Growth factor-free and antimicrobial elastic nanocomposites for bone regeneration

Abstract

Elastic platforms have been shown recently to be advantageous for bone tissue engineering. Specifically, PEGylated polyglycerol sebecate has recently been reported in literature as a modification for Poly glycerol sebacate (PGS). It is highly elastic with tunable mechanical properties, rendering it an interesting candidate for bone regeneration. However, it lacks the bioactive and antimicrobial properties that are essential for bone tissue engineering scaffolds. To address these issues, herein, PEGylated PGS with different PEG ratios was mixed with PCL to obtain uniform electrospun mats. Further functions were added to the structure to render it osteoinductive and antimicrobial through the addition of Laponite and naturally-based antimicrobial system. Nuclear magnetic resonance (NMR) and Fourier-transform infrared spectroscopy (FTIR) confirmed the successful synthesis of the polymer and the integration of the Laponite within the electrospun membranes. Field Emission scanning electron microscopy (FESEM) showed the electrospun membranes to have fibers with diameters in the microscale, which decrease upon increasing the concentration of Laponite. In vitro degradation showed the nanocomposites to be stable for 28 days. Uniaxial mechanical testing indicated the PEGylated PGS scaffolds to be highly elastomeric when the PEG ratio was increased. Nevertheless, the addition of Laponite leads to a drop in the elongation accompanied with a decrease in elastic modulus and an increase in ultimate tensile strength. Despite this drop in the mechanics, the nanocomposites were still within the range suitable for bone tissue engineering. Furthermore, the addition of the AMPs did not have any influence on the mechanical properties. Colony forming unit assay (CFU) showed a significate antimicrobial activity of the nanocomposites containing AMPs after 12 hours against both gram-positive S. aureus and gram-negative E. coli. These results were confirmed by optical density readings over 12 hours and FESEM imaging. In vitro biocompatibility test using preosteoblasts murine stromal cell line W-20-17 iii iv indicated that the addition of the Laponite and the AMPs did not influence the cells viability, with increasing the cells metabolic activity rover time. Moreover, the cells were able to attach and spread on the scaffolds. Further experiments such as Ca2+ deposition assay, alkaline phosphatase activity (ALP) and alizarin red staining (ARS) proved an enhancement in the osteogenic differentiation of the cells cultured on the nanocomposites. This was supported by gene expression quantification using real time PCR (RT-PCR), which showed up-regulation of genes involved in osteogenic differentiation such as ALP, RUNX2 and Axin2 in addition to genes involved in secretion of the extra cellular matrix such as COL1A1. Collectively, the nanocomposites containing AMPs were proven to have an osteoinductive and antimicrobial activity, which deem them desirable for bone tissue engineering.

Department

Biotechnology Program

Degree Name

MS in Biotechnology

Graduation Date

2-1-2019

Submission Date

September 2018

First Advisor

Allam, Nageh K.

Committee Member 1

Annabi, Nasim

Committee Member 2

El-Fawal, Hassan A.N.

Extent

72 p.

Document Type

Master's Thesis

Rights

The author retains all rights with regard to copyright. The author certifies that written permission from the owner(s) of third-party copyrighted matter included in the thesis, dissertation, paper, or record of study has been obtained. The author further certifies that IRB approval has been obtained for this thesis, or that IRB approval is not necessary for this thesis. Insofar as this thesis, dissertation, paper, or record of study is an educational record as defined in the Family Educational Rights and Privacy Act (FERPA) (20 USC 1232g), the author has granted consent to disclosure of it to anyone who requests a copy.

Institutional Review Board (IRB) Approval

Not necessary for this item

Comments

I am deeply grateful to my advisor, Professor Nageh Allam. I am beyond thankful for the guidance, sincere advice, unconditional and constant support. Thank you for pushing me always forward to be a better person and scientist and for considering the education and the v research of your students as a priority. I also would like to thank Dr. Nasim Annabi for hosting me in her lab and for providing all the possible guidance and facilities I needed to complete my work as presented. I really enjoyed the learning experience I gained by being part of a top-notch research team. Furthermore, I am also thankful to Dr. Ahmed Yousef from the National Research Center (NRC), Egypt for his kind help in the very early phase of this work. I acknowledge the financial support I received from Al Ghurair Foundation for Education for funding my studies at AUC. In addition to the financial support I received from AUC in terms of conference and study abroad grants that definitely contributed towards finalizing this work in the best possible way. I would like to express my deep appreciation and gratitude for the encouragement and support I received from my family without which I wouldn’t have been the person I am today. Thank you for believing in me. Finally, I greatly appreciate the fruitful discussions, trainings and support I received from my friends and colleagues at AUC, Boston and all members of EML and Annabi’s group specifically: Alaa Mohye, Mohammed Salama, Amgad Ouf, Ahmed Shehata, Nashat Ahmed, Mohammed Ramadan, Ahmed Khalifa, Kareem Kirallah, Marwan Yasser, Monira Yazeed, Alaa Ahmed, Amal Badr, Icell Sharf Eldin, Dr. Islam Khalil, Ehsan Shirzaei Sani, Noshine Zandi, Ebrahim Mostafavi, Dr. Habiba El fandi, Sarah Elmiligy and Radwa Hassan.

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