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Understanding the relationship between an amino acid sequence and its phase separation has important implications for analyzing cellular function, treating disease, and designing novel biomaterials. Several sequence features have been identified as drivers for protein liquid-liquid phase separation (LLPS), leading to the development of a “molecular grammar” for LLPS. In this work, we further probed how sequence modulates phase separation and the material properties of the resulting condensates. Specifically, we used a model intrinsically disordered polypeptide composed of an 8-residue repeat unit and performed systematic sequence manipulations targeting sequence features previously overlooked in the literature. We generated sequences with no charged residues, high net charge, no glycine residues, or devoid of aromatic or arginine residues. We report that all but one of the twelve variants we designed undergo LLPS, albeit to different extents, despite significant differences in composition. These results support the hypothesis that multiple interactions between diverse residue pairs work in tandem to drive phase separation. Molecular simulations paint a picture of underlying molecular details involving various atomic interactions mediated by not just a handful of residue types, but by most residues. We characterized the changes to inter-residue contacts in all the sequence variants, thereby developing a more complete understanding of the contributions of sequence features such as net charge, hydrophobicity, and aromaticity to phase separation. Further, we find that all condensates formed behave like viscous fluids, despite large differences in their viscosities. The results presented in this study significantly advance the current sequence-phase behavior and sequence-material properties relationships to help interpret, model, and design protein assembly.

INTRODUCTION The extracellular matrix (ECM) is vital for cell and tissue development. Given its importance, extensive work has been conducted to develop biomaterials and drug delivery vehicles that capture features of ECM structure and function. AREAS COVERED This review highlights recent developments of ECM-inspired nanocarriers and their exploration for drug and gene delivery applications. Nanocarriers that are inspired by or created from primary components of the ECM (e.g. elastin, collagen, hyaluronic acid (HA), or combinations of these) are explicitly covered. An update on current clinical trials employing elastin-like proteins is also included. EXPERT OPINION Novel ECM-inspired nanoscale structures and conjugates continue to be of great interest in the materials science and bioengineering communities. Hyaluronic acid nanocarrier systems in particular are widely employed due to the functional activity of HA in mediating a large number of disease states. In contrast, collagen-like peptide nanocarriers are an emerging drug delivery platform with potential relevance to a myriad of ECM-related diseases, making their continued study most pertinent. Elastin-like peptide nanocarriers have a well-established tolerability and efficacy track record in preclinical analyses that has motivated their recent advancement into the clinical arena.

Heterogeneities in hydrogel scaffolds are known to impact the performance of cells in cell-laden materials constructs, and we have employed the phase separation of resilin-like polypeptides (RLPs) ...

Injectable nanocarriers and hydrogels have found widespread use in a variety of biomedical applications such as local and sustained biotherapeutic cargo delivery, and as cell-instructive matrices for tissue engineering. Recent advances in the development and application of recombinant protein-based materials as injectable platforms under physiological conditions have made them useful platforms for the development of nanoparticles and tissue engineering matrices, which are reviewed in this work. Protein-engineered biomaterials are highly customizable, and they provide distinctly tunable rheological properties, encapsulation efficiencies, and delivery profiles. In particular, the key advantages of emerging technologies which harness the stimuli-responsive properties of recombinant polypeptide-based materials are highlighted in this review.

The development of hybrid hydrogels has been of great interest over recent decades, especially in the field of biomaterials. Such hydrogels provide various opportunities in tissue engineering, drug delivery, and regenerative medicine due to their ability to mimic cellular environments, sequester and release therapeutic agents, and respond to stimuli. Herein we report the synthesis and characterization of an injectable poly(ethylene glycol) hydrogel crosslinked via thiol-maleimide reactions and containing both chemically crosslinked temperature-sensitive liposomes (TSLs) and matrix metalloproteinase-sensitive peptide crosslinks. Rheological studies demonstrate that the hydrogel is mechanically stable and can be synthesized to achieve a range of physically applicable moduli. Experiments characterizing the in situ drug delivery and degradation of these materials indicate that the TSL-gel responds to both thermal and enzymatic stimuli in a local environment. Doxorubicin, a widely used anti-cancer drug, was loaded in the TSLs with a high encapsulation efficiency and the subsequent release was temperature dependent. Finally, TSLs did not compromise viability and proliferation of human and murine fibroblasts, supporting the use of these hydrogel-linked liposomes as a thermo-responsive drug carrier for controlled release.

Collagen-targeting strategies have proven to be an effective method for targeting drugs to pathological tissues for treatment of disease. The use of collagen-like peptides for controlling the assembly of drug delivery vehicles, as well as their integration into collagen-containing matrices, offers significant advantages for tuning the morphologies of assembled structures, their thermoresponsiveness, and the loading and release of both small-molecule and macro-molecular cargo. In this contribution, we summarize the design and development of collagen-peptide-based drug delivery systems introduced by the Kiick group and detail the expansion of our understanding and the application of these unique molecules through collaborations with experts in computational simulations (Jayaraman), osteoarthritis (Price), and gene delivery (Sullivan). Kiick was inducted as a Fellow of the National Academy of Inventors in 2019 and was to deliver an address describing the innovations of her research. Given the cancellation of the NAI Annual Meeting as a result of coronavirus travel restrictions, her work based on collagen-peptide-mediated assembly is instead summarized in this contribution.

There has been an increased interest in the use of protein therapeutics, especially antibodies, for the treatment of a variety of diseases due to their high specificity to tissues and biological pathways of interest. However, the use of antibodies can be hindered by physical aggregation, degradation, and diffusion when injected in vivo leading to the need for antibody-releasing depots for the controlled and localized delivery within tissues of interest. Here, we investigated photolabile hydrogel chemistries for creating on-demand and tunable antibody release profiles. Innovative, scalable synthetic procedures were established and applied for fabricating hydrogels with nitrobenzyl (NB) and coumarin (CMR) photolabile crosslinks that responded to clinically relevant doses of long-wavelength UV and short-wavelength visible light. This synthetic procedure includes a route to make a CMR linker possessing two functional handles at the same ring position with water-stable bonds. The photocleavage properties of NB and CMR crosslinked hydrogels were characterized, as well as their potential for translational studies by degradation through pig skin, a good human skin mimic. The mechanism of hydrogel degradation, bulk versus surface eroding, was determined to be dependent on the wavelength of light utilized and the molar absorptivity of the different photolabile linkers, providing a facile means for altering protein release upon hydrogel degradation. Further, the encapsulation and on-demand release of a model monoclonal antibody was demonstrated, highlighting the ability to control antibody release from these hydrogels through the application of light while retaining its bioactivity. In particular, the newly designed CMR hydrogels undergo surface erosion-based protein release using visible light, which is more commonly used clinically. Overall, this work establishes scalable syntheses and relevant pairings of formulation-irradiation conditions for designing on-demand and light-responsive material systems that provide controlled, tunable release of bioactive proteins toward addressing barriers to preclinical translation of light-based materials and ultimately improving therapeutic regimens.

