Human muscle stem cell responses to mechanical stress into tunable 3D alginate matrices.

Preclinical data acquired for human muscle stem (hMuStem) cells indicate their great repair capacity in the context of muscle injury. However, their clinical potential is limited by their moderate ability to survive after transplantation. To overcome these limitations, their encapsulation within protective environment would be beneficial. In this study, tunable calcium-alginate hydrogels obtained through molding method using external or internal gelation were investigated as a new strategy for hMuStem cell encapsulation. The mechanical properties of these hydrogels were characterized in their fully hydrated state by compression experiments using Atomic Force Microscopy. Measured elastic moduli strongly depended on the gelation mode and calcium/alginate concentrations. Values ranged from 1 to 12.5 kPa and 3.9 to 25 kPa were obtained for hydrogels prepared following internal and external gelation, respectively. Also, differences in mechanical properties of hydrogels resulted from their internal organization, with an isotropic structure for internal gelation, while external mode led to anisotropic one. It was further shown that viability, morphological and myogenic differentiation characteristics of hMuStem cells incorporated within alginate hydrogels were preserved after their release. These results highlight that hMuStem cells encapsulated in calcium-alginate hydrogels maintain their functionality, thus allowing to develop muscle regeneration protocols to improve their therapeutic efficacy.

Interactions between proteins and cellulose in a liquid crystalline media: Design of a droplet based experimental platform.

Model systems are needed to provide controlled environment for the understanding of complex phenomena. Interaction between polysaccharides and proteins in dense medium are involved in numerous complex systems such as biomass conversion or plant use for food processing or biobased materials. In this work, cellulose nanocrystals (CNCs) were used to study proteins in a dense and organized cellulosic environment. This environment was designed within microdroplets using a microfluidic setup, and applied to two proteins, bovine serum albumin (BSA) and a GH7 endoglucanase, relevant to food and plant science, respectively. The CNC at 56.5 g/L organized in liquid crystalline structure and the distribution of the proteins was probed using synchrotron deep-UV radiation. The proteins were homogeneously distributed throughout the volume, but BSA significantly disturbed the droplet global organization, preferring partition in hydrophilic external micelles. In contrast, GH7 partitioned with the CNCs showing stronger non-polar interaction but without disruption of the system organization. Such results pave the road for the development of more complex polysaccharides - proteins in-vitro models.

Micromolding-based encapsulation of mesenchymal stromal cells in alginate for intraarticular injection in osteoarthritis.

Osteoarthritis (OA) is an inflammatory joint disease that affects cartilage, subchondral bone, and joint tissues. Undifferentiated Mesenchymal Stromal Cells are a promising therapeutic option for OA due to their ability to release anti-inflammatory, immuno-modulatory, and pro-regenerative factors. They can be embedded in hydrogels to prevent their tissue engraftment and subsequent differentiation. In this study, human adipose stromal cells are successfully encapsulated in alginate microgels via a micromolding method. Microencapsulated cells retain their in vitro metabolic activity and bioactivity and can sense and respond to inflammatory stimuli, including synovial fluids from OA patients. After intra-articular injection in a rabbit model of post-traumatic OA, a single dose of microencapsulated human cells exhibit properties matching those of non-encapsulated cells. At 6 and 12 weeks post-injection, we evidenced a tendency toward a decreased OA severity, an increased expression of aggrecan, and a reduced expression of aggrecanase-generated catabolic neoepitope. Thus, these findings establish the feasibility, safety, and efficacy of injecting cells encapsulated in microgels, opening the door to a long-term follow-up in canine OA patients.

Droplet Microfluidics for Food and Nutrition Applications.

