Harnessing cortical plasticity via gabapentinoid administration promotes recovery after stroke.

Stroke causes devastating sensory-motor deficits and long-term disability due to disruption of descending motor pathways. Restoration of these functions enables independent living and therefore represents a high priority for those afflicted by stroke. Here, we report that daily administration of gabapentin, a clinically approved drug already used to treat various neurological disorders, promotes structural and functional plasticity of the corticospinal pathway after photothrombotic cortical stroke in adult mice. We found that gabapentin administration had no effects on vascular occlusion, haemodynamic changes nor survival of corticospinal neurons within the ipsilateral sensory-motor cortex in the acute stages of stroke. Instead, using a combination of tract tracing, electrical stimulation and functional connectivity mapping, we demonstrated that corticospinal axons originating from the contralateral side of the brain in mice administered gabapentin extend numerous collaterals, form new synaptic contacts and better integrate within spinal circuits that control forelimb muscles. Not only does gabapentin daily administration promote neuroplasticity, but it also dampens maladaptive plasticity by reducing the excitability of spinal motor circuitry. In turn, mice administered gabapentin starting 1 h or 1 day after stroke recovered skilled upper extremity function. Functional recovery persists even after stopping the treatment at 6 weeks following a stroke. Finally, chemogenetic silencing of cortical projections originating from the contralateral side of the brain transiently abrogated recovery in mice administered gabapentin, further supporting the conclusion that gabapentin-dependent reorganization of spared cortical pathways drives functional recovery after stroke. These observations highlight the strong potential for repurposing gabapentinoids as a promising treatment strategy for stroke repair.

Nanofiber-based paramagnetic probes for rapid, real-time biomedical oximetry.

EPR (electron paramagnetic resonance) based biological oximetry is a powerful tool that accurately and repeatedly measures tissue oxygen levels. In vivo determination of oxygen in tissues is crucial for the diagnosis and treatment of a number of diseases. Here, we report the first successful fabrication and remarkable properties of nanofiber sensors for EPR-oximetry applications. Lithium octa-n-butoxynaphthalocyanine (LiNc- BuO), an excellent paramagnetic oxygen sensor, was successfully encapsulated in 300-500 nm diameter fibers consisting of a core of polydimethylsiloxane (PDMS) and a shell of polycaprolactone (PCL) by electrospinning. This core-shell nanosensor (LiNc-BuO-PDMS-PCL) shows a linear dependence of linewidth versus oxygen partial pressure (pO2). The nanofiber sensors have response and recovery times of 0.35 s and 0.55 s, respectively, these response and recovery times are ~12 times and ~218 times faster than those previously reported for PDMS-LiNc-BuO chip sensors. This greater responsiveness is likely due to the high porosity and excellent oxygen permeability of the nanofibers. Electrospinning of the structurally flexible PDMS enabled the fabrication of fibers having tailored spin densities. Core-shell encapsulation ensures the non-exposure of embedded LiNc-BuO and mitigates potential biocompatibility concerns. In vitro evaluation of the fiber performed under exposure to cultured cells showed that it is both stable and biocompatible. The unique combination of biocompatibility due to the PCL 'shell,' the excellent oxygen transparency of the PDMS core, and the excellent oxygen-sensing properties of LiNc-BuO makes LiNc-BuO-PDMS-PCL platform promising for long-term oximetry and repetitive oxygen measurements in both biological systems and clinical applications.

Fabrication of functional nanofibers through post-nanoparticle functionalization.

A facile method is developed to functionalize nanofiber surfaces with nanoparticles (NPs) through dithiocarbamate chemistry. Gold nanoparticles (AuNPs) and quantum dots (QDs) are immobilized on the nanofiber surface. These surfaces provide scaffolds for further supramolecular functionalization, as demonstrated through the Förster resonance energy transfer (FRET) pairing of QD-decorated fibers and fluorescent proteins.

THA Retrievals: The Need to Mark the Anatomic Orientation of the Femoral Head.

