Self-Healing Injectable Hydrogels for Tissue Regeneration.

Biomaterials with the ability to self-heal and recover their structural integrity offer many advantages for applications in biomedicine. The past decade has witnessed the rapid emergence of a new class of self-healing biomaterials commonly termed injectable, or printable in the context of 3D printing. These self-healing injectable biomaterials, mostly hydrogels and other soft condensed matter based on reversible chemistry, are able to temporarily fluidize under shear stress and subsequently recover their original mechanical properties. Self-healing injectable hydrogels offer distinct advantages compared to traditional biomaterials. Most notably, they can be administered in a locally targeted and minimally invasive manner through a narrow syringe without the need for invasive surgery. Their moldability allows for a patient-specific intervention and shows great prospects for personalized medicine. Injected hydrogels can facilitate tissue regeneration in multiple ways owing to their viscoelastic and diffusive nature, ranging from simple mechanical support, spatiotemporally controlled delivery of cells or therapeutics, to local recruitment and modulation of host cells to promote tissue regeneration. Consequently, self-healing injectable hydrogels have been at the forefront of many cutting-edge tissue regeneration strategies. This study provides a critical review of the current state of self-healing injectable hydrogels for tissue regeneration. As key challenges toward further maturation of this exciting research field, we identify (i) the trade-off between the self-healing and injectability of hydrogels vs their physical stability, (ii) the lack of consensus on rheological characterization and quantitative benchmarks for self-healing injectable hydrogels, particularly regarding the capillary flow in syringes, and (iii) practical limitations regarding translation toward therapeutically effective formulations for regeneration of specific tissues. Hence, here we (i) review chemical and physical design strategies for self-healing injectable hydrogels, (ii) provide a practical guide for their rheological analysis, and (iii) showcase their applicability for regeneration of various tissues and 3D printing of complex tissues and organoids.

A Modular Platform for Cytocompatible Hydrogels with Tailored Mechanical Properties Based on Monolithic Matrices and Particulate Building Blocks.

We establish a versatile hydrogel platform based on modular building blocks that allows the design of hydrogels with tailored physical architecture and mechanical properties. We demonstrate its versatility by assembling (i) a fully monolithic gelatin methacryloyl (Gel-MA) hydrogel, (ii) a hybrid hydrogel composed of 1:1 Gel-MA and gelatin nanoparticles, and (iii) a fully particulate hydrogel based on methacryloyl-modified gelatin nanoparticles. The hydrogels were formulated to exhibit the same solid content and comparable storage modulus but different stiffness and viscoelastic stress relaxation. The incorporation of particles resulted in softer hydrogels with enhanced stress relaxation. Murine osteoblastic cells cultured in two-dimensional (2D) on hydrogels showed proliferation and metabolic activity comparable to established collagen hydrogels. Furthermore, the osteoblastic cells showed a trend of increased cell numbers, cell expansion, and more defined protrusions on stiffer hydrogels. Hence, modular assembly allows the design of hydrogels with tailored mechanical properties and the potential to alter cell behavior.

Surfactant Adsorption to Different Fluid Interfaces.

Surfactant adsorption to fluid interfaces is ubiquitous in biological systems, industrial applications, and scientific fields. Herein, we unravel the impact of the hydrophobic phase (air and oil) and the role of oil polarity on the adsorption of surfactants to fluid interfaces. We investigated the adsorption of anionic (sodium dodecyl sulfate), cationic (dodecyltrimethylammonium bromide), and non-ionic (polyoxyethylene-(23)-monododecyl ether) surfactants at different interfaces, including air and oils, with a wide range of polarities. The surfactant-induced interfacial tension decrease, called the interfacial pressure, correlates linearly with the initial interfacial tension of the clean oil-water interface and describes the experimental results of over 30 studies from the literature. The higher interfacial competition of surfactant and polar oil molecules caused the number of adsorbed molecules at the interface to drop. Further, we found that the critical micelle concentration of surfactants in water correlates to the solubility of the oil molecules in water. Hence, the nature of the oil affects the adsorption behavior and equilibrium state of the surfactant at fluid interfaces. These results broaden our understanding and enable better predictability of the interactions of surfactants with hydrophobic phases, which is essential for emulsion, foam, and capsule formation, pharmaceutical commodities, cosmetics, and many food products.

