Cell Adhesion Motif Research Articles

Blended alginate/collagen hydrogels promote neurogenesis and neuronal maturation.

Brain extracellular matrix (ECM) is complex, heterogeneous and often poorly replicated in traditional 2D cell culture systems. The development of more physiologically relevant 3D cell models capable of emulating the native ECM is of paramount importance for the study of human induced pluripotent stem cell (iPSC)-derived neurons. Due to its structural similarity with hyaluronic acid, a primary component of brain ECM, alginate is a potential biomaterial for 3D cell culture systems. However, a lack of cell adhesion motifs within the chemical structure of alginate has limited its application in neural culture systems. This study presents a simple and accessible method of incorporating collagen fibrils into an alginate hydrogel by physical mixing and controlled gelation under physiological conditions and tests the hypothesis that such a substrate could influence the behaviour of human neurons in 3D culture. Regulation of the gelation process enabled the penetration of collagen fibrils throughout the hydrogel structure as demonstrated by transmission electron microscopy. Encapsulated human iPSC-derived neurons adhered to the blended hydrogel as evidenced by the increased expression of α1, α2 and β1 integrins. Furthermore, immunofluorescence microscopy revealed that encapsulated neurons formed complex neural networks and matured into branched neurons expressing synaptophysin, a key protein involved in neurotransmission, along the neurites. Mechanical tuning of the hydrogel stiffness by modulation of the alginate ionic crosslinker concentration also influenced neuron-specific gene expression. In conclusion, we have shown that by tuning the physicochemical properties of the alginate/collagen blend it is possible to create different ECM-like microenvironments where complex mechanisms underpinning the growth and development of human neurons can be simulated and systematically investigated.

The mechanics of single cross-links which mediate cell attachment at a hydrogel surface.

The response of cultured cells to the mechanical properties of hydrogel substrates depends ultimately on the response of single crosslinks to external forces exerted at cell attachment points. We prepared hydrogels by co-polymerization of poly(ethylene glycol diacrylate) (PEGDA) and carboxy poly(ethylene glycol) acrylate (ACPEG-COOH) and confirmed fibroblast spreading on the hydrogel after the ACPEG linker was functionalized with the RGD cell adhesive motif. We performed specific force spectroscopy experiments on the same ACPEG linkers in order to probe the mechanics of single cross-links which mediate the cell attachment and spreading. Measurements were performed with tips of an atomic force microscope (AFM) functionalized with streptavidin and ACPEG linkers functionalized with biotin. We compared hydrogels of varying elastic modulus between 4 and 41 kPa which exhibited significant differences in cell spreading. An effective spring constant for the displacement of single cross-links at the hydrogel surface was derived from the distributions of rupture force and molecular stiffness. A factor of ten in the elastic modulus E of the hydrogel corresponded to a factor of five in the effective spring constant k of single crosslinks, indicating a transition in scaling with the mesh size ξ from the macroscopic E∝ξ-3 to the molecular k∝ξ-2. The quantification of stiffness and deformation at the molecular length scale contributes to the discussion of mechanisms in force-regulated phenomena in cell biology.

Fabrication of modular hyaluronan-PEG hydrogels to support 3D cultures of hepatocytes in a perfused liver-on-a-chip device

Liver cell culture models are attractive in both tissue engineering and for development of assays for drug toxicology research. To retain liver specific cell functions, the use of adequate cell types and culture conditions, such as a 3D orientation of the cells and a proper supply of nutrients and oxygen, are critical. In this article, we show how extracellular matrix mimetic hydrogels can support hepatocyte viability and functionality in a perfused liver-on-a-chip device. A modular hydrogel system based on hyaluronan and poly(ethylene glycol) (HA-PEG), modified with cyclooctyne moieties for bioorthogonal strain-promoted alkyne-azide 1, 3-dipolar cycloaddition (SPAAC), was developed, characterized, and compared for cell compatibility to hydrogels based on agarose and alginate. Hepatoma cells (HepG2) formed spheroids with viable cells in all hydrogels with the highest expression of albumin and urea in alginate hydrogels. By including an excess of cyclooctyne in the HA backbone, azide-modified cell adhesion motifs (linear and cyclic RGD peptides) could be introduced in order to enhance viability and functionality of human induced pluripotent stem cell derived hepatocytes (hiPS-HEPs). In the HA-PEG hydrogels modified with cyclic RGD peptides hiPS-HEPs migrated and grew in 3D and showed an increased viability and higher albumin production compared to when cultured in the other hydrogels. This flexible SPAAC crosslinked hydrogel system enabled fabrication of perfused 3D cell culture of hiPS-HEPs and is a promising material for further development and optimization of liver-on-a-chip devices.