The hypothesis of this study was that the rotational orientation of femoral head damage would greatly affect the volumetric wear rate of the opposing polyethylene (PE) liner. Damage on twenty retrieved cobalt-chromium femoral heads was simulated in a validated damage-feature-based finite element model. For each individual retrieval, the anatomic orientation of the femoral head about the femoral neck axis was systematically varied, in 30° increments. The PE wear rate differential between the maximum- versus minimum-wear orientations was often sizable, as high as 7-fold. Knowing the correct femoral head anatomic orientation is therefore important when analyzing the effects of femoral head damage on PE liner wear. Surgeons retrieving modular femoral heads should routinely mark the anatomic orientation of those components.

Preferential, enhanced breast cancer cell migration on biomimetic electrospun nanofiber 'cell highways'.

BACKGROUND: Aggressive metastatic breast cancer cells seemingly evade surgical resection and current therapies, leading to colonization in distant organs and tissues and poor patient prognosis. Therefore, high-throughput in vitro tools allowing rapid, accurate, and novel anti-metastatic drug screening are grossly overdue. Conversely, aligned nanofiber constitutes a prominent component of the late-stage breast tumor margin extracellular matrix. This parallel suggests that the use of a synthetic ECM in the form of a nanoscale model could provide a convenient means of testing the migration potentials of cancer cells to achieve a long-term goal of providing clinicians an in vitro platform technology to test the efficacy of novel experimental anti-metastatic compounds. METHODS: Electrospinning produces highly aligned, cell-adhesive nanofiber matrices by applying a strong electric field to a polymer-containing solution. The resulting fibrous microstructure and morphology closely resembles in vivo tumor microenvironments suggesting their use in analysis of migratory potentials of metastatic cancer cells. Additionally, a novel interface with a gel-based delivery system creates CXCL12 chemotactic gradients to enhance CXCR4-expressing cell migration. RESULTS: Cellular dispersions of MCF-10A normal mammary epithelial cells or human breast cancer cells (MCF-7 and MDA-MB-231) seeded on randomly-oriented nanofiber exhibited no significant differences in total or net distance traveled as a result of the underlying topography. Cells traveled ~2-5 fold greater distances on aligned fiber. Highly-sensitive MDA-MB-231 cells displayed an 82% increase in net distance traversed in the presence of a CXCL12 gradient. In contrast, MCF-7 cells exhibited only 31% increase and MCF-10A cells showed no statistical difference versus control or vehicle conditions. MCF-10A cells displayed little sensitivity to CXCL12 gradients, while MCF-7 cells displayed early sensitivity when CXCL12 concentrations were higher. MDA-MB-231 cells displayed low relative expression levels of CXCR4, but high sensitivity resulting in 55-fold increase at late time points due to CXCL12 gradient dissipation. CONCLUSIONS: This model could create clinical impact as an in vitro diagnostic tool for rapid assessment of tumor needle biopsies to confirm metastatic tumors, their invasiveness, and allow high-throughput drug screening providing rapid development of personalized therapies.

Polydimethylsiloxane Core-Polycaprolactone Shell Nanofibers as Biocompatible, Real-Time Oxygen Sensors.

Real-time, continuous monitoring of local oxygen contents at the cellular level is desirable both for the study of cancer cell biology and in tissue engineering. In this paper, we report the successful fabrication of polydimethylsiloxane (PDMS) nanofibers containing oxygen-sensitive probes by electrospinning and the applications of these fibers as optical oxygen sensors for both gaseous and dissolved oxygen. A protective 'shell' layer of polycaprolactone (PCL) not only maintains the fiber morphology of PDMS during the slow curing process but also provides more biocompatible surfaces. Once this strategy was perfected, tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) (Ru(dpp)) and platinum octaethylporphyrin (PtOEP) were dissolved in the PDMS core and the resulting sensing performance established. These new core-shell sensors containing different sensitivity probes showed slight variations in oxygen response but all exhibited excellent Stern-Volmer linearity. Due in part to the porous nature of the fibers and the excellent oxygen permeability of PDMS, the new sensors show faster response (<0.5 s) -4-10 times faster than previous reports - than conventional 2D film-based oxygen sensors. Such core-shell fibers are readily integrated into standard cell culture plates or bioreactors. The photostability of these nanofiber-based sensors was also assessed. Culture of glioma cell lines (CNS1, U251) and glioma-derived primary cells (GBM34) revealed negligible differences in biological behavior suggesting that the presence of the porphyrin dyes within the core carries with it no strong cytotoxic effects. The unique combination of demonstrated biocompatibility due to the PCL 'shell' and the excellent oxygen transparency of the PDMS core makes this particular sensing platform promising for sensing in the context of biological environments.