Droplet microfluidics revolutionizes the way experiments and analyses are conducted in many fields of science, based on decades of basic research. Applied sciences are also impacted, opening new perspectives on how we look at complex matter. In particular, food and nutritional sciences still have many research questions unsolved, and conventional laboratory methods are not always suitable to answer them. In this review, we present how microfluidics have been used in these fields to produce and investigate various droplet-based systems, namely simple and double emulsions, microgels, microparticles, and microcapsules with food-grade compositions. We show that droplet microfluidic devices enable unprecedented control over their production and properties, and can be integrated in lab-on-chip platforms for in situ and time-resolved analyses. This approach is illustrated for on-chip measurements of droplet interfacial properties, droplet-droplet coalescence, phase behavior of biopolymer mixtures, and reaction kinetics related to food digestion and nutrient absorption. As a perspective, we present promising developments in the adjacent fields of biochemistry and microbiology, as well as advanced microfluidics-analytical instrument coupling, all of which could be applied to solve research questions at the interface of food and nutritional sciences.

Manipulating and studying triglyceride droplets in microfluidic devices.

Triglyceride is the main lipid class in nature, found as droplets in both living systems and man-made products (such as manufactured foods and drugs). Characterizing triglyceride droplets in situ in these systems is complex due to many environmental interactions. To answer basic research questions about droplet formation, structuration, stability, or degradation, microfluidic strategies were developed, allowing well-controlled droplets to be formed, manipulated, and studied. In this review, these strategies are described, starting with the presentation of droplet production devices, with applications essentially related to microencapsulation and delivery, then detailing methods to monitor droplet degradation in situ and in real time, finishing with microfluidic platforms allowing the investigation of many aspects of biological lipid droplets simultaneously.

Studying the real-time interplay between triglyceride digestion and lipophilic micronutrient bioaccessibility using droplet microfluidics. 1 lab on a chip method.

This article is the first part of a series reporting on real-time digestion kinetics of triglyceride droplets containing different lipophilic micronutrients. This part focuses on the design, fabrication, and operation of a polydimethylsiloxane microfluidic device which enables the generation and digestion of oil droplets. The micro-channels were made hydrophilic to obtain oil droplets in an aqueous continuous phase. Optimized chip design and outlet control were implemented to provide efficient oil droplet generation, manipulation, and immobilization on a single chip. Highly monodisperse oil droplets were generated, immobilized in an array of traps and monitored in real time by fluorescence using a confocal microscopy method. The device was used to study the kinetics of beta-carotene release during tricaprylin digestion (intestinal lipolysis and micellar solubilization). The effect of the gastric phase on beta-carotene degradation was also investigated using the same method.

Studying the real-time interplay between triglyceride digestion and lipophilic micronutrient bioaccessibility using droplet microfluidics. 2 application to various oils and (pro)vitamins.

The kinetics of micellar solubilization of lipophilic micronutrients (bioaccessibility) in relation with triglyceride digestion remains poorly known. To study this interplay in real-time, a droplet microfluidic method was designed and used as reported in the first part of this article series. In this second part, the interplay between the micellar solubilization of (pro)vitamins (beta-carotene or retinyl palmitate) and the digestion of triglyceride oils (tricaprylin TC, or high-oleic sunflower seed oil HOSO, or fish oil FO) during simulated gastrointestinal digestion was investigated. The relation between the release of both micronutrients and of triglyceride lipolytic products was found to be non-linear. The kinetics of beta-carotene was found to follow the kinetics of lipolytic products, depending on the oil type (TC > HOSO > FO). The effect of the gastric phase on the intestinal phase was also found to follow this order, mostly due to partial lipolysis during the gastric phase.

Microcarriers Based on Glycosaminoglycan-Like Marine Exopolysaccharide for TGF-ß1 Long-Term Protection.