Elastin-like polypeptides (ELP) have been widely used in the biomaterials community due to their controllable, thermoresponsive properties and biocompatibility. Motivated by our previous work on the effect of tryptophan (W) substitutions on the LCST-like transitions of short ELPs, we studied a series of short ELPs containing tyrosine (Y) and/or phenylalanine (F) guest residues with only 5 or 6 pentapeptide repeat units. A combination of experiments and molecular dynamics (MD) simulations illustrated that the substitution of F with Y guest residues impacted the transition temperature (T(t)) of short ELPs when conjugated to collagen-like-peptides (CLP), with a reduction in the transition temperature observed only after substitution of at least two residues. Placement of the Y residues near the N-terminal end of the ELP, away from the tethering point to the CLP, resulted in a lower T(t) than that observed for peptides with the Y residues near the tethering point. Atomistic and coarse-grained MD simulations indicated an increase in intra- and inter- peptide hydrogen bonds in systems containing Y guest residues that are suggested to enhance the ability of the peptides to coacervate, with a concomitantly lower T(t). Simulations also revealed that the placement of Y-containing pentads near the N-terminus (i.e., away from CLP tethering point) versus C-terminus of the ELP led to more π-π stacking interactions at low temperatures, in agreement with our experimental observations of a lower T(t). Overall, this study provides mechanistic insights into the driving forces for the LCST-like transitions of ELPs and offers additional means for tuning the T(t) of short ELPs for biomedical applications such as on-demand drug delivery and tissue engineering.

Growth factor therapy has demonstrated great promise for chronic wound repair, but controlling growth factor activity and cell phenotype over desired time frames remains a critical challenge. In this study, we developed a gene-activated hyaluronic acid-collagen matrix (GAHCM) comprising DNA/polyethylenimine (PEI) polyplexes retained on hyaluronic acid (HA)-collagen hydrogels using collagen mimetic peptides (CMPs). We hypothesized that manipulating both the number of CMP-collagen tethers and the ECM composition would provide a powerful strategy to control growth factor gene transfer kinetics while regulating cell behavior, resulting in enhanced growth factor activity for wound repair. We observed that polyplexes with 50% CMP-modified PEI (50 CP) showed enhanced retention of polyplexes in HCM hydrogels by 2.7-fold as compared to non-CMP modified polyplexes. Moreover, the incorporation of HA in the hydrogel promoted a significant increase in gene transfection efficiency based upon analysis of Gaussia luciferase (GLuc) reporter gene expression, and gene expression could be attenuated by blocking HA-CD44 signaling. Furthermore, when fibroblasts were exposed to vascular endothelial growth factor-A (VEGF-A)-GAHCM, the 50 CP matrix facilitated sustained VEGF-A production for up to 7 days, with maximal expression at day 5. Application of these VEGF-A-50 CP samples stimulated prolonged pro-healing responses, including the TGF-β1-induced myofibroblast-like phenotypes and enhanced closure of murine splinted wounds. Overall, these findings demonstrate the use of ECM-based materials to stimulate efficient gene transfer and regulate cellular phenotype, resulting in improved control of growth factor activity for wound repair. GAHCM has significant potential to overcome key challenges in growth factor therapy for regenerative medicine. STATEMENT OF SIGNIFICANCE: Despite great promise for growth factor therapies in wound treatment, controlling growth factor activity and providing a microenvironment for cells that maximizes growth factor signaling have continued to limit the success of existing formulations. Our GAHCM strategy, combining CMP gene delivery and a hyaluronic acid-collagen matrix, enabled enhanced wound healing efficacy via the combination of controlled and localized growth factor expression and matrix-mediated regulation of cell behavior. Incorporation of CMPs and HA in the same matrix synergistically enhanced VEGF activity as compared with simpler matrices. Accordingly, GAHCM will advance our ability to leverage growth factor signaling for wound healing, resulting in new long-term treatments for recalcitrant wounds.

Induced pluripotent stem cells (iPSCs) provide an extraordinary tool for disease modeling owing to their potential to differentiate into the desired cell type. The differentiation of iPSCs is typically performed on 2-dimensional monolayers of stromal cell or animal tissue derived extracellular matrices. Recent advancements in disease modeling have utilized iPSCs in 3-dimensional (3D) cultures to study diseases such as muscular dystrophy, cardiomyopathy, and pulmonary fibrosis. However, these approaches are yet to be explored in modeling the hematological malignancies. Transient myeloproliferative disorder (TMD) is a preleukemic stage, which is induced in 10-20% of children with trisomy 21 possessing the pathognomonic mutation in the transcription factor GATA1. In this study, we established a synthetic 3D iPSC culture system for modeling TMD via hematopoietic differentiation of customized iPSCs. A chemically cross-linkable PEG hydrogel decorated with integrin binding peptide was found to be permissive of hematopoietic differentiation of iPSCs. It provided a cost-effective system for the generation of hematopoietic stem and progenitor cells (HSPCs) with higher yield of early HSPCs compared to traditional 2D culture on Matrigel coated dishes. Characterization of the HSPCs produced from the iPSC lines cultured in 3D showed that the erythroid population was reduced whereas the megakaryoid and myeloid populations were significantly increased in GATA1 mutant trisomic line compared to disomic or trisomic lines with wild-type GATA1, consistent with TMD characteristics. In conclusion, we have identified a cost-effective tunable 3D hydrogel system to model TMD.

The use of hydrogels in biomedical applications dates back multiple decades, and the engineering potential of these materials continues to grow with discoveries in chemistry and biology. The approaches have led to increasing complex hydrogels that incorporate both synthetic and natural polymers and functional domains for tunable release kinetics, mediated cell response, and ultimately use in clinical and research applications in biomedical practice. This review focuses on recent advances in hybrid hydrogels that incorporate nano/microstructures, their synthesis, and applications in biomedical research. Examples discussed include the implementation of click reactions, photopatterning, and 3D printing for the facile production of these hybrid hydrogels, the use of biological molecules and motifs to promote a desired cellular outcome, and the tailoring of kinetic and transport behavior through hybrid-hydrogel engineering to achieve desired biomedical outcomes. Recent progress in the field has established promising approaches for the development of biologically relevant hybrid hydrogel materials with potential applications in drug discovery, drug/gene delivery, and regenerative medicine.

