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The proper regulation of muscle stem cell (MuSC) fate by cues from the niche is essential for regeneration of skeletal muscle. How pro-regenerative niche factors control the dynamics of MuSC fate decisions remains unknown due to limitations of population-level endpoint assays. To address this knowledge gap, we developed a novel binary dynamic fluorescence time lapse imaging analysis (BDFA) approach that leverages machine learning classification strategies to track single cell fate decisions with high temporal resolution. Using two fluorescent reporters that read out maintenance of stemness and myogenic commitment, we constructed detailed lineage trees for individual MuSCs and their progeny, classifying each division event as symmetric self-renewing, asymmetric, or symmetric committed. Our analysis reveals that treatment with the lipid metabolite, prostaglandin E2 (PGE2), accelerates the rate of MuSC proliferation over time, while biasing division events toward symmetric self-renewal. In contrast, the IL6 family member, Oncostatin M (OSM) decreases the proliferation rate after the first generation, while blocking myogenic commitment. These insights into the dynamics of MuSC regulation by niche cues were uniquely enabled by our BDFA approach. We anticipate that similar binary live cell readouts derived from BDFA will markedly expand our understanding of how niche factors control tissue regeneration in real time.

Recent efforts to develop hydrogel biomaterials have focused on better recapitulating the dynamic properties of the native extracellular matrix. In hydrogel biomaterials, binding thermodynamics and cross-link kinetics directly affect numerous bulk dynamic properties such as strength, stress relaxation, and material clearance. However, despite the broad range of bulk dynamic properties observed in biological tissues, present strategies to incorporate dynamic linkages in cell-encapsulating hydrogels rely on a relatively small number of dynamic covalent chemical reactions and host-guest interactions. To expand this toolkit, we report the preparation of supramolecular gelatin hydrogels with cucurbit[8]uril (CB[8])-based cross-links that form on demand via thiol-ene reactions between preassembled CB[8]·FGGC peptide ternary complexes and grafted norbornenes. Human fibroblast cells encapsulated within these optically transparent, shear thinning, injectable hydrogels remained highly viable and exhibited a well-spread morphology in culture. These CB[8]-based gelatin hydrogels are anticipated to be useful in applications ranging from bioprinting to cell and drug delivery.

Skeletal muscle stem cells (MuSCs) are essential for efficacious muscle repair, making MuSCs promising therapeutic targets for tissue engineering and regenerative medicine. MuSCs are presented with a diverse and temporally defined set of cues from their microenvironment during regeneration that direct stem cell expansion, differentiation, and return to quiescence. Understanding the complex interplay among these biophysical and biochemical cues is necessary to develop therapies targeting or employing MuSCs. To probe the role of mechanical cues presented by the extracellular matrix, we leverage chemically defined hydrogel substrates with controllable stiffness and adhesive ligand composition to characterize the MuSC response to matrix cues presented during early and late phases of regeneration. We demonstrate that relatively soft hydrogels recapitulating healthy muscle stiffness promote MuSC activation and expansion, while relatively stiff hydrogels impair MuSC proliferation and arrest myogenic progression. These effects are seen on soft and stiff hydrogels presenting laminin-111 and exacerbated on hydrogels presenting RGD adhesive peptides. Soluble factors present in the MuSC niche during different phases of regeneration, prostaglandin E2 and oncostatin M, synergize with matrix-presented cues to enhance stem cell expansion on soft substrates and block myogenic progression on stiff substrates. To determine if temporally varied matrix stiffness reminiscent of the regenerating microenvironment alters MuSC fate, we developed a photoresponsive hydrogel system with accelerated reaction kinetics that can be rapidly softened on demand. MuSCs cultured on these materials revealed that the cellular response to a stiff microenvironment is fixed within the first three days of culture, as subsequent softening back to a healthy stiffness did not rescue MuSC proliferation or myogenic progression. These results highlight the importance of temporally controlled biophysical and biochemical cues in regulating MuSC fate that can be harnessed to improve regenerative medicine approaches to restore skeletal muscle tissue.

