Soft materials as biological and artificial membranes.

The past few decades have seen emerging growth in the field of soft materials for synthetic biology. This review focuses on soft materials involved in biological and artificial membranes. The biological membranes discussed here are mainly those involved in the structure and function of cells and organelles. As building blocks in medicine, non-native membranes including nanocarriers (NCs), especially liposomes and DQAsomes, and polymeric membranes for scaffolds are constructed from amphiphilic combinations of lipids, proteins, and carbohydrates. Artificial membranes can be prepared using synthetic, soft materials and molecules and then incorporated into structures through self-organization to form micelles or niosomes. The modification of artificial membranes can be realized using traditional chemical methods such as click reactions to target the delivery of NCs and control the release of therapeutics. The biomembrane, a lamellar structure inlaid with ion channels, receptors, lipid rafts, enzymes, and other functional units, separates cells and organelles from the environment. An active domain inserted into the membrane and organelles for energy conversion and cellular communication can target disease by changing the membrane's composition, structure, and fluidity and affecting the on/off status of the membrane gates. The biological membrane targets analyzing pathological mechanisms and curing complex diseases, which inspires us to create NCs with artificial membranes.

The effect of polarized light on the organization of collagen secreted by fibroblasts.

Recent studies have demonstrated the beneficial effect of low-power lasers and polarized light on wound healing, inflammation, and the treatment of rheumatologic and neurologic disorders. The overall effect of laser irradiation treatment is still controversial due to the lack of studies on the biochemical mechanisms and the optimal parameters for the incident light that should be chosen for particular applications. Here, we study how NIH/3T3 fibroblasts respond to irradiation with linearly polarized light at different polarization angles. In particular, we examined vascular endothelial growth factor (VEGF) secretion, differentiation to myofibroblasts, and collagen organization in response to 800 nm polarized light at 0°, 45°, 90°, and 135° with a power density of 40 mW/cm2 for 6 min every day for 6 days. Additional experiments were conducted in which the polarization angle of the incident was changed every day to induce an isotropic distribution of collagen. The data presented here shows that polarized light can upregulate VEGF production, myofibroblast differentiation, and induce different collagen organization in response to different polarization angles of the incident beam. These results are encouraging and demonstrate possible methods for controlling cell response through the polarization angle of the laser light, which has potential for the treatment of wounds.

Effect of surface modification and macrophage phenotype on particle internalization.

Material properties play a key role in the cellular internalization of polymeric particles. In the present study, we have investigated the effects of material characteristics such as water contact angle, zeta potential, melting temperature, and alternative activation of complement on particle internalization for pro-inflammatory, pro-angiogenic, and naïve macrophages by using biopolymers (∼600 nm), functionalized with 13 different molecules. Understanding how material parameters influence particle internalization for different macrophage phenotypes is important for targeted delivery to specific cell populations. Here, we demonstrate that material parameters affect the alternative pathway of complement activation as well as particle internalization for different macrophage phenotypes. Here, we show that the quantitative structure-activity relationship method (QSAR) previously used to predict physiochemical properties of materials can be applied to targeting different macrophage phenotypes. These findings demonstrated that targeted drug delivery to macrophages could be achieved by exploiting material parameters.

Improving selective targeting to cancer-associated fibroblasts by modifying liposomes with arginine based materials.

A library of arginine-like surface modifiers was tested to improve the targetability of DOPE:DOPC liposomes towards myofibroblasts in a tumour microenvironment. Liposomes were characterised using zeta potential and dynamic light scattering. Cell viability remained unchanged for all liposomes. Liposomes were encapsulated using doxorubicin (DOX) with an encapsulation efficiency >94%. The toxicity of DOX-loaded liposomes was calculated via half-maximal inhibitory concentration (IC50) for fibroblasts and myofibroblasts. These liposomes resulted in significantly lower IC50-values for myofibroblasts compared to fibroblasts, making them more toxic towards the myofibroblasts. Furthermore, a significant increase in cell internalisation was observed for myofibroblasts compared to fibroblasts, using fluorescein-loaded liposomes. Most importantly, a novel regression model was constructed to predict the IC50-values for different modifications using their physicochemical properties. Fourteen modifications (A-N) were used to train and validate this model; subsequently, this regression model predicted IC50-values for three new modifications (O, P and Q) for both fibroblasts and myofibroblasts. Predicted and measured IC50-values showed no significant difference for fibroblasts. For myofibroblasts, modification O showed no significant difference. This study demonstrates that the tested surface modifications can improve targeting to myofibroblasts in the presence of fibroblasts and hence are suitable drug delivery vehicles for myofibroblasts in a tumour microenvironment.