Articular cartilage is an avascular, non-innervated connective tissue with limited ability to regenerate. Articular degenerative processes arising from trauma, inflammation or due to aging are thus irreversible and may induce the loss of the joint function. To repair cartilaginous defects, tissue engineering approaches are under intense development. Association of cells and signalling proteins, such as growth factors, with biocompatible hydrogel matrix may lead to the regeneration of the healthy tissue. One current strategy to enhance both growth factor bioactivity and bioavailability is based on the delivery of these signalling proteins in microcarriers. In this context, the aim of the present study was to develop microcarriers by encapsulating Transforming Growth Factor-ß1 (TGF-ß1) into microparticles based on marine exopolysaccharide (EPS), namely GY785 EPS, for further applications in cartilage engineering. Using a capillary microfluidic approach, two microcarriers were prepared. The growth factor was either encapsulated directly within the microparticles based on slightly sulphated derivative or complexed firstly with the highly sulphated derivative before being incorporated within the microparticles. TGF-ß1 release, studied under in vitro model conditions, revealed that the majority of the growth factor was retained inside the microparticles. Bioactivity of released TGF-ß1 was particularly enhanced in the presence of highly sulphated derivative. It comes out from this study that GY785 EPS based microcarriers may constitute TGF-ß1 reservoirs spatially retaining the growth factor for a variety of tissue engineering applications and in particular cartilage regeneration, where the growth factor needs to remain in the target location long enough to induce robust regenerative responses.

Investigation of interactions between the marine GY785 exopolysaccharide and transforming growth factor-ß1 by atomic force microscopy.

Sulfated polysaccharides, such as glycosaminoglycans (GAG) regulate various biological activities through their interactions with growth factors. Investigating these interactions becomes the key to understand the structure-function relationship of GAG. Highly sulfated derivatives prepared from the marine GY785 exopolysaccharide (EPS) produced by the deep-sea hydrothermal vent bacterium Alteromonas infernus have previously shown to stimulate the chondrogenic differentiation of mesenchymal stem cells in the presence of Transforming Growth Factor-ß1 (TGF-ß1). Here, the interactions between the GAG-mimetic GY785 EPS derivatives and TGF-ß1 were investigated by Atomic Force Microscopy (AFM). The affinity between slightly sulfated or highly sulfated derivatives and TGF-ß1 was explored by AFM imaging and single-molecule force spectroscopy experiments. The number of measured interactions and the interaction strength were both higher for highly sulfated derivative compared to the slightly sulfated one. These results clearly emphasize the involvement of sulfate groups in the protein binding and open new ways to tune cellular processes by designing macromolecules with adjustable sulfate charge density.

Assembly of HE800 exopolysaccharide produced by a deep-sea hydrothermal bacterium into microgels for protein delivery applications.

Assembly of biopolymers into microgels is an elegant strategy for bioencapsulation with various potential biomedical applications. Such biocompatible and biodegradable microassemblies are developed not only to protect the encapsulated molecule but also to ensure its sustained local delivery. The present study describes the fabrication of microassemblies from a marine HE800 exopolysaccharide (EPS), which displays a glycosaminoglycan (GAG)-like structure and biological properties. HE800 EPS was assembled, through physical cross-linking with divalent ions, into microgel particles and microfibers using microfluidics. The microparticle morphology was highly affected by the polysaccharide concentration and its molecular weight. A model protein, namely Bovine Serum Albumin (BSA) was subsequently encapsulated within HE800 microparticles in one-step process using microfluidics. The protein release was tuned by the microparticle morphology with a lower protein amount released from the most homogeneous structures. Our findings demonstrate the high potential of HE800 EPS based microassemblies as innovative protein microcarriers for further biomedical applications.

Microfluidic Encapsulation of Pickering Oil Microdroplets into Alginate Microgels for Lipophilic Compound Delivery.

Alginate microgels are widely used as delivery systems in food, cosmetics, and pharmaceutical industries for encapsulation and sustained release of hydrophilic compounds and cells. However, the encapsulation of lipophilic molecules inside these microgels remains a great challenge because of the complex oil-core matrix required. The present study describes an original two-step approach allowing the easy encapsulation of several oil microdroplets within alginate microgels. In the first step, stable oil microdroplets were formed by preparing an oil-in-water (O/W) Pickering emulsion. To stabilize this emulsion, we used two solid particles, namely the cotton cellulose nanocrystals (CNC) and calcium carbonate (CaCO3). It was observed that the surface of the oil microdroplets formed was totally covered by a CNC layer, whereas CaCO3 particles were adsorbed onto the cellulose layer. This solid CNC shell efficiently stabilized the oil microdroplets, preventing them from undesired coalescence. In the second step, oil microdroplets resulting from the Pickering emulsion were encapsulated within alginate microgels using microfluidics. Precisely, the outermost layer of oil microdroplets composed of CaCO3 particles was used to initiate alginate gelation inside the microfluidic device, following the internal gelation mode. The released Ca2+ ions induced the gel formation through physical cross-linking with alginate molecules. This innovative and easy to carry out two-step approach was successfully developed to fabricate monodisperse alginate microgels of 85 µm in diameter containing around 12 oil microdroplets of 15 µm in diameter. These new oil-core alginate microgels represent an attractive system for encapsulation of lipophilic compounds such as vitamins, aroma compounds or anticancer drugs that could be applied in various domains including food, cosmetics, and medical applications.

