A Rare Case of Argatroban-Induced Anaphylaxis in a Patient With Intermediate-High Risk Pulmonary Embolism.

Heparin-induced thrombocytopenia is a rare and potentially devastating complication of heparin therapy. Patients with an absolute indication for anticoagulation, such as those with significant pulmonary embolism, must be switched to a different anticoagulant, such as argatroban, a direct thrombin inhibitor. We report a case of anaphylaxis to argatroban in a patient who was initially on heparin for intermediate-high risk pulmonary embolism but developed suspected type II heparin-induced thrombocytopenia. This case highlights the significance of recognizing and treating anaphylactic reactions and the diagnostic challenges associated with heparin-induced thrombocytopenia.

Keratose Hydrogel Drives Differentiation of Cardiac Vascular Smooth Muscle Progenitor Cells: Implications in Ischemic Treatment.

Peripheral artery disease (PAD) is a common vascular disorder in the extremity of limbs with limited clinical treatments. Stem cells hold great promise for the treatment of PAD, but their therapeutic efficiency is limited due to multiple factors, such as poor engraftment and non-optimal selection of cell type. To date, stem cells from a variety of tissue sources have been tested, but little information is available regarding vascular smooth muscle cells (VSMCs) for PAD therapy. The present study examines the effects of keratose (KOS) hydrogels on c-kit+/CD31- cardiac vascular smooth muscle progenitor cell (cVSMPC) differentiation and the therapeutic potential of the resultant VSMCs in a mouse hindlimb ischemic model of PAD. The results demonstrated that KOS but not collagen hydrogel was able to drive the majority of cVSMPCs into functional VSMCs in a defined Knockout serum replacement (SR) medium in the absence of differentiation inducers. This effect could be inhibited by TGF-ß1 antagonists. Further, KOS hydrogel increased expression of TGF-ß1-associated proteins and modulated the level of free TGF-ß1 during differentiation. Finally, transplantation of KOS-driven VSMCs significantly increased blood flow and vascular densities of ischemic hindlimbs. These findings indicate that TGF-ß1 signaling is involved in KOS hydrogel-preferred VSMC differentiation and that enhanced blood flow are likely resulted from angiogenesis and/or arteriogenesis induced by transplanted VSMCs.

Three-Dimensional Porous Trabecular Scaffold Exhibits Osteoconductive Behaviors In Vitro.

In the USA, approximately 500,000 bone grafting procedures are performed annually to treat injured or diseased bone. Autografts and allografts are the most common treatment options but can lead to adverse outcomes such as donor site morbidity and mechanical failure within 10 years. Due to this, tissue engineered replacements have emerged as a promising alternative to the biological options. In this study, we characterize an electrospun porous composite scaffold as a potential bone substitute. Various mineralization techniques including electrodeposition were explored to determine the optimal method to integrate mineral content throughout the scaffold. In vitro studies were performed to determine the biocompatibility and osteogenic potential of the nanofibrous scaffolds. The presence of hydroxyapatite (HAp) and brushite throughout the scaffold was confirmed using energy dispersive X-ray fluorescence, scanning electron microscopy, and ash weight analysis. The active flow of ions via electrodeposition mineralization led to a threefold increase in mineral content throughout the scaffold in comparison to static and flow mineralization. Additionally, a ten-layer scaffold was successfully mineralized and confirmed with an alizarin red assay. In vitro studies confirmed the mineralized scaffold was biocompatible with human bone marrow derived stromal cells. Additionally, bone marrow derived stromal cells seeded on the mineralized scaffold with embedded HAp expressed 30% more osteocalcin, a primary bone protein, than these cells seeded on non-mineralized scaffolds and only 9% less osteocalcin than mature pre-osteoblasts on tissue culture polystyrene. This work aims to confirm the potential of a biomimetic mineralized scaffold for full-thickness trabecular bone replacement.

Interaction of material stiffness and negative pressure to enhance differentiation of bone marrow-derived stem cells and osteoblast proliferation.