Organoids.

Organoids have attracted increasing attention because they are simple tissue-engineered cell-based in vitro models that recapitulate many aspects of the complex structure and function of the corresponding in vivo tissue. They can be dissected and interrogated for fundamental mechanistic studies on development, regeneration, and repair in human tissues. Organoids can also be used in diagnostics, disease modeling, drug discovery, and personalized medicine. Organoids are derived from either pluripotent or tissue-resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumors. To date, numerous organoid engineering strategies that support organoid culture and growth, proliferation, differentiation and maturation have been reported. This Primer serves to highlight the rationale underlying the selection and development of these materials and methods to control the cellular/tissue niche; and therefore, structure and function of the engineered organoid. We also discuss key considerations for generating robust organoids, such as those related to cell isolation and seeding, matrix and soluble factor selection, physical cues and integration. The general standards for data quality, reproducibility and deposition within the organoid community is also outlined. Lastly, we conclude by elaborating on the limitations of organoids in different applications, and key priorities in organoid engineering for the coming years.

Effect of RGD functionalization and stiffness of gellan gum hydrogels on macrophage polarization and function.

Macrophages, the primary effector cells in the immune response, respond rapidly to the physical or chemical properties of biomaterial implants. Balanced macrophage polarization, phagocytosis, and migration would be beneficial for implant success and tissue regeneration. Here, we investigated macrophage phenotypic changes, phagocytosis, and migration in response to RGD functionalized surfaces and changes in stiffness of gellan gum hydrogels. We also inhibited the RhoA pathway. The compressive moduli ranged from ~5 to 30 kPa. Cell population and cell spreading area of classically activated macrophages (M(LPS)) and alternatively activated macrophages (M(IL-4)) are promoted on RGD modified hydrogel. ROCK inhibitor induced the opposite effect on the cell spreading of both M(LPS) and M(IL-4) macrophages on RGD modified hydrogels. Macrophage polarization was found to be stiffness-driven and regulated by the RGD motif and blocked by the RhoA pathway. RGD functionalized hydrogel shifted M(IL-4) cells toward a more pro-inflammatory phenotype, while ROCK inhibition shifted M(LPS) cells to a more anti-inflammatory phenotype. Both M(LPS) and M(IL-4) cells on untreated hydrogels shifted to a more pro-inflammatory phenotype in the presence of aminated-PS particles. The RGD motif had a significant impact on cellular uptake, whereas cellular uptake was stiffness driven on untreated hydrogels. Cell migration of M(LPS) and M(IL-4) cells had ROCK-dependent migration. The stiffness of gellan gum hydrogels had no influence on macrophage migration rate. Collectively, our results showed that gellan gum hydrogels can be used to direct immune response, macrophage infiltration, and phagocytosis.

Macrophage Phenotypic Changes on FN-Coated Physical Gradient Hydrogels.

The chemical and physical properties are two crucial cues when designing tissue engineering scaffold to mimic living tissue. Macrophages, the major players in the immune response, react rapidly to microenvironmental signals, including gradients of physical or chemical cues. Spatiotemporal gradients can modulate cell behavior, such as polarization, proliferation, and adhesion. Here, we studied macrophage phenotypic changes on untreated and fibronectin (FN)-coated methacrylated gellan gum with varying stiffnesses. The compressive moduli of hydrogel with different stiffnesses ranged from ∼5 to 30 kPa. Fibronectin was chemically attached to the substrate to facilitate macrophage proliferation, adhesion, and polarization. Classically (M1) and alternatively (M2) activated macrophages were cultured on both untreated and FN-coated gels. FN-coated substrates elevated cell numbers and enhanced macrophage spreading. The urea/nitrite ratio indicated that untreated rigid substrates shifted both polarizations toward a more proinflammatory phenotype. FN-coated substrates had no impact on M1 polarization. In contrast, FN-coated stiffer gels polarized M2 cells toward an anti-proinflammatory state based on arginine activity and CD206 expression. In addition, macrophage polarization on the softer gel was not influenced by the neighboring cells cultured on the stiffer side of the gel. Using mechanical gradients to control macrophage polarization can be a useful tool in ensuring a proper healing response and for tissue engineering.