CELL LADEN ALGINATE/ALBUMIN HYDROGEL FIBERS FOR POTENTIAL SKIN TISSUE ENGINEERING APPLICATIONS

Alginate hydrogel fibers are receiving a great attention for tissue engineering applications. However, an important limitation of alginate is that it does not provide cell adhesion motifs. In this work, albumin was blended with alginate to improve the compatibility of alginate fibers with cells. Cell laden alginate/albumin fibers, potentially usable for skin regeneration, were obtained through a spinning process, by extruding an alginate/albumin solution containing cells into a calcium chloride solution. Cell laden pure alginate fibers were prepared for comparison. Plain alginate and alginate/albumin fibers were also produced. Morphological, mechanical and functional properties of the produced fibers were investigated. In addition, the ability of the fibers to release albumin and to support the viability and growth of A549 cells embedded into them was studied. Fibers with a uniform shape and an average diameter within the range 550–570[Formula: see text][Formula: see text]m were produced. The water content was [Formula: see text]% for alginate fibers, and [Formula: see text]% for alginate/albumin fibers. Stress–strain tests showed, up to a strain value of 20%, the same Young’s modulus for the produced fibers, regardless of the presence of albumin. Overall, obtained results demonstrated that morphology, size, hydrophilicity and mechanical properties were not affected by albumin. Albumin was gradually released over a period of 4 days, with a residual amount (13%) remaining into the fibers. Viability test was carried out on A549 cells, laden inside alginate and alginate/albumin fibers, to evaluate cell proliferation ability. A favorable effect of albumin on the loaded cells was evidenced by a faster kinetics of growth.

Enhanced Osteogenesis of Bone Marrow-Derived Mesenchymal Stem Cells by a Functionalized Silk Fibroin Hydrogel for Bone Defect Repair.

Silk fibroin (SF) from Bombyx mori is a promising natural material for the synthesis of biocompatible and biodegradable hydrogels for use in biomedical applications from tissue engineering to drug delivery. However, weak gelation performance and the lack of biochemical cues to trigger cell proliferation and differentiation currently significantly limit its application in these areas. Herein, a biofunctional hydrogel containing SF (2.0%) and a small peptide gelator (e.g., NapFFRGD = 1.0 wt%) is generated via cooperative molecular self-assembly. The introduction of NapFFRGD to SF is shown to significantly improve its gelation properties by lowering both its threshold gelation concentration to 2.0% and gelation time to 20 min under physiological conditions (pH = 7.4, 37 °C), as well as functionalizing the SF hydrogel with cell-adhesive motifs (e.g., RGD). Besides mediating cell adhesion, the RGD ligands incorporated within the SF-RGD gel promote the osteogenic differentiation of bone marrow-derived mesenchymal stem cells encapsulated within the gel matrix, leading to bone regeneration in a mouse calvarial defect model, compared with a blank SF gel (2.0%, pH = 7.4). This work suggests that SF could be easily tailored with bioactive peptide gelators to afford bioactive hydrogels with favorable microenvironments for tissue regeneration applications.

Novel Pathway for Efficient Covalent Modification of Polyester Materials of Different Design to Prepare Biomimetic Surfaces.

To form modern materials with biomimic surfaces, the novel pathway for surface functionalization with specific ligands of well-known and widely used polyester-based rigid media was developed and optimized. Two types of material bases, namely, poly(lactic acid) and poly(ε-caprolactone), as well as two types of material design, e.g., supermacroporous matrices and nanoparticles (NPs), were modified via covalent attachment of preliminary oxidized polyvinylsaccharide poly(2-deoxy-N-methacryloylamido-d-glucose) (PMAG). This polymer, being highly biocompatible and bioinspired, was used to enhance hydrophilicity of the polymer surface and to provide the elevated concentration of reactive groups required for covalent binding of bioligands of choice. The specialties of the interaction of PMAG and its preliminary formed bioconjugates with a chemically activated polyester surface were studied and thoroughly discussed. The supermacroporous materials modified with cell adhesion motifs and Arg-Gly-Asp-containing peptide (RGD-peptide) were tested in the experiments on bone tissue engineering. In turn, the NPs were modified with bioligands (“self-peptide” or camel antibodies) to control their phagocytosis that can be important, for example, for the preparation of drug delivery systems.