Elastin-like polypeptides (ELPs) are thermoresponsive biopolymers that undergo an LCST-like phase transition in aqueous solutions. The temperature of this LCST-like transition, Tt , can be tuned by varying the number of repeat units in the ELP, sequence and composition of the repeat units, the solution conditions, and via conjugation to other biomacromolecules. In this study, we show how and why the choice of guest (X) residue in the VPGXG pentad repeat tunes the Tt of short ELPs, (VPGXG)4, in the free state and when conjugated to collagen-like peptides (CLPs). In experiments, the (VPGWG)4 chain (in short, WWWW) has a Tt 353 K in both free ELP and ELP–CLP systems. The Tt for the FWWF ELP sequence decreases from being >353 K for free ELP to

Hydrogels have been broadly studied for applications in clinically motivated fields such as tissue regeneration, drug delivery, and wound healing, as well as in a wide variety of consumer and industry uses. While the control of mechanical properties and network structures are important in all of these applications, for regenerative medicine applications in particular, matching the chemical, topographical and mechanical properties for the target use/tissue is critical. There have been multiple alternatives developed for fabricating materials with microstructures with goals of controlling the spatial location, phenotypic evolution, and signaling of cells. The commonly employed polymers such as poly(ethylene glycol) (PEG), polypeptides, and polysaccharides (as well as others) can be processed by various methods in order to control material heterogeneity and microscale structures. We review here the more commonly used polymers, chemistries, and methods for generating microstructures in biomaterials, highlighting the range of possible morphologies that can be produced, and the limitations of each method. With a focus in liquid-liquid phase separation, methods and chemistries well suited for stabilizing the interface and arresting the phase separation are covered. As the microstructures can affect cell behavior, examples of such effects are reviewed as well. STATEMENT OF SIGNIFICANCE: Heterogeneous hydrogels with enhanced matrix complexity have been studied for a variety of biomimetic materials. A range of materials based on poly(ethylene glycol), polypeptides, proteins, and/or polysaccharides, have been employed in the studies of materials that by virtue of their microstructure, can control the behaviors of cells. Methods including microfluidics, photolithography, gelation in the presence of porogens, and liquid-liquid phase separation, are presented as possible strategies for producing materials, and their relative advantages and disadvantages are discussed. We also describe in more detail the various processes involved in LLPS, and how they can be manipulated to alter the kinetics of phase separation and to yield different microstructured materials.

Heterogeneities in hydrogel scaffolds are known to impact the performance of cells in cell-laden materials constructs, and we have employed the phase separation of resilin-like polypeptides (RLPs) as a means to generate such materials. Here, we study the compositional features of resilin-like polypeptides (RLPs) that further enable our control of their liquid-liquid phase separation (LLPS) and how such control impacts the formation of microstructured hydrogels. The evaluation of the phase separation of RLPs in solutions of ammonium sulfate offers insights into the sequence-dependent LLPS of the RLP solutions, and atomistic simulations, along with 2D-nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY)

The aggregation behavior and stability of a series of alanine-rich peptides, which are included as components of peptide-polymer conjugates, were characterized using a combination of biophysical techniques. Light scattering techniques were used to monitor changes in peptide morphology and size distributions as a function of time and temperature. The results show large particles immediately upon dissolution in buffer. At room temperature, these particles relaxed to reach a mostly monomeric peptide state, while at higher temperatures, they grew to form aggregates. Circular dichroism spectroscopy (CD) was used to monitor temperature- and time-dependent conformational changes as a function of peptide sequence and incubation time. CD measurements reveal that all of the sequences are helical at low temperatures with transitions to non-helical conformation with increased temperature. Samples incubated at room temperature were able to recover their original helicity. At increased temperature, the shorter and longer peptide sequences showed notable changes in conformation, and were not able to recover their original helicity after 72 h. After incubation for up to one week, β-sheet conformations were observed in these two cases, while only α-helical conformation loss was observed for the peptide of intermediate molecular weight. Transmission electron microscopy measurements reveal the formation of fibrils after 72 h of incubation at 60 °C for all samples, in agreement with the scattering measurements. Additional quenching experiments show that peptide aggregation can be stalled when solutions are cooled to room temperature.

Cord blood (CB) mononuclear cell populations have demonstrated significant promise in biomaterials-based regenerative therapies; however, the contributions of monocyte and macrophage subpopulations towards proper tissue healing and regeneration are not well understood, and the phenotypic responses of macrophage to microenvironmental cues have not been well-studied. In this work, we evaluated the effects of cytokine stimulation and altered substrate stiffness. Macrophage derived from CB CD14(+) monocytes adopted distinct inflammatory (M1) and anti-inflammatory (M2a and M2c) phenotypes in response to cytokine stimulation (M1: lipopolysaccharide (LPS) and interferon (IFN-γ); M2a: interleukin (IL)-4 and IL-13; M2c: IL-10) as determined through expression of relevant cell surface markers and growth factors. Cytokine-induced macrophage readily altered their phenotypes upon sequential administration of different cytokine cocktails. The impact of substrate stiffness on macrophage phenotype was evaluated by seeding CB-derived macrophage on 3wt%, 6wt%, and 14wt% poly(ethylene glycol)-based hydrogels, which exhibited swollen shear moduli of 0.1, 3.4, and 10.3 kPa, respectively. Surface marker expression and cytokine production varied depending on modulus, with anti-inflammatory phenotypes increasing with elevated substrate stiffness. Integration of specific hydrogel moduli and cytokine cocktail treatments resulted in the differential regulation of macrophage phenotypic biomarkers. These data suggest that CB-derived macrophages exhibit predictable behaviors that can be directed and finely tuned by combinatorial modulation of substrate physical properties and cytokine profiles.

The thermoresponsiveness and structures of peptide-based nanocarriers can be altered to enable versatile mechanisms of cargo delivery., The self-assembly of nanostructures from conjugates of elastin-like peptides and collagen-like peptides (ELP-CLP) has been studied as means to produce thermoresponsive, collagen-binding drug delivery vehicles. Motivated by our previous work in which ELP-CLP conjugates successfully self-assembled into vesicles and platelet-like nanostructures, here, we extend our library of ELP-CLP bioconjugates to a series of tryptophan/phenylalanine-containing ELPs and GPO-based CLPs [W2Fx-b-(GPO)y] with various domain lengths to determine the impact of these modifications on the thermoresponsiveness and morphology. The lower transition temperature of the conjugates with longer ELP or CLP domains enables the formation of well-defined nanoparticles near physiological temperature. Moreover, the morphological transition from vesicles to platelet-like nanostructures occurred when the ratio of the lengths of ELP/CLP decreased. Given the previously demonstrated ability of many ELP-CLP bioconjugates to bind to both hydrophobic drugs and collagen-containing materials, our results suggest new opportunities for designing specific thermoresponsive nanostructures for targeted biological applications.

