Real-time imaging of cell mineralization
Abstract
A calcificação ectópica tem-se revelado um problema emergente na área da saúde devido à sua correlação com várias doenças, como a doença renal crónica (DRC), hipertensão, diabetes mellitus (DM), doença cardiovascular aterotrombótica e envelhecimento. Este tipo de calcificação está associado principalmente à deposição anormal de complexos de sais de cálcio e fosfato– hidroxiapatite – em células do músculo liso nos vasos sanguíneos. Estudos atuais têm revelado o papel de proteínas plasmáticas que poderão inibir a calcificação tal como a fetuína-A. A presente dissertação visa estudar o papel da proteína plasmática – Fetuína-A – na calcificação ectópica, utilizando o dispositivo de aquisição de imagens em tempo real –CELLCYTE – para seguir e avaliar a sequência de eventos biológicos e a cinética celular. Desta forma, uma variante recombinante de fetuína-A com propriedade de fluorescência, mFa-mRuby, foi produzida e estudada, uma vez que esta proteína é caracterizada por se ligar a depósitos minerais intrínsecos da calcificação ectópica. Protocolos como expressão proteica em células de ovário de hamster chinês (CHO), purificação, transfecção de alto rendimento, Western Dot Blot e SDS-PAGE foram realizados para produzir as amostras de fetuína-A em estudo. Para mimetizar a calcificação patológica no corpo humano, cultura de duas linhas celulares de células do músculo liso vasculares (VSMC) – IM1 e IM3 - foram cultivadas assim como células de osteossarcoma (SaOs-2) e cementoblastos (OCCM), para mimetizar mineralização fisiológica. Todas as linhas celulares foram incubadas em meio de cultura ou de calcificação, para avaliar a proliferação celular e a mineralização ao longo de 7 dias, com ou sem adição de fetuína-A ao meio. Os depósitos minerais foram revelados utilizando o protocolo de coloração de fetuína-A e posterior coloração Alizarin Red. Os resultados observados revelaram uma maior taxa de calcificação das VSMC em comparação com os mineralizadores profissionais (SaOs-2 e OCCM) e uma ligeira inibição da calcificação e mineralização das quatro linhas celulares com adição de fetuína-A em ambos os meios de cultura. Em suma o presente trabalho, proporcionou uma base robusta para pesquisas aprofundadas sobre a calcificação ectópica e o papel da fetuína-A, assim como expandir as aplicações médicas desta proteína plasmática promissora.
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Universidade do Minho Escola de Engenharia Eduarda Margarida Alves Silva Real-Time Imaging Of Cell Mineralization Outubro de 2024 Real-Time Imaging Of Cell Mineralization Eduarda Margarida Alves Silva UMinho | 2024
Eduarda Margarida Alves Silva Real-Time Imaging Of Cell Mineralization Outubro de 2024 Dissertação de Mestrado Mestrado Integrado em Engenharia Biomédica Ramo de Engenharia Clínica Trabalho efetuado sob a orientação do Professor Doutor Wilhelm Jahnen-Dechent RWTH Aachen e do Professor Doutor Francisco Miguel Gama Universidade do Minho
ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição CC BY https://creativecommons.org/licenses/by/4.0/
iii “I was taught that the way of progress is neither swift nor easy.” - Marie Curie
iv ACKNOWLEDGMENTS This acknowledgment section expresses my deepest gratitude to everyone involved in this important path in my academic life. Firstly, I would like to express my deep gratitude to Prof.Wilhelm Jahnen-Dechent for the kindness of accepting me in his laboratory, for the resources and facilities made available to me and for sharing his immense knowledge. To Prof.Francisco Miguel Gama for being my supervisor, for helping and supporting my Erasmus journey, and for the constant availability, guidance, and insightful feedback. To Camilla for being the best supervisor I could ever ask for. For the unwavering support in every step of my work, for being available any time to help me, and for guiding me through the construction of a well-constructed master’s thesis. My deepest gratitude. To Aaron, Christian, and Robert for always being available to help me and share their wide knowledge. To my dearest Mónica for making a 360 much-needed change in my life, and for facilitating and introducing me to the opportunity to take part in the Erasmus mobility exchange, a life-changing experience. For your constant care and kindness, I will always cherish you. To my dearest friends who gave support even miles away from home, for being there for me all the time and for encouraging me when I needed it most. I'm deeply thankful for your friendship and a constant source of strength during this journey. To Maria, Juliana, and Helena for becoming my second family and making Aachen our little piece of home. To my family for supporting all my decisions through this path and during my Erasmus program and always providing emotional support and encouragement in every step. To my two little guiding stars in heaven, Grandpa, and Uncle, I did it!