Media-based effects on the hydrolytic degradation and crystallization of electrospun synthetic-biologic blends.

Tissue engineering scaffold degradation in aqueous environments is a widely recognized factor determining the fate of the associated anchorage-dependent cells. Electrospun blends of synthetic polycaprolactone (PCL) and a biological polymer, gelatin, of 25, 50, and 75 wt% were investigated for alterations in crystallinity, microstructure and morphology following widely used in vitro biological exposures. To our knowledge, the effects of these different aqueous-based biological media compositions on the degradation of these blends have never been directly compared. X-ray diffraction (XRD) analysis exposed that differences in PCL crystallinity were observed following exposures to phosphate buffered solution (PBS), Dulbecco's modified eagle medium (DMEM) cell culture media, and DI water following 7 days of exposure at 37 °C. XRD data suggested that in vitro medium exposures aid in providing chain mobility and rearrangement due to hydrolytic degradation of the gelatin phase, allowing previously constrained, poorly crystalline PCL regions to achieve more intense reflections resulting in the presence of crystalline peaks. The dry, as-spun modulus of relatively soft 100 % PCL fibers was approximately 10 % of any gelatin-containing composition. Tensile testing results indicate that hydrated gelatin containing scaffolds on average had a fivefold increase in elongation compared to as-spun scaffolds. After 24-h of aqueous exposure, the elastic modulus decreased in proportion to increasing gelatin content. After 1 day of exposure, the 75 and 100 % gelatin compositions largely ceased to display measurable values of modulus, elongation or tensile strength due to considerable hydrolytic degradation. On a relative basis, common aqueous in vitro medium exposures (deionized water, PBS, and DMEM) resulted in significantly divergent amounts of crystalline PCL, overall microstructure and fiber morphology in the blended compositions, subsequently 'shielding' scaffolds from significant changes in mechanical properties after 24-h of exposure. Understanding electrospun PCL-gelatin scaffold dynamics in different aqueous-based cell culture medias enables the ability to tailor scaffold composition to 'tune' degradation rate, microstructure, and long-term mechanical stability for optimal cellular growth, proliferation, and maturation.

Modeling polyethylene wear acceleration due to femoral head dislocation damage.

Scratching, scraping, and metal transfer to femoral heads commonly accompany acetabular shell contact during dislocation and closed reduction maneuvers. While head damage conceptually leads to accelerated wear, reports on this subject are mainly anecdotal, and differ widely on the potency of such effect. Towards better understanding this relationship, a physically validated finite element (FE) model was used to compute polyethylene wear acceleration propensity of specific head damage patterns on thirteen retrievals. These FE models estimated wear increases averaging half an order of magnitude when compared to simulations for undamaged heads. There was no correlation between the number of dislocations sustained and wear acceleration. These results underscore the importance of implant-gentle closed reduction, and heightened wear monitoring of successfully reduced dislocation patients.

Gene delivery to cultured embryonic stem cells using nanofiber-based sandwich electroporation.

Multiple gene transfections are often required to control the differentiation of embryonic stem cells. This is typically done by removing the cells from the culture substrate and conducting gene transfection via bulk electroporation (in suspension), which is then followed by further culture. Such repetitive processes could affect the growth and behavior of delicate/scarce adherent cells. We have developed a novel nanofiber-based sandwich electroporation device capable of in situ and in culture gene transfection. Electrospinning was used to deposit poly(ε-caprolactone)/gelatin nanofibers on the Al(2)O(3) nanoporous support membrane, on top of which a polystyrene microspacer was thermally bonded to control embryonic stem cell colony formation. The applicability of this system was demonstrated by culturing and transfecting mouse embryonic stem cells. Measurements of secreted alkaline phosphatase protein and metabolic activity showed higher transfection efficacy and cell viability compared to the conventional bulk electroporation approach.

Visualization of porosity and pore size gradients in electrospun scaffolds using laser metrology.

