Direct Comparison of Three Buckling-Based Methods to Measure the Elastic Modulus of Nanobiocomposite Thin Films.

To engineer tunable thin-film materials, the accurate measurement of their mechanical properties is crucial. However, characterizing the elastic modulus with current methods is particularly challenging for sub-micrometer thick films and hygroscopic materials because they are highly sensitive to environmental conditions and most methods require free-standing films which are difficult to prepare. In this work, we directly compared three buckling-based methods to determine the elastic moduli of supported thin films: (1) biaxial thermal shrinking, (2) uniaxial thermal shrinking, and (3) the mechanically compressed, strain-induced elastic buckling instability for mechanical measurements (SIEBIMM) method. Nanobiocomposite model films composed of cellulose nanocrystals (CNCs) and polyethyleneimine (PEI) were assembled using layer-by-layer deposition to control composition and thickness. The three buckling-based methods yielded the same trends and comparable values for the elastic moduli of each CNC-PEI film composition (ranging from 15 to 44 GPa, depending on film composition). This suggests that the methods are similarly effective for the quantification of thin-film mechanical properties. Increasing the CNC content in the films statistically increased the modulus; however, increasing the PEI content did not lead to significant changes. For the CNC-PEI system, the standard deviation of elastic moduli determined from SIEBIMM was 2-4 times larger than that for thermal shrinking, likely due to extensive cracking due to the different stress applied to the film when subjected to compression of a relaxed substrate versus the shrinking of a pre-strained substrate. These results show that biaxial thermal shrinking is a reliable method for the determination of the mechanical properties of thin films with a simple implementation and analysis and low sensitivity to small deviations in the input parameter values, such as film thickness or substrate modulus.

Fibrin and Transforming Growth Factor Alpha Affect Prostatic Smooth Muscle Cell's Phenotype and Motility.

Smooth muscle cells (SMCs) are dynamic and transition from a contractile to a synthetic phenotype under different circumstances. Plasma factors (fibrin and transforming growth factors, TGFs) are possible components affecting SMCs differentiation and behavior. Thus, the objective of this work was to investigate how the fibrin matrix and TGFs affect SMCs differentiation and motility behavior. SMCs invaded the fibrin gel and adopted a stellate phenotype while reducing the expression of differentiation markers (Acta2, Myh11, and Smtn). At the ultrastructural level, SMCs did not assemble a basal lamina and showed numerous blebs along the entire cell surface. This transition was not associated with changes in focal adhesion kinase (FAK) content and phosphorylation status but reflected a marked change in FAK distribution in the cytoplasm. After 48 h in culture, SMCs caused an active degradation of the fibrin gel. Additionally, we tested the SMCs response to TGFs in a cell layer wound repair assay. TGFα, but not TGFß1 or TGFß3, had significantly increased motility. In conclusion, prostatic SMCs present a phenotypical transition when cultured on fibrin, adopting a micro-blebbing based motility behavior and increasing migration in response to TGFα.

Polarization, migration, and homotypical interactions among prostatic smooth muscle cells in a laminin 111-rich extracellular matrix.

Prostate cancer is a life-threatening condition worldwide. As the tumor progresses, smooth muscle cells (SMCs) become atrophic/dedifferentiated, within a series of stromal changes named stromal reaction. Here, we tested whether a laminin 111-rich extracellular matrix (Lr-ECM) could affect SMCs phenotype and differentiation status. Using time-lapse microscopy, image analyses, quantitative real-time reverse transcription polymerase chain reaction, immunohistochemistry and immunoblotting, and transmission electron microscopy, we showed that SMCs acquires a migratory behavior with a decreased expression of differentiation markers and relocation of focal adhesion kinase. SMCs set homotypic cell junctions and were active in autophagy/phagocytosis. Analysis of the migratory behavior showed that SMCs polarized and migrated toward each other, recognizing long-distance signals such as matrix tensioning. However, half of the cell population were immotile, irrespective of the nearest neighbor distance, suggesting they do not engage in productive interactions, possibly as a result of back-to-back positioning. In conclusion, the Lr-ECM, mimics the effects of the proliferating and infiltrating tumor epithelium, causing SMCs phenotypical change similar to that observed in the stromal reaction, in addition to a hitherto undescribed, stereotyped pattern of cell motility resulting from cell polarization.

Cross-linked cellulose nanocrystal aerogels as viable bone tissue scaffolds.

Chemically cross-linked cellulose nanocrystal (CNC) aerogels possess many properties beneficial for bone tissue scaffolding applications. CNCs were extracted using sulfuric acid or phosphoric acid, to produce CNCs with sulfate and phosphate half-ester surface groups, respectively. Hydrazone cross-linked aerogels fabricated from the two types of CNCs were investigated using scanning electron microscopy, X-ray micro-computed tomography, X-ray photoelectron spectroscopy, nitrogen sorption isotherms, and compression testing. CNC aerogels were evaluatedin vitrowith osteoblast-like Saos-2 cells and showed an increase in cell metabolism up to 7 days while alkaline phosphatase assays revealed that cells maintained their phenotype. All aerogels demonstrated hydroxyapatite growth over 14 days while submerged in simulated body fluid solution with a 0.1 M CaCl2 pre-treatment. Sulfated CNC aerogels slightly outperformed phosphated CNC aerogels in terms of compressive strength and long-term stability in liquid environments, and were implanted into the calvarian bone of adult male Long Evans rats. Compared to controls at 3 and 12 week time points, sulfated CNC aerogels showed increased bone volume fraction of 33% and 50%, respectively, compared to controls, and evidence of osteoconductivity. These results demonstrate that cross-linked CNC aerogels are flexible, porous and effectively facilitate bone growth after they are implanted in bone defects. STATEMENT OF SIGNIFICANCE: Due to the potential complications associated with autografts, there is a need for synthetic bone tissue scaffolds. Here, we report a new naturally-based aerogel material for bone regeneration made solely from chemically cross-linked cellulose nanocrystals (CNC). These highly porous CNC aerogels were shown to promote the proliferation of bone-like cells and support the growth of hydroxyapatite on their surface in vitro. The first in vivo study on these materials was conducted in rats and showed their osteconductive properties and an increase in bone volume up to 50% compared to sham sites. This study demonstrates the potential of using functionalized cellulose nanocrystals as the basis for aerogel scaffolds for bone tissue engineering.

Optimization of cellulose nanocrystal length and surface charge density through phosphoric acid hydrolysis.

Cellulose nanocrystals (CNCs) are emerging nanomaterials with a large range of potential applications. CNCs are typically produced through acid hydrolysis with sulfuric acid; however, phosphoric acid has the advantage of generating CNCs with higher thermal stability. This paper presents a design of experiments approach to optimize the hydrolysis of CNCs from cotton with phosphoric acid. Hydrolysis time, temperature and acid concentration were varied across nine experiments and a linear least-squares regression analysis was applied to understand the effects of these parameters on CNC properties. In all but one case, rod-shaped nanoparticles with a high degree of crystallinity and thermal stability were produced. A statistical model was generated to predict CNC length, and trends in phosphate content and zeta potential were elucidated. The CNC length could be tuned over a relatively large range (238-475 nm) and the polydispersity could be narrowed most effectively by increasing the hydrolysis temperature and acid concentration. The CNC phosphate content was most affected by hydrolysis temperature and time; however, the charge density and colloidal stability were considered low compared with sulfuric acid hydrolysed CNCs. This study provides insight into weak acid hydrolysis and proposes 'design rules' for CNCs with improved size uniformity and charge density.This article is part of a discussion meeting issue 'New horizons for cellulose nanotechnology'.