Assessment of nanofibrous scaffold chemistry, design, and use of extracellular stimuli for bone tissue engineering.

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Title: Assessment of nanofibrous scaffold chemistry, design, and use of extracellular stimuli for bone tissue engineering.
Author: McCullen, Seth Dylan
Advisors: Laura Clarke, Committee Co-Chair
Russell Gorga, Committee Member
Greg McCarty, Committee Member
Lola Reid, Committee Member
Elizabeth Loboa, Committee Chair
Abstract: An emerging cell source for bone tissue engineering is human adipose-derived stem cells (hASCs). This research investigated the creation of biocompatible scaffolds through the electrospinning technique and their subsequent culture with hASCs. Electrospun nanofibrous scaffolds were fabricated from poly (l-lactic acid) and loaded with β-tricalcium phosphate particles (β-TCP). The physical properties and ability of the composite scaffolds to induce osteogenic differentiation of hASCs was assessed. Scaffolds loaded with β-TCP exhibited a significant decrease in tensile strength. As scaffolds underwent in vitro degradation, the amount of calcium residing within the scaffold significantly decreased. Human ASCs were able to adhere, proliferate, and differentiate on all scaffolds those cultured on the scaffolds with the largest loss in residual calcium also had significant increases in hASC-mediated mineralization. The production of this mineral was hypothesized to be based on the influence of the dissolution products of the β-TCP particles. To investigate the effect of the dissolution products of β-TCP, hASCs were cultured on electrospun nanofibrous PLA scaffolds in culture medium containing 1.8 (normal), 8, or 16 mM Ca2+ with or without the use of osteogenic supplements (β-glycerol phosphate, ascorbic acid, and dexamethasone). Materials deposited by hASCs were analyzed and mineral deposition was significantly enhanced under both growth and osteogenic medium conditions by increasing extracellular Ca2+. The greatest mineral deposition occurred in the osteogenic differentiating medium 8 mM Ca2+ treatment group. FTIR and X-ray diffraction indicated that elevate calcium concentration of 8 mM Ca2+ increased both PO4 amount and the crystalline structure of the mineral, respectively. To further functionalize the electrospun scaffolds, cylindrical pores of 150, 300, and 600 µm diameter were micro-machined through the scaffolds using a laser ablation technique. Laser ablation parameters were varied and it was determined that the aperture and z-travel direction of the laser linearly correlated with the ablated pore diameter. Scaffold morphology was assessed and it was determined that scaffolds with 600 µm diameter pores exhibited significant polymer redeposition. The addition of the 300 µm pore arrays was assessed and compared to non-ablated scaffolds by subsequent stacking and bonding together with collagen gel. To evaluate the benefit of assembled scaffolds with and without engineered pores, hASCs were seeded on individual electrospun scaffolds, hASC-seeded scaffolds were bonded with type I collagen, and the entire constructs cultured to examine their potential as bone tissue engineering scaffolds. Assembled electrospun scaffolds/collagen gel constructs using electrospun scaffolds with pores resulted in enhanced hASC viability, proliferation, and mineralization of the scaffolds after three weeks in vitro compared to constructs using electrospun scaffolds without pores. Electron microscopy and histological examination revealed that the assembled constructs with laser ablated electrospun scaffolds maintained a contracted structure and not delaminate, unlike assembled constructs containing non-ablated electrospun scaffolds. A final study examined the influence of environmental stimuli, specifically low frequency electric fields on hASCs. Electric stimulation is known to initiate signaling pathways and provides a technique to enhance osteogenic differentiation of stem and/or progenitor cells. Human ASCs were cultured directly on custom interdigitated electrodes and exposed to either acute or chronic electric fields. Acute exposure to sinusoidal electric fields of 1 Hz induced hASC calcium signaling that increased in response to electric field magnitude. Human ASCs were cultured under osteogenic medium and hASCs that were chronically exposed to electric field treatment of 1 V/cm led to a significant increase in hASC mineralization. This is the first study to evaluate the effects of sinusoidal electric fields on hASCs and to demonstrate that both acute and chronic electric field exposure can significantly increase intracellular calcium signaling and the amount of mineralized calcium under osteogenic differentiation conditions, respectively.
Date: 2010-03-04
Degree: PhD
Discipline: Biomedical Engineering

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