Recent efforts in the design of hydrogel biomaterials have focused on better mimicking the native cellular microenvironment to direct cell fate. To simultaneously control multiple material parameters, several orthogonal chemistries may be needed. However, present strategies to prepare cell-encapsulating hydrogels make use of relatively few chemical reactions. To expand this chemical toolkit, we report the preparation of hydrogels based on a Diels-Alder reaction between fulvenes and maleimides with markedly improved gelation kinetics and hydrolytic stability. Fulvene-maleimide gels cross-link up to 10-times faster than other commonly used DA reaction pairs and remain stable for months under physiological conditions. Furthermore, fulvene-maleimide gels presenting relevant biochemical cues, such as cell-adhesive ligands and proteolytic degradability, support the culture of human mesenchymal stromal cells. Finally, this rapid DA reaction was combined with an orthogonal click reaction to demonstrate how the use of selective chemistries can provide new avenues to incorporate multiple functionalities in hydrogel materials.

Neural progenitor cells (NPCs) are promising therapeutic candidates for nervous system regeneration. Significant efforts focus on developing hydrogel-based approaches to facilitate the clinical translation of NPCs, from scalable platforms for stem cell production to injectable carriers for cell transplantation. However, fundamental questions surrounding NPC-hydrogel interactions remain unanswered. While matrix degradability is known to regulate the stemness and differentiation capacity of NPCs, how degradability impacts NPC epigenetic regulation and secretory phenotype remains unknown. To address this question, NPCs encapsulated in recombinant protein hydrogels with tunable degradability are assayed for changes in chromatin organization and neurotrophin expression. In high degradability gels, NPCs maintain expression of stem cell factors, proliferate, and have large nuclei with elevated levels of the stemness-associated activating histone mark H3K4me3. In contrast, NPCs in low degradability gels exhibit more compact, rounded nuclei with peripherally localized heterochromatin, are non-proliferative yet non-senescent, and maintain expression of neurotrophic factors with potential therapeutic relevance. This work suggests that tuning matrix degradability may be useful to direct NPCs toward either a more-proliferative, stem-like phenotype for cell replacement therapies, or a more quiescent-like, pro-secretory phenotype for soluble factor-mediated therapies.

Degradation of three-dimensional hydrogels is known to regulate many cellular behaviors. Accordingly, several elegant approaches have been used to render hydrogels degradable by cell-secreted proteases. However, existing hydrogel systems are limited in their ability to simultaneously and quantitatively tune two aspects of hydrogel degradability: cleavage rate (the rate at which individual chemical bonds are cleaved) and degraded hydrogel architecture (the network structure during degradation). Using standard peptide engineering approaches, we alter the proteolytic kinetics of the polymer cleavage rate to tune gel degradation time from less than 12 h to greater than 9 days. Independently, we vary the cross-linker functionality to achieve network architectures that initially have identical molecular weight between cross-links but upon degradation are designed to release between 5% and 100% of the polymer. Confirming the biological relevance of both parameters, formation of vascular-like structures by endothelial cells is regulated both by bond cleavage rate and by degraded hydrogel architecture. This strategy to fine-tune different aspects of hydrogel degradability has applications in cell culture, regenerative medicine, and drug delivery.

Although only a few stem cell-based therapies are currently available to patients, stem cells hold tremendous regenerative potential, and several exciting clinical applications are on the horizon. Biomaterials with tuneable mechanical and biochemical properties can preserve stem cell function in culture, enhance survival of transplanted cells and guide tissue regeneration. Rapid progress with three-dimensional hydrogel culture platforms provides the opportunity to grow patient-specific organoids, and has led to the discovery of drugs that stimulate endogenous tissue-specific stem cells and enabled screens for drugs to treat disease. Therefore, bioengineering technologies are poised to overcome current bottlenecks and revolutionize the field of regenerative medicine.

Neural progenitor cell (NPC) culture within three-dimensional (3D) hydrogels is an attractive strategy for expanding a therapeutically relevant number of stem cells. However, relatively little is known about how 3D material properties such as stiffness and degradability affect the maintenance of NPC stemness in the absence of differentiation factors. Over a physiologically relevant range of stiffness from â 1/40.5 to 50 kPa, stemness maintenance did not correlate with initial hydrogel stiffness. In contrast, hydrogel degradation was both correlated with, and necessary for, maintenance of NPC stemness. This requirement for degradation was independent of cytoskeletal tension generation and presentation of engineered adhesive ligands, instead relying on matrix remodelling to facilitate cadherin-mediated cell-cell contact and promote ?-catenin signalling. In two additional hydrogel systems, permitting NPC-mediated matrix remodelling proved to be a generalizable strategy for stemness maintenance in 3D. Our findings have identified matrix remodelling, in the absence of cytoskeletal tension generation, as a previously unknown strategy to maintain stemness in 3D.

