Dissolvable microgel-templated macroporous hydrogels for controlled cell assembly

Mesenchymal stem cells (MSCs)-based therapies have been widely used to promote tissue regeneration and to modulate immune/inflammatory response. The therapeutic potential of MSCs can be further improved by forming multi-cellular spheroids. Meanwhile, hydrogels with macroporous structures are advantageous for improving mass transport properties for the cell-laden matrices. Herein, we report the fabrication of MSC-laden macroporous hydrogel scaffolds through incorporating rapidly dissolvable spherical cell-laden microgels. Dissolvable microgels were fabricated by tandem droplet-microfluidics and thiol-norbornene photopolymerization using a novel fast-degrading macromer poly(ethylene glycol)-norbornene-dopamine (PEGNB-Dopa). The cell-laden microgels were subsequently encapsulated within another bulk hydrogel matrix, whose porous structure was generated efficiently by the rapid degradation of the PEGNB-Dopa microgels. The cytocompatibility of this in situ pore-forming approach was demonstrated with multiple cell types. Furthermore, adjusting the stiffness and cell adhesiveness of the bulk hydrogels afforded the formation of solid cell spheroids or hollow spheres. The assembly of solid or hollow MSC spheroids led to differential activation of AKT pathway. Finally, MSCs solid spheroids formed in situ within the macroporous hydrogels exhibited robust secretion of HGF, VEGF-A, IL-6, IL-8, and TIMP-2. In summary, this platform provides an innovative method for forming cell-laden macroporous hydrogels for a variety of future biomedical applications.

Design of a custom-made device for real-time optical measurement of differential mineral concentrations in three-dimensional scaffolds for bone tissue engineering

Monitoring bone tissue engineered (TEed) constructs during their maturation is important to ensure the quality of applied protocols. Several destructive, mainly histochemical, methods are conventionally used to this aim, requiring the sacrifice of the investigated samples. This implies (i) to plan several scaffold replicates, (ii) expensive and time consuming procedures and (iii) to infer the maturity level of a given tissue construct from a cognate replica. To solve these issues, non-destructive techniques such as light spectroscopy-based methods have been reported to be useful. Here, a miniaturized and inexpensive custom-made spectrometer device is proposed to enable the non-destructive analysis of hydrogel scaffolds. Testing involved samples with a differential amount of calcium salt. When compared to a reference standard device, this custom-made spectrometer demonstrates the ability to perform measurements without requiring elaborate sample preparation and/or a complex instrumentation. This preliminary study shows the feasibility of light spectroscopy-based methods as useful for the non-destructive analysis of TEed constructs. Based on these results, this custom-made spectrometer device appears as a useful option to perform real-time/in-line analysis. Finally, this device can be considered as a component that can be easily integrated on board of recently prototyped bioreactor systems, for the monitoring of TEed constructs during their conditioning.

Carbohydrate/protein hydrogels as responsive scaffolds in controlling inorganic crystallization