The Influence of Polysaccharides-Based Material on Macrophage Phenotypes.

Macrophage polarization is a key factor in determining the success of implanted tissue engineering scaffolds. Polysaccharides (derived from plants, animals, and microorganisms) are known to modulate macrophage phenotypes by recognizing cell membrane receptors. Numerous studies have developed polysaccharide-based materials into functional biomaterial substrates for tissue regeneration and pharmaceutical application due to their immunostimulatory activities and anti-inflammatory response. They are used as hydrogel substrates, surface coatings, and drug delivery carriers. In addition to their innate immunological functions, the newly endowed physical and chemical properties, including substrate modulus, pore size/porosity, surface binding chemistry, and the mole ratio of polysaccharides in hybrid materials may regulate macrophage phenotypes more precisely. Growing evidence indicates that the sulfation pattern of glycosaminoglycans and proteoglycans expressed on polarized macrophages leads to the changes in protein binding, which may alter macrophage phenotype and influence the immune response. A comprehensive understanding of how different types of polysaccharide-based materials alter macrophage phenotypic changes can be beneficial to predict transplantation/implantation outcomes. This review focuses on recent advances in promoting wound healing and balancing macrophage phenotypes using polysaccharide-based substrates/coatings and new directions to address the limitations in the current understanding of macrophage responses to polysaccharides.

Fibroblasts treated with macrophage conditioned medium results in phenotypic shifts and changes in collagen organization.

In tissue regeneration, the goal is to regenerate tissue similar to what was damaged or missing while preventing fibrotic scarring, which may lead to decreased mechanical strength and dissimilar tissue characteristics compared to native tissue. We believe collagen orientation plays a critical role in wound contraction and scarring and that it is modulated by myofibroblasts. We used macrophage conditioned medium to simulate complex events that can influence the fibroblast phenotype during the wound healing process. In addition to examining the effect of macrophage phenotype on fibroblasts, we inhibited focal adhesion kinase (FAK), Rho-associated protein kinase (ROCK), and myosin II for fibroblasts cultured on both tissue culture plastic and methacrylated gellan gum to understand how different pathways and materials influence fibroblast responses. Collagen orientation, α-SMA expression, focal adhesion area, and cell migration were altered by inhibition of FAK, ROCK, or myosin II and macrophage phenotype, along with the substrate. An increase in either focal adhesion area or α-smooth muscle actin (α-SMA) expression correlated with an aligned collagen orientation. Gellan gum hydrogels upregulated α-SMA expression in ROCK inhibited conditioned media and downregulated the FAK area in FAK and ROCK inhibited conditioned media. Myosin II had no impact on the α-SMA expression on the substrate compared to coverslip except for M2 conditioned medium. Gellan gum hydrogel significantly increased cell migration under FAK and Myosin II mediated conditioned media and unconditioned media. Collectively, our study examined how macrophage phenotype influences fibroblast response, which would be beneficial in controlling scar tissue formation.

Gellan gum-gelatin viscoelastic hydrogels as scaffolds to promote fibroblast differentiation.