Complex fluids in animal survival strategies.

Animals have evolved distinctive survival strategies in response to constant selective pressure. In this review, we highlight how animals exploit flow phenomena by manipulating their habitat (exogenous) or by secreting (endogenous) complex fluids. Ubiquitous endogenous complex fluids such as mucus demonstrate rheological versatility and are therefore involved in many animal behavioral traits ranging from sexual reproduction to protection against predators. Exogenous complex fluids such as sand can be used either for movement or for predation. In all cases, time-dependent rheological properties of complex fluids are decisive for the fate of the biological behavior and vice versa. To exploit these rheological properties, it is essential that the animal is able to sense the rheology of their surrounding complex fluids in a timely fashion. As timing is key in nature, such rheological materials often have clearly defined action windows matching the time frame of their direct biological behavior. As many rheological properties of these biological materials remain poorly studied, we demonstrate with this review that rheology and material science might provide an interesting quantitative approach to study these biological materials in particular in context towards ethology and bio-mimicking material design.

Globular protein assembly and network formation at fluid interfaces: effect of oil.

The formation of viscoelastic networks at fluid interfaces by globular proteins is essential in many industries, scientific disciplines, and biological processes. However, the effect of the oil phase on the structural transitions of proteins, network formation, and layer strength at fluid interfaces has received little attention. Herein, we present a comprehensive study on the effect of oil polarity on globular protein networks. The formation dynamics and mechanical properties of the interfacial networks of three different globular proteins (lysozyme, ß-lactoglobulin, and bovine serum albumin) were studied with interfacial shear and dilatational rheometry. Furthermore, the degree of protein unfolding at the interfaces was evaluated by subsequent injection of disulfide bonds reducing dithiothreitol. Finally, we measured the interfacial layer thickness and protein immersion into the oil phase with neutron reflectometry. We found that oil polarity significantly affects the network formation, the degree of interfacial protein unfolding, interfacial protein location, and the resulting network strength. These results allow predicting emulsion stabilization of proteins, tailoring interfacial layers with desired mechanical properties, and retaining the protein structure and functionality upon adsorption.

Crystallization-Induced Network Formation of Tri- and Monopalmitin at the Middle-Chain Triglyceride Oil/Air Interface.

Crystalline glycerides play an important role in the formation of multiphase systems such as emulsions and foams. The stabilization of oil/water interfaces by glyceride crystals has been extensively studied compared to only few studies which have been dedicated to oil/air interfaces. This study investigates the crystallization and network formation of tripalmitin (TP) and monopalmitin (MP) at the middle-chain triglyceride (MCT) oil/air interface. TP crystals were found to crystallize in the bulk before aggregating as large rectangular crystal conglomerates at the MCT oil/air interface. This leads to the slow formation of a plastic deformable, macroscopic crystal layer with high interfacial rheological moduli. MP crystals form directly at the MCT oil/air interface resulting in a comparatively fast formation of an elastic deformable network. Crystals with tentacle-like morphology were found to be responsible for the network elasticity. In this work, we show how interfacial crystallization dynamics and mechanical strength can be linked to the molecular structure and crystallization behavior of glyceride crystals.

Interfacial Rheology of Charged Anisotropic Cellulose Nanocrystals at the Air-Water Interface.

Cellulose nanocrystals (CNCs) have received attention as a biological alternative for the stabilization of fluid interfaces, yielding biocompatible and sustainable emulsions, foams, and aerogels. The interfacial behavior of nanoparticles with shape anisotropy and surface charge like CNCs is still poorly understood, although it ultimately dictates the mechanical properties and stability of the macroscopic colloidal material. Here, we report on the linear and nonlinear interfacial dilatational and shear rheology of CNCs at the air-water interface. We observed the formation of viscoelastic CNC layers at comparably low surface coverage, which was attributed to the shape anisotropy of CNCs. Further, the interfacial elasticity of CNC layers can be modulated by salt-induced charge screening, thereby shifting the interplay of repulsive and attractive CNC interactions. CNC layers had a viscous character without salt, followed by increasing viscoelasticity upon salt addition. CNC layers display strain hardening during compression and show a yield stress followed by flow under shear. The observed interfacial behavior is discussed in the context of CNC-stabilized foam and emulsion properties. We conclude that understanding the CNC interfacial behavior may help improve the performance of CNC-stabilized colloidal materials.