Native vascular extracellular matrices (vECM) consist of elastic fibers that impart varied topographical properties, yet most in vitro models designed to study the effects of topography on cell behavior are not representative of native architecture. Here, we engineer an electrospun elastin-like protein (ELP) system with independently tunable, vECM-mimetic topography and demonstrate that increasing topographical variation causes loss of endothelial cell-cell junction organization. This loss of VE-cadherin signaling and increased cytoskeletal contractility on more topographically varied ELP substrates in turn promote YAP activation and nuclear translocation, resulting in significantly increased endothelial cell migration and proliferation. Our findings identify YAP as a required signaling factor through which fibrous substrate topography influences cell behavior and highlights topography as a key design parameter for engineered biomaterials.

Hydrogels are commonly used as engineered extracellular matrix (ECM) mimics in applications ranging from tissue engineering to in vitro disease models. Ideal mechanisms used to crosslink ECM-mimicking hydrogels do not interfere with the biology of the system. However, most common hydrogel crosslinking chemistries exhibit some form of cross-reactivity. The field of bio-orthogonal chemistry has arisen to address the need for highly specific and robust reactions in biological contexts. Accordingly, bio-orthogonal crosslinking strategies have been incorporated into hydrogel design, allowing for gentle and efficient encapsulation of cells in various hydrogel materials. Furthermore, the selective nature of bio-orthogonal chemistries can permit dynamic modification of hydrogel materials in the presence of live cells and other biomolecules to alter matrix mechanical properties and biochemistry on demand. In this review, we provide an overview of bio-orthogonal strategies used to prepare cell-encapsulating hydrogels and highlight the potential applications of bio-orthogonal chemistries in the design of dynamic engineered ECMs.

Covalently-crosslinked hydrogels are commonly used as 3D matrices for cell culture and transplantation. However, the crosslinking chemistries used to prepare these gels generally cross-react with functional groups present on the cell surface, potentially leading to cytotoxicity and other undesired effects. Bio-orthogonal chemistries have been developed that do not react with biologically relevant functional groups, thereby preventing these undesirable side reactions. However, previously developed biomaterials using these chemistries still possess less than ideal properties for cell encapsulation, such as slow gelation kinetics and limited tuning of matrix mechanics and biochemistry. Here, engineered elastin-like proteins (ELPs) are developed that cross-link via strain-promoted azide-alkyne cycloaddition (SPAAC) or Staudinger ligation. The SPAAC-crosslinked materials form gels within seconds and complete gelation within minutes. These hydrogels support the encapsulation and phenotypic maintenance of human mesenchymal stem cells, human umbilical vein endothelial cells, and murine neural progenitor cells. SPAAC-ELP gels exhibit independent tuning of stiffness and cell adhesion, with significantly improved cell viability and spreading observed in materials containing a fibronectin-derived arginine-glycine-aspartic acid (RGD) domain. The crosslinking chemistry used permits further material functionalization, even in the presence of cells and serum. These hydrogels are anticipated to be useful in a wide range of applications, including therapeutic cell delivery and bioprinting.

Dimensionality can have a profound impact on stiffness-mediated differentiation of mesenchymal stem cells (MSCs). However, while we have begun to understand cellular response when encapsulated within 3D substrates, the behavior of cells within macro-porous substrates is relatively underexplored. The goal of this study was to determine the influence of macro-porous topographies on stiffness-mediated differentiation of MSCs. We developed macro-porous recombinant elastin-like protein (ELP) substrates that allow independent control of mechanical properties and ligand chemistry. We then used computational modeling to probe the impact of pore topography on the mechanical stimulus that cells are exposed to within these substrates, and finally we investigated stiffness induced biases towards adipogenic and osteogenic differentiation of MSCs within macro-porous substrates. Computational modeling revealed that there is significant heterogeneity in the mechanical stimuli that cells are exposed to within porous substrates and that this heterogeneity is predominantly due to the wide range of possible cellular orientations within the pores. Surprisingly, MSCs grown within 3D porous substrates respond to increasing substrate stiffness by up-regulating both osteogenesis and adipogenesis. These results demonstrate that within porous substrates the behavior of MSCs diverges from previously observed responses to substrate stiffness, emphasizing the importance of topography as a determinant of cellular behavior.

