Identifying Specific Combinations of Matrix Properties that Promote Controlled and Sustained Release of a Hydrophobic Drug from Electrospun Meshes

Despite advances in the development of degradable polymers for drug delivery, effective translation of drug-loaded materials is often hindered due to a poor understanding of matrix property combinations that promote controlled and sustained release. In this study, we investigated the influence of dominant factors on the release of a hydrophobic glucocorticoid dexamethasone (DEX) from electrospun meshes. Polycaprolactone meshes released 98% of the drug within 24 h, while poly(l-lactide) meshes exhibited negligible release even after 28 days despite both polymers being slow-degrading. Differences in drug-polymer interactions and drug-polymer miscibility—but neither matrix degradation nor differences in bulk hydrophobicity—influenced DEX release from these semi-crystalline matrices. Poly(d,l-lactide-co-glycolide) 50:50 meshes possessing two different fiber diameters exhibited a sequential burst and sustained release, while poly(d,l-lactide-co-glycolide) 85:15 meshes cumulatively released 26% drug in a controlled manner. Although initial drug release from these matrices was driven by differences in matrix architecture and solid-state drug solubility, release toward the later stages was influenced by a combination of fiber swelling and matrix degradation as evidenced by gross and microstructural changes to the mesh network. We suggest that drug release from polymeric matrices can be better understood via investigation of critical matrix characteristics influencing release, as well as concomitant examination of drug-polymer interactions and miscibility. Our findings offer rational matrix design criteria to achieve controlled/extended drug release for promoting sustained biological responses

Indirect Tissue Scaffold Fabrication via Fused Deposition Modeling and Biomimetic Mineralization

To alleviate material limitations of the additive manufacture of tissue scaffolds, researchers have looked to indirect fabrication approaches. The feature resolution of these processes is limited however, due to the viscous ceramic slurries that are typically employed. To alleviate these limitations, the authors look to an indirect fabrication process wherein a pattern, created using Fused Deposition Modeling, is biomimetically mineralized with an aqueous simulated body fluid, which forms a bonelike hydroxyapatite throughout the scaffold pattern. Mineralized patters are then heat treated to pyrolyze the pattern and sinter the minerals. With this process, scaffolds were created with wall thicknesses as small as 150 m and internal channel diameters of 280-340 m, an appropriate range for bone tissue engineering.Mechanical Engineerin

Potent Particle-Based Vehicles for Growth Factor Delivery from Electrospun Meshes: Fabrication and Functionalization Strategies for Effective Tissue Regeneration

Functionalization of electrospun meshes with growth factors (GFs) is a common strategy for guiding specific cell responses in tissue engineering. GFs can exert their intended biological effects only when they retain their bioactivity and can be subsequently delivered in a temporally controlled manner. However, adverse processing conditions encountered in electrospinning can potentially disrupt GFs and diminish their biological efficacy. Further, meshes prepared using conventional approaches often promote an initial burst and rely solely on intrinsic fiber properties to provide extended release. Sequential delivery of multiple GFs-a strategy that mimics the natural tissue repair cascade-is also not easily achievable with traditional fabrication techniques. These limitations have hindered the effective use and translation of mesh-based strategies for tissue repair. An attractive alternative is the use of carrier vehicles (e.g., nanoparticles, microspheres) for GF incorporation into meshes. This review presents advances in the development of particle-integrated electrospun composites for safe and effective delivery of GFs. Compared to traditional approaches, we reveal how particles can protect GF activity, permit the incorporation of multiple GFs, decouple release from fiber properties, help achieve spatiotemporal control over delivery, enhance surface bioactivity, exert independent biological effects, and augment matrix mechanics. In presenting innovations in GF functionalization and composite engineering strategies, we also discuss specific in vitro and in vivo biological effects and their implications for diverse tissue engineering applications. © 2021 American Chemical Society

Robust strategies to reduce burst and achieve tunable control over extended drug release from uniaxially electrospun composites

In this study, robust strategies were developed to prepare uniaxially electrospun composites that reduced initial burst and provided controlled release of budesonide (a clinically important corticosteroid) over an extended timeframe in vitro. First, poly(caprolactone) (PCL) meshes were shown to exhibit significant burst while poly(D,L-lactide-co-glycolide) 85:15 (PLGA) meshes intrinsically promoted zero-order release over 28d. In-depth characterization of the architectural, physico-chemical and thermal properties revealed differences in gross morphological behavior, water uptake capacity, polymer Tg, and drug affinity to the matrix as factors governing release. Based on this mechanistic understanding of release from fast- and slow-releasing polymers, PCL and PLGA were next judiciously blended in a specific ratio i.e., 20PCL/80PLGA to achieve controlled first-order release over 28d at a faster rate than PLGA but without an initial burst. Interestingly, increasing the PCL content to 30% (i.e., a 30PCL/70PLGA blend) resulted in a sharp burst release. Since the release from the 30PCL/70PLGA blend depended on intrinsic system characteristics, a dual-spinneret co-electrospinning approach was employed to effectively decouple fiber properties and achieve precise drug distribution across independently integrated PCL and PLGA fibers. Consequently, the co-electrospun meshes — possessing identical polymer compositions and drug/polymer ratios as the 30PCL/70PLGA blend — exhibited no burst and resulted in predictable release characterized by 10d zero-order kinetics. Notably, neither the rationally guided blending nor the co-electrospinning strategy involved the use of cytotoxic cross-linkers, bioactivity-reducing excipients, delamination-prone barrier layers and complex set-ups. Based on the application, a rational choice of blend ratios or co-electrospinning parameters may be used to tune the rate of release from the composites. © 2022 Elsevier Lt

