Sarah E. Stabenfeldt

Ultrasoft microgels displaying emergent platelet-like behaviours

Efforts to create platelet-like structures for the augmentation of haemostasis have focused solely on recapitulating aspects of platelet adhesion; more complex platelet behaviours such as clot contraction are assumed to be inaccessible to synthetic systems. Here, we report the creation of fully synthetic platelet-like particles (PLPs) that augment clotting in vitro under physiological flow conditions and achieve wound-triggered haemostasis and decreased bleeding times in vivo in a traumatic injury model. PLPs were synthesized by combining highly deformable microgel particles with molecular-recognition motifs identified through directed evolution. In vitro and in silico analyses demonstrate that PLPs actively collapse fibrin networks, an emergent behaviour that mimics in vivo clot contraction. Mechanistically, clot collapse is intimately linked to the unique deformability and affinity of PLPs for fibrin fibres, as evidenced by dissipative particle dynamics simulations. Our findings should inform the future design of a broader class of dynamic, biosynthetic composite materials.

In Vitro Neural Injury Model for Optimization of Tissue-Engineered Constructs

Stem cell transplantation is a promising approach for the treatment of traumatic brain injury, although the therapeutic benefits are limited by a high degree of donor cell death. Tissue engineering is a strategy to improve donor cell survival by providing structural and adhesive support. However, optimization prior to clinical implementation requires expensive and time‐consuming in vivo studies. Accordingly, we have developed a three‐dimensional (3‐D) in vitro model of the injured host–transplant interface that can be used as a test bed for high‐throughput evaluation of tissue‐engineered strategies. The neuronal‐astrocytic cocultures in 3‐D were subjected to mechanical loading (inducing cell death and specific astrogliotic alterations) or to treatment with transforming growth factor‐β1 (TGF‐β1), inducing astrogliosis without affecting viability. Neural stem cells (NSCs) were then delivered to the cocultures. A sharp increase in the number of TUNEL+ donor cells was observed in the injured cocultures compared to that in the TGF‐β1‐treated and control cocultures, suggesting that factors related to mechanical injury, but not strictly astrogliosis, were detrimental to donor cell survival. We then utilized the mechanically injured cocultures to evaluate a methylcellulose‐laminin (MC‐LN) scaffold designed to reduce apoptosis. When NSCs were codelivered with MC alone or MC‐LN to the injured cocultures, the number of caspase+ donor cells significantly decreased compared to that with vehicle delivery (medium). Collectively, these results demonstrate the utility of an in vitro model as a preanimal test bed and support further investigation of a tissue‐engineering approach for chaperoned NSC delivery targeted to improve donor cell survival in neural transplantation.

Engineering fibrin polymers through engagement of alternative polymerization mechanisms.

Fibrin is an attractive material for regenerative medicine applications. It not only forms a polymer but also contains cryptic matrikines that are released upon its activation/degradation and enhance the regenerative process. Despite this advantageous biology associated with fibrin, commercially available systems (e.g. TISSEEL) display limited regenerative capacity. This limitation is in part due to formulations that are optimized for tissue sealant applications and result in dense fibrous networks that limit cell infiltration. Recent evidence suggests that polymerization knob B engagement of polymerization hole b activates an alternative polymerization mechanism in fibrin, which may result in altered single fiber mechanical properties. We hypothesized that augmenting fibrin polymerization through the addition of PEGylated knob peptides with specificity to hole b (AHRPYAAC-PEG) would result in distinct fibrin polymer architectures with grossly different physical properties. Polymerization dynamics, polymer architecture, diffusivity, viscoelasticity, and degradation dynamics were analyzed. Results indicate that specific engagement of hole b with PEGylated knob B conjugates during polymerization significantly enhances the porosity of and subsequent diffusivity through fibrin polymers. Paradoxically, these polymers also display increased viscoelastic properties and decreased susceptibility to degradation. As a result, fibrin polymer strength was significantly augmented without any adverse effects on angiogenesis within the modified polymers.