Fabricating hydrogel scaffolds that are both bioreactive toward fibroblasts while still mechanically compatible with surrounding tissue is a major challenge in tissue engineering. This is because the outcome of scaffold implantation is largely determined by fibroblasts differentiating toward myofibroblasts, which is characterized by the expression of α-smooth muscle actin (α-SMA). Previous studies promoted fibroblasts differentiation by increasing scaffold substrate stiffness. However, the stiffness of scaffold has to be compatible with surrounding tissue, as mismatched stiffness may cause initial hyperplasia and inappropriate endothelial layer development. Therefore, we adjusted the hydrogel chemical component, and thus viscoelasticity to affect the mechano-signaling of fibroblasts and promote fibroblasts differentiation. Elastic gellan gum and viscoelastic gelatin were hybridized at different ratios to fabricate hydrogel scaffold with varied stress-relaxation. Vitronectin (VN) was used to further regulate the interaction between fibroblasts and the substrate. Fibroblast differentiation, characterized by α-SMA area per cell, increased from~3000-4000 µm2/cell on less viscoelastic gels to ~5000 µm2/cell on the most viscoelastic gel. Fibroblasts seeded on hydrogels had a slower migration rate on more viscoelastic hydrogels (slowest at 38 ± 14 µm/h) compared to the migration speed on less viscoelastic hydrogels (74 ± 20 µm/h). VN slowed the migration speed on all hydrogels. The organization of collagen deposited by fibroblasts cultured on the hydrogels was characterized by second harmonic generation (SHG), which showed that collagen was more organized (parallel) on more viscoelastic hydrogels. In summary, we provided a novel strategy to fabricate hydrogel scaffolds that can promote fibroblasts differentiation while keeping the stiffness compatible with blood vessels. The most viscoelastic hydrogel studied here meets these requirements best.

Manipulate intestinal organoids with niobium carbide nanosheets.

Multifunctional two-dimensional nanosheet materials have attracted attention in biomedical fields due to their unique physiochemical and biological properties. Interactions between intestinal stem cells and Engineered Nanomaterials (ENMs) are an essential area in research with the growing diagnosis of gastrointestinal (GI) diseases. One unique type of two-dimensional metal carbide nanomaterial, niobium carbide (Nb2 C), has shown promising properties for potential applications in this field, such as biocompatibility, stability, and high photothermal conversion efficiency. In this study, Nb2 C nanosheets were prepared by spark plasma sintering and HF etching. Various concentrations of Nb2 C nanosheets were placed inside intestinal organoids, which mimic the real functions of an intestinal system. These organoids were formed from intestinal crypts that were isolated from mice and grew into self-maintained systems. Through growth analysis, surface area calculations, and cell viability tests, it was concluded that an optimal concentration of nanosheets exists that may offer stimulation to intestinal cells while having no toxic effects. A high concentration of nanosheets in the organoids inhibited growth, whereas the control and low concentration of nanosheets showed no reduced growth rate. When placed under infrared exposure, the organoids with nanosheets offered stimulation and showed more viability after time as compared to the control organoids with no nanosheets. These results show overall potential benefits of placing low concentration Nb2 C nanosheets in intestinal systems to protect and stimulate cell survivability when undergoing various treatments.

Poly-l-arginine modifications alter the organization and secretion of collagen in SKH1-E mice.

Functionalized biomaterials interface with tissue upon implantation. There is a growing need to understand how materials properties influence this interaction so that efficient tissue engineering systems can be developed. In this study, we characterize collagen organization in response to functionalized glass beads implanted in SKH1-E mice. Poly-l-arginine (PLR) was modified with arginine derivatives to create a functionalized surface and was coated on glass beads. Tissue sections were removed 28 days post-implantation and were imaged using second harmonic generation (SHG) microscopy. These chemical modifications were able to alter the collagen distribution from highly aligned to disordered (17 ±â€¯6 to 78 ±â€¯1° full width at half-maximum (FWHM)) and the collagen III/I ratio (0.02 to 0.42). Principal component analysis (PCA) comparing the physical properties of the modifiers (e.g. hydrophobicity, molar volume, freely rotating bonds, polarizability) with the SHG analytically derived parameters (e.g. collagen III/I ratio, collagen orientation) was performed. Chemical properties of the PLR-like modifications including lipophilicity, along with the number of freely rotating bonds and the polarizability had significant effects on the collagen surrounding the implant, both in terms of collagen orientation as well as the production of collagen III. These findings demonstrate the possibility of tuning the foreign body response, in terms of collagen deposition and organization, to positively influence the acceptance of implanted biomaterials.