We applied a recently developed method, laser metrology, to characterize the influence of collector rotation on porosity gradients of electrospun polycaprolactone (PCL) widely investigated for use in tissue engineering. The prior- and post-sintering dimensions of PCL scaffolds were compared to derive quantitative, spatially-resolved porosity 'maps' from net shrinkage. Deposited on a rotating mandrel (200 RPM), the central region of deposition reaches the highest porosity, ~92%, surrounded by approximately symmetrical decreases to ~89% at the edges. At 1100 RPM, a uniform porosity of ~88-89% is observed. At 2000 RPM, the lowest porosity, ~87%, is found in the middle of the deposition, rebounding to ~89% at the edges. Using a statistical model of random fiber network, we demonstrated that these relatively small changes in porosity values produce disproportionately large variations in pore size. The model predicts an exponential dependence of pore size on porosity when the scaffold is highly porous (e.g., >80%) and, accordingly, the observed porosity variation is associated with dramatic changes in pore size and ability to accommodate cell infiltration. Within the thickest regions most likely to 'bottleneck' cell infiltration, pore size decreases from ~37 to 23 µm (38%) when rotational speeds increased from 200 to 2000 RPM. This trend is corroborated by electron microscopy. While faster rotational speeds ultimately overcome axial alignment induced by cylindrical electric fields associated with the collector geometry, it does so at the cost of eliminating larger pores favoring cell infiltration. This puts the bio-mechanical advantages associated with collector rotation-induced alignment at odds with biological goals. A more significant decrease in pore size from ~54 to ~19 µm (65%), well below the minimum associated with cellular infiltration, is observed from enhanced collector biases. Finally, similar predictions show that sacrificial fiber approaches are inefficient in achieving cell-permissive pore sizes.

Micro/nanoscale technologies for the development of hormone-expressing islet-like cell clusters.

Insulin-expressing islet-like cell clusters derived from precursor cells have significant potential in the treatment of type-I diabetes. Given that cluster size and uniformity are known to influence islet cell behavior, the ability to effectively control these parameters could find applications in the development of anti-diabetic therapies. In this work, we combined micro and nanofabrication techniques to build a biodegradable platform capable of supporting the formation of islet-like structures from pancreatic precursors. Soft lithography and electrospinning were used to create arrays of microwells (150-500 µm diameter) structurally interfaced with a porous sheet of micro/nanoscale polyblend fibers (~0.5-10 µm in cross-sectional size), upon which human pancreatic ductal epithelial cells anchored and assembled into insulin-expressing 3D clusters. The microwells effectively regulated the spatial distribution of the cells on the platform, as well as cluster size, shape and homogeneity. Average cluster cross-sectional area (~14000-17500 µm(2)) varied in proportion to the microwell dimensions, and mean circularity values remained above 0.7 for all microwell sizes. In comparison, clustering on control surfaces (fibers without microwells or tissue culture plastic) resulted in irregularly shaped/sized cell aggregates. Immunoreactivity for insulin, C-peptide and glucagon was detected on both the platform and control surfaces; however, intracellular levels of C-peptide/cell were ~60 % higher on the platform.

Micropatterning and characterization of electrospun poly(ε-caprolactone)/gelatin nanofiber tissue scaffolds by femtosecond laser ablation for tissue engineering applications.

Experimental investigations aimed at assessing the effectiveness of femtosecond (FS) laser ablation for creating microscale features on electrospun poly(ε-caprolactone) (PCL)/gelatin nanofiber tissue scaffold capable of controlling cell distribution are described. Statistical comparisons of the fiber diameter and surface porosity on laser-machined and as-spun surface were made and results showed that laser ablation did not change the fiber surface morphology. The minimum feature size that could be created on electrospun nanofiber surfaces by direct-write ablation was measured over a range of laser pulse energies. The minimum feature size that could be created was limited only by the pore size of the scaffold surface. The chemical states of PCL/gelatin nanofiber surfaces were measured before and after FS laser machining by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) and showed that laser machining produced no changes in the chemistry of the surface. In vitro, mouse embryonic stem cells (mES cells) were cultured on as-spun surfaces and in laser-machined microwells. Cell densities were found to be statistically indistinguishable after 1 and 2 days of growth. Additionally, confocal microscope imaging confirmed that spreading of mES cells cultured within laser-machined microwells was constrained by the cavity walls, the expected and desired function of these cavities. The geometric constraint caused statistically significant smaller density of cells in microwells after 3 days of growth. It was concluded that FS laser ablation is an effective process for microscale structuring of these electrospun nanofiber tissue scaffold surfaces.