Oil core microcapsules by inverse gelation technique.

A promising technique for oil encapsulation in Ca-alginate capsules by inverse gelation was proposed by Abang et al. This method consists of emulsifying calcium chloride solution in oil and then adding it dropwise in an alginate solution to produce Ca-alginate capsules. Spherical capsules with diameters around 3 mm were produced by this technique, however the production of smaller capsules was not demonstrated. The objective of this study is to propose a new method of oil encapsulation in a Ca-alginate membrane by inverse gelation. The optimisation of the method leads to microcapsules with diameters around 500 µm. In a search of microcapsules with improved diffusion characteristics, the size reduction is an essential factor to broaden the applications in food, cosmetics and pharmaceuticals areas. This work contributes to a better understanding of the inverse gelation technique and allows the production of microcapsules with a well-defined shell-core structure.

A microfluidic device to study the digestion of trapped lipid droplets.

At the junction of chemistry, physics and biology, digestion involves many processes. Studying the mechanisms in such a complex system is challenging because numerous interactions coexist. Even in an apparently simple system such as an emulsion, many physicochemical characteristics affect lipid digestion. Moreover, these characteristics are difficult to control using conventional in vitro techniques. The goal of this work was to design a microfluidic device allowing the study of well-controlled individual oil droplets under gastrointestinal digestion conditions. Different parameters were investigated in order to validate the relevance of this device compared to conventional in vitro techniques using emulsions. Various triglycerides and digestion conditions were tested with droplets of the same initial diameter generated by a flow focusing device, then placed in individual traps of a microfluidic chamber for digestion with continuous digestive juice renewal. The results are in good agreement with those obtained with conventional in vitro techniques and open the way to screening of lipid digestion, in particular, bioaccessibility of lipophilic molecules, a prerequisite for bioavailability studied in nutrition, pharmacology, and toxicology.

Microfluidics-assisted diffusion self-assembly: toward the control of the shape and size of pectin hydrogel microparticles.

We demonstrated the generation of pectin hydrogel microparticles having complex shapes either by combining the phenomenon of gelation and water diffusion-induced self-assembly in microfluidic channels (on-chip) or by the deformation of the pregelled droplets outside the channels (off-chip) at a fluid-fluid interface. We proved that by tuning the mode of pectin cross-linking (CaCl2 vs CaCO3) and the degree of shrinking (water content in the dimethyl carbonate (DMC) organic continuous phase) we can control the shape of the final particle. Sphere, doughnut, oblate ellipsoid, or mushroom-type morphologies were thus produced, demonstrating the ability to control the formation of anisotropic biopolymer-based hydrogel microparticles using microfluidics. Shape changes were explained by the redistribution of calcium ions in combination with the local Peclet number experienced by the microdroplets during the on-chip process. Moreover, during the off-chip process, the interplay between elastic and viscous forces for microdroplets entering the CaCl2-DMC interface caused deformation of the pregelled droplets to occur and therefore resulted in the formation of microparticles with a mushroom-like morphology.

Microfluidic generation and selective degradation of biopolymer-based Janus microbeads.