How Cross-Linking Mechanisms of Methacrylated Gellan Gum Hydrogels Alter Macrophage Phenotype.

In tissue engineering scaffolds, macrophages play a critical role in determining the host response to implanted biomaterials. Macrophage phenotype is dynamic throughout the host response, and a balance of phenotypes is essential for timely progression from injury to proper wound healing. Therefore, it is important to predict how materials will modulate the response of macrophages. In this study, we investigated the effect of methacrylated gellan gum (GG) hydrogels on macrophage phenotype and proliferation with the ultimate goal of improving rational design of biomedical implants. Naïve, along with classically and alternatively activated RAW 264.7 macrophages were seeded on methacrylated gellan gum hydrogels that were fabricated with different thiol-ene ratios and cross-linking mechanisms. Live/dead assays showed that all hydrogels supported cell attachment and proliferation. Stiffer substrates enhanced anti-inflammatory production of nitrites from both naïve and classically activated macrophages compared to the softer substrates. Moreover, arginine and CD206 expression-markers for alternatively activated macrophages-were inhibited by higher thiol content. Introducing ionic cross-links using calcium did not influence the proliferation or polarization for any of the three macrophage phenotypes. Our results suggest that the macrophage phenotype shift from M1 to M2 is controlled by the different cross-linking mechanisms, physical properties, and the chemistry of methacrylated gellan gum hydrogels.

Second harmonic generation microscopy of collagen organization in tunable, environmentally responsive alginate hydrogels.

We fabricated photocrosslinked, environmentally responsive alginate hydrogels for tissue engineering applications. Methacrylated alginate (ALGMA) hydrogels were prepared across a variety and combination of ionic and covalent (chain growth, step growth, and mixed mode) crosslinking strategies to obtain a range of compressive moduli from 9.3 ± 0.2 kPa for the softest ionically crosslinked hydrogels to 22.6 ± 0.3 kPa for the dually crosslinked ionic mixed mode gels. The swelling behavior of the alginate hydrogels was significantly higher under basic pH conditions. Stiffer gels consistently swelled to a lesser degree compared to softer gels for all conditions. These hydrogels were stable - retaining >80% of their original mass for three weeks - when incubated in a basic solution of diluted sodium hydroxide, which mimicked accelerated degradation conditions. Encapsulated NIH/3T3 fibroblasts remained viable and proliferated significantly more in stiffer hydrogel substrates compared to softer gels. Additionally, the collagen secreted by encapsulated fibroblasts was quantifiably compared using second harmonic generation (SHG) imaging. Fibroblasts encapsulated in the softer hydrogels secreted significantly less collagen than the stiffer gels. The collagen in these softer gels was also more aligned than the stiffer gels. The ability to tune collagen organization using hydrogels has potential applications ranging from corneal wound healing where organized collagen is desired to epithelial wound scaffolds where a random organization is preferable.

Degradation and Remodeling of Epitaxially Grown Collagen Fibrils.

INTRODUCTION­: The extracellular matrix (ECM) in the tumor microenvironment contains high densities of collagen that are highly aligned, resulting in directional migration called contact guidance that facilitates efficient migration out of the tumor. Cancer cells can remodel the ECM through traction force controlled by myosin contractility or proteolytic activity controlled by matrix metalloproteinase (MMP) activity, leading to either enhanced or diminished contact guidance. METHODS­: Recently, we have leveraged the ability of mica to epitaxially grow aligned collagen fibrils in order to assess contact guidance. In this article, we probe the mechanisms of remodeling of aligned collagen fibrils on mica by breast cancer cells. RESULTS­: We show that cells that contact guide with high fidelity (MDA-MB-231 cells) exert more force on the underlying collagen fibrils than do cells that contact guide with low fidelity (MTLn3 cells). These high traction cells (MDA-MB-231 cells) remodel collagen fibrils over hours, pulling so hard that the collagen fibrils detach from the surface, effectively delaminating the entire contact guidance cue. Myosin or MMP inhibition decreases this effect. Interestingly, blocking MMP appears to increase the alignment of cells on these substrates, potentially allowing the alignment through myosin contractility to be uninhibited. Finally, amplification or dampening of contact guidance with respect to a particular collagen fibril organization is seen under different conditions. CONCLUSIONS­: Both myosin II contractility and MMP activity allow MDA-MB-231 cells to remodel and eventually destroy epitaxially grown aligned collagen fibrils.