Injectable, dispersible polysulfone-polysulfone core-shell particles for optical oxygen sensing.

Injectable sensors can significantly improve the volume of critical biomedical information emerging from the human body in response to injury or disease. Optical oxygen sensors with rapid response times can be achieved by incorporating oxygen-sensitive luminescent molecules within polymeric matrices with suitably high surface area to volume ratios. In this work, electrospraying utilizes these advances to produce conveniently injectable, oxygen sensing particles made up of a core-shell polysulfone-polysulfone structure containing a phosphorescent oxygen-sensitive palladium porphyrin species within the core. Particle morphology is highly dependent on solvent identity and electrospraying parameters; DMF offers the best potential for the creation of uniform, sub-micron particles. Total internal reflection fluorescence (TIRF) microscopy confirms the existence of both core-shell structure and oxygen sensitivity. The dissolved oxygen response time is rapid (<0.30 s), ideal for continuous real-time monitoring of oxygen concentration. The incorporation of Pluronic F-127 surfactant enables efficient dispersion; selection of an appropriate electrospraying solvent (DMF) yields particles readily injected even through a <100 µm diameter needle.

High throughput assembly of spatially controlled 3D cell clusters on a micro/nanoplatform.

Guided assembly of microscale tissue subunits (i.e. 3D cell clusters/aggregates) has found applications in cell therapy/tissue engineering, cell and developmental biology, and drug discovery. As cluster size and geometry are known to influence cellular responses, the ability to spatially control cluster formation in a high throughput manner could be advantageous for many biomedical applications. In this work, a micro- and nanofabricated platform was developed for this purpose, consisting of a soft-lithographically fabricated array of through-thickness microwells structurally bonded to a sheet of electrospun fibers. The microwells and fibers were manufactured from several polymers of biomedical interest. Human hepatocytes were used as model cells to demonstrate the ability of the platform to allow controlled cluster formation. In addition, the ability of the device to support studies on semi-controlled heterotypic interactions was demonstrated by co-culturing hepatocytes and fibroblasts. Preliminary experiments with other cells of interest (pancreatic cells, embryonic stem cells, and cardiomyocytes) were also conducted. Our platform possesses several advantages over previously developed microwell arrays: a more in vivo-like topographical stimulation of cells; better nutrient/waste exchange through the underlying nanofiber mat; and easy integration into standard two-chamber cell culture well systems.

Injectable biodegradable bi-layered capsule for sustained delivery of bevacizumab in treating wet age-related macular degeneration.

Vascular endothelial growth factor (VEGF) is a key regulator of abnormal blood vessel growth. As such, bevacizumab-based inhibition of VEGF has been the clinically adopted strategy to treat colorectal and breast cancers as well as age-related macular degeneration (AMD). However, as the treatment of vascular diseases often requires a high drug concentration for a long period, the burst release of bevacizumab remains a critical limitation in anti-VEGF-based therapies. Maintaining bevacizumab at high concentrations over extended periods remains challenging due to insufficient drug loading capacity and drug-device interactions. We report the development of a polymeric based bi-layered capsule that could address these challenges by extending the release over one year, thereby providing an effective platform enabling treatment of chronic vascular diseases. Remarkably, the developed capsules have a bi-layered structure which ensures the structural integrity of the injectable capsules and appropriate diffusion of bevacizumab by providing optimal physical trapping and electrostatic interaction. Meanwhile, the central hollow design enables a higher drug loading to meet the need for long-term release of bevacizumab for several months to one year. Using an in vitro drug release assay, we demonstrated that the bi-layered capsule could produce longer-term local drug administration by intravitreal injection compared to previously reported devices. The capsules also present minimal toxicity and maintain anti-VEGF potency, suggesting that our approach may have the potential to treat vascular-related diseases using bevacizumab.

Effects of orthopedic implants with a polycaprolactone polymer coating containing bone morphogenetic protein-2 on osseointegration in bones of sheep.