<p>The strategies that both invertebrate and vertebrate organism use to produce organic-inorganic composite materials for different purposes such as mechanical support and protection for the body are fascinating. While extensive research has been done on understanding the basic principles of biomineral formation, mimicking the critical principles of the mechanisms of biomineralization in vitro and fully capturing the structural information and characteristics remain challenging issues for scientists. Calcium is an essential element in biological systems. It plays a central role in the mineralization and maintenance of the skeleton as well as in fundamental physiological processes including growth and development in vertebrates. Within a biological organism calcium ions are stored, delivered, or released in the presence of different anions such as phosphate, carbonate and citrate. Competition between the different anions which interact with calcium ions in different hydrogel matrices leads to manipulation of the various composite materials produced such as bone and nacre. Soluble anionic acidic macromolecules associated with biominerals play a vital role in modulating the mineral morphology and hierarchy of the organized composite. Understanding the interaction between the constituent ions and the organic matrix is crucial if we are to make synthetic materials, the structure and properties of which replicate those of native biominerals, or materials that have the storage and/or release characteristics of foods, for example. Carbohydrate-based hydrogels versus protein-based hydrogels are used here as scaffolds for the synthesis of calcium carbonate and calcium phosphate biominerals. Water soluble acidic additives are used to modulate the nucleation and growth of the minerals. In particular chitosan and gelatin hydrogel templates were used as the mineralization scaffolds. Three different mineralization methods were used: the Kitano, alternate soaking and McGrath methods. Monomeric vs. polymeric additives (acrylic acid, glutamic acid, aspartic acid and their corresponding polymers) were introduced into all systems in order to control the nucleation and growth of the so-formed minerals. The morphology, crystallinity, polymorphism and composition of the synthesized organic-inorganic composites were investigated. Analyses were carried out using a number of techniques including Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD), micro-Raman spectroscopy and solid-sate NMR. In the case of calcium carbonate the morphology and crystallinity were more affected by the use of polymeric additives compared with their monomeric equivalents. In particular the calcium carbonate preferentially grew laterally within and on the periphery of the chitosan or gelatin hydrogel scaffold. This results from the formation of a polyelectrolyte complex between the chitosan or gelatin hydrogels and the added polymer. The presence of the polyelectrolyte complex modifies the nucleation of the mineral. Nanoparticles are preferentially formed which then aggregate together maintaining a lateral perspective with the scaffold. In the case of calcium phosphate mostly spherical and platelet-shaped morphologies composed of amorphous calcium phosphate and poorly crystalline hydroxylapatite respectively were always formed within both chitosan and gelatin hydrogel scaffolds. pH was also found to be a key factor in controlling which polymorph of calcium phosphate precipitates. The crystallinity is influenced by the presence of additives for chitosan scaffold. For systems with added aspartic or polyaspartic acid platelet-shaped CaP forms. These crystals are more highly crystalline compared to those where predominately the porous spherical calcium phosphate morphology is observed which form when L-glutamic acid is added. In the presence of polymeric additives nanoparticles form which then aggregate to yield larger crystals. Such aggregation was preferentially observed for gelatin scaffolds. Citrate anions are particularly important in calcium phosphate precipitation in bone. Biomimetic hydroxylapatite-chitosan and hydroxylapatite-gelatin nanocomposite were synthesized where citrate ions were used to control the size and crystallinity of the hydroxylapatite crystals. TEM data show that the size of the hydroxylapatite crystals decreases upon introducing citrate ions into the systems. Solid-state NMR dipolar dephasing data indicate the hydroxylapatite precipitation can be stabilized with 2.5 wt% sodium citrate with respect to the chitosan and gelatin mass. The data included within this thesis illustrate that both gelatin and chitosan hydrogel scaffolds display similar ability in modulating calcium carbonate or calcium phosphate crystallization in the absence and presence of additives. The role of soluble acidic additives is significant in the formation of biominerals. These results reveal therefore the possibility that carbohydrate-based systems, which have many advantages over protein-based systems, could be used to provide more options for fabricating new implantable materials for humans and animals. The results from the combination of techniques used including XRD, SSNMR and TEM showed the possibility of in vitro synthesis of a bio-nanocomposite material in the presence of citrate similar to that of natural bone (in terms of composition and morphology). The achievement of this work demonstrates that new advanced materials with various composite structures and morphologies can be synthesized through a biomimetic biomineralization mechanism under ambient conditions similar to natural materials such as bone and nacre. These advancements have potential application in biomedical research and more specifically in fabrication of implantable materials.</p>

Carbohydrate/protein hydrogels as responsive scaffolds in controlling inorganic crystallization