Transglutaminase-Triggered Gelation and Functionalization of Designed Self-Assembling Peptides for Guiding Cell Migration.

Due to the structural and compositional similarity to the extracellular matrix (ECM), self-assembling peptide hydrogels (SAPHs) have shown potential as three-dimensional (3D) scaffolds in regenerative medicine. However, how to fabricate SAPHs with controlled bioactivity for specific applications still remains greatly challenging. In this study, we develop a viable strategy to prepare bioactive SAPHs based on designed short amphiphilic peptides and cell-adhesive motifs, through the combination of enzymatic functionalization and gelation methods. Transglutaminase (TGase) can successfully catalyze the tethering of bioactive features onto the peptide nanofiber surface, and the TGase-meditated conjugation is found to rapidly trigger peptide gelation. Confocal microscopy indicates a uniform ligand distribution within the fibrous hydrogel, and mass spectrometry measurements reveal the involved enzymatic reactions and the gelation mechanism. Furthermore, the preosteoblast cells introduced on the bioactive gel surface are found to be able to migrate into the gel to form 3D distribution and further undergo differentiation, primarily directed by the cell-adhesive motifs within the gel. Such a TGase-based strategy for reconstituting bioactive hydrogels can be readily applied to any other self-assembling peptides and active motifs given rational molecular design, thus showing broad-spectrum application potentials in biological and biomedical fields.

Influence of collagen-based integrin α1 and α2 mediated signaling on human mesenchymal stem cell osteogenesis in three dimensional contexts.

Collagen I interactions with integrins α1 and α2 are known to support human mesenchymal stem cell (hMSC) osteogenesis. Nonetheless, elucidating the relative impact of specific integrin interactions has proven challenging, in part due to the complexity of native collagen. In the present work, we employed two collagen-mimetic proteins-Scl2-2 and Scl2-3- to compare the osteogenic effects of integrin α1 versus α2 signaling. Scl2-2 and Scl2-3 were both derived from Scl2-1, a triple helical protein lacking known cell adhesion, cytokine binding, and matrix metalloproteinase sites. However, Scl2-2 and Scl2-3 were each engineered to display distinct collagen-based cell adhesion motifs: GFPGER (binding integrins α1 and α2 ) or GFPGEN (binding only integrin α1 ), respectively. hMSCs were cultured within poly(ethylene glycol) (PEG) hydrogels containing either Scl2-2 or Scl2-3 for 2 weeks. PEG-Scl2-2 gels were associated with increased hMSC osterix expression, osteopontin production, and calcium deposition relative to PEG-Scl2-3 gels. These data indicate that integrin α2 signaling may have an increased osteogenic effect relative to integrin α1 . Since p38 is activated by integrin α2 but not by integrin α1 , hMSCs were further cultured in PEG-Scl2-2 hydrogels in the presence of a p38 inhibitor. Results suggest that p38 activity may play a key role in collagen-supported hMSC osteogenesis. This knowledge can be used toward the rational design of scaffolds which intrinsically promote hMSC osteogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2594-2604, 2018.

The three dimensional cues-integrated-biomaterial potentiates differentiation of human mesenchymal stem cells

Alginate (Alg) hydrogels, the most popular natural biomaterials, mimic the extracellular matrix (ECM) microenvironment and offer potential biomedical applications. Despite their excellent properties such as biocompatibility, hydrophilicity and ionic crosslinking, the absence of an intrinsic cell adhesion domain lessens their cell-carrier applications in tissue engineering. Herein, we suggest a three-dimensional (3D) cell culture system by integrating Alg hydrogel and fibroblast-derived matrix (FDM). FDM including cell-adhesion motifs, signaling, and physico-mechanical cues is prepared by the decellularization process by avoiding unfavorable chemical reactions. This cues-integrated-biomaterials (CiB) 3D platform shows increased cell viability, proliferation, chondrogenic and osteogenic differentiation of human bone-marrow-derived mesenchymal stem cells (hMSCs) in situ. The results show that the Alg/FDM hydrogel (CiB) matrix provides an excellent microenvironment for cell adhesion and can control the differentiation of hMSCs into specific lineages. Thus, these results suggest the potential applications of the Alg/FDM hydrogel matrix as a viable 3D culture system for tissue regeneration.