OBJECTIVE: To determine elution characteristics of bone morphogenetic protein (BMP)-2 from a polycaprolactone coating applied to orthopedic implants and determine effects of this coating on osseointegration. ANIMALS: 6 sheep. PROCEDURES: An in vitro study was conducted to determine BMP-2 elution from polycaprolactone-coated implants. An in vivo study was conducted to determine the effects on osseointegration when the polycaprolactone with BMP-2 coating was applied to bone screws. Osseointegration was assessed via radiography, measurement of peak removal torque and bone mineral density, and histomorphometric analysis. Physiologic response was assessed by measuring serum bone-specific alkaline phosphatase activity and uptake of bone markers. RESULTS: Mean +/- SD elution on day 1 of the in vitro study was 263 +/- 152 pg/d, which then maintained a plateau at 59.8 +/- 29.1 pg/d. Mean peak removal torque for screws coated with polycalprolactone and BMP-2 (0.91 +/- 0.65 dN x m) and screws coated with polycaprolactone alone (0.97 +/- 1.30 dN.m) did not differ significantly from that for the control screws (2.34 +/- 1.62 dN x m). Mean bone mineral densities were 0.535 +/- 0.060 g/cm(2), 0.596 +/- 0.093 g/cm(2), and 0.524 +/- 0.142 g/cm(2) for the polycaprolactone-BMP-2-coated, polycaprolactone-coated, and control screws, respectively, and did not differ significantly among groups. Histologically, bone was in closer apposition to the implant with the control screws than with either of the coated screws. CONCLUSIONS AND CLINICAL RELEVANCE: BMP-2 within the polycaprolactone coating did not stimulate osteogenesis. The polycaprolactone coating appeared to cause a barrier effect that prevented formation of new bone. A longer period or use of another carrier polymer may result in increased osseointegration.

Sintered electrospun polycaprolactone for controlled model drug delivery.

Electrospinning has been used widely for drug delivery applications due to its versatility and ease of modification of spun fiber properties. Net drug loading and release is typically limited by the inherent surface-area of the sample. In a relatively novel approach, sintering of electrospun fiber was used to create a capsule favoring long-term delivery. We showed that electrospun polycaprolactone (PCL) retained its initial morphology out to 1042 days of in vitro exposure, illustrating its potential for extended performance. Sintering decreased the electrospun pore size by 10- and 28-fold following 56 and 57 °C exposures, respectively. At 58 and 59 °C, the PCL capsules lost all apparent surface porosity, but entrapped pores were observed in the 58 °C cross-section. The use of Rhodamine B (RhB, 479.02 g mol-1), Rose Bengal (RB, 1017.64 g mol-1) and albumin-fluorescein isothiocyanate conjugate from bovine serum (BSA-FITC, ~66,000 g mol-1) as model compounds demonstrated that release (RhB > RB ≫ BSA-FITC) is controlled both by molecular weight and available porosity. Interestingly, the ranking of release following sintering was 57 > 56 > 59 > 58 °C; COMSOL simulations explored the effects of capsule wall thickness and porosity on release rate. It was hypothesized that model drug adsorption on the available fiber surface-area (57 versus 56 °C) and entrapped porosity (59 versus 58 °C) could have also attributed to the observed ranking of release rates. While the 56 and 57 °C exposures allowed the bulk of the release to occur in <1 day, the capsules sintered at 58 and 59 °C exhibited release that continued after 12 days of exposure.

Comparison of polyglycolic acid, polycaprolactone, and collagen as scaffolds for the production of tissue engineered intestine.