Wound healing is a complex biological process that requires coordinated cell proliferation, migration, and extracellular matrix production/remodeling, all of which are inhibited/delayed in chronic wounds. In this study, a formulation was developed that marries a fibrin-based, provisional-like matrix with collagen mimetic peptide (CMP)/PDGF gene-modified collagens, leading to the formation of robust gels that supported temporally controlled PDGF expression and facile application within the wound bed. Analysis employing in vitro co-gel scaffolds confirmed sustained and temporally controlled gene release based on matrix metalloproteinase (MMP) activity, with ~30% higher PDGF expression in MMP producing fibroblasts as-compared with non-MMP-expressing cells. The integration of fibrin with the gene-modified collagens resulted in co-gels that strongly supported both fibroblast cell recruitment/invasion as well as multiple aspects of the longer-term healing process. The excisional wound healing studies in mice established faster wound closure using CMP-modified PDGF polyplex-loaded co-gels, which exhibited up to 24% more wound closure (achieved with ~2 orders of magnitude lower growth factor dosing) after 9 days as compared to PDGF-loaded co-gels, and 19% more wound closure after 9 days as compared to CMP-free polyplex loaded co-gels. Moreover, minimal scar formation as well as improved collagen production, myofibroblast activity, and collagen orientation was observed following CMP-modified PDGF polyplex-loaded co-gel application on wounds. Taken together, the combined properties of the co-gels, including their stability and capacity to control both cell recruitment and cell phenotype within the murine wound bed, strongly supports the potential of the co-gel scaffolds for improved treatment of chronic non-healing wounds.

The use of collagen-based biomaterials in regenerative medicine has rapidly increased over the past decade. The unique structural and biochemical properties of collagen make it a particularly promising material for delivering both protein and DNA-based therapeutics. Although many collagen modification techniques have been developed, the majority of them require multi-step chemical treatments that can modify the natural favourable properties of collagen. We have developed a promising biomimetic modification technique employing collagen-mimetic peptides (CMP)s to control the retention and delivery of DNA polyplexes from collagen structures, including both monomeric 2-D collagen films and fibrous, 3-D gels. Variations in the concentration of CMPs displayed on polyplexes enabled tuning of polyplex retention vs. release over periods of at least 2 weeks on films and a month on gels. Retention of CMP-modified polyplexes (20 days) was substantially improved compared to non-modified polyplexes, which were retained for only 2 days. The activity of bound polyplex in collagen gels was shown, through a series of transfection studies, to be maintained in the presence of serum for a minimum of 2 weeks. Only matrix metalloproteinase (MMP)-stimulated cells exhibited significant levels of transfection suggesting that cell mobility within the gel was vital and that collagen remodelling played a role in stimulating gene release and expression. To our knowledge, this study is the first to deliver genes with CMP-modified polyplexes and to examine the effects of CMP display on DNA release. The results suggest that this technique may be used more broadly to create tuneable, collagen-based delivery systems.

Current cell expansion methods for tissue engineering and regenerative medicine applications rely on the use of enzymatic digestion passaging and 2D platforms. However, this enzymatic treatment significantly reduces cell quality, due to the destruction of important cell-surface proteins. In addition, culture in 2D results in undesired de-differentiation of the cells caused by the lack of 3D similarity to the natural extracellular matrix (ECM) environment. Research has led to the development of thermo-responsive surfaces for the continuous culture of cells. These thermo-responsive materials properties can be used to passage cells from the surface when the cell culture temperature is reduced. Here we report the development of a PLA/thermo-responsive (PDEGMA) blend 3D electrospun fibre-based scaffold to create an enzymatic-free 3D cell culture platform for the expansion of mammalian cells with the desired phenotype for clinical use. Human corneal stromal cells (hCSCs) were used as an exemplar as they have been observed to de-differentiate to an undesirable myo-fibroblastic phenotype when cultured by conventional 2D cell culture methods. Scaffolds were functionalised with a cell adherence peptide sequence GGG-YIGSR by thiol–ene chemistry to improve cell adherence and phenotype support. This was obtained by functionalising the thermo-responsive polymer with a thiol (PDEGMA/PDEGSH) by co-polymerisation. These incorporated thiols react with the norbornene acid functionalised peptide (Nor-GGG-YIGSR) under UV exposure. Presence of the thiol in the scaffold and subsequent peptide attachment on the scaffolds were confirmed by fluorescence labelling, ToF-SIMS and XPS analysis. The biocompatibility of the peptide containing scaffolds was assessed by the adhesion, proliferation and immuno-staining of hCSCs. Significant increase in hCSC adherence and proliferation was observed on the peptide containing scaffolds. Immuno-staining showed maintained expression of the desired phenotypic markers ALDH, CD34 and CD105, while showing no or low expression of the undesired phenotype marker α-SMA. This desired expression was observed to be maintained after thermo-responsive passaging and higher when cells were cultured on PLA scaffolds with 10 wt% PDEGMA/4 mol% PDEGS-Nor-GGG-YIGSR. This paper describes the fabrication and application of a first generation, biocompatible peptide conjugated thermo-responsive fibrous scaffold. The ease of fabrication, successful adherence and expansion of a therapeutically relevant cell type makes these scaffolds a promising new class of materials for the application of cell culture expansion platforms in the biomaterials and tissue engineering field.

Covalent co-assembly holds great promise for the fabrication of hydrogels with controllable nanostructure, versatile chemical composition, and enhanced mechanical properties given its relative simplicity, high efficiency, and bond stability. This report describes our approach to designing functional multicomponent hydrogels based on photo-induced chemical interactions between an acrylamide-functionalized resilin-like polypeptide (RLP) and a peptide amphiphile (PA). Circular dichroism (CD) spectroscopy, electron microscopy, and amplitude sweep rheology were used to demonstrate that the co-assembled hydrogel systems acquired distinct structural conformations, tunable nanostructures, and enhanced elasticity in a PA concentration-dependent manner. We envisage the use of these materials in numerous biomedical applications such as controlled drug release systems, microfluidic devices, and scaffolds for tissue engineering.