Electrospun meshes intrinsically promote M2 polarization of microglia under hypoxia and offer protection from hypoxia-driven cell death

In this study, we offer new insights into the contrasting effects of electrospun fiber orientation on microglial polarization under normoxia and hypoxia, and establish for the first time, the intrinsically protective roles of electrospun meshes against hypoxia-induced microglial responses. First, resting microglia were cultured under normoxia on poly(caprolactone) fibers possessing two distinctly different fiber orientations. Matrix-guided differences in cell shape/orientation and differentially expressed Rho GTPases (RhoA, Rac1, Cdc42) were well-correlated with the randomly oriented fibers inducing a pro-inflammatory phenotype and the aligned fibers sustaining a resting phenotype. Upon subsequent hypoxia induction, both sets of meshes offered protection from hypoxia-induced damage by promoting a radical phenotypic switch and beneficially altering the M2/M1 ratio to different extents. Compared to 2D hypoxic controls, meshes significantly suppressed the expression of pro-inflammatory markers (IL-6, TNF-α) and induced drastically higher expression of anti-inflammatory (IL-4, IL-10, VEGF-189) and neuroprotective (Nrf-2) markers. Consistent with this M2 polarization, the expression of Rho GTPases was significantly lower in the mesh groups under hypoxia compared to normoxic culture. Moreover, meshes - particularly with aligned fibers - promoted higher cell viability, suppressed caspase 3/8 and LC-3 expression and promoted LAMP-1 and LAMP-2 expression, which suggested the mitigation of apoptotic/autophagic cell death via a lysosomal membrane-stabilization mechanism. Notably, all protective effects under hypoxia were observed in the absence of additional soluble cues. Our results offer promise for leveraging the intrinsic therapeutic potential of electrospun meshes in degenerative diseases where microglial dysfunction, hypoxia and inflammation are implicated

Evaluating the protective role of carrier microparticles in preserving protein secondary structure within electrospun meshes

Direct incorporation of proteins into electrospun meshes using approaches such as blend electrospinning can promote adverse interactions with hydrophobic polymers, organic solvents and high voltage, potentially leading to loss of protein activity. However, pre-encapsulation within a protective carrier phase can preserve protein conformation by avoiding exposure to harsh processing conditions. In this study, bovine serum albumin (BSA) was loaded within cellulose microparticles (MPs) and the BSA-loaded MPs were dispersed in a solution of poly(ethylene oxide) (PEO). Particle-mesh composites were created using a sacrificial fiber/co-electrospinning approach in which the BSA/MP/PEO solution was simultaneously electrospun against a poly(caprolactone) (PCL) solution. Post-fabrication, sacrificial PEO fibers were selectively dissolved by treatment with ethanol. Microscopy, weight loss analysis and FTIR spectroscopy together confirmed selective dissolution of PEO fibers and the retention of BSA-loaded MPs within the PCL network without significant loss of either the MPs or the protein. Circular dichroism spectroscopy and intrinsic fluorescence measurements on BSA extracted from the co-electrospun meshes indicated minimal disruption to secondary structure, although partial sheet induction was observed. In contrast, direct exposure of BSA to four commonly used electrospinning solvents resulted in a large decrease in helical content and significant induction of sheets, revealing significant changes to the secondary structure. In summary, our results demonstrate the protective role of MPs in minimizing adverse effects of electrospinning on the secondary structure of incorporated protein. © 2020 Wiley Periodicals LLC

Electrospun fiber-based strategies for controlling early innate immune cell responses: Towards immunomodulatory mesh designs that facilitate robust tissue repair

