Aaron S. Goldstein

Polypyrrole-Coated Electrospun PLGA Nanofibers for Neural Tissue Applications

Electrospinning is a promising approach to create nanofiber structures that are capable of supporting adhesion and guiding extension of neurons for nerve regeneration. Concurrently, electrical stimulation of neurons in the absence of topographical features also has been shown to guide axonal extension. Therefore, the goal of this study was to form electrically conductive nanofiber structures and to examine the combined effect of nanofiber structures and electrical stimulation. Conductive meshes were produced by growing polypyrrole (PPy) on random and aligned electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers, as confirmed by scanning electron micrographs and X-ray photon spectroscopy. PPy-PLGA electrospun meshes supported the growth and differentiation of rat pheochromocytoma 12 (PC12) cells and hippocampal neurons comparable to non-coated PLGA control meshes, suggesting that PPy-PLGA may be suitable as conductive nanofibers for neuronal tissue scaffolds. Electrical stimulation studies showed that PC12 cells, stimulated with a potential of 10 mV/cm on PPy-PLGA scaffolds, exhibited 40-50% longer neurites and 40-90% more neurite formation compared to unstimulated cells on the same scaffolds. In addition, stimulation of the cells on aligned PPy-PLGA fibers resulted in longer neurites and more neurite-bearing cells than stimulation on random PPy-PLGA fibers, suggesting a combined effect of electrical stimulation and topographical guidance and the potential use of these scaffolds for neural tissue applications.

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.

Microporous bacterial cellulose as a potential scaffold for bone regeneration

Nanoporous cellulose biosynthesized by bacteria is an attractive biomaterial scaffold for tissue engineering due to its biocompatibility and good mechanical properties. However, for bone applications a microscopic pore structure is needed to facilitate osteoblast ingrowth and formation of a mineralized tissue. Therefore, in this study microporous bacterial cellulose (BC) scaffolds were prepared by incorporating 300-500 microm paraffin wax microspheres into the fermentation process. The paraffin wax microspheres were subsequently removed, and scanning electron microscopy confirmed a microporous surface of the scaffolds while Fourier transform infrared spectroscopy verified the elimination of paraffin and tensile measurements showed a Youngs modulus of approximately 1.6 MPa. Microporous BC and nanoporous (control) BC scaffolds were seeded with MC3T3-E1 osteoprogenitor cells, and examined by confocal microscopy and histology for cell distribution and mineral deposition. Cells clustered within the pores of microporous BC, and formed denser mineral deposits than cells grown on control BC surfaces. This work shows that microporous BC is a promising biomaterial for bone tissue engineering applications.

Effect of Intermittent Shear Stress on Mechanotransductive Signaling and Osteoblastic Differentiation of Bone Marrow Stromal Cells

Perfusion culture of osteoprogenitor cells seeded within porous scaffolds suitable for bone tissue engineering is known to enhance deposition of a bone-like extracellular matrix, and the underlying mechanism is thought to involve flow-induced activation of mechanotransductive signaling pathways. Basic studies have shown that mechanotransduction is enhanced by impulse flow and may be mediated through autocrine signaling pathways. To test this, an intermittent flow regimen (5 min on/5 min off ) that exerts impulses on adherent cells and permits accumulation of secreted factors in the cell microenvironment was compared to continuous flow for its ability to stimulate phosphorylation of ERK and p38, synthesis of prostaglandin E2 (PGE2), and expression of mRNA for collagen 1alpha1 (Col-1alpha1), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN). Studies were performed using bone marrow stromal cells cultured in osteogenic media, and parallel-plate flow chambers were used to exert a shear stress of 2.3 dyn/cm2 on cell layers. Results show that continuous flow significantly enhanced phosphorylation of ERK and p38 after 30 min relative to intermittent flow, while intermittent flow significantly increased accumulation of PGE2 in the circulating medium by 24 h relative to continuous flow. Neither continuous nor intermittent flow affected mRNA expression of Col-1alpha1 and OPN after 4 h, but when monolayers were stimulated for 24 h and then allowed to differentiate under static conditions for an additional 13 days, expression of Col-1alpha1, OPN, BSP, and OCN under continuous and intermittent flow was similar and significantly elevated relative to static controls. This study demonstrates that the variation of perfusion regimen modulates mechanotransductive signaling.

Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells.

Dexamethasone is capable of directing osteoblastic differentiation of bone marrow stromal cells (BMSCs) in vitro, but its effects are not lineage‐specific, and sustained exposure has been shown to down‐regulate collagen synthesis and induce maturation of an adipocyte subpopulation within BMSC cultures. Such side effects might be reduced if dexamethasone is applied in a regimented manner, but the discrete steps in osteoblastic maturation that are stimulated by dexamethasone are not known. To examine this, dexamethasone was added to medium to initiate differentiation of rat BMSCs cultures and then removed after a varying number of days. Cell layers were analyzed for cell number, rate of collagen synthesis, expression of osteocalcin (OC), bone sialoprotein (BSP) and lipoprotein lipase (LpL), and matrix mineralization. Withdrawal of dexamethasone at 3 and 10 days was found to enhance cell number relative to continuous exposure, but did not affect to decrease collagen synthesis slightly. Late markers of osteoblastic differentiation, BSP expression and matrix mineralization, were also sensitive to dexamethasone and increased systematically with exposure while LpL systematically decreased. These results indicate that dexamethasone acts at both early and late stages to direct proliferative osteoprogenitor cells toward terminal maturation. J. Cell. Biochem. 90: 13–22, 2003.