Adventitial fibroblasts (AFs) are critical mediators of vascular remodeling. However, the contributions of AFs towards development of vasculature and the specific mechanisms by which these cells regulate physiological expansion of the vasa vasorum, the specialized microvasculature that supplies nutrients to the vascular wall, are not well understood. To determine the regulatory role of AFs in microvascular endothelial cell (MVEC) neovasculogenesis and to investigate the regulatory pathways utilized for communication between the two cell types, AFs and MVECs were cultured together in poly(ethylene glycol)-based hydrogels. Following preliminary evaluation of a set of cell adhesion peptides (AG10, AG73, A2G78, YIGSR, RGD), 7.5wt% hydrogels containing 3 mM RGD were selected as these substrates did not initiate primitive tubule structures in 3D MVEC monocultures, thus providing a passive platform to study AF-MVEC interaction. The addition of AFs to hydrogels promoted MVEC viability; however, increasing AF density within hydrogels stimulated MVEC proliferation, increased microvessel density and size, and enhanced deposition of basement membrane proteins, collagen IV and laminin. Importantly, AF-MVEC communication through the transforming growth factor beta (TGF-β)/activin receptor-like kinase 5 (ALK5) signaling pathway was observed to mediate microvessel formation, as inhibition of ALK5 significantly decreased MVEC proliferation, microvessel formation, mural cell recruitment, and basement membrane production. These data indicate that AFs regulate MVEC neovasculogenesis and suggest that therapeutics targeting the TGF-β/ALK5 pathway may be useful for regulation of vasculogenic and anti-vasculogenic responses.

Templating methods, combined with colloidal chemistry synthesis, provide a strategy to fabricate inorganic nanomaterials with controllable architectures. Computationally designed peptide assemblies...

Computationally designed peptides form desired antiparallel, tetrameric coiled-coil bundles that hierarchically assemble into a variety of well-controlled nanostructures depending on aqueous solution conditions. The bundles selectively self-assemble into different structures: nanotubes, platelets, or needle-like structures at solution pH values of 4.5, 7, and 10, respectively. The self-assembly produces hollow tubes or elongated needle-like structures at pH conditions associated with charged bundles (pH 4.5 or 10); at neutral pH, near the pI of the bundle, a plate-like self-assembled structure forms. Transmission electron microscopy and small-angle X-ray scattering show the nanotubes to be uniform with a tube diameter of ∼13 nm and lengths of up to several μm, yielding aspect ratios1000. Combining the measured nanostructure geometry with the apparent charged states of the constituent amino acids, a tilted-bundle packing model is proposed for the formation of the homogeneous nanotubes. This work demonstrates the successful use of assembly pathway control for the construction of nanostructures with diverse, well-structured morphologies associated with the folding and self-association of a single type of molecule.

Vocal folds are connective tissues housed in the larynx, which can be subjected to various injuries and traumatic stimuli that lead to aberrant tissue structural alterations and fibrotic-induced biomechanical stiffening observed in patients with voice disorders. Much effort has been devoted to generate soft biomaterials that are injectable directly to sites of injury. To date, materials applied toward these applications have been largely focused on natural extracellular matrix-derived materials such as collagen, fibrin or hyaluronic acid; these approaches have suffered from the fact that materials are not sufficiently robust mechanically nor offer sufficient flexibility to modulate material properties for targeted injection. We have recently developed multiple resilin-inspired elastomeric hydrogels that possess similar mechanical properties as those reported for vocal fold tissues, and that also show promising in vitro cytocompatibility and in vivo biocompatibility. Here we report studies that test the delivery of resilin-based hydrogels through injection to the subcutaneous tissue in a wild-type mice model; histological and genetic expression outcomes were monitored. The rapid kinetics of crosslinking enabled facile injection and ensured the rapid transition of the viscous resilin precursor solution to a solid-like hydrogel in the subcutaneous space in vivo; the materials exhibited storage shear moduli in the range of 1000-2000 Pa when characterized through oscillatory rheology. Histological staining and gene expression profiles suggested minimal inflammatory profiles three weeks after injection, thereby demonstrating the potential suitability for site-specific in vivo injection of these elastomeric materials. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2229-2242, 2018.

The sequence and helical content of two alanine-rich peptides (AQK18 and GpAQK18, Gp: l-propargylglycine) and their conjugates with poly(ethylene glycol) (PEG) have been investigated by multidimensional mass spectrometry (MS), encompassing electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) interfaced with tandem mass spectrometry (MS2) fragmentation and shape-sensitive separation via ion mobility mass spectrometry (IM-MS). The composition, sequence, and molecular weight distribution of the peptides and bioconjugates were identified by MS and MS2 experiments, which also confirmed the attachment of PEG at the C-terminus of the peptides. ESI coupled with IM-MS revealed the existence of random coil and α-helical conformers for the peptides in the gas phase. More importantly, the proportion of the helical conformation increased substantially after PEG attachment, suggesting that conjugation adds stability to this conformer. The conformational assemblies detected in the gas pha...

Detailed understanding of the local structure–property relationships in soft biopolymeric hydrogels can be instrumental for applications in regenerative tissue engineering. Resilin-like polypeptide (RLP) hydrogels have been previously demonstrated as useful biomaterials with a unique combination of low elastic moduli, excellent resilience, and cell-adhesive properties. However, comprehensive mechanical characterization of RLP hydrogels under both low-strain and high-strain conditions has not yet been conducted, despite the unique information such measurements can provide about the local structure and macromolecular behavior underpinning mechanical properties. In this study, mechanical properties (elastic modulus, resilience, and fracture initiation toughness) of equilibrium swollen resilin-based hydrogels were characterized via oscillatory shear rheology, small-strain microindentation, and large-strain puncture tests as a function of polypeptide concentration. These methods allowed characterization, for the first time, of the resilience and failure in hydrogels with low polypeptide concentrations (