Electrospun fibrous meshes are widely used for tissue repair due to their ability to guide a host of cell responses including phenotypic differentiation and tissue maturation. A critical factor determining the eventual biological outcomes of mesh-based regeneration strategies is the early innate immune response following implantation. The natural healing process involves a sequence of tightly regulated, temporally varying and delicately balanced pro-/anti-inflammatory events which together promote mesh integration with host tissue. Matrix designs that do not account for the immune milieu can result in dysregulation, chronic inflammation and fibrous capsule formation, thus obliterating potential therapeutic outcomes. In this review, we provide systematic insights into the effects of specific fiber/mesh properties and mechanical stimulation on the responses of early innate immune modulators viz., neutrophils, monocytes and macrophages. We identify matrix characteristics that promote anti-inflammatory immune phenotypes, and we correlate such responses with pro-regenerative in vivo outcomes. We also discuss recent advances in 3D fabrication technologies, bioactive functionalization approaches and biomimetic/bioinspired immunomodulatory mesh design strategies for tissue repair and wound healing. The mechanobiological insights and immunoregulatory strategies discussed herein can help improve the translational outcomes of fiber-based regeneration. Statement of significance: The crucial role played by immune cells in promoting biomaterial-based tissue regeneration is being increasingly recognized. In this review focusing on the interactions of innate immune cells with electrospun fibrous meshes, we systematically elucidate the effects of the fiber microenvironment and mechanical stimulation on biological responses, and build upon these insights to inform the rational design of immunomodulatory meshes for effective tissue repair. We discuss state-of-the-art fabrication methods and mechanobiological advances that permit the orchestration of temporally controlled phenotypic switches in immune cells during different phases of healing. The design strategies discussed herein can also be leveraged to target several complex autoimmune and inflammatory diseases. © 202

Towards Indirect Tissue Scaffold Fabrication via Additive Manufacturing and Hydroxyapatite Mineralization

Unlike traditional stochastic scaffold fabrication techniques, additive manufacturing (AM) can be used to create tissue-specific three-dimensional scaffolds with controlled porosity and pore geometry. Directly fabricating scaffolds through AM methods is limited because of the relatively few biocompatible materials available for processing in AM machines. To alleviate these material limitations, an indirect fabrication method is proposed. Specifically, the authors investigate the use of Fused Deposition Modeling to fabricate scaffold patterns of varied pore size and geometry. The scaffold patterns are then mineralized with a biocompatible ceramic (hydroxyapatite). A heat treatment is then used to pyrolyze the pattern and to sinter the thin ceramic coating. The result is a biocompatible ceramic scaffold composed of hollow tubes, which may promote attachment of endothelial cells and vascularization [1]. In this paper, the authors explore the scaffold pattern fabrication and mineralization processes. Two scaffold pattern materials are tested [acrylonitrile butadiene styrene (ABS) and investment cast wax (ICW)] to determine which material is the most appropriate for scaffold mineralization and sintering. While the ICW could not be thoroughly mineralized despite a sodium hydroxide surface treatment, the ABS patterns were successfully mineralized following an oxygen plasma surface treatment. A 0.004 mm mineral coating was found on the ABS patterns that featured a strut offset of 0.3 mm, which is in the range of appropriate pore size for bone tissue engineering [2].Mechanical Engineerin

Coupling between voltage and tip-to-collector distance in polymer electrospinning: Insights from analysis of regimes, transitions and cone/jet features

In this study, we shed light on the coupling between voltage (V) and tip-to-collector distance (T) in polymer electrospinning. First, an operating map in the V-T plane – with the potential to facilitate real-time control – reveals four electrospinning regimes including a newly identified rotational regime that serves as a transition from the cone-jet to the multi-jet regime. Next, across experiments in which V and T are independently varied, we image and comprehensively investigate regime-specific cone and jet dynamics using quantifiable and universally recognizable features. Our results demonstrate for the first time that theoretical potential drop (V/T) ─ although crucial in determining electrospinning outcomes ─ is not a fundamental V-T coupling parameter. Further, the nature of coupling is shown to dynamically vary with specific V and T combinations, with correlations indicating stronger dependence of cone/jet features on V than on T. Significantly, small changes to the collector position orchestrates regime transitions at the needle tip despite the large separation distance, although the effects of V dominate at large T values. Supporting simulations implicate the combined roles of effective field strength, charge density and field line distribution near the cone apex as critical factors influencing V-T coupling

Three‐dimensional imaging and quantification of real‐time cytosolic calcium oscillations in microglial cells cultured on electrospun matrices using laser scanning confocal microscopy

The development of a minimally invasive, robust, and inexpensive technique that permits real-time monitoring of cell responses on biomaterial scaffolds can improve the eventual outcomes of scaffold-based tissue engineering strategies. Towards establishing correlations between in situ biological activity and cell fate, we have developed a comprehensive workflow for real-time volumetric imaging of spatiotemporally varying cytosolic calcium oscillations in pure microglial cells cultured on electrospun meshes. Live HMC3 cells on randomly oriented electrospun fibers were stained with a fluorescent dye and imaged using a laser scanning confocal microscope. Resonance scanning provided high-resolution in obtaining the time-course of intracellular calcium levels without compromising spatial and temporal resolution. Three-dimensional reconstruction and depth-coding enabled the visualization of cell location and intracellular calcium levels as a function of sample thickness. Importantly, changes in cell morphology and in situ calcium spiking were quantified in response to a soluble biochemical cue and varying matrix architectures (i.e., randomly oriented and aligned fibers). Importantly, raster plots generated from spiking data revealed calcium signatures specific to culture conditions. In the future, our approach can be used to elucidate correlations between calcium signatures and cell phenotype/activation, and facilitate the rational design of scaffolds for biomedical applications