Pre-osteoblastic cell response on porous ceramic biomaterials and nanoparticles for bone regeneration
Authors: Χατζηχαραλάμπους Χρυσταλλένη 
Published: September 2021

Due to common bone defects arising from trauma, tumors, infection or bone diseases, techniques to replace, restore, or regenerate bone have become a major clinical challenge [1]. Ceramic biomaterials (calcium phosphates, zirconia, and alumina) hold a prominent position among the strategies employed to address bone repair, while undergoing a continuous evolution to improve both mechanical and biological properties. Dense zirconia (ZrO₂) and alumina (Al₂O₃) based ceramics are widely used for load-bearing applications in bone repair due to their excellent mechanical properties and biocompatibility. Unfortunately, they are often regarded as ‘bio inert’ materials since no direct bone-material interface is created, which is crucial for implant fixation [2]. However, improved bone integration and long-term stability of the implant, may be achieved by facilitating the initial attachment of osteoblast precursor cells to orthopedic implant surfaces and several studies using diverse materials have demonstrated the importance of surface properties into this matter [3]. In this regard, the introduction of a porous structure is believed to enhance cell migration and proliferation thus enabling bone tissue growth and implant stability, but the biological properties of macroporous (pore size > 50 nm) zirconia and alumina ceramics remain largely unexplored. Therefore, investigating interactions between cells and porous ceramics through in vitro experimentation, is of great interest, as it may lead to identifying beneficial characteristics for improved biological behavior [4]. Taking into consideration the inherent strong mechanical properties of alumina and zirconia, their porous counterparts may be interesting as scaffolds for load bearing segmental bone defects. Also, among ceramic biomaterials, calcium phosphates constitute a major class of compounds for synthetic bone substitution materials, due to their chemical similarity to the inorganic matrix of bone. During the last decade, inorganic nanoparticles based on calcium phosphate are emerging as new bone substitution materials that can be used as growth factor gene delivery systems, promoting the process of bone regeneration locally [5, 6]. Currently, bone morphogenetic proteins are clinically used for the treatment of long-bone fractures, but their delivery needs improvement. Gene transfer is an improved way of delivering such growth factors, as it is possible to achieve high concentrations locally for an extended period of time. Consequently, suitable ceramic scaffolds with improved mechanical and biological characteristics, in combination with a bone morphogenetic growth factor delivery system, are integral parts of the strategy to heal large defects in load bearing sites [7]. Since zirconia and alumina ceramics are promising biomaterials for bone regeneration, we first investigated the effect of porosity on cell adhesion and proliferation (Chapter 3). The results from this part of the study indicated that highly porous ceramics exhibit better cell behavior compared to low porosity ceramics and this effect was more pronounced among zirconia rather than alumina ceramics. Therefore we focused on the investigation of the pre-osteoblastic osteogenic response employing highly porous zirconia, alumina as well as their zirconia-alumina composite (Chapter 4). We showed that the zirconia-containing ceramics supported a more pronounced cell response, compared to alumina. To further investigate the effect of chemical composition on cell response, we focused on two formulations of zirconia ceramics, namely the yttria-and the magnesia-stabilized zirconia, and investigated the pre-osteoblastic cell adhesion, proliferation and osteogenic potential on them (Chapter 5). Overall in this thesis, eight ceramic scaffolds of different composition and chemistry were employed, and the biological investigation was carried out using MC3T3-E1 pre-osteoblastic cells and focused on the cell adhesion, proliferation and differentiation potential of the cells within the scaffolds. The objective was to identify beneficial characteristics of porosity and chemistry for improved biological behavior in these new porous alumina and zirconia scaffolds, which possessed strong mechanical properties similar to cortical bone. Furthermore, the potential of calcium phosphate nanoparticles functionalized with plasmid DNA encoding for bone morphogenetic protein 7 (BMP-7), as a suitable gene delivery system for pre-osteoblasts was investigated. The investigation focused on transfection efficiency and the induction of a subsequence osteogenic response (Chapter 6). More specifically, an introduction in bone biology and biomaterial properties is given in Chapter 1. Ceramic biomaterials as well as recent advances of nanoparticles for bone applications are reviewed with emphasis on the materials used in this thesis. In Chapter 2, the experimental procedures for the in vitro biological investigation are described. Information is given on method principle and experimental steps taken for the optimization of various bioanalytical, molecular biology, and microscopic methods used in this study. Chapters 3, 4 and 5 describe and discuss results on the in vitro investigation onto porous alumina and zirconia ceramics. Specifically, Chapter 3 presents and discusses results on the effect of substrate