The generation of hematopoietic stem and progenitor cells (HSPCs) from induced pluripotent stem cells (iPSCs) provides an extraordinary tool for hematological disease modeling of rare disorders such as Down syndrome (DS) associated transient myeloproliferative disorder (TMD). TMD is a preleukemic condition observed in 10-20% of children with trisomy 21 possessing the pathognomonic mutation in the transcription factor GATA1. Hematopoiesis in the bone marrow (BM) is affected by cell-cell and cell-matrix interactions. The current methods for iPSC differentiation into HSPCs utilize either 2-dimensional (2D) monolayer of mouse stromal cells or animal tissue derived extracellular matrices. Generation of a 3-dimensional (3D) culture environment attempts to facilitate both cell-cell and cell-matrix interactions during iPSC differentiation. This study reports the development of a 3D culture system for hematopoietic differentiation of iPSCs to model TMD. iPSC colonies were encapsulated in 3D polyethylene glycol (PEG) based hydrogels containing synthetic integrin binding peptide (GRGDSPC) and enzymatically degradable peptide (GGPQGIWGQGKG) (Fig. 1A) and cultured in maintenance medium (mTeSR1™, Stem Cell Technology) without feeder cells. There were notable morphological differences between the 3D encapsulated and 2D cultured iPSC colonies (Fig. 1B). The 3D encapsulation did not have an adverse effect on the viability of the iPSC colonies evaluated by in situ staining with viability dye (Fig. 1C). The 3D encapsulated colonies were more compact with a spheroid morphology in PEG whereas colonies in 2D were more flattened (Fig. 1D). The pluripotency of the 3D encapsulated iPSCs was confirmed alkaline phosphatase staining (purple colonies) and by the presence of >96% population expressing pluripotency markers, Tra-1-60 and SSEA-4 (Fig. 1E). To test the efficiency of the 3D model system to generate HSPCs, the encapsulated iPSCs were subjected to hematopoietic differentiation using STEMdiff Hematopoietic Kit. Following differentiation, immunophenotype analysis of single cells by flow cytometry revealed a 1.7-fold higher CD34+CD45+CD38-CD45RA- cell percentage in 3D hydrogels compared to 2D. Further delineation of sub-populations in HSPC compartment from 2D and 3D hydrogel revealed a 1.9-fold and 2.1-fold higher population of early HSPCs and multipotent progenitors (MPPs) in 3D compared to 2D respectively (Fig 1F, *P We used this 3D system to model TMD by utilizing isogenic iPSCs with disomy 21 (D21), trisomy 21 (T21), and trisomy 21 bearing pathologic mutation in GATA1 (T21-G1). The megakaryoid population in the HSPCs generated by hematopoietic differentiation of 3D encapsulated iPSCs was characterized by the percentage of CD34+CD41+ population within the total CD41+ population, myeloid population as CD18+CD45+ and erythroid population as CD71+CD235+. T21 HSPCs showed increased erythroid and megakaryoid populations as compared to isogenic D21, consistent with the role of trisomy 21 in perturbing hematopoiesis. T21-G1 had elevated megakaryoid (93±6% vs 71±1%,) and myeloid (32±16% vs 8±4%) populations with reduced erythroid (27±12% vs 79±6%) population as compared to T21 HSPCs implicating GATA1s in altered hematopoiesis (Fig. 1H). T21-G1 HSPCs only produced CFU-GM colonies as compared to a high number of CFU-GEMM and BFU-E in T21 and D21 HSPCs (Fig. 1I). The expression of GATA1s in T21-G1 megakaryoid population was confirmed (Fig. 1J). The immunophenotype marker analysis of T21-G1 megakaryoid blasts showed expression of megakaryoid/erythroid antigens (CD41, CD61, CD42b, CD71) along with myeloid markers (CD11b, CD33, CD13) and increased expression of CD56 and CD117 consistent with TMD patients (Fig. 1K). In conclusion, our cost-effective tunable 3D hydrogel system promoted hematopoietic differentiation of iPSCs and generated TMD model mimicking the salient features of the disease. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Gopalakrishnapillai: Geron: Research Funding.

Gene therapies have great potential in regenerative medicine; however, clinical translation has been inhibited by low stability and limited transfection efficiencies. Herein, we incorporate collagen-mimetic peptide (CMP)-linked polyplexes in collagen scaffolds to increase DNA stability by up to 400% and enable tailorable in vivo transgene expression at 100-fold higher levels and 10-fold longer time periods. These improvements were directly linked to a sustained interaction between collagen and polyplexes that persisted during cellular remodeling, polyplex uptake, and intracellular trafficking. Specifically, incorporation of CMPs into polyethylenimine (PEI) polyplexes preserved serum-exposed polyplex-collagen activity over a period of 14 days, with 4 orders-of-magnitude more intact DNA present in CMP-modified polyplex-collagen relative to unmodified polyplex-collagen after a 10 day incubation under cell culture conditions. CMP-modification also altered endocytic uptake, as indicated by gene silencing studies showing a nearly 50% decrease in transgene expression in response to caveolin-1 silencing in modified samples versus only 30% in unmodified samples. Furthermore, cellular internalization studies demonstrated that polyplex-collagen association persisted within cells in CMP polyplexes, but not in unmodified polyplexes, suggesting that CMP linkage to collagen regulates intracellular transport. Moreover, experiments in an in vivo repair model showed that CMP modification enabled tailoring of transgene expression from 4 to 25 days over a range of concentrations. Overall, these findings demonstrate that CMP decoration provides substantial improvements in gene retention, altered release kinetics, improved serum-stability, and improved gene activity in vivo. This versatile technique has great potential for multiple applications in regenerative medicine. Statement of Significance In this work, we demonstrate a novel approach for stably integrating DNA into collagen scaffolds to exploit the natural process of collagen remodelling for high efficiency non-viral gene delivery. The incorporation of CMPs into DNA polyplexes, coupled with the innate affinity between CMPs and collagen, not only permitted improved control over polyplex retention and release, but also provided a series of substantial and highly unique benefits via the stable and persistent linkage between CMP-polyplexes and collagen fragments. Specifically, CMP-modification of polyplexes was demonstrated to (i) control release for nearly a month, (ii) improve vector stability under physiological-like conditions, and (iii) provide ligands able to efficiently transfer genes via endocytic collagen pathways. These unique properties overcome key barriers inhibiting non-viral gene therapy.

Arteries for bypass grafting are harvested either with neighboring tissue attached or as skeletonized vessels that are free of surrounding tissue. There are significant benefits to skeletonization, but reports suggest that skeletonized vessels may develop structural defects and are at risk for atherosclerosis. We investigated the specific short-term effects of skeletonization on carotid artery biomechanics and microanatomy in a rabbit model. Six carotid arteries were surgically skeletonized. To support healing, three of these received polyethylene glycol hydrogel injected along their exterior surfaces. M-mode ultrasonography was used to track circumferential cyclic strain in the skeletonized, hydrogel-treated, and contralateral vessels. On day 21, the arteries were harvested, and vessel structure was assessed by histology, immunofluorescence microscopy, two-photon elastin autofluorescence, and second harmonic generation (SHG) microscopy. Intimal-medial thickness appeared unaffected by skeletonization, but the SHG signals indicated significant changes in collagen turnover in the adventitia. Skeletonized arteries also exhibited significantly decreased radial compliance (circumferential cyclic strain dropped ∼30%) and decreased numbers of elastic laminae (9.1 ± 2.0 to 2.3 ± 1.4). Hydrogel treatment protected against these effects with treated vessels maintaining normal mechanical properties. These results indicate that arterial skeletonization triggers immediate effects on vessel remodeling and reduced vessel compliance resulting in specific tissue alterations within 21 days, but that these effects can be attenuated by the placement of hydrogel on the exterior surface of the skeletonized vessel.