Negative pressure wound therapy (NPWT) results in improved wound repair and the combined use of NPWT with elastomeric materials may further stimulate and accelerate tissue repair. No firmly established treatment modalities using both NPWT and biomaterials exist for orthopedic application. The goal of this study was to investigate the response of osteoblasts and bone marrow-derived mesenchymal stem cells to negative pressure and to determine whether a newly developed elastic osteomimetic bone repair material (BRM), a blend of type I collagen, chondroitin 6-sulfate, and poly (octanediol citrate) could enhance the osteoblastic phenotype. The results indicate that proliferation and alkaline phosphatase activity of hFOB1.19 osteoblasts were significantly increased with exposure to 12 hr of negative pressure (-125 mmHg). Follow-on studies with rat and human mesenchymal stem cells confirmed that negative pressure enhanced osteoblastic maturation. In addition, a significant interaction of negative pressure and electrospun BRM resulted in increased mRNA expression of alkaline phosphatase, osteopontin, collagen1α2, and HIF1α, whereas little or no effect on these genes was observed on electrospun collagen or tissue culture plastic. Together, these results suggest that the use of this novel biomaterial, BRM, with NPWT may ultimately translate into a safe and cost-effective clinical application to accelerate bone repair.

Improving the Osteogenicity of PCL Fiber Substrates by Surface-Immobilization of Bone Morphogenic Protein-2.

Polycaprolactone (PCL) fiber scaffolds are attractive, albeit inert, substrates for ligament regeneration, that may be improved by incorporating trophic factors to guide tissue remodeling in vivo. In particular, immobilization of bone morphogenic protein-2 (BMP-2) to the scaffold surface may facilitate rapid and robust integration of the scaffold with adjacent bone tissues. As a first step toward testing this, model PCL surfaces were modified by the addition of heparin (Hep) and BMP-2 to facilitate osteoblastic differentiation. Specifically, Hep was combined with PCL at 0, 0.5, and 1 wt% (denoted as PCL, PCL-0.5Hep, and PCL-1Hep), cast into films, and then BMP-2 was immobilized to surfaces by either adsorption and covalent conjugation. Here, BMP-2 concentration increased systematically with incorporation of Hep, and higher concentrations were achieved by covalent conjugation. Next, blends were electrospun to form thin meshes with fiber diameters of 0.92, 0.62, and 0.54 µm for PCL, PCL-0.5Hep, and PCL-1Hep, respectively. Mesenchymal stem cells (MSCs) had no difficulty attaching to and proliferating on all meshes. Lastly, PCL-1Hep meshes were prepared with adsorbed or covalently conjugated BMP-2 and cultured with MSCs in the absence of osteogenic factors. Under these conditions, alkaline phosphatase activity and deposition of bone sialoprotein, osteopontin, and calcium minerals-markers of osteoblastic differentiation-were significantly higher on surfaces with immobilized BMP-2. Together, these data indicate that covalent immobilization of trophic factors confers bioactivity to scaffolds, which may be applied in a spatially controlled manner for ligament regeneration and bone integration.

Polymer microfiber meshes facilitate cardiac differentiation of c-kit(+) human cardiac stem cells.

Electrospun microfiber meshes have been shown to support the proliferation and differentiation of many types of stem cells, but the phenotypic fate of c-kit(+) human cardiac stem cells (hCSCs) have not been explored. To this end, we utilized thin (~5µm) elastomeric meshes consisting of aligned 1.7µm diameter poly (ester-urethane urea) microfibers as substrates to examine their effect on hCSC viability, morphology, proliferation, and differentiation relative to cells cultured on tissue culture polystyrene (TCPS). The results showed that cells on microfiber meshes displayed an elongated morphology aligned in the direction of fiber orientation, lower proliferation rates, but increased expressions of genes and proteins majorly associated with cardiomyocyte phenotype. The early (NK2 homeobox 5, Nkx2.5) and late (cardiac troponin I, cTnI) cardiomyocyte genes were significantly increased on meshes (Nkx=2.5 56.2±13.0, cTnl=2.9±0.56,) over TCPS (Nkx2.5=4.2±0.9, cTnl=1.6±0.5, n=9, p<0.05 for both groups) after differentiation. In contrast, expressions of smooth muscle markers, Gata6 and myosin heavy chain (SM-MHC), were decreased on meshes. Immunocytochemical analysis with cardiac antibody exhibited the similar pattern of above cardiac differentiation. We conclude that aligned microfiber meshes are suitable for guiding cardiac differentiation of hCSCs and may facilitate stem cell-based therapies for treatment of cardiac diseases.