Ion-Induced Formation of Nanocrystalline Cellulose Colloidal Glasses Containing Nematic Domains.

Controlling the assembly of colloids in dispersion is a fundamental approach toward the production of functional materials. Nanocrystalline cellulose (NCC) is a charged nanoparticle whose colloidal interactions can be modulated from repulsive to attractive by increasing ionic strength. Here, we combine polarized optical microscopy, rheology, and small-angle scattering techniques to investigate (i) the concentration-driven transition from isotropic dispersion to cholesteric liquid crystals and (ii) salt-induced NCC phase transitions. In particular, we report on the formation of NCC attractive glasses containing nematic domains. At increasing NCC concentration, a structure peak was observed in small-angle X-ray scattering (SAXS) patterns. The evolution of the structure peak demonstrates the decrease in NCC interparticle distance, favoring orientational order during the isotropic-cholesteric phase transition. Small amounts of salt reduce the cholesteric volume fraction and pitch by a decrease in excluded volume. Beyond a critical salt concentration, NCC forms attractive glasses due to particle caging and reduced motility. This results in a sharp increase in viscosity and formation of viscoelastic glasses. The presence of nematic domains is suggested by the appearance of interference colors and the Cox-Merz rule failure and was confirmed by an anisotropic SAXS scattering pattern at q ranges associated with the presence of nematic domains. Thus, salt addition allows the formation of NCC attractive glasses with mechanical properties similar to those of gels while remaining optically active owed to entrapped nematic domains.

Designing Cellulose Nanofibrils for Stabilization of Fluid Interfaces.

Particles of biological origin are of increasing interest for the Pickering stabilization of biocompatible and environmentally friendly foams and emulsions. Cellulose nanofibrils (CNFs) are readily employed in that respect; however, the underlying mechanisms of interfacial stabilization remain widely unknown. For instance, it has not been resolved why CNFs are unable to stabilize foams while efficiently stabilizing emulsions. Here, we produce CNFs with varying contour lengths and charge densities to investigate their behavior at the air-water phase boundary. CNFs adsorbing at the air-water interface reduce surface tension and form interfacial layers with high viscoelasticity, which are attributed to the thermodynamic and kinetic stability of CNF-stabilized colloids, respectively. CNF adsorption is accelerated and higher surface pressures are attained at lower charge densities, indicating that CNF surface charges limit both adsorption and surface coverage. CNFs form monolayers with ∼40% coverage and are primarily wetted by the aqueous phase indicating a contact angle <90°, as demonstrated by neutron reflectometry. The low contact angle at the air-water interface is energetically unfavorable for adsorbed CNFs, which is proposed as a potential explanation why CNFs show poor foaming capacity.

Transient measurement and structure analysis of protein-polysaccharide multilayers at fluid interfaces.

The formation of electrostatic protein-polysaccharide multilayers has attracted attention for the design of fluid interfaces with enhanced stability and functionality. However, current techniques are often limited to measuring final multilayer properties. We present an interfacial shear rheology setup with simultaneous subphase exchange, allowing the transient measurement of biopolymer multilayers by their viscoelasticity. The successive and simultaneous adsorption of ß-lactoglobulin (ß-lg) and low-methoxyl pectin were investigated at the n-dodecane/water interface at pH 4. The successive injection of pectin increased the viscoelasticity of an adsorbed ß-lg layer by electrostatic complexation. On the other hand, simultaneous adsorption impeded adsorption kinetics and interfacial layer strength due to complexation in the bulk phase prior to adsorption. Neutron reflectometry at the air-water interface confirmed the formation of an initial ß-lg layer and electrostatic complexation of a secondary pectin layer, which desorbed upon pH-induced charge inversion. The layer formed by simultaneous adsorption mainly consisted of ß-lg. We conclude that protein-polysaccharide complexes show limited surface activity and result in a lower effective protein concentration available for adsorption.