PAMAM dendrimer grafted cellulose paper scaffolds as a novel in vitro 3D liver model for drug screening applications.

Development of cheap and portable 3D liver models is a need of an hour for drug screening applications in the healthcare and diagnostic sector. An appropriate 3D liver model would support adhesion, proliferation, viability and functionality of the liver cells. With this perspective, the present study delineates the fabrication and characterization of PAMAM conjugated cellulose filter paper-based 3D liver model. Due to the absence of any cell adhesion motif, paper cannot directly be used for cell culture applications, thus, it was functionalized with PAMAM dendrimer using glutaraldehyde. PAMAM derivatized paper (PF) supported significantly higher adhesion, proliferation, and viability of the HepG2 cells in comparison to non-functionalized paper. Moreover, HepG2 cells maintained their functional aspects i.e., expression of mature hepatocyte markers albumin, CK19, CYP3 A4, MDR1 and SULT2 A1 on PF. Further, we showed the application of the PF-based 3D liver model for drug toxicity evaluations against hepatotoxins. The results showed a significantly higher sensitivity of the HepG2 cells in paper-based 3D culture as compared to 2D monolayer culture, which is similar to that of physiological drug-induced responses. In conclusion, PAMAM functionalized filter paper could serve as an efficient biomaterial platform for the development of microscale technologies, liver-associated diagnostics, and drug screening applications.

Tuning RGD Motif and Hyaluronan Density to Study Integrin Binding.

Well-controlled surfaces with immobilized substrates enable novel approaches to investigate specific aspects of biological processes related to cell adhesion or motility. A subset of integrins, cellular transmembrane glycoproteins, recognize the evolutionarily conserved tripeptide sequence RGD, and anchor cells to their surrounding proteins as well as mediate bidirectional signaling. In this study, the main question was how co-presentation of hyaluronan (HA), an essential component of the extracellular matrix (ECM), and the RGD motif affect integrin binding. We report a method to prepare self-assembled monolayers on gold surfaces, co-presenting the cell adhesive RGD motif and small HA molecules, to investigate integrin containing proteoliposome binding. This technique enables an independent adjustment of the RGD motif and HA density while maintaining a passivating background: Layer formation and subsequent interactions with αIIbβ3 integrins, which are reconstituted in liposomes, was monitored by label-free quartz crystal microbalance with dissipation monitoring (QCM-D). Exceeding a critical RGD motif density of 40% results in enhanced binding of proteoliposomes. Co-presentation studies with varying HA and constant RGD motif density demonstrate that marginal amounts of HA are sufficient to prevent integrin binding. These findings are of specific importance in relation to cancer cell microenvironments, which show highly enriched HA in the surrounding ECM to reduce adhesion properties.

Cardiac differentiation of induced pluripotent stem cells on elastin-like protein-based hydrogels presenting a single-cell adhesion sequence

Substrate-dependent cardiac differentiation of induced pluripotent stem cells (iPSCs) has been studied on various extracellular matrix (ECM)-derived substrates, such as collagen type I (Col-I). However, ECM-derived substrates have multiple cell-adhesive amino acid sequences and stimulate various signaling pathways in cells, making it difficult to clarify the mechanism of substrate-dependent stem cell differentiation. A substrate presenting one of these sequences is a powerful tool for elucidating the mechanism. We designed elastin-like proteins (ELPs) composed of repetitive VPGIG sequences with or without the RGD cell adhesion motif (ELP-RGD/ELP-Ctrl) and used a chemical crosslinker to generate hydrogels. By adjusting the ELP and crosslinker concentrations, we obtained ELP-Ctrl and ELP-RGD hydrogels with a Young’s modulus of 0.3 kPa. The ELP-Ctrl and ELP-RGD gels were used as a substrate for the cardiac differentiation of cultured murine iPSCs. Cells on the ELP-RGD gel showed four times higher gene expression of the contractile protein troponin T type 2 than those on a Col-I gel, which is an effective substrate for iPSC cardiac differentiation. The ELP-RGD gel might stimulate integrin-derived signaling pathways in the cells to promote cardiac differentiation. This study showed the potential of ELP hydrogels for studying substrate-dependent iPSC cardiac differentiation by enabling the control of cell-adhesive sequence presentation.