Stress and binge drinking: A toxic combination for the teenage brain.

Young adult university students frequently binge on alcohol and have high stress levels. Based on findings in rodents, we predicted that heavy current alcohol use and elevated stress and depression scores would be associated with deficits on high interference memory tasks, while early onset, prolonged binge patterns would lead to broader cognitive deficits on tests of associative encoding and executive functions. We developed the Concentration Memory Task, a novel computerized version of the Concentration card game with a high degree of interference. We found that young adults with elevated stress, depression, and alcohol consumption scores were impaired in the Concentration Memory Task. We also analyzed data from a previous study, and found that higher alcohol consumption scores were associated with impaired performance on another high interference memory task, based on Kirwan and Stark's Mnemonic Similarity Test. On the other hand, adolescent onset of binge drinking predicted poorer performance on broader range of memory tests, including a more systematic test of spatial recognition memory, and an associative learning task. Our results are broadly consistent with findings in rodents that acute alcohol and stress exposure suppress neurogenesis in the adult hippocampus, which in turn impairs performance in high interference memory tasks, while adolescent onset binge drinking causes more extensive brain damage and cognitive deficits.

Fiber/collagen composites for ligament tissue engineering: influence of elastic moduli of sparse aligned fibers on mesenchymal stem cells.

Electrospun microfibers are attractive for the engineering of oriented tissues because they present instructive topographic and mechanical cues to cells. However, high-density microfiber networks are too cell-impermeable for most tissue applications. Alternatively, the distribution of sparse microfibers within a three-dimensional hydrogel could present instructive cues to guide cell organization while not inhibiting cell behavior. In this study, thin (∼5 fibers thick) layers of aligned microfibers (0.7 µm) were embedded within collagen hydrogels containing mesenchymal stem cells (MSCs), cultured for up to 14 days, and assayed for expression of ligament markers and imaged for cell organization. These microfibers were generated through the electrospinning of polycaprolactone (PCL), poly(ester-urethane) (PEUR), or a 75/25 PEUR/PCL blend to produce microfiber networks with elastic moduli of 31, 15, and 5.6 MPa, respectively. MSCs in composites containing 5.6 MPa fibers exhibited increased expression of the ligament marker scleraxis and the contractile phenotype marker α-smooth muscle actin versus the stiffer fiber composites. Additionally, cells within the 5.6 MPa microfiber composites were more oriented compared to cells within the 15 and 31 MPa microfiber composites. Together, these data indicate that the mechanical properties of microfiber/collagen composites can be tuned for the engineering of ligament and other target tissues. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1894-1901, 2016.

Cell layer-electrospun mesh composites for coronary artery bypass grafts.

This work investigates the potential of cell layer-electrospun mesh constructs as coronary artery bypass grafts. These cell-mesh constructs were generated by first culturing a confluent layer of 10T½ smooth muscle progenitor cells on a high strength electrospun mesh with uniaxially aligned fibers. Cell-laden mesh sheets were then wrapped around a cylindrical mandrel such that the mesh fibers were aligned circumferentially. The resulting multi-layered constructs were then cultured for 4 wks in media supplemented with TGF-ß1 and ascorbic acid to support 10T½ differentiation toward a smooth muscle cell-like fate as well as to support elastin and collagen production. The underlying hypothesis of this work was that extracellular matrix (ECM) deposited by the cell layers would act as an adhesive agent between the individual mesh layers, providing strength to the construct as well as a source for structural elasticity at low strains. In addition, the structural anisotropy of the mesh would inherently guide desired circumferential cell and ECM alignment. Results demonstrate that the cell-mesh constructs exhibited a J-shaped circumferential stress-strain response similar to that of native coronary artery, while also displaying acceptable tensile strength. Furthermore, associated 10T½ cells and deposited collagen fibers showed a high degree of circumferential alignment. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2200-2209, 2016.