v DECLARAÇÃO DE INTEGRIDADE Declaro ter atuado com integridade na elaboração do presente trabalho académico e confirmo que não recorri à prática de plágio, nem a qualquer forma de utilização indevida ou falsificação de informações ou resultados em nenhuma das etapas conducente à sua elaboração. Mais declaro que conheço e que respeitei o Código de Conduta Ética da Universidade do Minho.
vi RESUMO Imagem em tempo real de Calcificação Celular A calcificação ectópica tem-se revelado um problema emergente na área da saúde devido à sua correlação com várias doenças, como a doença renal crónica (DRC), hipertensão, diabetes mellitus (DM), doença cardiovascular aterotrombótica e envelhecimento. Este tipo de calcificação está associado principalmente à deposição anormal de complexos de sais de cálcio e fosfato– hidroxiapatite – em células do músculo liso nos vasos sanguíneos. Estudos atuais têm revelado o papel de proteínas plasmáticas que poderão inibir a calcificação tal como a fetuína-A. A presente dissertação visa estudar o papel da proteína plasmática – Fetuína-A – na calcificação ectópica, utilizando o dispositivo de aquisição de imagens em tempo real –CELLCYTE – para seguir e avaliar a sequência de eventos biológicos e a cinética celular. Desta forma, uma variante recombinante de fetuína-A com propriedade de fluorescência, mFa-mRuby, foi produzida e estudada, uma vez que esta proteína é caracterizada por se ligar a depósitos minerais intrínsecos da calcificação ectópica. Protocolos como expressão proteica em células de ovário de hamster chinês (CHO), purificação, transfecção de alto rendimento, Western Dot Blot e SDS-PAGE foram realizados para produzir as amostras de fetuína-A em estudo. Para mimetizar a calcificação patológica no corpo humano, cultura de duas linhas celulares de células do músculo liso vasculares (VSMC) – IM1 e IM3 - foram cultivadas assim como células de osteossarcoma (SaOs-2) e cementoblastos (OCCM), para mimetizar mineralização fisiológica. Todas as linhas celulares foram incubadas em meio de cultura ou de calcificação, para avaliar a proliferação celular e a mineralização ao longo de 7 dias, com ou sem adição de fetuína-A ao meio. Os depósitos minerais foram revelados utilizando o protocolo de coloração de fetuína-A e posterior coloração Alizarin Red. Os resultados observados revelaram uma maior taxa de calcificação das VSMC em comparação com os mineralizadores profissionais (SaOs-2 e OCCM) e uma ligeira inibição da calcificação e mineralização das quatro linhas celulares com adição de fetuína-A em ambos os meios de cultura. Em suma o presente trabalho, proporcionou uma base robusta para pesquisas aprofundadas sobre a calcificação ectópica e o papel da fetuína-A, assim como expandir as aplicações médicas desta proteína plasmática promissora. Palavras-chave: Calcificação ectópica; CELLCYTE; Fetuína-A; SDS-PAGE; VSMC
vii ABSTRACT Real-Time Imaging of Cell Mineralization Ectopic calcification has become an emerging problem in the health field due to its correlation to various diseases such as chronic kidney disease (CKD), hypertension, diabetes mellitus (DM), atherothrombotic cardiovascular disease, and aging. This type of calcification is associated mainly with the abnormal deposition of calcium and phosphate complexes– hydroxyapatite – onto smooth muscle cells in blood vessels. Current studies rely on the comprehension of the role of proteins that can play a role in inhibiting calcification, such as mineral chaperones like fetuin-A. Therefore, this thesis aims to study the role of plasma protein - fetuin-A - in ectopic calcification, by using the real-time acquisition imaging device CELLCYTE, to follow and evaluate the sequence of biological events and cell kinetics. Therefore, recombinant fluorescent Fetuin-A probe, mFa-mRuby, was produced and studied, as Fetuin-A is known to bind to calcium phosphates. Protocols like protein expression in Chinese hamster ovary cells (CHO), purification, high-yield transfection, Western Dot Blot and SDS-PAGE were performed to produce the fetuin-A probes. To mimic the pathologic calcification in the human body, two cell lines of vascular smooth muscle cells (VSMC) were cultured (IM1 and IM3). To mimic physiological mineralization osteosarcoma cells (SaOs-2) and cementoblasts (OCCM) were cultured. All cell lines were incubated in a culture or calcification medium to assess cell proliferation and mineralization throughout a 7-day study, with or without fetuin-A addition to the medium. Mineral deposits were revealed by using fetuin-A staining protocol and Alizarin red staining. Those results revealed a higher calcification rate of the VSMC in comparison to the professional mineralizers (SaOs-2 and OCCM) and a slight inhibition of calcification and mineralization of the four cell lines with both mediums with the addition of Fetuin-A. In conclusion the present work, provided a robust foundation for in-depth research on ectopic calcification and the role of fetuin-A, as well as expand the medical application of these promising plasma protein. Keywords: CELLCYTE; Ectopic Calcification; Fetuin-A; SDS-PAGE; VSMC