Stem cells are a powerful resource for many applications including regenerative medicine, patient-specific disease modeling, and toxicology screening. However, eliciting the desired behavior from stem cells, such as expansion in a naïve state or differentiation into a particular mature lineage, remains challenging. Drawing inspiration from the native stem cell niche, hydrogel platforms have been developed to regulate stem cell fate by controlling microenvironmental parameters including matrix mechanics, degradability, cell-adhesive ligand presentation, local microstructure, and cell–cell interactions. We survey techniques for modulating hydrogel properties and review the effects of microenvironmental parameters on maintaining stemness and controlling differentiation for a variety of stem cell types. Looking forward, we envision future hydrogel designs spanning a spectrum of complexity, ranging from simple, fully defined materials for industrial expansion of stem cells to complex, biomimetic systems for organotypic cell culture models.

The effectiveness of stem-cell therapies has been hampered by cell death and limited control over fate1. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype2–4. Stem cell behavior can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials5–7, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel's elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel's elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem-cell behaviors in situ.

Interactions between cells and the extracellular matrix (ECM) are known to play critical roles in regulating cell phenotype. The identity of ECM ligands presented to mesenchymal stem cells (MSCs) has previously been shown to direct the cell fate commitment of these cells. To enhance osteogenic differentiation of MSCs, alginate hydrogels were prepared that present the DGEA ligand derived from collagen I. When presented from hydrogel surfaces in 2D, the DGEA ligand did not facilitate cell adhesion, while hydrogels presenting the RGD ligand derived from fibronectin did encourage cell adhesion and spreading. However, the osteogenic differentiation of MSCs encapsulated within alginate hydrogels presenting the DGEA ligand was enhanced when compared with unmodified alginate hydrogels and hydrogels presenting the RGD ligand. MSCs cultured in DGEA-presenting gels exhibited increased levels of osteocalcin production and mineral deposition. These data suggest that the presentation of the collagen I-derived DGEA ligand is a feasible approach for selectively inducing an osteogenic phenotype in encapsulated MSCs.

In this issue of Chem , Ulijn, Dalby, and co-workers have developed a novel biomaterials platform as a screening tool for identifying metabolites that can bias differentiation of mesenchymal stem cells.

In-situ tissue regeneration aims to utilize the body’s endogenous healing capacity through the recruitment of host stem or progenitor cells to an injury site. Stromal cell-derived factor-1α (SDF-1α) is widely discussed as a potent chemoattractant. Here we use a cell-free biomaterial-based approach to (i) deliver SDF-1α for the recruitment of endogenous bone marrow-derived stromal cells (BMSC) into a critical-sized segmental femoral defect in rats and to (ii) induce hydrogel stiffness-mediated osteogenic differentiation in-vivo. Ionically crosslinked alginate hydrogels with a stiffness optimized for osteogenic differentiation were used. Fast-degrading porogens were incorporated to impart a macroporous architecture that facilitates host cell invasion. Endogenous cell recruitment to the defect site was successfully triggered through the controlled release of SDF-1α. A trend for increased bone volume fraction (BV/TV) and a significantly higher bone mineral density (BMD) were observed for gels loaded with SDF-1α, compared to empty gels at two weeks. A trend was also observed, albeit not statistically significant, towards matrix stiffness influencing BV/TV and BMD at two weeks. However, over a six week time-frame, these effects were insufficient for bone bridging of a segmental femoral defect. While mechanical cues combined with ex-vivo cell encapsulation have been shown to have an effect in the regeneration of less demanding in-vivo models, such as cranial defects of nude rats, they are not sufficient for a SDF-1α mediated in-situ regeneration approach in segmental femoral defects of immunocompetent rats, suggesting that additional osteogenic cues may also be required. Statement of Significance Stromal cell-derived factor-1α (SDF-1α) is a chemoattractant used to recruit host cells for tissue regeneration. The concept that matrix stiffness can direct mesenchymal stromal cell (MSC) differentiation into various lineages was described a decade ago using in-vitro experiments. Recently, alginate hydrogels with an optimized stiffness and ex-vivo encapsulated MSCs were shown to have an effect in the regeneration of skull defects of nude rats. Here, we apply this material system, loaded with SDF-1α and without encapsulated MSCs, to (i) recruit endogenous cells and (ii) induce stiffness-mediated osteogenic differentiation in-vivo, using as model system a load-bearing femoral defect in immunocompetent rats. While a cell-free approach is of great interest from a translational perspective, the current limitations are described.