A role for adult hippocampal neurogenesis at multiple time scales: A study of recent and remote memory in humans.

Adult hippocampal neurogenesis (AHN) is downregulated by numerous lifestyle factors including chronic stress. While the functional significance of AHN remains elusive, computational models and empirical evidence implicate immature neurons in minimizing interference between similar memories-a process termed pattern separation. The role of neurogenesis in remote memory is less clear. Some have proposed that neurogenesis promotes the clearance of old memories from the hippocampus, while others have proposed that neurogenesis promotes long-term retention of memories within the hippocampus. We used a modified version of the behavioral pattern separation task originally described by Kirwan and Stark (2007). In this task, some objects are repeated across trials, some are similar lures and the rest are novel. Participants are asked to classify each object as old, new, or similar. The correct classification of lures as similar may tax pattern separation processes in the hippocampus and AHN. To investigate the potential role of AHN in remote memory, we introduced a 2-week delay between the presentation and recognition of certain stimuli. As in previous studies, we found that those with higher depression scores made significantly more errors at identifying lures as similar when presentation and recognition were separated by a brief delay. When presentation and recognition trials were separated by a longer delay, the correct classification of lures dropped to chance levels for all groups, but now lower stress and depression scores were associated with superior identification of exact repetitions. Our data suggest a role for AHN in the stabilization of remote memories.

Static and cyclic mechanical loading of mesenchymal stem cells on elastomeric, electrospun polyurethane meshes.

Biomaterial substrates composed of semi-aligned electrospun fibers are attractive supports for the regeneration of connective tissues because the fibers are durable under cyclic tensile loads and can guide cell adhesion, orientation, and gene expression. Previous studies on supported electrospun substrates have shown that both fiber diameter and mechanical deformation can independently influence cell morphology and gene expression. However, no studies have examined the effect of mechanical deformation and fiber diameter on unsupported meshes. Semi-aligned large (1.75 µm) and small (0.60 µm) diameter fiber meshes were prepared from degradable elastomeric poly(esterurethane urea) (PEUUR) meshes and characterized by tensile testing and scanning electron microscopy (SEM). Next, unsupported meshes were aligned between custom grips (with the stretch axis oriented parallel to axis of fiber alignment), seeded with C3H10T1/2 cells, and subjected to a static load (50 mN, adjusted daily), a cyclic load (4% strain at 0.25 Hz for 30 min, followed by a static tensile loading of 50 mN, daily), or no load. After 3 days of mechanical stimulation, confocal imaging was used to characterize cell shape, while measurements of deoxyribonucleic acid (DNA) content and messenger ribonucleic acid (mRNA) expression were used to characterize cell retention on unsupported meshes and expression of the connective tissue phenotype. Mechanical testing confirmed that these materials deform elastically to at least 10%. Cells adhered to unsupported meshes under all conditions and aligned with the direction of fiber orientation. Application of static and cyclic loads increased cell alignment. Cell density and mRNA expression of connective tissue proteins were not statistically different between experimental groups. However, on large diameter fiber meshes, static loading slightly elevated tenomodulin expression relative to the no load group, and tenascin-C and tenomodulin expression relative to the cyclic load group. These results demonstrate the feasibility of maintaining cell adhesion and alignment on semi-aligned fibrous elastomeric substrates under different mechanical conditions. The study confirms that cell morphology is sensitive to the mechanical environment and suggests that expression of select connective tissue genes may be enhanced on large diameter fiber meshes under static tensile loads.

Electrospun meshes possessing region-wise differences in fiber orientation, diameter, chemistry and mechanical properties for engineering bone-ligament-bone tissues.