Engineering fibrin matrices: the engagement of polymerization pockets through fibrin knob technology for the delivery and retention of therapeutic proteins

Engineering extracellular matrices that utilize the bodys natural healing capacity enable the progression of regenerative therapies. Fibrin, widely used as a surgical sealant, is one such matrix that may be augmented by the addition of protein factors to promote cell infiltration and differentiation. The thrombin-catalyzed conversion of fibrinogen to fibrin exposes N-terminal fibrin knobs that bind to C-terminal pockets to form the fibrin network. Here, we have created a platform system for the production of therapeutic proteins that capitalize on these native knob:pocket interactions for protein delivery within fibrin matrices. This system enables the retention of therapeutic proteins within fibrin without additional enzymatic or synthetic crosslinking factors. Using an integrin-binding fibronectin fragment as a model protein, we demonstrate that engineered knob-protein fusions bind consistently and specifically to fibrin(ogen). Equilibrium dissociation constants (K(D)) obtained using surface plasmon resonance indicate that these fusions have mum binding affinities, comparable to the native knob-containing fibrin fragments. The specificity of these interactions was verified by ELISA in the presence of molar excess of competing knob mimics. Release profiles and real-time confocal imaging demonstrate that the fusions were retained within fibrin matrices, even under the stringent continuous perfusion conditions used in the latter. In summary, this work explores the benefits and limitations of engaging native, biologically-inspired, non-covalent knob:pocket interactions within fibrin(ogen) for the retention of therapeutic proteins in fibrin matrices and provides insight into the stability of native knob:pocket interactions within fibrin networks.

Variations in rigidity and ligand density influence neuronal response in methylcellulose-laminin hydrogels.

Cells are continuously sensing their physical and chemical environment, generating dynamic interactions with the surrounding microenvironment and neighboring cells. Specific to neurons, neurite outgrowth is influenced by many factors, including the mechanical properties and adhesive signals of the growth substrata. In designing biomaterials for neural regeneration, it is important to understand the influence of substrate material, rigidity and bioadhesion on neurite outgrowth. To this end, we developed and characterized a tunable 3-D methylcellulose (MC) hydrogel polymeric system tethered to laminin-1 (MC-x-LN) across a range of substrate rigidities (G* range = 50-565 Pa) and laminin densities. Viability and neurite outgrowth of primary cortical neurons plated within 3-D MC hydrogels were used as cell outcome measures. After 4 days in culture, neuronal viability was significantly augmented with increasing rigidity for MC-x-LN as compared to control non-bioactive MC; however, neurite outgrowth was only observed in MC hydrogels with complex moduli of 565 Pa. Varying LN while maintaining a constant MC formulation (G* = 565 Pa) revealed a threshold response for neuronal viability, whereas a direct dose-dependent response to LN density was observed for neurite outgrowth. Collectively, these data demonstrate the synergistic play between material compliance and bioactive ligand concentrations within MC hydrogels. Such results can be used to better understand the adhesive and mechanical factors that mediate neuronal response to MC-based, tissue-engineered materials.

The role of SDF-1α-ECM crosstalk in determining neural stem cell fate

The consequences of central nervous system injury are far-reaching and debilitating and, while an endogenous repair response to neural injury has been observed in recent years, the mechanisms behind this response remain unclear. Neural progenitor/stem cell (NPSC) migration to the site of injury from the neural stem cell niches (e.g. subventricular zone and hippocampus) has been observed to be vasophilic in nature. While the chemotactic stimuli directing NPSC homing to injury is not well established, it is thought to be due in part to an increasing gradient of chemotactic cytokines, such as stromal cell-derived factor 1α (SDF-1α). Based on these recent findings, we hypothesize that critical crosstalk between SDF-1α and the extracellular matrix (ECM) drives injury-induced NPSC behavior. In this study, we investigated the effect of SDF-1α and ECM substrates (Matrigel, laminin, and vitronectin) on the migration, differentiation, and proliferation of NPSCs inxa0vitro using standard assays. The results demonstrated that SDF-1α and laminin-based ECM (Matrigel and laminin) significantly and synergistically enhanced NPSC migration and acute neuronal differentiation. These effects were significantly attenuated with the addition of AMD3100 (an antagonist against the SDF-1α receptor, CXCR4). SDF-1α alone significantly increased NPSC proliferation regardless of ECM substrate, however no synergy was observed between SDF-1α and the ECM. These results serve to elucidate the relationship between adhesive and soluble signaling factors of interest and their effect on NPSC behavior following neural injury. Furthermore, these results better inform the next generation of biomaterials aimed at stimulating endogenous neural regeneration for neural injury and neurodegenerative diseases.

