Publication Date

2018-04-10

Availability

Open access

Embargo Period

2018-04-10

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biomedical Engineering (Engineering)

Date of Defense

2017-11-15

First Committee Member

Fotios M. Andreopoulos

Second Committee Member

Alicia R. Jackson

Third Committee Member

Gianluca D'Ippolito

Abstract

Back pain affects more than 80% of the adult population between the ages of 45- 54 years old, and it is a leading cause of disabilities in developing countries. For the most part, low back pain is associated with degeneration of the intervertebral disc (IVD) called degenerative disc disorder (DDD) or degenerative IVD disorder (IDD). Current treatment options are all symptomatic and lead to further degeneration of adjacent vertebrae. Although tissue engineering approaches show potential for treatment of DDD, IVD cells are not readily available, rendering their use for implantation difficult. This study focuses on the use of marrow-isolated adult multilineage inducible (MIAMI) cells co-cultured together with native annulus fibrosus (AF) cells as alternative source for IVD tissue engineering. Moreover, the paper proposes the use of electrospun, polycaprolactone (PCL) scaffolds as substrates for the culture of the cells. The hypothesis was that co-culturing AF and MIAMI cells on PCL scaffolds would lead to differentiation of the latter ones into native disc lineages effectively. PCL scaffolds with random fiber orientation were fabricated using different solvents (DCM and HFIP) and polymer concentrations (12%, 15% and 20%). Scaffolds were characterized using scanning electron microscopy (SEM) for their fiber diameter and uniformity. PCL/HFIP constructs possessed superior properties, with uniform fibers and diameters of 0.88±0.27, 0.94±0.35, and 2.07±0.18μm for 12%, 15% and 20% scaffolds respectively. Porcine AF and MIAMI cells were obtained and co-cultured on scaffolds for 1, 2 and 3 weeks, by which time samples were obtained for RT-PCR. Cells were tested for the expression of markers Col I, II, Acan, as well as Oct-4 and Nanog. AF cells alone were also seeded on tissue culture plates and their ability to differentiate into osteogenic lineages was studied through the addition of osteogenic media. Cells on the scaffolds did not yield consistent Ct values after PCR, however, co-cultured cells seeded on scaffolds for three weeks had distinct expression of Col 1 and Acan, with a significant increase of Col 1 compared to MIAMI-only controls. AF that underwent osteogenesis tested positive for osteogenic markers Alk-P, Osteocalcin and Osterix unlike the AF controls (normal culture), which tested negative.

Keywords

Biomaterials; Tissue Engineering; Electrospinning

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