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Identifier 000417976
Title Biological response of innovative biomaterials for bone tissue engineering applications
Alternative Title Μελέτη βιοσυμβατότητας καινοτόμων βιοϋλικών με χρήση κυττάρων για αναγέννηση οστίτη ιστού
Author Γεωργοπούλου, Ανθή
Thesis advisor Χατζηνικολαϊδου, Μαρία
Reviewer Βαμβακάκη, Μαρία
Ποντίκογλου, Χαράλαμπος
Μητράκη, Άννα
Βενυχάκη, Μαρία
Μπακοπούλου, Αθηνά
Ηλιόπουλος, Αριστείδης
Abstract Bone is a multi-scale, hierarchically structured composite tissue with the ability to regenerate when damaged. However, when bone defects are large enough or critical-sized, they cannot regenerate and the intervention in the form of bone grafts is required [1]. Due to the large economic impact of bone grafts and their companion materials, the investigation of suitable alternatives is a major clinical challenge in medicine. Bone tissue engineering strategies guide tissue regeneration via osteoconductive and highly porous scaffolds. Different classes of materials have been utilized for scaffold fabrication including a variety of ceramics and polymers. Polymers, either natural or chemically synthesized, should be biodegradable with great design flexibility molecules. Chitosan is a natural, biocompatible and biodegradable polymer derived from alkaline deacetylation of chitin. It’s an important biomaterial as it evokes minimal foreign body response and fibrous encapsulation as well as it promotes the wound healing. Moreover, as a cationic polymer, chitosan possesses antibacterial properties and supports the adhesion and proliferation of osteogenic cells due to its hydrophilicity. Taking into consideration all the above advantages of this natural polymer, the main objective during this thesis was to combine chitosan with other synthetic (section 3.1) or natural (section 3.2) polymers and examine their biocompatibility and their potential to promote osteogenesis. In section 3.1, the results on the biocompatibility and osteogenic assessment of a chitosan-graft-polycaprolactone (CS-g-PCL) copolymer are presented, and in section 4.1 these results are discussed. These copolymers were synthesized via a multi-step process and were evaluated as a potential biomaterial for the adhesion, proliferation and differentiation of MC3T3-E1 pre-osteoblastic cells. CS-g-PCL material surfaces were synthesized and characterized by the research group of Prof. Vamvakaki. PCL is a synthetic, biocompatible, biodegradable and non-toxic polyester with good mechanical properties and high plasticity. Previous studies have included CS/PCL blends in skin and osteochondral tissue engineering, by combining the biocompatibility and biological properties of chitosan with the suitable mechanical properties of PCL [2]. Moreover, our group has shown that CS-g-PCL copolymers promote the viability and proliferation of Wharton’s jelly mesenchymal stromal cells and could potentially be used in myocardium regeneration. Our results indicated a strong adhesion of MC3T3-E1 pre-osteoblastic cells with a characteristic spindle-shaped morphology from the first day of culture onto the copolymer surfaces. The viability and proliferation of the cells on the CS-g-PCL surfaces, after 3 and 7 days in culture, were significantly higher compared to the cells cultured on the tissue culture treated polystyrene (TCPS) control. The osteogenic potential of the pre-osteoblastic cells cultured on CS-g-PCL surfaces was evaluated by determining various osteogenic differentiation markers. Specifically, alkaline phosphatase activity levels show significantly higher values at both time points compared to TCPS, while secreted collagen into the extracellular matrix was found to be higher on day 7. Calcium biomineralization deposited into the matrix was significantly higher for the CS-g-PCL copolymer after 14 days in culture, while intracellular osteopontin expression was increased in cells grown on CS-g-PCL surfaces compared to TCPS. The expression levels of alkaline phosphatase (alp), collagen type I (collaI) and bone sialoprotein (bsp) genes were found to be similar for cells cultured either on CS-g-PCL or on TCPS. In section 3.2, the results on the fabrication of 3D porous scaffolds combining chitosan with another natural polymer, gelatin, as well as their biocompatibility and osteogenic potential are presented, and these