Mesenchymal Stem Cell Responses to Bone-Mimetic Electrospun Matrices Composed of Polycaprolactone, Collagen I and Nanoparticulate Hydroxyapatite

The performance of biomaterials designed for bone repair depends, in part, on the ability of the material to support the adhesion and survival of mesenchymal stem cells (MSCs). In this study, a nanofibrous bone-mimicking scaffold was electrospun from a mixture of polycaprolactone (PCL), collagen I, and hydroxyapatite (HA) nanoparticles with a dry weight ratio of 50/30/20 respectively (PCL/col/HA). The cytocompatibility of this tri-component scaffold was compared with three other scaffold formulations: 100% PCL (PCL), 100% collagen I (col), and a bi-component scaffold containing 80% PCL/20% HA (PCL/HA). Scanning electron microscopy, fluorescent live cell imaging, and MTS assays showed that MSCs adhered to the PCL, PCL/HA and PCL/col/HA scaffolds, however more rapid cell spreading and significantly greater cell proliferation was observed for MSCs on the tri-component bone-mimetic scaffolds. In contrast, the col scaffolds did not support cell spreading or survival, possibly due to the low tensile modulus of this material. PCL/col/HA scaffolds adsorbed a substantially greater quantity of the adhesive proteins, fibronectin and vitronectin, than PCL or PCL/HA following in vitro exposure to serum, or placement into rat tibiae, which may have contributed to the favorable cell responses to the tri-component substrates. In addition, cells seeded onto PCL/col/HA scaffolds showed markedly increased levels of phosphorylated FAK, a marker of integrin activation and a signaling molecule known to be important for directing cell survival and osteoblastic differentiation. Collectively these results suggest that electrospun bone-mimetic matrices serve as promising degradable substrates for bone regenerative applications

Place, Politics and Ethnicity in the Contemporary American City

The purpose of this essay is to discuss the impacts of ethnicity in the politics of the contemporary American city. The importance of ethnicity is seen as derivative of the structure of local politics in the United States and the processes which have resulted in the establishment and maintenance of ethnic identities and communities in American cities. As such, the paper is intended to provide a context for the remaining papers in this session which focus on the recent political behavior of specific ethnic groups in particular cities

Live cell imaging of GFP-expressing MSCs seeded onto electrospun scaffolds.

<p>A) Cells were seeded onto scaffolds and imaged over varying time points. Panels a–c: PCL scaffolds; panels d–f: PCL/HA scaffolds; panels g–i: PCL/col/HA scaffolds and panels j–l: col scaffolds. Scale bar = 100 µm. B) Higher magnification images of GFP-expressing MSCs at seven hours on electrospun scaffolds (panels m–p).</p

Immunostaining for phosphorylated Focal Adhesion Kinase.

<p>MSCs were seeded onto glass coverslips coated with electrospun nanofibers, or with FBS as a control. After 5 hours, cells were fixed and stained for phosphorylated Focal Adhesion Kinase (red). Cells were counterstained with DAPI to show cell nuclei (blue). Cells seeded onto PCL/col/HA scaffolds were better spread, and exhibited greater amounts of punctuate pFAK staining (site pY397) as compared with cells on PCL or PCL/HA. Cells seeded onto FBS-coated glass coverslips displayed pFAK staining in focal adhesion-type structures (white arrows), as expected for cells grown on 2D surfaces.</p

Tensile Properties of dry and hydrated scaffolds.

<p>Values represent the average ± standard deviation calculated in the linear portion at 10% strain. The hydrated collagen scaffolds have very low mechanical properties and could not be measured by this technique.</p

Adsorption of FN and VN by electrospun scaffolds.

<p>Scaffolds were coated with fetal bovine serum (A), or implanted into rat tibial osteotomies for 30 min (B). Scaffolds were then washed to remove loosely bound proteins, and proteins were subsequently desorbed by incubation in boiling SDS-containing solution. The amounts of FN and VN were evaluated by Western blot.</p

SEM images of MSCs cultured on nanofibrous scaffolds for 24 hours.

<p>A) Cell spreading was observed on PCL, PCL/HA, and PCL/col/HA scaffolds, but not on 100% collagen I (col). B) Col scaffolds (without cells) were incubated in culture media for 24 hrs to allow the potential release of soluble factors, and then the solution was collected. MSCs were suspended into this conditioned media, seeded onto PCL scaffolds, and allowed adhere in the media for 24 h. Under these conditions cell spreading was extensive, suggesting that lack of cell spreading on col substrates was not due to any soluble factors released from these scaffolds.</p

MTS assay quantifying cell proliferation on electrospun scaffolds of PCL, PCL/HA or PCL/col/HA.

<p>At day one, cell number was significantly higher on PCL/HA and PCL/col/HA scaffolds in comparison to PCL. By day four, PCL/HA was still significantly higher than PCL, and PCL/col/HA was significantly higher than PCL/HA and PCL. In addition, cell number on PCL/col/HA was significantly higher on day four than day one. An * denotes p<0.05</p