Engineered protein hydrogels have shown promise as artificial extracellular matrix materials for the 3D culture of stem cells due to the ability to decouple hydrogel biochemistry and mechanics. The modular design of these proteins allows for incorporation of various bioactive sequences to regulate cellular behavior. However, the chemistry used to cross-link the proteins into hydrogels can limit what bioactive sequences can be incorporated, in order to prevent nonspecific cross-linking within the bioactive region. Bio-orthogonal cross-linking chemistries may allow for the incorporation of any arbitrary bioactive sequence, but site-selective and scalable incorporation of bio-orthogonal reactive groups such as azides that do not rely on commonly used amine-reactive chemistry is often challenging. In response, we have optimized the reaction of an azide-bearing 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) with engineered elastin-like proteins (ELPs) to selectively azide-functionalize tyrosine residues within the proteins. The PTAD-azide functionalized ELPs cross-link with bicyclononyne (BCN) functionalized ELPs via the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction to form hydrogels. Human mesenchymal stem cells and murine neural progenitor cells encapsulated within these hydrogels remain highly viable and maintain their phenotypes in culture. Tyrosine-specific modification may expand the number of bioactive sequences that can be designed into protein-engineered materials by permitting incorporation of lysine-containing sequences without concern for nonspecific cross-linking.

Protein-engineered biomaterials are designed and synthesized using recombinant protein technology and offer an alternative to both harvested natural biomaterials and synthetic polymeric biomaterials. A defining feature of such protein-engineered biomaterials is the precise control over macromolecular composition and function, made possible by modular peptide domain design and exact specification of the encoding DNA sequence. This approach allows multiple functional domains to be encoded directly into the protein backbone, creating a plethora of biomaterials with tunable mechanical, biochemical, and biodegradation properties. This chapter describes the design, recombinant synthesis, and fabrication strategies for protein-engineered biomaterials for use in tissue engineering applications. Analytical tools for characterizing the molecular and bulk properties of these materials are also discussed. Building upon modular domain design and by expanding peptide functionality, a variety of protein biomaterials have been engineered which serve as versatile platforms for studying and addressing various challenges in regenerative medicine.

Neural progenitor cells (NPCs) are a promising cell source to repair damaged nervous tissue. However, expansion of therapeutically relevant numbers of NPCs and their efficient differentiation into desired mature cell types remains a challenge. Material‐based strategies, including culture within 3D hydrogels, have the potential to overcome these current limitations. An ideal material would enable both NPC expansion and subsequent differentiation within a single platform. It has recently been demonstrated that cell‐mediated remodeling of 3D hydrogels is necessary to maintain the stem cell phenotype of NPCs during expansion, but the role of matrix remodeling on NPC differentiation and maturation remains unknown. By culturing NPCs within engineered protein hydrogels susceptible to degradation by NPC‐secreted proteases, it is identified that a critical amount of remodeling is necessary to enable NPC differentiation, even in highly degradable gels. Chemical induction of differentiation after sufficient remodeling time results in differentiation into astrocytes and neurotransmitter‐responsive neurons. Matrix remodeling modulates expression of the transcriptional co‐activator Yes‐associated protein, which drives expression of NPC stemness factors and maintains NPC differentiation capacity, in a cadherin‐dependent manner. Thus, cell‐remodelable hydrogels are an attractive platform to enable expansion of NPCs followed by differentiation of the cells into mature phenotypes for therapeutic use.

We report the synthesis of trans -dichlorobis(1,3-diaminopropane)ruthenium(III) chloride. The molecular structure consists of a ruthenium metal complex and a chloride counterion. Electronic spectra of the solid complexes indicate an octahedral geometry around the ruthenium(III) metal center. We observe a UV-broadened band at 350 nm, a common absorption band in trans -ruthenium(III) amine and haloamine complexes. We also report, the single-crystal X-ray structures of the titled complex. The complex was shown to form a self assembled monolayer on quartz, which is a suitable substrate for deposition of conducting polymer PANI, PEDOT and PPy. We discuss key parameters which can be adjusted to tune both the physical properties of the Ru(III) surfaces and the sheet conductivity of the deposited conducting polymers thin films.

