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.

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.

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.

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.

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.

Transient in situ measurement of kombucha biofilm growth and mechanical properties.

Kombucha is a traditional beverage obtained by the fermentation of sugared tea by a symbiotic culture of bacteria and yeast which has recently re-emerged as a popular lifestyle product with potential health benefits. The characteristic feature of kombucha is the formation of a cellulosic biofilm due to the excretion of bacterial cellulose with high purity and crystallinity. Despite the growing industrial and technological interest in kombucha, current characterization techniques rely on the periodic sampling of tea broth or biofilm and ex situ analysis of its biochemical or microbial composition. Here, we use interfacial shear rheology (ISR) for the transient in situ determination of kombucha biofilm growth directly at the interface. ISR revealed that kombucha biofilm formation is a two step process with clearly distinguishable growth phases. The first phase can be attributed to the initial adsorption of bacteria at the air-water interface and shows great variability, probably due to varying bacteria content and composition. The second phase is initiated by bacterial cellulose excretion and shows astonishing reproducibility regarding onset and final mechanical properties. Hence, ISR qualifies as a new in situ characterization technique for kombucha biofilm growth and bacterial cellulose production.

Injectable Biocompatible Hydrogels from Cellulose Nanocrystals for Locally Targeted Sustained Drug Release.

Injectable hydrogels from biocompatible materials are in demand for tissue engineering and drug delivery systems. Here, we produce hydrogels from mere cellulose nanocrystals (CNCs) by salt-induced charge screening. The injectability of CNC hydrogels was assessed by a combination of shear and capillary rheology, revealing that CNC hydrogels are conveyed via plug flow in capillaries allowing injection with minimal impact on mechanical properties. The potential of CNC hydrogels as drug carriers was elaborated by the in vitro release of the model protein bovine serum albumin (BSA), poorly water soluble tetracycline (TC), and readily soluble doxorubicin (DOX) into physiological saline and simulated gastric juice. For TC, a burst release was observed within 2 days, whereas BSA and DOX both showed a sustained release for 2 weeks. Only DOX was released fully from the hydrogels. The different release patterns were attributed to drug size, solubility, and specific drug-CNC interactions. The biocompatibility of CNC hydrogels and maintained bioactivity of released DOX were confirmed in a HeLa cell assay. The drug release was modulated by the incorporation of sucrose or xanthan gum in CNC hydrogels, whereas altering CNC concentration showed minor effects. The release into simulated gastric juice at pH 2 ceased for BSA due to charge inversion and electrostatic complexation, but not for smaller TC. Thus, CNC hydrogels may act as pH-responsive delivery systems that preserve drugs under gastric conditions followed by pH-triggered release in the duodenum.