Rapid Bioorthogonal Chemistry Enables in Situ Modulation of the Stem Cell Behavior in 3D without External Triggers.

Chemical modification of engineered microenvironments surrounding living cells represents a means for directing cellular behaviors through cell-matrix interactions. Presented here is a temporally controlled method for modulating the properties of biomimetic, synthetic extracellular matrices (ECM) during live cell culture employing the rapid, bioorthogonal tetrazine ligation with trans-cyclooctene (TCO) dienophiles. This approach is diffusion-controlled, cytocompatible, and does not rely on light, catalysts, or other external triggers. Human bone-marrow-derived mesenchymal stem cells (hMSCs) were initially entrapped in a hydrogel prepared using hyaluronic acid carrying sulfhydryl groups (HA-SH) and a hydrophilic polymer bearing both acrylate and tetrazine groups (POM-AT). Inclusion of a matrix metalloprotease (MMP)-degradable peptidic cross-linker enabled hMSC-mediated remodeling of the synthetic environment. The resultant network displayed dangling tetrazine groups for subsequent conjugation with TCO derivatives. Two days later, the stiffness of the matrix was increased by adding chemically modified HA carrying multiple copies of TCO (HA-TCO) to the hMSC growth media surrounding the cell-laden gel construct. In response, cells developed small processes radially around the cell body without a significant alteration of the overall shape. By contrast, modification of the 3D matrix with a TCO-tagged cell-adhesive motif caused the resident cells to undergo significant actin polymerization, changing from a rounded shape to spindle morphology with long cellular processes. After additional 7 days of culture in the growth media, quantitative analysis showed that, at the mRNA level, RGD tagging upregulated cellular expression of MMP1, but downregulated the expression of collagen I/III and tenascin C. RGD tagging, however, was not sufficient to induce the classic osteoblastic, chondrogenic, adipogenic, or fibroblastic/myofibroblastic differentiation. The modular approach allows facile manipulation of synthetic ECM to modulate cell behavior, thus potentially applicable to the engineering of functional tissues or tissue models.

Effects of cell adhesion motif, fiber stiffness, and cyclic strain on tenocyte gene expression in a tendon mimetic fiber composite hydrogel

We recently developed a fiber composite consisting of tenocytes seeded onto discontinuous fibers embedded within a hydrogel, designed to mimic physiological tendon micromechanics of tension and shear. This study examined if cell adhesion peptide (DGEA or YRGDS), fiber modulus (50 or 1300 kPa) and/or cyclic strain (5% strain, 1 Hz) influenced bovine tenocyte gene expression. Ten genes were analyzed and none were sensitive to peptide or fiber modulus in the absence of cyclic tensile strain. Genes associated with tendon (SCX and TNMD), collagens (COL1A1, COL3A1, COL11A1), and matrix remodelling (MMP1, MMP2, and TIMP3) were insensitive to cyclic strain. Contrarily, cyclic strain up-regulated IL6 by 30-fold and MMP3 by 10-fold in soft YRGDS fibers. IL6 expression in soft YRGDS fibers was 5.7 and 3.3-fold greater than in soft DGEA fibers and stiff RGD fibers, respectively, under cyclic strain. Our findings suggest that changes in the surrounding matrix can influence catabolic genes in tenocytes when cultured in a complex strain environment mimicking that of tendon, while having minimal effects on tendon and homeostatic genes.

Design and Evaluation of Short Self-Assembling Depsipeptides as Bioactive and Biodegradable Hydrogels.