results are discussed in section 4.2. Gelatin is a biocompatible and biodegradable polymer that promotes cell adhesion, proliferation, migration and differentiation as it retains the Arg-Gly-Asp [RGD] motif, an important sequence found in collagen, well known for mediating cell attachment [3]. The produced chitosan/gelatin scaffolds (CS:Gel) were tested for their capacity to promote osteogenic response. CS:Gel scaffolds have also been used in many tissue engineering applications such as skin [4], cartilage [5] and bone [6] regeneration. CS:Gel scaffolds were fabricated by chemical crosslinking using either glutaraldehyde or genipin as a crosslinker. In both cases the scaffolds were produced by freeze-drying. Our results suggested that CS:Gel scaffolds crosslinked with either glutaraldehyde or genipin elicit a similar structure morphology with a small difference in the pore size ranging between 40-120 μm and 70-170 μm respectively. Due to the more efficient cell adhesion and infiltration that was observed for the glutaraldehyde crosslinked CS:Gel scaffolds, a more detailed in vitro evaluation was set. Among the four different ratios of CS:Gel scaffolds (20%-80% CS:Gel, 80%-20% CS:Gel, 40%-60% CS:Gel and 60%-40% CS:Gel), the viability and proliferation was significantly increased for cells cultured on 40%-60% CS:Gel scaffolds for 5 and 7 days. Moreover, 40%-60% CS:Gel scaffolds crosslinked with two different concentrations of glutaraldehyde (0.1% v/v and 1% v/v) were used to examine the biological response of MC3T3-E1 pre-osteoblastic cells. These scaffolds show significant cell viability and proliferation increase of MC3T3-E1 pre-osteoblastic cells after 7 days in culture compared to 2D CS:Gel substrates. Collagen secreted into the extracellular matrix by the pre-osteoblasts cultured for 4 and 7 days on the CS:Gel scaffolds, indicate a significant increase when compared to TCPS control surface. Moreover in order to test the stability of these scaffolds, degradation assay showed that the degradation rate of 40%-60% CS:Gel scaffolds crosslinked with 0.1% v/v or 1% v/v glutaraldehyde was 48% and 18% of weight loss after 21 days, respectively. CS:Gel scaffolds crosslinked with 0.1% glutaraldehyde were also tested for their ability in sustaining viability, proliferation and extracellular matrix formation of primary cells. Human bone marrow (hBM) derived mesenchymal stem cells (MSCs), abbreviated as hBM-MSCs, were cultured on these scaffolds and were able to adequately grow, proliferate and secrete collagen in our conditions. The above observations extend also to co-culture setups. Co-culture of hBM-MSCs with human umbilical vein endothelial cells (HUVECs) on CS:Gel scaffolds resulted in increased proliferation rates for both cell types compared to monocultures, which is attributed to the availability of secreted signal molecules that promote their proliferative behaviour. This PhD thesis focuses on the potential of chitosan-based biomaterials, through their investigation in appropriate cell systems in vitro, to be used in bone repair as biocompatible and biodegradable scaffolds. Both CS-g-PCL and CS:Gel material surfaces support the viability, proliferation and differentiation of MC3T3-E1 pre-osteoblastic cells and hBM-MSCs, demonstrating their potential use in cancellous bone tissue regeneration. This thesis is structured as follows: Section 1 provides an introduction to this thesis, comprising background, related work, and a short summary of thesis contributions. Section 2 describes the materials and methods used for the investigation of both chitosan-based biomaterials. Section 3 describes the main thesis results, including the data obtained from the in vitro biocompatibility and osteogenic differentiation capacity of both biomaterials. Section 4 discusses the results of this study. Section 5 outlines the thesis conclusions, and Section 6 discusses some perspectives and future work, with focus on preliminary results on nanohydroxyapatite containing chitosan/gelatin scaffolds (appendix, section 7).
Language English
Subject Natural and chemical polymers
Φυσικά και χημικά πολυμερή
Issue date 2018-07-20
Collection   Faculty/Department--Faculty of Sciences and Engineering--Department of Materials Science and Technology--Doctoral theses
  Type of Work--Doctoral theses
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