Computational predictions of the tensile properties of electrospun fibre meshes: Effect of fibre diameter and fibre orientation

The mechanical properties of biomaterial scaffolds are crucial for their efficacy in tissue engineering and regenerative medicine. At the microscopic scale, the scaffold must be sufficiently rigid to support cell adhesion, spreading, and normal extracellular matrix deposition. Concurrently, at the macroscopic scale the scaffold must have mechanical properties that closely match those of the target tissue. The achievement of both goals may be possible by careful control of the scaffold architecture. Recently, electrospinning has emerged as an attractive means to form fused fibre scaffolds for tissue engineering. The diameter and relative orientation of fibres affect cell behaviour, but their impact on the tensile properties of the scaffolds has not been rigorously characterized. To examine the structure-property relationship, electrospun meshes were made from a polyurethane elastomer with different fibre diameters and orientations and mechanically tested to determine the dependence of the elastic modulus on the mesh architecture. Concurrently, a multiscale modelling strategy developed for type I collagen networks was employed to predict the mechanical behaviour of the polyurethane meshes. Experimentally, the measured elastic modulus of the meshes varied from 0.56 to 3.0 MPa depending on fibre diameter and the degree of fibre alignment. Model predictions for tensile loading parallel to fibre orientation agreed well with experimental measurements for a wide range of conditions when a fitted fibre modulus of 18 MPa was used. Although the model predictions were less accurate in transverse loading of anisotropic samples, these results indicate that computational modelling can assist in design of electrospun artificial tissue scaffolds.

Hydrodynamic Shear Stimulates Osteocalcin Expression But Not Proliferation of Bone Marrow Stromal Cells

Bone marrow stromal cells (BMSCs) are a promising component for engineered bone tissues, but in vitro formation of a bonelike tissue requires culture conditions that direct these multipotent cells toward osteoblastic maturation. Fluid flow has been postulated to stimulate bone tissue development in vivo, but the effect of shear stress on proliferation and differentiation of osteoprogenitor cell cultures in vitro has not been examined closely. In this study BMSCs were cultured on fibronectin-coated substrates and exposed intermittently (for 30 min 3, 5, 7, 9, 11, and 13 days after seeding) to a spatially dependent range of shear stresses (0.36 to 2.7 dyn/cm(2)) using a radial-flow chamber. After 7 days cell density did not vary between sheared and control cell layers. In contrast, after 21 days the accumulation of osteocalcin protein (OC) in cell layers was increased significantly relative to static controls, while the quantity of multilayer cell aggregates (i.e., bone nodules) was diminished. Neither of these effects varied systematically with shear magnitude. Finally, pretreatment of cultures with the cyclooxygenase (COX)-2-specific inhibitor NS-398 blocked prostaglandin secretion in response to shearing flow and significantly reduced OC accumulation in cell layers. These results provide evidence that flow stimulates osteoblastic maturation but not proliferation of bone marrow stromal cells and that prostaglandin signaling is involved in this effect.

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. Copyright

Fabrication of a model continuously graded co-electrospun mesh for regeneration of the ligament–bone interface

Current scaffolds for the regeneration of anterior cruciate ligament injuries are unable to capture intricate mechanical and chemical gradients present in the natural ligament-bone interface. As a result, stress concentrations can develop at the scaffold-bone interface, leading to poor osseointegration. Hence, scaffolds that possess appropriate mechano-chemical gradients would help establish normal loading properties at the interface, while promoting scaffold integration with bone. With the long-term goal of investigating regeneration of the ligament-bone interface, this feasibility study aimed to fabricate a continuously graded mesh. Specifically, graded meshes were fabricated by co-electrospinning nanohydroxyapatite/polycaprolactone (nHAP-PCL) and poly(ester urethane) urea elastomer solutions from offset spinnerets. Next, mineral crystallites were selectively deposited on the nHAP-PCL fibers by treatment with a 5× simulated body fluid (5× SBF). X-ray diffraction and energy-dispersive spectroscopy indicated calcium-deficient hydroxyapatite-like mineral crystallites with an average Ca/P ratio of 1.48. Tensile testing demonstrated the presence of a mechanical gradient, which became more pronounced upon treatment with 5× SBF. Finally, biocompatibility of the graded meshes was verified using an MC3T3-E1 osteoprogenitor cell line. The study demonstrates that graded meshes, for potential application in interfacial tissue engineering, can be fabricated by co-electrospinning.

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.Oil.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.