Date of Award

5-2021

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biomedical and Chemical Engineering and Sciences

First Advisor

Vipuil Kishore

Second Advisor

Venkat Keshav Chivukula

Third Advisor

Csaba Palotai

Fourth Advisor

D. Andrew Knight

Abstract

Biomimetic composite scaffolds comprising of bioactive ceramic-based materials incorporated within a polymeric framework have shown considerable promise for use in bone tissue engineering (BTE) applications. Although bioceramics have shown immense promise for use in BTE, few studies have performed a head-to-head comparison of the osteogenic properties of the most promising bioceramic materials. The goal of this study was to compare three different bioceramics – Bioglass 45S5 (BG), Laponite XLG (LAP), and β-Tricalcium Phosphate (TCP) – on the physical properties (i.e., swelling, stability, compressive modulus, bone bioactivity) of methacrylated collagen (CMA) hydrogels and osteogenic differentiation of human MSCs (hMSCs) encapsulated within CMA hydrogels. The study also evaluated the effect of different bioceramic materials on osteogenic differentiation of hMSCs cultured in two different culture media – osteoconductive (without Dexamethasone) and osteoinductive (with Dexamethasone). CMA only hydrogels (i.e., no bioceramic) were used as control. Bioceramic incorporated CMA hydrogels were prepared by homogenizing phosphate buffered saline (PBS) suspended bioceramics - BG, TCP or LAP with 3 mg/ml CMA at a concentration of 10% w/w. UV crosslinking (365 nm, 1 min) of bioceramic-laden CMA hydrogels was performed post gelation using a water soluble azo-photoinitator (VA-086). Results from the study revealed that bioceramic incorporation had no effect on the swelling capability and in vitro stability of CMA hydrogels. Compressive modulus of CMA hydrogels decreased upon addition of bioceramics. Specifically, incorporation of BG and LAP resulted in a significant decrease (p < 0.05) in the compressive modulus of CMA hydrogels. Bone bioactivity of bioceramic-laden CMA hydrogels was assessed by incubating the hydrogels in simulated body fluid (SBF) and the results showed that incorporation of TCP and LAP resulted in a dense cauliflower like deposition of hydrocycarbonate apatite (HCA) layer on the surface of these hydrogels by day 7 suggesting that these hydrogels are bone bioactive. Cell metabolic activity assessed using the Alamar blue assay was comparable between different bioceramic-laden hydrogels and the control; albeit, higher cell metabolic activity was observed in cultures without Dexamethasone which may be indicative of higher rates of cell proliferation in osteoconductive culture medium. Cell cytoskeleton staining results showed that hMSCs exhibited a flattened and cuboidal morphology in bioceramic-laden CMA hydrogels at day 7 which is representative of osteoblastic cells. Osteogenic differentiation of human MSCs in bioceramic-laden CMA hydrogels was assessed via measurement of alkaline phosphatase (ALP) activity over time. TCP incorporation resulted in a significant increase (p < 0.05) in ALP activity compared to the control in both osteoconductive and osteoinductive culture medium. Additionally, TCP incorporation was observed to accelerate ALP activity in cultures without Dexamethasone indicating that addition of TCP stimulates osteogenic differentiation of hMSCs without the addition of external factors. Overall, these results indicate that TCP has superior osteostimulative properties compared to BG and LAP and hence has significant potential for use in the biomimetic design of collagen-based composite constructs for BTE applications.

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