Although bone-patellar tendon-bone (B-PT-B) autografts are the gold standard for repair of anterior cruciate ligament ruptures, they suffer from drawbacks such as donor site morbidity and limited supply. Engineered tissues modeled after B-PT-B autografts are promising alternatives because they have the potential to regenerate connective tissue and facilitate osseointegration. Towards the long-term goal of regenerating ligaments and their bony insertions, the objective of this study was to construct 2D meshes and 3D cylindrical composite scaffolds - possessing simultaneous region-wise differences in fiber orientation, diameter, chemistry and mechanical properties - by electrospinning two different polymers from off-set spinnerets. Using a dual drum collector, 2D meshes consisting of an aligned polycaprolactone (PCL) fiber region, randomly oriented poly(lactide-co-glycolide) (PLGA) fiber region and a transition region (comprised of both PCL and PLGA fibers) were prepared, and region-wise differences were confirmed by microscopy and tensile testing. Bone marrow stromal cells (BMSCs) cultured on these meshes exhibited random orientations and low aspect ratios on the random PLGA regions, and high aspect ratios and alignment on the aligned PCL regions. Next, meshes containing an aligned PCL region flanked by two transition regions and two randomly oriented PLGA regions were prepared and processed into 3D cylindrical composite scaffolds using an interpenetrating photo-crosslinkable polyethylene glycol diacrylate hydrogel to recapitulate the shape of B-PT-B autografts. Tensile testing indicated that cylindrical composites were mechanically robust, and eventually failed due to stress concentration in the aligned PCL region. In summary, this study demonstrates a process to fabricate electrospun meshes possessing region-wise differences in properties that can elicit region-dependent cell responses, and be readily processed into scaffolds with the shape of B-PT-B autografts.

Electrospun fibre diameter, not alignment, affects mesenchymal stem cell differentiation into the tendon/ligament lineage.

Efforts to develop engineered tendons and ligaments have focused on the use of a biomaterial scaffold and a stem cell source. However, the ideal scaffold microenvironment to promote stem cell differentiation and development of organized extracellular matrix is unknown. Through electrospinning, fibre scaffolds can be designed with tailorable architectures to mimic the intended tissue. In this study, the effects of fibre diameter and orientation were examined by electrospinning thin mats, consisting of small (< 1 µm), medium (1-2 µm) or large (> 2 µm) diameter fibres with either random or aligned fibre orientation. C3H10T1/2 model stem cells were cultured on the six different electrospun mats, as well as smooth spin-coated films, and the morphology, growth and expression of tendon/ligament genes were evaluated. The results demonstrated that fibre diameter affects cellular behaviour more significantly than fibre alignment. Initially, cell density was greater on the small fibre diameter mats, but similar cell densities were found on all mats after an additional week in culture. After 2 weeks, gene expression of collagen 1α1 and decorin was increased on all mats compared to films. Expression of the tendon/ligament transcription factor scleraxis was suppressed on all electrospun mats relative to spin-coated films, but expression on the large-diameter fibre mats was consistently greater than on the medium-diameter fibre mats. These results suggest that larger-diameter fibres (e.g. > 2 µm) may be more suitable for in vitro development of a tendon/ligament tissue.

Cellularized cylindrical fiber/hydrogel composites for ligament tissue engineering.

Electrospun meshes suffer from poor cell infiltration and limited thickness, which restrict their use to thin tissue applications. Herein, we demonstrate two complementary processes to overcome these limitations and achieve elastomeric composites that may be suitable for ligament repair. First, C3H10T1/2 mesenchymal stem cells were incorporated into electrospun meshes using a hybrid electrospinning/electrospraying process. Second, electrospun meshes were rolled and formed into composites with an interpenetrating polyethylene glycol (PEG) hydrogel network. Stiffer composites were formed from poly(lactic-co-glycolic acid) (PLGA) meshes, while softer and more elastic composites were formed from poly(ester-urethane urea) (PEUUR) meshes. As-spun PLGA and PEUUR rolled meshes had tensile moduli of 19.2 ± 1.9 and 0.86 ± 0.34 MPa, respectively, which changed to 11.6 ± 4.8 and 1.05 ± 0.39 MPa with the incorporation of a PEG hydrogel phase. In addition, cyclic tensile testing indicated that PEUUR-based composites deformed elastically to at least 10%. Finally, C3H10T1/2 cells incorporated into electrospun meshes survived the addition of the PEG phase and remained viable for up to 5 days. These results indicate that the fabricated cellularized composites are support cyclic mechanical conditioning, and have potential application in ligament repair.