Real-Time Imaging of Cell Mineralization xiv Figure 15:Blast Alignment of the amino acids of the protein chains of fetuin-A variants from mammals: Mouse, Cotja and two different protein chains lengths from chicken using the Uniprot Software. ........ 80 Figure 16:Percentage Identity Matrix from Uniprot Software of the four different fetuin-A protein variants from mammals: Mouse, Cotja and Chicken. ...................................................................................... 80 Figure 17: Potential representation of a 3D-Model of the protein structure of Fetuin-A derived from chicken using the ChimeraX software. ........................................................................................................... 81 Figure 18:Neubauer Chamber Layout for counting live/dead cells. .................................................... 82 Figure 19: General representation of a Cell Growth Curve .................................................................. 83 Figure 20: 24-well-plate display used for the calcification assay. ........................................................ 84
Real-Time Imaging of Cell Mineralization xv LIST OF SYMBOLS °C µm g/L % w/v mM M Rpm mL µL ng g mA kDa Degrees Celsius Micrometer Grams per Liter Percentage Weight per volume Micromolar Molar Rounds per minute Milliliter Microliter Nanograms Gram Milliampere Kilo Dalton
xvi LIST OF TABLES Table 1:Description of the medium composition of the 3 media necessary for culture, growth and proliferation of E.Coli . ....................................................................................................................... 29 Table 2: Reagents required for the western dot blot protocol. ............................................................. 35 Table 3:List of buffers and solutions used in the affinity chromatography process as well as each reagent concentration needed. ...................................................................................................................... 37 Table 4:List of reagents for the production of the gels required for the SDS-PAGE method. This table represents the compounds necessary to produce 2 gels. ................................................................... 38 Table 5: Constitution of each solution and concentrations of the reagents utilized in the calcium-phosphate precipitation inhibition assay. ............................................................................................................ 41 Table 6: Range of concentration of protein used in the calcium phosphate precipitation inhibition assay. ........................................................................................................................................................ 42 Table 7: List of reagents and each volume used during the preparation of both mediums necessary for the different phases of the calcification assay. ................................................................................... 46
Real-Time Imaging of Cell Mineralization 17 1. MOTIVATION AND GOALS Ectopic calcification has become an emerging alert in the health field as its treatment is complex. This pathologic condition is characterized by the inappropriate deposit and precipitation of calcium phosphate in the soft tissues along the human body that leads to calcification of the smooth muscle cells (Hutcheson et al., 2015; Leopold, 2015; Snijders et al., 2023). Various disorders such as hypertension, chronic kidney disease (CKD), diabetes mellitus (DM), atherothrombotic cardiovascular disease and aging can be associated with ectopic calcification (Hutcheson et al., 2015; Leopold, 2015; Millán et al., 2021; Snijders et al., 2023). This type of calcification is associated mainly with the final form of phosphocalcic crystals known as hydroxyapatite in smooth muscle cells (Giachelli, 1999; Kempf et al., 2021). Smooth muscle cells are responsible for the contraction and relaxation process of the vessels along the body to control blood flow