Author(s)

Pamela Kubinski

Date Approved

6-5-2013

Document Type

Thesis

Degree Name

M.S. Chemical Engineering

Department

Chemical Engineering

College

Henry M. Rowan College of Engineering

First Advisor

Vernengo, Jennifer

Subject(s)

Spinal implants;Tissue scaffolds;Spinal cord injuries

Disciplines

Chemical Engineering

Abstract

The primary goal of this work is to investigate the potential of poly(Nisopropylacrylamide)- based scaffolds in tissue engineering applications, specifically spinal injury and nucleus pulposus replacement. Poly(N-isopropylacrylamide), denoted PNIPAAm, is a synthetic polymer with hydrophilic properties at room temperature, but above 32C the chains become hydrophobic and entangle to form physical crosslinks. This thermoresponsive property enables PNIPAAm to be implanted as a free-flowing solution at room temperature and collapse into a gel in physiological conditions. However, as a homopolymer, the hydrophobicity of PNIPAAm in vivo causes the hydrogel to expel water and exhibit inelastic properties that do not mimic those of natural tissue. These limitations have been previously addressed by copolymerizing NIPAAm monomer with a synthetic hydrophilic polymer, such as polyethylene glycol, or PEG. Incorporation of hydrophilic PEG chains enables the hydrogel to absorb more water, generating an elastic hydrogel with high water content. This copolymer is suitable for spinal cord applications because the scaffold can fill irregularly shaped spinal cord defects non-invasively and its mechanical properties can be easily tailored. In this work, PNIPAAm-PEG is evaluated in vitro as a candidate scaffold for repair of the injured spinal cord. Biocompatibility of the scaffold is characterized using two cell lines, human embryonic kidney cells (HEK) and rat fibroblasts expressing green fluorescent protein (RF-GFP). The cells were suspended in a 10 wt% PNIPAAm-PEG solution and cultured at 37°C. The viability of HEK was assessed over 25 days using a qualitative dual fluorescent stain to distinguish between live and dead cells, and a quantitative MTT cell proliferation assay. RF-GFP viability was assessed over 15 days in vitro using fluorescence microscopy. Results from these studies suggest that PNIPAAm-PEG is non-toxic to cells and should be evaluated further in vivo for treatment of spinal cord injuries. Although PNIPAAm-PEG copolymers have also been studied for nucleus pulposus replacement in the intervertebral disc, a more suitable PNIPAAm-based copolymer scaffold is proposed here. In this work, hydrogels composed of PNIPAAm and natural chondroitin sulfate-A (CS) are studied. CS is a glycosaminoglycan naturally found in connective tissue and cartilage that is negatively charged and degraded by chondroitinase ABC enzymes. Incorporating CS into PNIPAAm will create a three-dimensional hydrated copolymer network with increased elasticity and biocompatibility. As before, the thermoresponsive properties of PNIPAAm enable the copolymer to be injected noninvasively. In addition, the biodegradability of CS will enhance the porosity of the hydrogel and create interconnected pores that aid in tissue ingrowth, nutrient transport to and waste products from cells within the network. In this work, the swelling and degradation behavior, and the mechanical properties of PNIPAAm-CS copolymers are characterized. Preliminary cytocompatibility is also performed in vitro using human mesenchymal stem cells. Results from these analyses indicate that PNIPAAm-CS has potential as a scaffold to replace the damaged nucleus pulposus and recreate the biomechanical function of the disc.

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