Calcium phosphate ceramics in bone tissue engineering: a review of properties and their influence on cell behavior.

Calcium phosphate ceramics (CPCs) have been widely used as biomaterials for the regeneration of bone tissue because of their ability to induce osteoblastic differentiation in progenitor cells. Despite the progress made towards fabricating CPCs possessing a range of surface features and chemistries, the influence of material properties in orchestrating cellular events such as adhesion and differentiation is still poorly understood. Specifically, questions such as why certain CPCs may be more osteoinductive than others, and how material properties contribute to osteoinductivity/osteoconductivity remain unanswered. Therefore, this review article systematically discusses the effects of the physical (e.g. surface roughness) and chemical properties (e.g. solubility) of CPCs on protein adsorption, cell adhesion and osteoblastic differentiation in vitro. The review also provides a summary of possible signaling pathways involved in osteoblastic differentiation in the presence of CPCs. In summary, these insights on the contribution of material properties towards osteoinductivity and the role of signaling molecules involved in osteoblastic differentiation can potentially aid the design of CPC-based biomaterials that support bone regeneration without the need for additional biochemical supplements.

Degradable segmented polyurethane elastomers for bone tissue engineering: effect of polycaprolactone content.

Segmented polyurethanes (PURs), consisting of degradable poly(a-hydroxy ester) soft segments and aminoacid-derived chain extenders, are biocompatible elastomers with tunable mechanical and degradative properties suitable for a variety of tissue-engineering applications. In this study, a family of linear PURs synthesized from poly(ϵ-caprolactone) (PCL) diol, 1,4-diisocyanobutane and tyramine with theoretical PCL contents of 65-80 wt% were processed into porous foam scaffolds and evaluated for their ability to support osteoblastic differentiation in vitro. Differential scanning calorimetry and mechanical testing of the foams indicated increasing polymer crystallinity and compressive modulus with increasing PCL content. Next, bone marrow stromal cells (BMSCs) were seeded into PUR scaffolds, as well as poly(lactic-co-glycolic acid) (PLGA) scaffolds, and maintained under osteogenic conditions for 14 and 21 days. Analysis of cell number indicated a systematic decrease in cell density with increasing PUR stiffness at both 14 and 21 days in culture. However, at these same time points the relative mRNA expression for the bone-specific proteins osteocalcin and the growth factors bone morphogenetic protein-2 and vascular endothelial growth factor gene expression were similar among the PURs. Finally, prostaglandin E2 production, alkaline phosphatase activity and osteopontin mRNA expression were highly elevated on the most-crystalline PUR scaffold as compared to the PLGA and PUR scaffolds. These results suggest that both the modulus and crystallinity of the PUR scaffolds influence cell proliferation and the expression of osteoblastic proteins.

Response of bone marrow stromal cells to graded co-electrospun scaffolds and its implications for engineering the ligament-bone interface.

Biomaterial scaffolds with gradients in architecture, mechanical and chemical properties have the potential to improve the osseointegration of ligament grafts by recapitulating phenotypic gradients that exist at the natural ligament-bone (L-B) interface. Towards the larger goal of regenerating the L-B interface, this in vitro study was performed to investigate the potential of two scaffolds with mineral gradients in promoting a spatial gradient of osteoblastic differentiation. Specifically, the first graded scaffold was fabricated by co-electrospinning two polymer solutions (one doped with nano-hydroxyapatite particles) from offset spinnerets, while the second was created by immersing the first scaffold in a 5 × simulated body fluid. Rat bone marrow stromal cells, cultured in the presence of osteogenic supplements, were found to be metabolically active on all regions of both scaffolds after 1 and 7 days of culture. Gene expression of bone morphogenic protein-2 and osteopontin was elevated on mineral-containing regions as compared to regions without mineral, while the expression of alkaline phosphatase mRNA revealed the opposite trend. Finally, the presence of osteopontin and bone sialoprotein confirmed osteoblastic phenotypic maturation by day 28. This study indicates that co-electrospun scaffolds with gradients in mineral content can guide the formation of phenotypic gradients and may thus promote the regeneration of the L-B interface.