Recently, conducting polymer thin films have been investigated as transparent electrodes in photovoltaic devices and organic light emitting diodes. Due to its relatively high conductivity and excellent transmission in the visible region, poly (3, 4-ethyelenedioxythiophene) (PEDOT) has been shown to be a viable option for such applications. Herein described is a method for the vapor phase polymerization (VPP) of transparent PEDOT thin film electrodes on flexible polyethylene naphthalate (PEN) substrates and the comparison of this VPP method with two current approaches to PEDOT deposition: solution-based in situ polymerization and spin coating a dispersion of PEDOT:PSS. Electrical conductivities and UV–vis transmittances were measured for films produced by each of these methods, with VPP PEDOT showing both the highest conductivity (approx. 600 S/cm) and transmittance (>94% at 550 nm). The surface morphologies of the films were compared using AFM and SEM imaging. The stability of these PEDOT films, stored under ambient conditions, was investigated by monitoring the conductivity and transmittance of the thin films over time.

The innate biological response to peripheral nerve injury involves a complex interplay of multiple molecular cues to guide neurites across the injury gap. Many current strategies to stimulate regeneration take inspiration from this biological response. However, little is known about the balance of cell–matrix and Schwann cell–neurite dynamics required for regeneration of neural architectures. We present an engineered extracellular matrix (eECM) microenvironment with tailored cell–matrix and cell–cell interactions to study their individual and combined effects on neurite outgrowth. This eECM regulates cell–matrix interactions by presenting integrin-binding RGD (Arg–Gly–Asp) ligands at specified densities. Simultaneously, the addition or exclusion of nerve growth factor (NGF) is used to modulate L1CAM-mediated Schwann cell–neurite interactions. Individually, increasing the RGD ligand density from 0.16 to 3.2 mM resulted in increasing neurite lengths. In matrices presenting higher RGD ligand densities, neurite outgrowth was synergistically enhanced in the presence of soluble NGF. Analysis of Schwann cell migration and co-localization with neurites revealed that NGF enhanced cooperative outgrowth between the two cell types. Interestingly, neurites in NGF-supplemented conditions were unable to extend on the surrounding eECM without the assistance of Schwann cells. Blocking studies revealed that L1CAM is primarily responsible for these Schwann cell–neurite interactions. Without NGF supplementation, neurite outgrowth was unaffected by L1CAM blocking or the depletion of Schwann cells. These results underscore the synergistic interplay between cell–matrix and cell–cell interactions in enhancing neurite outgrowth for peripheral nerve regeneration.

During the early stages of bone wound healing, mesenchymal stem cells (MSCs) in the bone marrow migrate to an injury site and differentiate into osteoblasts, which actively participate in the bone repair process. We proposed that a biomaterial system which enhances migration of endogenous MSCs may accelerate the tissue healing process. The aims of this study were to 1) identify and characterize an MSC chemoattractant, and 2) quantify the release kinetics of the chemoattractant from alginate hydrogels. Timelapse microscopy of 3D MSC migration was used for real time tracking of cell movement. Quantitative analysis of chemotaxis parameters identified Thymus Chemokine-1 (TCK-1) to be a potent MSC chemoattractant (p-values less than .01 for velocity, directionality, and forward migration index compared to controls). Qualitative receptor staining of MSCs in a transwell membrane assay showed that MSCs which respond chemotactically to gradient of TCK-1 express the TCK-1 cell receptor, CXCR2. An alginate hydrogel was used for controlled release of TCK-1. To characterize the kinetics of this system, an enzyme-linked immunosorbent assay (ELISA) was used to quantify TCK-1 release rates. This approach may be widely applicable to the regeneration of other tissue types and may be more efficient than the current multistep procedures of cell transplantation.