Cell-seeded scaffolds play critical roles in the production of tissue engineered intestine (TEI), a potential strategy for the treatment of short bowel syndrome. The current study compares polyglycolic acid (PGA), polycaprolactone (PCL), and collagen as scaffolds for TEI production. Tubular PGA scaffolds were prepared from nonwoven BIOFELT® . Tubular PCL scaffolds were fabricated by electrospinning. Tubular collagen scaffolds were prepared using CollaTape, a wound dressing material. Both PGA and collagen were coated with poly-l-lactic acid (PLLA) to improve scaffold mechanical properties. Pore size, porosity, microstructure, mechanical properties (suture retention strength and ultimate compressive force) were determined. The scaffolds were first seeded with crypt stem cells isolated from 1 to 3 day old rat pups and then implanted into the peritoneal cavity of nude rats. After 4 weeks of in vivo incubation, these cell-seeded scaffolds were harvested for assessment of the TEI produced. Of the three materials compared, PLLA coated tubular PGA scaffolds had the appropriate pore size, mechanical properties and degradation rate leading to the production of TEI with an architecture similar to that of native rat intestine. © 2018 Wiley Periodicals, Inc. J. Biomed. Mater. Res. Part B, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 750-760, 2019.

Effect of Electrospun Fiber Mat Thickness and Support Method on Cell Morphology.

Electrospun fiber mats (EFMs) are highly versatile biomaterials used in a myriad of biomedical applications. Whereas some facets of EFMs are well studied and can be highly tuned (e.g., pore size, fiber diameter, etc.), other features are under characterized. For example, although substrate mechanics have been explored by several groups, most studies rely on Young's modulus alone as a characterization variable. The influence of fiber mat thickness and the effect of supports are variables that are often not considered when evaluating cell-mechanical response. To assay the role of these features in EFM scaffold design and to improve understanding of scaffold mechanical properties, we designed EFM scaffolds with varying thickness (50-200 µm) and supporting methodologies. EFM scaffolds were comprised of polycaprolactone and were either electrospun directly onto a support, suspended across an annulus (3 or 10 mm inner diameter), or "tension-released" and then suspended across an annulus. Then, single cell spreading (i.e., Feret diameter) was measured in the presence of these different features. Cells were sensitive to EFM thickness and suspended gap diameter. Overall, cell spreading was greatest for 50 µm thick EFMs suspended over a 3 mm gap, which was the smallest thickness and gap investigated. These results are counterintuitive to conventional understanding in mechanobiology, which suggests that stiffer materials, such as thicker, supported EFMs, should elicit greater cell polarization. Additional experiments with 50 µm thick EFMs on polystyrene and polydimethylsiloxane (PDMS) supports demonstrated that cells can "feel" the support underlying the EFM if it is rigid, similar to previous results in hydrogels. These results also suggest that EFM curvature may play a role in cell response, separate from Young's modulus, possibly because of internal tension generated. These parameters are not often considered in EFM design and could improve scaffold performance and ultimately patient outcomes.

Modulation of biomimetic mineralization of collagen by soluble ectodomain of discoidin domain receptor 2.

Collagen fibrils serve as the major template for mineral deposits in both biologically derived and engineered tissues. In recent years certain non-collagenous proteins have been elucidated as important players in differentially modulating intra vs. extra-fibrillar mineralization of collagen. We and others have previously shown that the expression of the collagen receptor, discoidin domain receptor 2 (DDR2) positively correlates with matrix mineralization. The objective of this study was to examine if the ectodomain (ECD) of DDR2 modulates intra versus extra-fibrillar mineralization of collagen independent of cell-signaling. For this purpose, a decellularized collagenous substrate, namely glutaraldehyde fixed porcine pericardium (GFPP) was subjected to biomimetic mineralization protocols. GFPP was incubated in modified simulated body fluid (mSBF) or polymer-induced liquid precursor (PILP) solutions in the presence of recombinant DDR2 ECD (DDR2-Fc) to mediate extra or intra-fibrillar mineralization of collagen. Thermogravimetric analysis revealed that DDR2-Fc increased mineral content in GFPP calcified in mSBF while no significant differences were observed in PILP mediated mineralization. Electron microscopy approaches were used to evaluate the quality and quantity mineral deposits. An increase in the matrix to mineral ratio, frequency of particles and size of mineral deposits was observed in the presence of DDR2-Fc in mSBF. Von Kossa staining and immunohistochemistry analysis of adjacent sections indicated that DDR2-Fc bound to both the matrix and mineral phase of GFPP. Further, DDR2-Fc was found to bind to hydroxyapatite (HAP) particles and enhance the nucleation of mineral deposits in mSBF solutions independent of collagen. Taken together, our results elucidate DDR2 ECD as a novel player in the modulation of extra-fibrillar mineralization of collagen.