Biomaterials have been extensively used to leverage beneficial outcomes in various therapeutic applications, such as providing spatial and temporal control over the release of therapeutic agents in drug delivery as well as engineering functional tissues and promoting the healing process in tissue engineering and regenerative medicine. This Perspective presents important milestones in the development of polymeric biomaterials with defined structures and properties. Contemporary studies of biomaterial design have been reviewed with focus on constructing materials with controlled structure, dynamic functionality, and biological complexity. Examples of these polymeric biomaterials enabled by advanced synthetic methodologies, dynamic chemistry/assembly strategies, and modulated cell–material interactions have been highlighted. As the field of polymeric biomaterials continues to evolve with increased sophistication, current challenges and future directions for the design and translation of these materials are a...

Natural biomolecular self-assembly typically occurs under a narrow range of solution conditions, and the design of sequences that can form prescribed structures under a range of such conditions would be valuable in the bottom-up assembly of predetermined nanostructures. We present a computationally designed peptide that robustly self-assembles into regular arrays under a wide range of solution pH and temperature conditions. Controling the solution conditions provides the opportunity to exploit a simple and reproducible approach for altering the pathway of peptide solution self-assembly. The computationally designed peptide forms a homotetrameric coiled-coil bundle that further self-assembles into 2-D plate structures with well-defined inter-bundle symmetry. Herein, we present how modulation of solution conditions, such as pH and temperature, can be used to control the kinetics of the inter-bundle assembly and manipulate the final morphology. Changes in solution pH primarily influence the inter-bundle assembly by affecting the charged state of ionizable residues on the bundle exterior while leaving the homotetrameric coiled-coil structure intact. At low pH, repulsive interactions prevent 2-D lattice nanostructure formation. Near the estimated isoelectric point of the peptide, bundle aggregation is rapid and yields disordered products, which subsequently transform into ordered nanostructures over days to weeks. At elevated temperatures (T = 40 °C or 50 °C), the formation of disordered, kinetically-trapped products largely can be eliminated, allowing the system to quickly assemble into plate-like nanostructured lattices. Moreover, subtle changes in pH and in the peptide charge state have a significant influence on the thickness of formed plates and on the hierarchical manner in which plates fuse into larger material structures with observable grain boundaries. These findings confirm the ability to finely tune the peptide assembly process to achieve a range of engineered structures with one simple 29-residue peptide building block.

Wound infections are a significant clinical problem affecting millions of people worldwide. Topically applied antibacterial formulations with longer residence time and controlled antimicrobial release would offer significant benefits for improved prevention and treatment of infected wounds. In this study, we developed collagen mimetic peptide (CMP) tethered vancomycin (Van)-containing liposomes (Lipo) (CMP-Van-Lipo) hybridized to collagen-based hydrogels ('co-gels,' e.g., collagen/fibrin combination hydrogels) for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections in vitro and in vivo. Tethering CMP-Van-Lipo nanostructures to co-gels enabled sustained Van release and enhanced in vitro antibacterial effects against MRSA as compared to Van loaded co-gels or Van-Lipo loaded co-gels following multiple fresh bacterial inoculations over a period of 48 h. These results were successfully translated in vivo wherein MRSA infected wounds were effectively treated with CMP-Van-Lipo loaded co-gels for up to 9 days, whereas the activity of Van loaded co-gels and Van-Lipo loaded co-gels were limited to2 days. Moreover, CMP-Van-Lipo retained in vivo antibacterial activity even after re-inoculation with bacteria; however, Van loaded co-gels and Van-Lipo loaded co-gels allowed significant bacterial growth demonstrating their limited efficacy. Altogether, these results provide proof-of-concept that CMP-Van-Lipo loaded co-gels can be effective topical formulations for preventive treatment of MRSA wound infections. STATEMENT OF SIGNIFICANCE: Current topical antimicrobial formulations (e.g., creams, gels, and ointments) do not control release, leaving antimicrobial concentrations either too high or too low at different time points, and provoking the development of antibacterial resistance and recurrence of wound infections. Here, collagen mimetic peptides (CMPs) were used to stably hybridize vancomycin-containing liposomal nanocarriers (CMP-Van-Lipo) within collagen-fibrin co-gels via triple-helical integration with collagen, enabling control over Van release for prolonged time periods and minimizing the adverse effects of the Lipo formulations on fibroblast cell viability in the wound bed. The CMP-Van-Lipo loaded co-gel's higher antibacterial effects in vitro were successfully translated in vivo for treatment of MRSA-infected mouse wounds, and thus the co-gels can be a potentially translatable treatment for improved clinical wound management.

Adventitial fibroblasts (AFs) are major contributors to vascular remodeling and maladaptive cascades associated with arterial disease, where AFs both contribute to and respond to alterations in their surrounding matrix. The relationships between matrix modulus and human aortic AF (AoAF) function are investigated using poly(ethylene glycol)-based hydrogels designed with matrix metalloproteinase (MMP)-sensitive and integrin-binding peptides. Initial equilibrium shear storage moduli for the substrates examined are 0.33, 1.42, and 2.90 kPa; after 42 days of culture, all hydrogels exhibit similar storage moduli (0.3-0.7 kPa) regardless of initial modulus, with encapsulated AoAFs spreading and proliferating. In 10 and 7.5 wt% hydrogels, modulus decreases monotonically throughout culture; however, in 5 wt% hydrogels, modulus increases after an initial 7 days of culture, accompanied by an increase in myofibroblast transdifferentiation and expression of collagen I and III through day 28. Thereafter, significant reductions in both collagens occur, with increased MMP-9 and decreased tissue inhibitor of metalloproteinase-1/-2 production. Releasing cytoskeletal tension or inhibiting cellular protein secretion in 5 wt% hydrogels block the stiffening of the polymer matrix. Results indicate that encapsulated AoAFs initiate cell-mediated matrix remodeling and demonstrate the utility of dynamic 3D systems to elucidate the complex interactions between cell behavior and substrate properties.