Described herein is the design of a cell-adherent and degradable hydrogel. Our goal was to create a self-assembling, backbone ester-containing analogue of the cell adhesion motif, arginine–glycine–aspartic acid (RGD). Two depsipeptides containing Fmoc (N-(fluorenyl)-9-methoxycarbonyl), Fmoc-FR-Glc-D, and Fmoc-F-Glc-RGD (where “Glc” is glycolic acid) were designed based on the results of integrin-binding affinity and cell interaction analyses. Two candidate molecules were synthesized, and their gelation characteristics, degradation profiles, and ability to promote cell attachment were analyzed. We found that ester substitution within the RGD sequence significantly decreases the integrin-binding affinity and subsequent cell attachment, but when the ester moiety flanks the bioactive sequence, the molecule can maintain its integrin-binding function while permitting nonenzymatic hydrolytic degradation. A self-assembled Fmoc-F-Glc-RGD hydrogel showed steady, linear degradation over 60 days, and when mixed with Fmoc-diphenylalanine (Fmoc-FF) for improved mechanical stiffness, the depsipeptide gel exhibited improved cell attachment and viability. Though the currently designed depsipeptide has several inherent limitations, our results indicate the potential of depsipeptides as the basis for biologically functional and degradable self-assembling hydrogel materials.

Silk fibroin/collagen protein hybrid cell-encapsulating hydrogels with tunable gelation and improved physical and biological properties

Cell encapsulating hydrogels with tunable mechanical and biological properties are of special importance for cell delivery and tissue engineering. Silk fibroin and collagen, two typical important biological proteins, are considered potential as cell culture hydrogels. However, both have been used individually, with limited properties (e.g., collagen has poor mechanical properties and cell-mediated shrinkage, and silk fibroin from Bombyx mori (mulberry) lacks cell adhesion motifs). Therefore, the combination of them is considered to achieve improved mechanical and biological properties with respect to individual hydrogels. Here, we show that the cell-encapsulating hydrogels of mulberry silk fibroin / collagen are implementable over a wide range of compositions, enabled simply by combining the different gelation mechanisms. Not only the gelation reaction but also the structural characteristics, consequently, the mechanical properties and cellular behaviors are accelerated significantly by the silk fibroin / collagen hybrid hydrogel approach. Of note, the mechanical and biological properties are tunable to represent the combined merits of individual proteins. The shear storage modulus is tailored to range from 0.1 to 20 kPa along the iso-compositional line, which is considered to cover the matrix stiffness of soft-to-hard tissues. In particular, the silk fibroin / collagen hydrogels are highly elastic, exhibiting excellent resistance to permanent deformation under different modes of stress; without being collapsed or water-squeezed out (vs. not possible in individual proteins) – which results from the mechanical synergism of interpenetrating networks of both proteins. Furthermore, the role of collagen protein component in the hybrid hydrogels provides adhesive sites to cells, stimulating anchorage and spreading significantly with respect to mulberry silk fibroin gel, which lacks cell adhesion motifs. The silk fibroin / collagen hydrogels can encapsulate cells while preserving the viability and growth over a long 3D culture period. Our findings demonstrate that the silk / collagen hydrogels possess physical and biological properties tunable and significantly improved (vs. the individual protein gels), implying their potential uses for cell delivery and tissue engineering. Statement of SignificanceDevelopment of cell encapsulating hydrogels with excellent physical and biological properties is important for the cell delivery and cell-based tissue engineering. Here we communicate for the first time the novel protein composite hydrogels comprised of ‘Silk’ and ‘Collagen’ and report their outstanding physical, mechanical and biological properties that are not readily achievable with individual protein hydrogels. The properties include i) gelation accelerated over a wide range of compositions, ii) stiffness levels covering 0.1 kPa to 20 kPa that mimic those of soft-to-hard tissues, iii) excellent elastic behaviors under various stress modes (bending, twisting, stretching, and compression), iv) high resistance to cell-mediated gel contraction, v) rapid anchorage and spreading of cells, and vi) cell encapsulation ability with a long-term survivability. These results come from the synergism of individual proteins of alpha-helix and beta-sheet structured networks. We consider the current elastic cell-encapsulating hydrogels of silk-collagen can be potentially useful for the cell delivery and tissue engineering in a wide spectrum of soft-to-hard tissues.