Effect of Oil Hydrophobicity on the Adsorption and Rheology of ß-Lactoglobulin at Oil-Water Interfaces.

The adsorption of protein layers at oil-water interfaces is critical to the formation and stability of various emulsions in, for example, technical applications as well as in biological lipid storage. Effects of ionic strength, pH, temperature, and pretreatments of the proteins are well-known. However, the oil phase has been regarded as exchangeable and its role in protein adsorption has been widely ignored. Herein, the influence of systematically selected oil interfaces of high purity on the formation and properties of ß-lactoglobulin (ß-lg) adsorption layers was evaluated. Droplet profile tensiometry and interfacial rheometry were employed to determine the adsorption kinetics and dilatational and interfacial shear moduli. We show that depending on the molecular size, flexibility, hydrophobicity, polarity, and polarizability of the oils, globular proteins adsorb distinctively. Stronger interactions of polar oils with the hydrophilic exterior of the native ß-lg lead to decelerated protein unfolding. This results in lower surface pressures and slower formation of viscoelastic networks. In addition, polar oils interact stronger with the protein network by hydrophilic bonding and thereby act as softening agents. The observed effects of hydrophobic subphases on the adsorbed protein layers provide knowledge, which promotes higher reproducibility in rheological studies and precise tailoring of interfacial films for enhanced formation and stability of emulsions.

Adsorption and Interfacial Layer Structure of Unmodified Nanocrystalline Cellulose at Air/Water Interfaces.

Nanocrystalline cellulose (NCC) is a promising biological nanoparticle for the stabilization of fluid interfaces.  However, the adsorption and interfacial layer structure of NCC are poorly understood as it is currently unknown how to form  NCC interfacial layers. Herein, we present parameters for the adsorption of unmodified NCC at the air-water (A/W) interface. Initial NCC adsorption is limited by diffusion, followed by monolayer saturation and decrease in surface tension at the time scale of hours. These results confirm the current hypothesis of a Pickering stabilization. NCC interfacial performance can be modulated by salt-induced charge screening, enhancing adsorption kinetics, surface load, and interfacial viscoelasticity. Adsorbed NCC layers were visualized by atomic force microscopy at planar Langmuir films and curved air bubbles, whereat NCC coverage was higher at curved interfaces. Structural analysis by neutron reflectometry revealed that NCC forms a discontinuous monolayer with crystallites oriented in the interfacial plane at a contact angle < 90°, favoring NCC desorption upon area compression. This provides the fundamental framework on the formation and structure of NCC layers at the A/W interface, paving the way for exploiting NCC interfacial stabilization for tailored colloidal materials.

Ion-Induced Hydrogel Formation and Nematic Ordering of Nanocrystalline Cellulose Suspensions.

Nanocrystalline cellulose (NCC) is a promising material for formation of hydrogels and nematic liquid crystals. While salt addition is known to facilitate hydrogel formation, it remains unclear whether this originates from cationic bridging or charge screening effects. Herein, we demonstrate the effect of mono- and divalent salts on NCC gelation and nematic ordering. A strong correlation of NCC suspension zeta-potential and rheological behavior was found. Lower concentrations of divalent cations were needed to decrease NCC zeta-potential and form hydrogels. The same zeta-potentials and gel strengths were achieved at higher concentrations of monovalent salts. Salt-induced NCC aggregation is thus caused by intermolecular attractive forces rather than cationic bridging. Against excluded volume argumentation, salt addition was found to promote NCC nematic phase formation. Increased nematic ordering was observed in a transition regime of moderate salt addition before complete aggregation occurs. This regime is governed by an equilibrium of repulsive and attractive forces. Small angle neutron scattering suggests lateral orientation of NCC. Hence, NCC gelation and nematic ordering can be modulated via its zeta-potential by targeted salt addition.