Materials that respond to temporally defined exogenous cues continue to be an active pursuit of research toward on‐demand nanoparticle drug delivery applications, and using one or more exogenous temperature stimuli could significantly expand the application of nanoparticle‐based drug delivery formulations under both hyperthermal and hypothermal conditions. Previously we have reported the development of a biocompatible and thermoresponsive elastin‐b‐collagen‐like polypeptide (ELP‐CLP) conjugate that is capable of self‐assembling into vesicles and encapsulating small molecule therapeutics that can be delivered at different rates via a single temperature stimulus. Herein we report the evaluation of multiple ELP‐CLP conjugates, demonstrating that the inverse transition temperature (T t) of the ELP‐CLPs can be manipulated by modifying the melting temperature (T m) of the CLP domain, and that the overall hydrophilicity of the ELP‐CLP conjugate also may alter the T t. Based on these design parameters, we demonstrate that the ELP‐CLP sequence (VPGFG)6‐(GPO)7GG can self‐assemble into stable vesicles at 25°C and dissociate at elevated temperatures by means of the unfolding of the CLP domain above its T m. We also demonstrate here for the first time the ability of this ELP‐CLP vesicle to dissociate via a hypothermic temperature stimulus by means of exploiting the inverse transition temperature (T t) phenomena found in ELPs. The development of design rules for manipulating the thermal properties of these bioconjugates will enable future modifications to either the ELP or CLP sequences to more finely tune the transitions of the conjugates for specific biomedical applications.

Vocal fold scar, characterized by alterations in the lamina propria extracellular matrix, disrupts normal voice quality and function. Due to a lack of satisfactory clinical treatments, there is a need for tissue engineering strategies to restore voice. Candidate biomaterials for vocal fold tissue engineering must match the unique biomechanical and viscoelastic properties of native tissue without provoking inflammation. We sought to introduce elastomeric properties to hyaluronic acid (HA)-based biomaterials by incorporating resilin-like polypeptide (RLP) into hybrid hydrogels. Physically crosslinked RLP/HA and chemically crosslinked RLP-acrylamide/thiolated HA (RLP-AM/HA-SH) hydrogels were fabricated using cytocompatible chemistries. Mechanical properties of hydrogels were assessed in vitro using oscillatory rheology. Hybrid hydrogels were injected into rabbit vocal folds and tissues were assessed using rheology and histology. A small number of animals underwent acute vocal fold injury followed by injection of RLP-AM/HA-SH hydrogel alone or as a carrier for human bone marrow mesenchymal stem cells (BM-MSCs). Rheological testing confirmed that mechanical properties of materials in vitro resembled native vocal fold tissue and that viscoelasticity of vocal fold mucosa was preserved days 5 and 21 after injection. Histological analysis revealed that hybrid hydrogels provoked only mild inflammation in vocal fold lamina propria with demonstrated safety in the airway for up to 3 weeks, confirming acute biocompatibility of crosslinking chemistries. After acute injury, RLP-AM/HA-SH gel with and without BM-MSCs did not result in adverse effects or increased inflammation. Collectively, results indicate that RLP and HA-based hybrid hydrogels are highly promising for engineering the vocal fold lamina propria.

The self-assembly of nanostructures from elastin-like (poly)peptide (ELP) containing block copolymers has been a subject of intense investigation over decades. However, short synthetic ELPs have rarely been used due to their high inverse transition temperature; the use of short ELPs has largely been limited to polymer conjugates. Motivated by our previous work which successfully overcame this barrier by simply conjugating short ELPs with a triple-helix-forming collagen-like peptide, in this study, we further extend the ELP library to a series of ELPs equipped with aromatic residues and having sequences as short as four pentapeptide motifs. The resulting elastin-like peptide-collagen-like peptide (ELP-CLP) bioconjugates unexpectedly self-assembled into nanosized platelets likely by forming a bilayer structure. Given the previously demonstrated ability of many other CLP conjugates to target collagens and the potential for encapsulation of hydrophobic drugs in collapsed ELPs, these ELP-CLP nanoplatelets may offer similar opportunities for targeted delivery in biomedical and other arenas.

The delivery of drugs and genes plays an integral role in regenerative engineering approaches to restore living tissue through integration of materials science and biochemical signaling. The efficient administration of such agents aids in the activation or inhibition of specific reparative pathways vital for regeneration of various tissues. However, delivery is complicated by poor therapeutic stability within the body, poor therapeutic retention in the delivery/active site, and off-target effects. Drug-delivery systems have been engineered to overcome these obstacles by preserving therapeutic bioactivity and enabling spatiotemporal control over delivery. Moreover, the advent of stimuli-responsive materials has provided a means for preengineering desirable release profiles in response to physiologically relevant conditions and/or remote controlling delivery kinetics through externally administered triggers. In this article, the various mechanisms used to control release within a diverse array of delivery systems are presented and compared. The common therapeutic categories within the regenerative engineering field are subsequently discussed, and finally, an in-depth analysis of system design in the context of chronic wound repair is provided. While chronic wound repair is only one application, the delivery obstacles and principles discussed are relevant to a broad range of regenerative engineering applications.

Modulation of inter- and intramolecular interactions between bioinspired designer molecules can be harnessed for developing functional structures that mimic the complex hierarchical organization of multicomponent assemblies observed in nature. Furthermore, such multistimuli-responsive molecules offer orthogonal tunability for generating versatile multifunctional platforms via independent biochemical and biophysical cues. In this review, the remarkable physicochemical and mechanical properties of genetically engineered protein polymers derived from intrinsically disordered proteins, specifically elastin and resilin, are discussed. This review highlights emerging technologies which use them as building blocks in the fabrication of highly programmable structured biomaterials for applications in delivery of biotherapeutic cargo and regenerative medicine.

Vocal folds are soft laryngeal connective tissues with distinct layered structures and complex multicomponent matrix compositions that endow phonatory and respiratory functions. This delicate tissue is easily damaged by various environmental factors and pathological conditions, altering vocal biomechanics and causing debilitating vocal disorders that detrimentally affect the daily lives of suffering individuals. Modern techniques and advanced knowledge of regenerative medicine have led to a deeper understanding of the microstructure, microphysiology, and micropathophysiology of vocal fold tissues. State-of-the-art materials ranging from extracecullar-matrix (ECM)-derived biomaterials to synthetic polymer scaffolds have been proposed for the prevention and treatment of voice disorders including vocal fold scarring and fibrosis. This review intends to provide a thorough overview of current achievements in the field of vocal fold tissue engineering, including the fabrication of injectable biomaterials to mimic in vitro cell microenvironments, novel designs of bioreactors that capture in vivo tissue biomechanics, and establishment of various animal models to characterize the in vivo biocompatibility of these materials. The combination of polymeric scaffolds, cell transplantation, biomechanical stimulation, and delivery of antifibrotic growth factors will lead to successful restoration of functional vocal folds and improved vocal recovery in animal models, facilitating the application of these materials and related methodologies in clinical practice.