Biomimetic and enzyme-responsive dynamic hydrogels for studying cell-matrix interactions in pancreatic ductal adenocarcinoma

The tumor microenvironment (TME) governs all aspects of cancer progression and in vitro 3D cell culture platforms are increasingly developed to emulate the interactions between components of the stromal tissues and cancer cells. However, conventional cell culture platforms are inadequate in recapitulating the TME, which has complex compositions and dynamically changing matrix mechanics. In this study, we developed a dynamic gelatin-hyaluronic acid hybrid hydrogel system through integrating modular thiol-norbornene photopolymerization and enzyme-triggered on-demand matrix stiffening. In particular, gelatin was dually modified with norbornene and 4-hydroxyphenylacetic acid to render this bioactive protein photo-crosslinkable (through thiol-norbornene gelation) and responsive to tyrosinase-triggered on-demand stiffening (through HPA dimerization). In addition to the modified gelatin that provides basic cell adhesive motifs and protease cleavable sequences, hyaluronic acid (HA), an essential tumor matrix, was modularly and covalently incorporated into the cell-laden gel network. We systematically characterized macromer modification, gel crosslinking, as well as enzyme-triggered stiffening and degradation. We also evaluated the influence of matrix composition and dynamic stiffening on pancreatic ductal adenocarcinoma (PDAC) cell fate in 3D. We found that either HA-containing matrix or a dynamically stiffened microenvironment inhibited PDAC cell growth. Interestingly, these two factors synergistically induced cell phenotypic changes that resembled cell migration and/or invasion in 3D. Additional mRNA expression array analyses revealed changes unique to the presence of HA, to a stiffened microenvironment, or to the combination of both. Finally, we presented immunostaining and mRNA expression data to demonstrate that these irregular PDAC cell phenotypes were a result of matrix-induced epithelial-mesenchymal transition (EMT).

Small Bioactive Peptides for Biomaterials Design and Therapeutics.

This review is aimed to provide a concise yet extensive survey of key short bioactive peptide sequences for a range of applications ranging from biomaterials development to peptides with therapeutic uses. The following are considered: cell adhesion motifs, structural peptides, cell-penetrating and tumor-homing peptides, antimicrobial peptides, peptide hormones, growth factors and matrix metalloprotease substrates, neuropeptides, amyloid peptides, antioxidant peptides, peptide affinity tags, anticancer peptides, and others. This review provides a convenient resource, summarizing a broad range of important sequences with great utility as a resource concerning both small peptide drugs and also novel biofunctional peptide-based materials.

Functional hepatocyte clusters on bioactive blend silk matrices towards generating bioartificial liver constructs.

An end stage liver disease called cirrhosis perturbs the self-healing ability and physiological functions of liver. Due to the scarcity of healthy donors, a functional in vitro hepatic construct retaining the liver-specific functions is in great demand for its prospects in bioartificial liver (BAL) and cell-based tissue engineering. Physicochemical attributes of a matrix influence the behavior of cultured hepatocytes in terms of attachment, morphology and functionality. Mulberry and non-mulberry silk fibroin presents unique amino acid sequence with difference in hydrophobicity and crystallinity. Considering this, the present study focuses on the development of a suitable three-dimensional (3D) bioactive matrix incorporating both mulberry silk fibroin and cell adhesion motif (RGD) rich non-mulberry silk fibroin. Porous silk blend scaffolds facilitated the formation of hepatocyte clusters with enhanced liver-specific functions emphasizing both cell-cell and cell-matrix interactions. Hemocompatibility and integral property of blend scaffolds offers a biological niche for seeding functional liver cells that would have future prospects in biohybrid devices.

Engineered Collagen: A Redox Switchable Framework for Tunable Assembly and Fabrication of Biocompatible Surfaces.

Collagen, processed into several morphologies and originating from various sources, has long since been used as a biocompatible material that can assist wound healing and tissue regeneration. With the advent of biotechnology and solid-phase peptide synthesis, new possibilities arise to create rationally designed biomaterials based on collagen sequences incorporating new functionalities while maintaining the beneficial properties of natural collagen. In this study a new class of synthetic collagen materials is presented, defined by its simplistic core structure and its therefore predictable behavior. These so-called eCols (engineered collagens) consist of a varying number of Gly-Pro-Pro repeats, a redox-switchable aminoterminal nucleation site and an optional carboxyterminal cell adhesion motif. We show which of these proteins are able to self-assemble into triple helices and cross-linked gelatinous networks and provide insights into their cytocompatibility in vitro.