Date of Award

1-1-2011

Document Type

Campus Access Dissertation

Department

Chemical Engineering

First Advisor

Esmaiel Jabbari

Abstract

Over the years, the repair of bone defect is an increasing challenge for reconstructive surgery. Due to disadvantage of autograft and allograft, including limit of bone supply, high morbidity and potential immune response, synthetic scaffold have been studied as a promising strategy for tissue engineered bone regeneration. The properties of synthetic scaffold are controllable and reproducible compared to natural materials. In addition, synthetic materials can be functionalized to enhance biocompatibility, biodegradability and osteoinductivity. These characteristics make the synthetic scaffold and ideal bone analog for bone reconstruction. This work will focus on the establishment of a multifunctional synthetic scaffold which are enzymatically degradable and osteoinductive for bone regeneration.

In the first part, the effect of conjugation of cell adhesion RGD peptide and osteogenic peptide KIPKA SSVPT ELSAI STLYL to a model biodegradable hydrogel on osteogenic differentiation of BMS cells and production of mineralized matrix was studied. RGD peptide was grafted to the hydrogel by adding acrylated RGD (Ac-GRGD) to the polymerization mixture consisting of poly (lactide-co-ethylene oxide fumarate) (PLEOF) macromer and propargyl acrylate. The azide functionalized osteogenic peptide modified with a mini-PEG spacer to increase solubility in aqueous solution, Az-mPEG-BMP peptide, was conjugated to the hydrogel by "click chemistry" between the reactive azide group of the peptide and propargyl group of the hydrogel. To determine the osteogenic activity, alkaline phosphatase activity and the production of mineralized matrix of BMS cells seeded on the hydrogels were measured. These results clearly demonstrate that RGD and BMP peptides, grafted to a model hydrogel substrate, act synergistically to increase osteogenic differentiation of BMS cells and the production of mineralized matrix.

In the second part, we develop a novel bioactive 3D hydrogel scaffold functionalized with RGD, BMP peptide and Osteopontin-derived peptide SVVYGLR (ODP) to enhance the adhesion and concurrent osteogenic and vasculogenic differentiation of bone marrow stromal stem (BMS) cells. The effect on osteogenesis and vasculogenesis of BMS cells were evaluated by measuring ALPase activity, calcium content, osteogenic marker osteopontin, (OP) osteocalcin (OC), osteonectin (ON) and vasculogenic marker platelet endothelial cell adhesion molecule (pecam1) mRNA expression level with incubation time. These results demonstrate that RGD, BMP and ODP peptides, conjugated to PLEOF-based 3D hydrogel scaffold, act synergistically to enhance the adhesion, proliferation and concurrent osteogenic and vasculogenic differentiation of BMS cells.

Furthermore, we investigate the effect of fiber alignment on osteogenic differentiation of bone marrow stromal (BMS) cells. Morphology and alignment of the BMS cells seeded on the fibers were characterized by SEM. The effect of fiber orientation on osteogenic differentiation of BMS cells was determined by measuring alkaline phosphatase (ALPase) activity, calcium content, and mRNA expression levels of osteogenic markers. The results indicate that BMS cells aligned in the direction of PLA fibers to form long cell extensions, and fiber orientation affected the extent of mineralization, but it had no effect on cell proliferation or mRNA expression of osteogenic markers.

Finally, enzymatically degradable Poly (lactic acid acrylate ) (PLAA) hydrogel containing a cell adhesive signal was developed as a synthetic analog of extracellular matrix (ECM). This hydrogel was synthesis with MMP sensitive peptide crosslinker which contains a matrix metalloproteinase-13 (MMP-13) degradable domain, AC-GRGD and PLAA macromers. Encapsulation procedure was used to assess the biocompatibility of the hydrogel. The effect of RGD and MMP peptide on the cell invasion was evaluated by a modified boyden's chamber. The results indicated that BMS cells migration was independent of RGD concentration in absence of MMP sensitive peptide. Meanwhile, MMP sensitive peptide enhanced the BMS cell migration with presence of RGD peptide. The synthetic enzymatically degradable PLAA hydrogel we developed may be potential employed as a tissue engineered bone substitute.

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