porosity to the attachment and proliferation of pre-osteoblastic MC3T3-E1 cells on zirconia or alumina ceramics using three porosities for each composition. Cell proliferation was measured by assessing the cellular metabolic activity whereas Scanning Electron Microscopy (SEM) and fluorescence microscopy were employed to qualitatively support the results and evaluate cell morphology. Cell adhesion and metabolic activity was found comparable among low porosity zirconia or alumina ceramics. As porosity increased, cell response to alumina ceramics was found unaffected. In contrast, cell response to zirconia ceramics was sensitive to changes in porosity, and higher porosity favored better cell spreading and growth, consequently leading to the formation of a uniform extracellular matrix as observed by SEM. Therefore, between the highest porosity materials in this study, cell response on zirconia (50% porosity) was found superior to alumina (61% porosity). Results showed that an average pore size of ~150 μm and 50% porosity can be considered beneficial to cellular growth on zirconia ceramics. In Chapter 4, we focused on ceramics of similar high porosity but different composition; alumina (61% porosity), zirconia (50% porosity) and a composite material synthesized of 80% zirconia-20% alumina (60% porosity). We performed in vitro cell-material investigations comparing the adhesion, longer term proliferation (up to 21 days) and differentiation of mouse pre-osteoblasts MC3T3-E1 on the materials. While all three ceramics demonstrate a strong cell attachment, better cell spreading was evidently observed on zirconia-containing substrates. Significantly higher cell growth was quantified on the latter ceramics, revealing an increased alkaline phosphatase activity, higher collagen production and increased calcium mineralization compared to alumina. Hence, these porous zirconia-containing ceramics elicit superior biological responses over porous alumina of similar porosity, promoting not only enhanced cell proliferation but also differentiation. Since two different formulations of zirconia ceramics, namely yttria stabilized zirconia (YSZ) and magnesia stabilized zirconia (MgSZ) are often used in biomedical applications, we investigated potential differences between cell response in porous YSZ and MgSZ in terms of growth and osteogenic response, including gene expression of key osteogenic markers. As presented in Chapter 5, MC3T3-E1 pre-osteoblasts were used to investigate the proliferation, alkaline phosphatase (ALP) activity, collagen secretion and expression profile of four genes involved in bone metabolism of cells on porous ceramics. Scanning electron microscopy and fluorescence microscopy were employed to visualize cell morphology and growth. Pre-osteoblasts adhered well on both ceramics but cell numbers on YSZ were higher. Cells exhibited an increase in ALP activity and collagen deposition after 14 days on both MgSZ and YSZ, with higher levels on YSZ. Real-time quantitative polymerase chain reaction (qPCR) was used to investigate gene expression of osteogenic markers. Results showed that the expression of bone sialoprotein (Bsp) and collagen type I (col1aI) were significantly higher on YSZ. No significant differences were found in their ability to regulate the early gene expression of Runx2and Alp. Nevertheless, the bio mineralized calcium content was similar on both ceramics after 21 days, indicating that despite chemical differences, both scaffolds direct the pre-osteoblasts towards a mature state capable of mineralizing the extracellular matrix. Finally, Chapter 6 concerns with calcium phosphate (CaP) nanoparticles (NPs) and their potential to transfect pre-osteoblastic cells and trigger an osteogenic response via the secretion of hBMP-7. Polyethylenimine-stabilized calcium phosphate nanoparticles loaded with plasmid DNA, which encodes either for bone morphogenetic protein 7 (BMP-7) or for enhanced green fluorescent protein (EGFP) were employed. They were used for the transfection of the pre-osteoblastic cells MC3T3-E1 and showed high transfection efficiency (25%) together with low cytotoxicity. Their potential to induce an osteogenic response by transfection was demonstrated by measuring ALP activity and calcium deposition. The expression of the osteogenic markers Alp, Runx2, ColIa1and Bsp was investigated by means of real-time quantitative polymerase chain reaction (qPCR). It was shown that phBMP-7-loaded nanoparticles can provide a means of transient transfection and localized production of BMP-7 in MC3T3-E1 cells, with a subsequent increase of two osteogenic markers, specifically alkaline phosphatase activity and calcium matrix bio-mineralization. In conclusion, this PhD work highlights the potential of macroporous alumina and zirconia to be used in bone repair as non-degradable scaffolds of high mechanical strength. The results bring out differences in the pre-osteoblastic cell response arising from varying porosity or chemistry that would be valuable for future in vivo studies. Also in this study, calcium phosphate nanoparticles are revealed as a non-toxic system for the delivery of nucleic acids in pre-osteoblasts with the ability to induce temporary synthesis of BMP-7 and the potential for a subsequent osteogenic response. The system showed good transfection efficiency and appears promising for future in vivo studies. Successful application of nucleic acid delivery for bone regeneration would be a major breakthrough in modern medicine.