College of Dental Medicine Student Theses, Dissertations and Capstones

Document Type

Thesis

Degree Name

Master of Science (M.S.) in Dentistry

Department

College of Dental Medicine

First Advisor

Singer, Richard

Publication Date / Copyright Date

2014

Publisher

Nova Southeastern University

Abstract

Craniofacial tissue loss due to traumatic injuries and congenital defects is a major clinical problem around the world. Cleft palate is the second most common congenital malformation in the United States occurring with an incidence of 1 in 700. Some of the problems associated with this defect are feeding difficulties, speech abnormalities and dentofacial anomalies. Current treatment protocol offers repeated surgeries with extended healing time. Our long-term goal is to regenerate bone in the palatal region using tissue-engineering approaches. Bone tissue engineering utilizes osteogenic cells, osteoconductive scaffolds and osteoinductive signals. Mesenchymal stem cells derived from human umbilical cord (HUMSCs) are highly proliferative with the ability to differentiate into osteogenic precursor cells. The primary objective of the study was to characterize HUMSCs and culture them in a 3D hydrogel scaffold and investigate their osteogenic potential. PuraMatrixTM is an injectable 3D nanofiber scaffold capable of self-assembly when exposed to physiologic conditions. Our second objective was to investigate the effect of Bone Morphogenic Protein 2 (BMP2) in enhancing the osteogenic differentiation of HUMSCs encapsulated in PuraMatrixTM. We isolated cells isolated from Wharton's Jelly region of the umbilical cord obtained from NDRI (New York, NY). Isolated cells satisfied the minimal criteria for mesenchymal stem cells (MSCs) as defined by International Society of Cell Therapy in terms of plastic adherence, fibroblastic phenotype, surface marker expression and osteogenic differentiation. Flow Cytometry analysis showed that cells were positive for CD73, CD90 and CD105 while negative for hematopoietic marker CD34. Alkaline phosphatase activity (ALP) of HUMSCs showed peak activity at 2 weeks (p<0.05). Cells were encapsulated in 0.2% PuraMatrixTM at cell densities of 10x104, 20x104, 40x104 and 80x104. Cell viability with WST and proliferation with Live-Dead cell assays showed viable cells at all cell concentrations (p<0.05). A two- fold upregulation of ALP gene was seen for cells encapsulated in PuraMatrixTM with osteogenic medium compared to cells in culture medium (p<0.05). HUMSCs encapsulated in PuraMatrixTM were treated with BMP2 at doses of 50ng/ml, 100ng/ml and 200ng/ml. A significant upregulation of ALP gene in BMP2 treated cells was seen compared to HUMSCs treated in osteogenic medium (p<0.05). Peak osteogenic activity was noted at BMP2 dose of 100ng/ml (p<0.05). We have developed a composite system of HUMSCs, PuraMatrixTM and BMP2 for repair of bone defects that is injectable precluding additional surgeries.

Disciplines

Dentistry

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Dentistry Commons

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