<p>The strategies that both invertebrate and vertebrate organism use to produce organic-inorganic composite materials for different purposes such as mechanical support and protection for the body are fascinating. While extensive research has been done on understanding the basic principles of biomineral formation, mimicking the critical principles of the mechanisms of biomineralization in vitro and fully capturing the structural information and characteristics remain challenging issues for scientists. Calcium is an essential element in biological systems. It plays a central role in the mineralization and maintenance of the skeleton as well as in fundamental physiological processes including growth and development in vertebrates. Within a biological organism calcium ions are stored, delivered, or released in the presence of different anions such as phosphate, carbonate and citrate. Competition between the different anions which interact with calcium ions in different hydrogel matrices leads to manipulation of the various composite materials produced such as bone and nacre. Soluble anionic acidic macromolecules associated with biominerals play a vital role in modulating the mineral morphology and hierarchy of the organized composite. Understanding the interaction between the constituent ions and the organic matrix is crucial if we are to make synthetic materials, the structure and properties of which replicate those of native biominerals, or materials that have the storage and/or release characteristics of foods, for example. Carbohydrate-based hydrogels versus protein-based hydrogels are used here as scaffolds for the synthesis of calcium carbonate and calcium phosphate biominerals. Water soluble acidic additives are used to modulate the nucleation and growth of the minerals. In particular chitosan and gelatin hydrogel templates were used as the mineralization scaffolds. Three different mineralization methods were used: the Kitano, alternate soaking and McGrath methods. Monomeric vs. polymeric additives (acrylic acid, glutamic acid, aspartic acid and their corresponding polymers) were introduced into all systems in order to control the nucleation and growth of the so-formed minerals. The morphology, crystallinity, polymorphism and composition of the synthesized organic-inorganic composites were investigated. Analyses were carried out using a number of techniques including Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD), micro-Raman spectroscopy and solid-sate NMR. In the case of calcium carbonate the morphology and crystallinity were more affected by the use of polymeric additives compared with their monomeric equivalents. In particular the calcium carbonate preferentially grew laterally within and on the periphery of the chitosan or gelatin hydrogel scaffold. This results from the formation of a polyelectrolyte complex between the chitosan or gelatin hydrogels and the added polymer. The presence of the polyelectrolyte complex modifies the nucleation of the mineral. Nanoparticles are preferentially formed which then aggregate together maintaining a lateral perspective with the scaffold. In the case of calcium phosphate mostly spherical and platelet-shaped morphologies composed of amorphous calcium phosphate and poorly crystalline hydroxylapatite respectively were always formed within both chitosan and gelatin hydrogel scaffolds. pH was also found to be a key factor in controlling which polymorph of calcium phosphate precipitates. The crystallinity is influenced by the presence of additives for chitosan scaffold. For systems with added aspartic or polyaspartic acid platelet-shaped CaP forms. These crystals are more highly crystalline compared to those where predominately the porous spherical calcium phosphate morphology is observed which form when L-glutamic acid is added. In the presence of polymeric additives nanoparticles form which then aggregate to yield larger crystals. Such aggregation was preferentially observed for gelatin scaffolds. Citrate anions are particularly important in calcium phosphate precipitation in bone. Biomimetic hydroxylapatite-chitosan and hydroxylapatite-gelatin nanocomposite were synthesized where citrate ions were used to control the size and crystallinity of the hydroxylapatite crystals. TEM data show that the size of the hydroxylapatite crystals decreases upon introducing citrate ions into the systems. Solid-state NMR dipolar dephasing data indicate the hydroxylapatite precipitation can be stabilized with 2.5 wt% sodium citrate with respect to the chitosan and gelatin mass. The data included within this thesis illustrate that both gelatin and chitosan hydrogel scaffolds display similar ability in modulating calcium carbonate or calcium phosphate crystallization in the absence and presence of additives. The role of soluble acidic additives is significant in the formation of biominerals. These results reveal therefore the possibility that carbohydrate-based systems, which have many advantages over protein-based systems, could be used to provide more options for fabricating new implantable materials for humans and animals. The results from the combination of techniques used including XRD, SSNMR and TEM showed the possibility of in vitro synthesis of a bio-nanocomposite material in the presence of citrate similar to that of natural bone (in terms of composition and morphology). The achievement of this work demonstrates that new advanced materials with various composite structures and morphologies can be synthesized through a biomimetic biomineralization mechanism under ambient conditions similar to natural materials such as bone and nacre. These advancements have potential application in biomedical research and more specifically in fabrication of implantable materials.</p>

Forskolin-Loaded Halloysite Nanotubes as Osteoconductive Additive for the Biopolymer Tissue Engineering Scaffolds

Here we report the use of forskolin-modified halloysite nanotubes (HNTs) as a dopant for biopolymer porous hydrogel scaffolds to impart osteoinductive properties. Forskolin is a labdane diterpenoid isolated from the Indian Coleus plant. This small molecule is widely used as a supplement in molecular biology for cell differentiation. It has been reported in some earlier publications that forskolin can activate osteodifferentiation process by cyclic adenosine monophosphate (c-AMP) signalling activation in stem cells. In presented study it was demonstrated that forskolin release from halloysite-doped scaffolds induced the osteodifferentiation of equine mesenchymal stem cells (MSCs) in vitro without addition of any specific growth factors. The reinforcement of mechanical properties of cells and intercellular space during the osteodifferentiation was demonstrated using atomic force microscopy (AFM). These clay-doped scaffolds may find applications to accelerate the regeneration of horse bone defects by inducing the processes of osteodifferentiation of endogenous MSCs.