Vitamin C and B3 as new biomaterials to alter intestinal stem cells.

Vitamin C (ascorbic acid) and vitamin B3 (niacin) have been extensively studied since the 20th century. In the area of stem cell biology, vitamin C has shown its direct impact toward homeostasis and epigenetic changes (D'Aniello et al., Stem Cells International, 2017, 1-16). Vitamin B3 aids in maintaining healthy intestinal homeostasis and reducing gut inflammation by participating in the rapamycin signaling pathway (Kumar et al., The American Journal of Physiology-Gastrointestinal and Liver Physiology, 2013). In this study, vitamin C and vitamin B3 (600 and 1,200 µg/mL) have been explored as potential new biomaterials to study their effects on four types of intestinal stem cells which are isolated from mice bearing different microbiota. We observed that C3H ASF and 129 ASF IL-10 are more sensitive towardB7 600 µg/mL vitamin B3 and 1,200 µg/mL vitamin C. The lowest growth rate and viability for all types of organoids was with 1,200 µg/mL vitamin C. From quantitative polymerase chain reaction analysis (qPCR analysis), MUC2 was upregulated for 129 ASF and C3H Conv when exposed to 600 µg/mL and 1,200 µg/mL vitamin C. It suggests that large amounts of glycoprotein may be produced after adding high concentrations of vitamin C. Since inflammatory bowel disease has low level of MUC2, this finding may be helpful in restoring mucosal health by upregulating the MUC2 gene while altering patient's microbiota (Sibila et al., Annals of the American Thoracic Society, 2016). These results are expected to have a positive translational impact because this bottom-up strategy would be instrumental in developing Vitamin C and B3 based orally available therapeutic strategies and formula for advancing the fields of gastrointestinal regenerative medicine.

Click Chemistry and Material Selection for in Situ Fabrication of Hydrogels in Tissue Engineering Applications.

This Review gives a brief introduction to hydrogels formed through click chemistry for applications in tissue engineering. Specifically, we focus on three representative click chemistry mechanisms: Diels-Alder reactions, azide-alkyne cycloaddition, and thiol-ene chemistry. Apart from that, we also discuss photoinitiated chain growth polymerization, which also has fast kinetics. The chemical mechanisms of these reactions, along with their advantages and disadvantages, are presented. These gelation methods are compared and contrasted with other methods of forming hydrogels. Further, we offer an insight on the fabrication of click chemistry hydrogels from a material selection perspective. Commonly used materials from both synthetic and naturally derived polymer families were selected and discussed with their special features and drawbacks in fabricating hydrogels used in tissue engineering applications. At the end, the impact of cross-linking mechanisms and hydrogels properties on the host response is discussed. In conclusion, click reactions are modular and stereospecific. These reactions proceed rapidly with high selectivity, which means they have the potential to be formed in situ with minimal interference on biological processes. In order to achieve optimal hydrogels for tissue engineering applications, it is important to consider different design principles and material fabrication strategies to develop optimal hydrogels for regenerative medicine applications.

Collagen organization deposited by fibroblasts encapsulated in pH responsive methacrylated alginate hydrogels.

