Dissertation
Bone tissue engineering: nucleic acids and surface coatings
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2021
DOI: 10.17077/etd.006236
Abstract
Tissue engineering offers new treatments for disorders such as temporomandibular joint disorder which among others frequently afflicts patients. In this thesis, tissue engineering strategies are explored with an emphasis on addressing bone tissue repair. The primary technique used was gene therapy in conjunction with a matrix to provide therapeutic growth factors and a foundation for new bone to form on, respectively. The first chapter describes the previous research of tissue engineering to address TMD; the next four chapters then describe optimizations and combination of gene therapy to improve current bone tissue engineering outcomes. The final chapter looks at the future of tissue engineering in the field of bone tissue repair with a focus on TMD.Damage or disease in the TMJ adversely affects masticatory function and speaking, reducing patients' quality of life. Effective treatment options for patients suffering from severe temporomandibular joint disorders are in high demand because surgical options are restricted to removal of damaged tissue or complete replacement of the joint with prosthetics. Tissue engineering approaches for the temporomandibular joint are a promising alternative to the limited clinical treatment options. However, tissue engineering is still a developing field and only in its formative years for the temporomandibular joint. This chapter outlines the anatomical and physiological characteristics of the temporomandibular joint, clinical management of temporomandibular joint disorder, and current perspectives in the tissue engineering approach for the temporomandibular joint disorder.
In the subsequent chapters, the goal was to investigate different bone tissue engineering approaches with gene therapy. The first was to identify if calcium ion (Ca2+) concentration affects the transfection of bone marrow stromal cells because these cells play a major role in bone healing and can infiltrate gene‐activated matrices designed to promote bone growth. The results indicate that Ca2+ levels between 8 and 12 mM positively impacted transfection of BMSCs with PEI‐pDNA complexes in terms of cell viability and transfection efficiency, and a Ca2+ concentration of 10 mM also increased the expression of an osteogenic gene, osteocalcin, when the cells were transfected with plasmid DNA encoding bone morphogenetic protein 2 (BMP‐2).
In the third chapter, gene-activated matrices (GAMs) mineralized with a simulated body fluid (SBF) were prepared and implanted in rat calvarial defects. The optimal GAM consisted of a collagen sponge with PEI-pDNA complexes embedded in a calcium phosphate coating produced by SBF, which increased total bone formation by 39% compared to 19% for control samples. A follow up in vivo study was performed to optimize the ratio of growth factors included in the GAM. The optimal ratio for supporting bone formation after 6 weeks of implantation was 5 parts of pBMP-2 to 3 parts pFGF-2. These studies demonstrated that collagen matrices biomimetically mineralized and activated with plasmids encoding FGF-2 and BMP-2 can optimally improve bone regeneration outcomes.
The next chapter focuses on the development of a system to control the formation of bone to complement developments that have enabled potent regeneration of bony tissue. Scaffolds were fabricated with chemically modified RNA encoding for bone morphogenetic protein-9 (cmBMP9) and capped with salicylic acid (SA)-containing polymer (SAPAE) with the goal to determine if SAPAE could inhibit the formation of bone in a pilot animal study since cmBMP9 has been demonstrated to be highly effective in regenerating bone in a rat calvarial defect model. The results indicated that cmBMP9 increased bone formation (30% increase in area covered compared to control) and that SAPAE trended toward reducing the bone formation. These results suggest SAPAE could be useful as a chemical agent in reducing unwanted bone formation in implants loaded with cmBMP9.
The final chapter investigates synthesis of chemically modified RNA and the application of these cmRNAs to bone defects. EGFP cmRNA was first synthesized to measure transfection efficiency and viability of BMSCs transfected with PEI-cmRNA complexes. Then BMP-2 and FGF-2 cmRNAs were created from plasmid DNA. These cmRNAs were demonstrated to increase protein expression in vitro. The cmRNAs were then loaded into GAMs and used to treat rat calvarial defects.
In conclusion, gene therapy is a promising method for improving outcomes related to bone tissue engineering, and the techniques and data described in this thesis are paramount in furthering this field. The main takeaways from this thesis are calcium can improve gene therapy efficiency, and chemically modified is a more potent form of gene therapy as compared to plasmid DNA when target bone tissue.
Details
- Title: Subtitle
- Bone tissue engineering: nucleic acids and surface coatings
- Creators
- Timothy Acri
- Contributors
- Aliasger Salem (Advisor)Kyungsup Shin (Committee Member)Satheesh Elangovan (Committee Member)Lewis Stevens (Committee Member)Jonathan Doorn (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Pharmacy
- Date degree season
- Spring 2021
- DOI
- 10.17077/etd.006236
- Publisher
- University of Iowa
- Number of pages
- xvi, 160 pages
- Copyright
- Copyright 2021 Timothy Acri
- Language
- English
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references
- Public Abstract (ETD)
The goal of my dissertation is to improve bone tissue engineering through innovations in gene delivery and scaffold design. Autografts, the surgical practice of harvesting bone from the patient and transferring the bone to fill in the damaged bone at another location, are currently the gold standard for forming bone in cases such as dental implants, bony fusions, and traumatic fractures. However, having the harvest site in addition to the wound site increases pain, the risk of infection, and the costs experienced by the patient. Developing synthetic implants that efficiently regenerate bone will eliminate the need for autografts, ultimately improving surgical outcomes and patients’ quality of life.
Bone tissue engineering approaches have been held back due to safety concerns, cost of manufacturing, and efficacy of the implants. Through my dissertation work I have explored multiple avenues to enhance bone tissue regeneration including non-viral gene therapy, synthetic polymers, and mineral coatings. The results from this dissertation establish that the combination of calcium with a DNA-based therapeutic agent can improve bone tissue engineering outcomes and will provide evidence for the potential of using an RNA-based therapy for bone tissue engineering.
- Academic Unit
- Pharmacy; Craniofacial Anomalies Research Center
- Record Identifier
- 9984188579302771
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