We describe a microfluidic approach for generating Janus microbeads from biopolymer hydrogels. A flow-focusing device was used to emulsify the coflow of aqueous solutions of one or two different biopolymers in an organic phase to synthesize homo or hetero Janus microbeads. Biopolymer gelation was initiated, in the chip, by diffusion-controlled ionic cross-linking of the biopolymers. Pectin-pectin (homo Janus) and, for the first time, pectin-alginate (hetero Janus) microbeads were produced. The efficiency of separation of the two hemispheres, which reflected mixing and convection phenomena, was investigated by confocal scanning laser microscopy (CSLM) of previously labeled biopolymers. The interface of the hetero Janus structure was clearly defined, whereas that of the homo Janus microbeads was poorly defined. The Janus structure was confirmed by subjecting each microbead hemisphere to specific enzymatic degradation. These new and original microbeads from renewable resources will open up opportunities for studying relationships between combined enzymatic hydrolysis and active compound release.

Patterning surface by site selective capture of biopolymer hydrogel beads.

This communication describes the fabrication of microstructured biopolymer surfaces by the site-selective capture of pectin hydrogel beads. A positively charged surface consisting of poly-L-lysine (PLL) was subjected to site-selective enzymatic degradation using patterned polydimethylsiloxane (PDMS) stamps covalently modified with trypsin, according to the recently described method. The patterned surface was used to capture ionically cross-linked pectin beads. The desired patterning of the hydrogel surfaces was generated by site-selective immobilization of these pectin beads. The ability of the hydrogels to be dried and swollen in water was assessed.

Cell wall modifications in Arabidopsis plants with altered alpha-L-arabinofuranosidase activity.

Although cell wall remodeling is an essential feature of plant growth and development, the underlying molecular mechanisms are poorly understood. This work describes the characterization of Arabidopsis (Arabidopsis thaliana) plants with altered expression of ARAF1, a bifunctional alpha-L-arabinofuranosidase/beta-D-xylosidase (At3g10740) belonging to family 51 glycosyl-hydrolases. ARAF1 was localized in several cell types in the vascular system of roots and stems, including xylem vessels and parenchyma cells surrounding the vessels, the cambium, and the phloem. araf1 T-DNA insertional mutants showed no visible phenotype, whereas transgenic plants that overexpressed ARAF1 exhibited a delay in inflorescence emergence and altered stem architecture. Although global monosaccharide analysis indicated only slight differences in cell wall composition in both mutant and overexpressing lines, immunolocalization experiments using anti-arabinan (LM6) and anti-xylan (LM10) antibodies indicated cell type-specific alterations in cell wall structure. In araf1 mutants, an increase in LM6 signal intensity was observed in the phloem, cambium, and xylem parenchyma in stems and roots, largely coinciding with ARAF1 expression sites. The ectopic overexpression of ARAF1 resulted in an increase in LM10 labeling in the secondary walls of interfascicular fibers and xylem vessels. The combined ARAF1 gene expression and immunolocalization studies suggest that arabinan-containing pectins are potential in vivo substrates of ARAF1 in Arabidopsis.

FT-IR investigation of cell wall polysaccharides from cereal grains. Arabinoxylan infrared assignment.

The FT-IR fingerprint of wheat endosperm arabinoxylan (AX) was investigated using a set of polysaccharides exhibiting variation of their degree of substitution and xylo-oligosaccharides comprising xylose units mono- or disubstituted by arabinose residues. Substitution of the xylose backbone by arabinose side units was more particularly studied in the 1000-800 cm(-1) spectral region, by taking advantage of second-derivative enhancement. The 920-1020 cm(-1) spectral region revealed two absorption bands at 984 and 958 cm(-1), the intensities of which varied according to the degree of substitution. Whereas the intensity of the band at 958 cm(-1) increased with the degree of substitution, that at 984 cm(-1) decreased. The second-derivative spectral data of xylo-oligosaccharides indicated that these changes could be attributed to substitution of the xylan backbone by arabinose residues, and the band at 958 cm(-1) was ascribed to the presence of disubstituted xylose residues. Principal component analysis of FT-IR spectra of model mixtures of AX, beta-glucans, and arabinogalactans suggested that it is possible to evaluate the relative proportions of the polymers and degree of substitution of AX in complex mixtures such as the cell wall of cereal grains.