factor), the establishment of focal adhesions and the expression of RhoA, a protein involved in cell contractility. In conclusion, our work provides a deeper insight on the in vitro response of human osteoblastic cells to poly(dopamine) by closing in on specific aspects of cell-PDA interactions, ultimately reaffirming the potential of this bio-inspired polymer as a functional coating for bone tissue engineering applications.