Hydrogels can promote desirable cellular phenotype by mimicking tissue-like stiffness or serving as a gene delivery depot. However, nonviral gene delivery inside three-dimensional (3D) hydrogels remains a great challenge, and increasing hydrogel stiffness generally results in further decrease in gene delivery efficiency. Here we report a method to enhance nonviral gene delivery efficiency inside 3D hydrogels across a broad range of stiffness using sacrificial microfibers for co-releasing cells and polymeric nanoparticles (NPs). We fabricated hydrolytically degradable alginate as sacrificial microfibers, and optimized the degradation profile of alginate by varying the degree of oxidization. Scanning electron microscopy confirmed degradation of alginate microfibers inside hydrogels, leaving behind microchannel-like structures within 3D hydrogels. Sacrificial microfibers also serve as a delivery vehicle for co-releasing encapsulated cells and NPs, allowing cell attachment and spreading within the microchannel surface upon microfiber degradation. To examine the effects of sacrificial microfibers on nonviral gene delivery inside 3D hydrogels, alginate microfibers containing human embryonic kidney 293 cells and polymeric NPs were encapsulated within 3D hydrogel scaffolds with varying stiffness (9, 58, and 197 kPa). Compared with cells encapsulated in bulk hydrogels, we observed up to 15-fold increase in gene delivery efficiency using sacrificial microfibers, and gene delivery efficiency increased as hydrogel stiffness increased. The platform reported herein provides a strategy for enhancing nonviral gene delivery inside 3D hydrogels across a broad range of stiffness, and may aid tissue regeneration by engaging both mechanotransduction and nonviral gene delivery.

Many strategies for controlling the fate of transplanted stem cells rely on the concurrent delivery of soluble growth factors that have the potential to produce undesirable secondary effects in surrounding tissue. Such off target effects could be eliminated by locally presenting growth factor peptide mimics from biomaterial scaffolds to control stem cell fate. Peptide mimics of bone morphogenetic protein 2 (BMP-2) were synthesized by solid phase Fmoc-peptide synthesis and covalently bound to alginate hydrogels via either carbodiimide or sulfhydryl-based coupling strategies. Successful peptide conjugation was confirmed by (1)H NMR spectroscopy and quantified by fluorescently labeling the peptides. Peptides derived from the knuckle epitope of BMP-2, presented from both 2D surfaces and 3D alginate hydrogels, were shown to increase alkaline phosphatase activity in clonally derived murine osteoblasts. Furthermore, when presented in 3D hydrogels, these peptides were shown to initiate Smad signaling, upregulate osteopontin production, and increase mineral deposition with clonally derived murine mesenchymal stem cells. These data suggest that these peptide-conjugated hydrogels may be effective alternatives to local BMP-2 release in directly and spatially eliciting osteogenesis from transplanted or host osteoprogenitors in the future.

We have been developing a new approach to layered hybrid (inorganic/organic) photovoltaic materials for fabrication by Roll-to-Roll (R2R) manufacturing. In this report, we combine the low cost and processability of organic electrically conducting polymers with the efficiency of dye sensitized titanium dioxide, semi-conductor quantum dots (CdSe) self-assembled on layered clay materials (Laponite) onto indium tin oxide coated flexible substrate polyethylene terephthalate (PET) substrates. We have shown electron transfer, guest-guest and host-guest interactions, charge separation, spectral line broadening, and quenching of fluorescence signals which indicate electronic coupling of the dye [Ru(bpy)3]2+ on a CdSe nanocrystal and titanium dioxide nanoparticles. Scanning electron microscopy and atomic force microscopy demonstrate successful nanoparticle formation and thin film self-assembly, as well as surface morphology and polymer thickness.

Both matrix biochemistry and neurotrophic factors are known to modulate neurite outgrowth and pathfinding; however, the interplay between these two factors is less studied. While previous work has shown that the biochemical identity of the matrix can alter the outgrowth of neurites in response to neurotrophins, the importance of the concentration of cell-adhesive ligands is unknown. Using engineered elastin-like protein matrices, we recently demonstrated a synergistic effect between matrix-bound cell-adhesive ligand density and soluble nerve growth factor treatment on neurite outgrowth from dorsal root ganglia. This synergism was mediated by Schwann cell-neurite contact through L1CAM. Cell-adhesive ligand density was also shown to alter the pathfinding behavior of dorsal root ganglion neurites in response to a gradient of nerve growth factor. While more cell-adhesive matrices promoted neurite outgrowth, less cell-adhesive matrices promoted more faithful neurite pathfinding. These studies emphasize the importance of considering both matrix biochemistry and neurotrophic factors when designing biomaterials for peripheral nerve regeneration.