Preliminary experimental results from a MARS Micro-CT system.

The Medipix All Resolution System (MARS) system is a commercial spectral/multi-energy micro-CT scanner designed and assembled by the MARS Bioimaging, Ltd. in New Zealand. This system utilizes the state-of-the-art Medipix photon-counting, energy-discriminating detector technology developed by a collaboration at European Organization for Nuclear Research (CERN). In this paper, we report our preliminary experimental results using this system, including geometrical alignment, photon energy characterization, protocol optimization, and spectral image reconstruction. We produced our scan datasets with a multi-material phantom, and then applied ordered subset-simultaneous algebraic reconstruction technique (OS-SART) to reconstruct images in different energy ranges and principal component analysis (PCA) to evaluate spectral deviation among the energy ranges.

Nerve growth factor-immobilized electrically conducting fibrous scaffolds for potential use in neural engineering applications.

Engineered scaffolds simultaneously exhibiting multiple cues are highly desirable for neural tissue regeneration. To this end, we developed a neural tissue engineering scaffold that displays submicrometer-scale features, electrical conductivity, and neurotrophic activity. Specifically, electrospun poly(lactic acid-co-glycolic acid) (PLGA) nanofibers were layered with a nanometer thick coating of electrically conducting polypyrrole (PPy) presenting carboxylic groups. Then, nerve growth factor (NGF) was chemically immobilized onto the surface of the fibers. These NGF-immobilized PPy-coated PLGA (NGF-PPyPLGA) fibers supported PC12 neurite formation ( 28.0±3.0% of the cells) and neurite outgrowth (14.2 µm median length), which were comparable to that observed with NGF (50 ng/mL) in culture medium ( 29.0±1.3%, 14.4 µm). Electrical stimulation of PC12 cells on NGF-immobilized PPyPLGA fiber scaffolds was found to further improve neurite development and neurite length by 18% and 17%, respectively, compared to unstimulated cells on the NGF-immobilized fibers. Hence, submicrometer-scale fibrous scaffolds that incorporate neurotrophic and electroconducting activities may serve as promising neural tissue engineering scaffolds such as nerve guidance conduits.

Fabrication and characterization of poly(lactic-co-glycolic acid) microsphere/amorphous calcium phosphate scaffolds.

Although hydroxyapatite (HAP) and ß-tricalcium phosphate have been used extensively as osteoconductive minerals in biomaterial scaffolds for bone regeneration, they lack the capacity to stimulate osteoblastic differentiation of progenitor cells. In contrast, amorphous calcium phosphates (ACPs), which convert to HAP under aqueous conditions, have the potential to facilitate osteoblastic differentiation through the transient local release of calcium and phosphate ions. Therefore, in this study ACPs were synthesized using zinc and zirconia divalent cations as stabilizers (denoted ZnACP and ZrACP, respectively) and compared to HAP. Analysis of ion release into serum-containing cell culture medium revealed transiently elevated levels of calcium and phosphorous, consistent with the enhanced solubility of ZrACP and ZnACP relative to HAP. In addition, X-ray diffraction analysis revealed partial conversion of ZrACP to HAP but no conversion of ZnACP after 96 h. Next, scaffolds were fabricated by sintering mixtures of 300-500 µm poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres and 0.5 wt% calcium phosphate mineral (HAP, ZrACP or ZnACP) at 70 °C for 24 h. Scanning electron microscopy revealed a porous microsphere matrix with calcium phosphate particulates clinging to the microsphere surfaces both prior to and after 14 days in culture medium. Finally, the incorporation of calcium phosphate resulted in a lower compressive modulus in the range 127 to 74-89 MPa. Taken together, these results indicate that ZrACP, ZnACP and HAP minerals exhibit very different properties, and therefore may elicit different osteoblastic responses in vitro.