3D Hydrogel Encapsulation Regulates Nephrogenesis in Kidney Organoids.

Stem cell-derived kidney organoids contain nephron segments that recapitulate morphological and functional aspects of the human kidney. However, directed differentiation protocols for kidney organoids are largely conducted using biochemical signals to control differentiation. Here, the hypothesis that mechanical signals regulate nephrogenesis is investigated in 3D culture by encapsulating kidney organoids within viscoelastic alginate hydrogels with varying rates of stress relaxation. Tubular nephron segments are significantly more convoluted in kidney organoids differentiated in encapsulating hydrogels when compared with those in suspension culture. Hydrogel viscoelasticity regulates the spatial distribution of nephron segments within the differentiating kidney organoids. Consistent with these observations, a particle-based computational model predicts that the extent of deformation of the hydrogel-organoid interface regulates the morphology of nephron segments. Elevated extracellular calcium levels in the culture medium, which can be impacted by the hydrogels, decrease the glomerulus-to-tubule ratio of nephron segments. These findings reveal that hydrogel encapsulation regulates nephron patterning and morphology and suggest that the mechanical microenvironment is an important design variable for kidney regenerative medicine.

Versatile Mechanically Tunable Hydrogels for Therapeutic Delivery Applications.

Hydrogels provide a versatile platform for biomedical material fabrication that can be structurally and mechanically fine-tuned to various tissues and applications. Applications of hydrogels in biomedicine range from highly dynamic injectable hydrogels that can flow through syringe needles and maintain or recover their structure after extrusion to solid-like wound-healing patches that need to be stretchable while providing a selective physical barrier. In this study, a toolbox is designed using thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) polymeric matrices and nanocelluloses as reinforcing agent to obtain biocompatible hydrogels with altering mechanical properties, from a liquid injectable to a solid-like elastic hydrogel. The liquid hydrogels possess low viscosity and shear-thinning properties at 25 °C, which allows facile injection at room temperature, while they become viscoelastic gels at body temperature. In contrast, the covalently cross-linked solid-like hydrogels exhibit enhanced viscoelasticity. The liquid hydrogels are biocompatible and are able to delay the in vitro release and maintain the bioactivity of model drugs. The antimicrobial agent loaded solid-like hydrogels are effective against typical wound-associated pathogens. This work presents a simple method of tuning hydrogel mechanical strength to easily adapt to applications in different soft tissues and broaden the potential of renewable bio-nanoparticles in hybrid biomaterials with controlled drug release capabilities.

Controlling lipid crystallization across multiple length scales by directed shear flow.

The crystallization behavior of lipids is relevant in many fields such as adipose tissue formation and regeneration, forensic investigations and food production. Using a lipid model system composed of triacylglycerols, we study the formation of crystalline structures under laminar shear flows across various length scales by polarized light-, scanning electron-, and atomic force microscopy, as well as laser diffraction spectroscopy. The shear rate during crystallization γ̇cryst influences the acyl-chain length structure and promotes domain growth into the flow direction thereby transforming the crystallites from oblate into prolate particles. Concentration dependent aggregation of crystallites into clusters is the rate limiting step for floc and floc network formation. At high γ̇cryst, fast crystallite cluster formation at smaller equilibrium diameters is promoted. The high crystallite cluster concentration induces their aggregation into flocs which form weak networks. At low γ̇cryst, floc generation is limited by the low amount of crystallite clusters leading to slow growth of larger flocs and forming of strong networks. The findings in this work have potential implications ranging from the design of injectable soft tissue fillers for adipose tissue regeneration, to the crystalline network formation in microorganism derived lipids, up to a more energy-efficient production of chocolate confectionery.

Colloidal hydrogels made of gelatin nanoparticles exhibit fast stress relaxation at strains relevant for cell activity.