The pH of dermal wounds shifts from neutral during the inflammatory phase to slightly basic in the tissue remodeling phase. Stage specific wound treatment can be developed using environmentally responsive alginate hydrogels. The chemistry of these networks dictates swelling behavior. Here, we fabricated alginate hydrogels using chain growth, step growth, and combined mixed mode gelation methods to crosslink methacrylated alginate (ALGMA) and gain control over swelling responses. Methacrylation of the alginate network was confirmed through NMR spectroscopy. Strontium cations were introduced to fabricate stiffer, dually crosslinked hydrogels. Dual crosslinking significantly decreased the swelling response over the pH range of 3-9 for step growth and chain growth hydrogels, with no impact on mixed mode hydrogels. The extent of crosslinking altered the hydrogel degradation profiles under accelerated degradation conditions. Encapsulated NIH/3T3 fibroblasts in the different ALGMA hydrogels remained viable with greater cell proliferation in the stiffer gels. Collagen organization deposited by the NIH/3T3 fibroblasts was monitored using second harmonic generation (SHG) microscopy and was influenced by the crosslinking mechanism. Ionic chain growth and ionic mixed mode crosslinked ALGMA hydrogels caused relatively isotropic collagen organization, particularly 10 days post-cell encapsulation. Principal component analysis (PCA) was employed to uncover correlations between the observed properties. The ability of these environmentally responsive gels to induce isotropic collagen and respond to pH changes means they hold promise as phase specific wound dressings. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2934-2943, 2018.

Chemically Modified Gellan Gum Hydrogels with Tunable Properties for Use as Tissue Engineering Scaffolds.

Gellan gum is a naturally occurring polymer that can cross-link in the presence of divalent cations to form biocompatible hydrogels. However, physically cross-linked gellan gum hydrogels lose their stability under physiological conditions, thus restricting the applications of these hydrogels in vivo. To improve the mechanical strength of the gels, we incorporated methacrylate into the gellan gum and chemically cross-linked the hydrogel through three polymerization methods: step growth through thiol-ene photoclick chemistry, chain-growth via photopolymerization, and mixed model in which both mechanisms were employed. Methacrylation was confirmed and quantified by proton nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy. The mechanical properties and chemistry of the cross-linked gels were systematically altered by varying the reaction conditions. The compression moduli of the resulting hydrogels ranged between 6.4 and 17.2 kPa. The swelling ratios of the hydrogels were correlated with the compression moduli and affected by the addition of calcium. In vitro enzymatic degradation rate was found to depend on the degree of methacrylation. NIH/3T3 fibroblast cell proliferation and morphology were related to substrate stiffness, with a high stiffness leading generally to higher proliferation. The proliferation is further affected by the thiol-ene ratio. These results suggest that a hydrogel platform based on the gellan gum can offer versatile chemical modifications and tunable mechanical properties. The influence of these substrates on cell behavior suggests that the gellan gum hydrogels have the flexibility to be engineered for a variety of biomaterials applications.

Transfer of assembled collagen fibrils to flexible substrates for mechanically tunable contact guidance cues.

Contact guidance or bidirectional migration along aligned fibers modulates many physiological and pathological processes such as wound healing and cancer invasion. Aligned 2D collagen fibrils epitaxially grown on mica substrates replicate many features of contact guidance seen in aligned 3D collagen fiber networks. However, these 2D collagen self-assembled substrates are difficult to image through, do not have known or tunable mechanical properties and cells degrade and mechanically detach collagen fibrils from the surface, leading to an inability to assess contact guidance over long times. Here, we describe the transfer of aligned collagen fibrils from mica substrates to three different functionalized target substrates: glass, polydimethylsiloxane (PDMS) and polyacrylamide (PA). Aligned collagen fibrils can be efficiently transferred to all three substrates. This transfer resulted in substrates that were to varying degrees resistant to cell-mediated collagen fibril deformation that resulted in detachment of the collagen fibril field, allowing for contact guidance to be observed over longer time periods. On these transferred substrates, cell speed is lowest on softer contact guidance cues for both MDA-MB-231 and MTLn3 cells. Intermediate stiffness resulted in the fastest migration. MTLn3 cell directionality was low on soft contact guidance cues, whereas MDA-MB-231 cell directionality marginally increased. It appears that the stiffness of the contact guidance cue regulates contact guidance differently between cell types. The development of this collagen fibril transfer method allows for the attachment of aligned collagen fibrils on substrates, particularly flexible substrates, that do not normally promote aligned collagen fibril growth, increasing the utility of this collagen self-assembly system for the fundamental examination of mechanical regulation of contact guidance.