Building better fibrin knob mimics: an investigation of synthetic fibrin knob peptide structures in solution and their dynamic binding with fibrinogen/fibrin holes

Fibrin polymerizes via noncovalent and dynamic association of thrombin-exposed knobs with complementary holes. Synthetic knob peptides have received significant interest as a means for understanding fibrin assembly mechanisms and inhibiting fibrin polymerization. Nevertheless, the inability to crystallize short peptides significantly limits our understanding of knob peptide structural features that regulate dynamic knob:hole interactions. In this study, we used molecular simulations to generate the first predicted structure(s) of synthetic knobs in solution before fibrin hole engagement. Combining surface plasmon resonance (SPR), we explored the role of structural and electrostatic properties of knob A mimics in regulating knob:hole binding kinetics. SPR results showed that association rates were most profoundly affected by the presence of both additional prolines as well as charged residues in the sixth to seventh positions. Importantly, analyzing the structural dynamics of the peptides through simulation indicated that the 3Arg side chain orientation and peptide backbone stability each contribute significantly to functional binding. These findings provide insights into early fibrin protofibril assembly dynamics as well as establishing essential design parameters for high-affinity knob mimics that more efficiently compete for hole occupancy, parameters realized here through a novel knob mimic displaying a 10-fold higher association rate than current mimics.

Endogenous Repair Signaling after Brain Injury and Complementary Bioengineering Approaches to Enhance Neural Regeneration

Traumatic brain injury (TBI) affects 5.3 million Americans annually. Despite the many long-term deficits associated with TBI, there currently are no clinically available therapies that directly address the underlying pathologies contributing to these deficits. Preclinical studies have investigated various therapeutic approaches for TBI: two such approaches are stem cell transplantation and delivery of bioactive factors to mitigate the biochemical insult affiliated with TBI. However, success with either of these approaches has been limited largely due to the complexity of the injury microenvironment. As such, this review outlines the many factors of the injury microenvironment that mediate endogenous neural regeneration after TBI and the corresponding bioengineering approaches that harness these inherent signaling mechanisms to further amplify regenerative efforts.

Integrin α3β1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat.

Background: The fibronectin (Fn) ninth type III repeat can modulate integrin binding and resulting cell spreading. Results: Mutations within the Fn integrin binding domains affect integrin α3β1 binding. Conclusion: Integrin α3β1-fibronectin binding depends on the presence and spacing of the RGD and synergy sites within Fn. Significance: α3β1-fibronectin binding may modulate epithelial cell wound healing responses. Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbβ3 and α5β1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3β1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu → Pro) stabilizing mutant (FnIII9′10), (ii) an Arg to Ala synergy site mutation (FnIII9R→A10), (iii) a two-Gly (FnIII92G10) insertion, and (iv) a four-Gly (FNIII94G10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3β1 to the Fn fragments. Integrin α3β1 had the highest affinity for FnIII9′10 and FnIII92G10. Mutation within the synergy site decreased integrin α3β1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9′10. Cell attachment studies demonstrate that α3β1-mediated epithelial cell binding is greater on FnIII9′10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3β1 binding to Fn.

Enhancing neural stem cell response to SDF-1α gradients through hyaluronic acid-laminin hydrogels.

Traumatic brain injury (TBI) initiates an expansive biochemical insult that is largely responsible for the long-term dysfunction associated with TBI; however, current clinical treatments fall short of addressing these underlying sequelae. Pre-clinical investigations have used stem cell transplantation with moderate success, but are plagued by staggeringly low survival and engraftment rates (2-4%). As such, providing cell transplants with the means to better dynamically respond to injury-related signals within the transplant microenvironment may afford improved transplantation survival and engraftment rates. The chemokine stromal cell-derived factor-1α (SDF-1α) is a potent chemotactic signal that is readily present after TBI. In this study, we sought to develop a transplantation vehicle to ultimately enhance the responsiveness of neural transplants to injury-induced SDF-1α. Specifically, we hypothesize that a hyaluronic acid (HA) and laminin (Lm) hydrogel would promote 1. upregulated expression of the SDF-1α receptor CXCR4 in neural progenitor/stem cells (NPSCs) and 2. enhanced NPSC migration in response to SDF-1α gradients. We demonstrated successful development of a HA-Lm hydrogel and utilized standard protein and cellular assays to probe NPSC CXCR4 expression and NPSC chemotactic migration. The findings demonstrated that NPSCs significantly increased CXCR4 expression after 48xa0h of culture on the HA-Lm gel in a manner critically dependent on both HA and laminin. Moreover, the HA-Lm hydrogel significantly increased NPSC chemotactic migration in response to SDF-1α at 48xa0h, an effect that was critically dependent on HA, laminin and the SDF-1α gradient. Therefore, this hydrogel serves to 1. prime NPSCs for the injury microenvironment and 2. provide the appropriate infrastructure to support migration into the surrounding tissue, equipping cells with the tools to more effectively respond to the injury microenvironment.