Viscoelastic properties of hydrogels such as stress relaxation or plasticity have been recognized as important mechanical cues that dictate the migration, proliferation, and differentiation of embedded cells. Stress relaxation rates in conventional hydrogels are usually much slower than cellular processes, which impedes rapid cellularization of these elastic networks. Colloidal hydrogels assembled from nanoscale building blocks may provide increased degrees of freedom in the design of viscoelastic hydrogels with accelerated stress relaxation rates due to their strain-sensitive rheology which can be tuned via interparticle interactions. Here, we investigate the stress relaxation of colloidal hydrogels from gelatin nanoparticles in comparison to physical gelatin hydrogels and explore the particle interactions that govern stress relaxation. Colloidal and physical gelatin hydrogels exhibit comparable rheology at small deformations, but colloidal hydrogels fluidize beyond a critical strain while physical gels remain primarily elastic independent of strain. This fluidization facilitates fast exponential stress relaxation in colloidal gels at strain levels that correspond to strains exerted by cells embedded in physiological extracellular matrices (10-50%). Increased attractive particle interactions result in a higher critical strain and slower stress relaxation in colloidal gels. In physical gels, stress relaxation is slower and mostly independent of strain. Hence, colloidal hydrogels offer the possibility to modulate viscoelasticity via interparticle interactions and obtain fast stress relaxation rates at strains relevant for cell activity. These beneficial features render colloidal hydrogels promising alternatives to conventional monolithic hydrogels for tissue engineering and regenerative medicine. STATEMENT OF SIGNIFICANCE: In the endeavor to design biomaterials that favor cell activity, research has long focused on biochemical cues. Recently, the time-, stress-, and strain-dependent mechanical properties, i.e. viscoelasticity, of biomaterials has been recognized as important factor that dictates cell fate. We herein present the viscoelastic stress relaxation of colloidal hydrogels assembled from gelatin nanoparticles, which show a strain-dependent fluidization at strains relevant for cell activity, in contrast to many commonly used monolithic hydrogels with primarily elastic behavior.

Lipid emulsion interfacial design modulates human in vivo digestion and satiation hormone response.

Lipid emulsions (LEs) with tailored digestibility have the potential to modulate satiation or act as delivery systems for lipophilic nutrients and drugs. The digestion of LEs is governed by their interfacial emulsifier layer which determines their gastric structuring and accessibility for lipases. A plethora of LEs that potentially modulate digestion have been proposed in recent years, however, in vivo validations of altered LE digestion remain scarce. Here, we report on the in vivo digestion and satiation of three novel LEs stabilized by whey protein isolate (WPI), thermo-gelling methylcellulose (MC), or cellulose nanocrystals (CNCs) in comparison to an extensively studied surfactant-stabilized LE. LE digestion and satiation were determined in terms of gastric emptying, postprandial plasma hormone and metabolite levels characteristic for lipid digestion, perceived hunger/fullness sensations, and postprandial food intake. No major variations in gastric fat emptying were observed despite distinct gastric structuring of the LEs. The plasma satiation hormone and metabolite response was fastest and highest for WPI-stabilized LEs, indicating a limited capability of proteins to prevent lipolysis due to fast hydrolysis under gastric conditions and displacement by lipases. MC-stabilized LEs show a similar gastric structuring as surfactant-stabilized LEs but slightly reduced hormone and metabolite responses, suggesting that thermo-gelling MC prevents lipase adsorption more effectively. Ultimately, CNC-stabilized LEs showed a drastic reduction (>70%) in plasma hormone and metabolite responses. This confirms the efficiency of particle (Pickering) stabilized LEs to prevent lipolysis proposed in literature based on in vitro experiments. Subjects reported more hunger and less fullness after consumption of LEs stabilized with MC and CNCs which were able to limit satiation responses. We do not find evidence for the widely postulated ileal brake, i.e. that delivery of undigested nutrients to the ileum triggers increased satiation. On the contrary, we find decreased satiation for LEs that are able to delay lipolysis. No differences in food intake were observed 5 h after LE consumption. In conclusion, LE interfacial design modulates in vivo digestion and satiation response in humans. In particular, Pickering LEs show extraordinary capability to prevent lipolysis and qualify as oral delivery systems for lipophilic nutrients and drugs.