and tissue oxygenation(Webb, 2003). This type of cell is mostly affected by unwanted calcification, as described previously(Hutcheson et al., 2015; Millán et al., 2021; Snijders et al., 2023). However, biological processes such as the molecular pathogenesis of ectopic calcification are poorly understood and are still largely unknown(Snijders et al., 2023). Therefore, an early diagnosis is possible through the quantification of amorphous calcium phosphate (ACP), as this is the main precursor of calcification. The 𝐶𝑎2+ (calcium ions) and 𝑃𝑂43− (phosphate ions) from ACP are converted in hydroxyapatite (HAP) crystals associated to calcification (Millán et al., 2021). Currently, the treatments available are based on surgical removal of the calcified tissues or breaking the calcification apart with sound waves. Independently of the treatment chosen, both are extremely invasive for the patient and can be dangerous to the point of putting the patient´s life at risk. Nevertheless, the heterogeneity of the disease also hinders the establishment of efficient treatment and the irreversibility of calcified tissues (Dzhanaev et al., 2023; Snijders et al., 2023) Ectopic vascular calcification has been reported to be an extremely complex biological process that implicates cell regulation processes like activation of cellular signalling pathways, presence of calcification inhibitors, genetic interference and hormonal activity (Leopold, 2015). Current studies rely on the comprehension of the role of proteins that play a role in inhibiting calcification, such as mineral chaperones, which are important to stabilize minerals in solution in the case of supersaturated solutions (Dzhanaev et al., 2023).
Real-Time Imaging of Cell Mineralization 18 Therefore, an emerging mineral chaperone protein has been studied and reported to be an active factor in the inhibition of cell calcification(Dzhanaev et al., 2023). The plasma protein fetuin-A/ 𝛼2HSglycoprotein has been found to be a key element in the inhibition of ectopic calcification by preventing undesirable biomineralization in the plasma on the blood and in smooth tissue fluids, due to its early state calcification inhibition activity and its capacity to bind and stabilize precursor of calcium phosphate deposits, by forming colloidal calciprotein particles (CPPs)(Dzhanaev et al., 2023; Jahnen-Dechent et al., 2008; Schinke et al., 1996; Westenfeld et al., 2007). This thesis aims to study the role of the plasma protein fetuin-A in cell calcification and mineralization and the production of recombinant protein with fluorescence proprieties entitled murine Ruby Fetuin-A, also referred to as mFa-mRuby. Furthermore, the real-time imaging of the differences between mineralization and calcification, by using osteosarcoma cells (SaOs-2) and cementoblast (OCCM) for mimicking professional mineralizers and two cell lines of smooth muscle cells (IM1 and IM3, same cell type, but from different patients) to mimic calcifying cells. A recombinant chicken Fetuin-A was equally produced to be delivered to Berlin BioGenes for Antibodies production, therefore not included in the subsequent methods. Thus, the study had the following aims: • Amplification of Fetuin-A plasmids - mFa-mRuby and Chicken Fetuin-A - in Escherichia Coli cells; • Transformation and expression of Fetuin-A recombinant proteins: mFa-mRuby and Chicken Fetuin-A - in Chinese Hamster Ovary (CHO) cells; • Purification and quantification of Fetuin-A protein variants produced; • Calcification assays on vascular smooth muscle cells, cementoblasts, and osteosarcoma cells under culture or calcification conditions and with or without the addition of the mFa-mRuby, the recombinant protein produced previously; • Live cell imaging with the fluorescence Fetuin-A probe – mFa-mRuby; • Cellular calcification and mineralization assessment.
Real-Time Imaging of Cell Mineralization 19 2. STATE OF ART 2.1 Physiologic Mineralization The human blood is a complex, dynamic fluid composed of various elements including minerals that allow it to function properly. Therefore, minerals like calcium and phosphate stand out as being one of the most vital ones, due to their role in different biological processes, like maintaining the normal cardiovascular and neuro-muscular function, enzyme-mediated and cellular signalling processes, and ensuring structural integrity to the skeleton, which serves as the primary deposition site of mineral ions in the body (Evenepoel & Wolf, 2013; Gharibzahedi & Jafari, 2017). These minerals can be found mainly in teeth and bones, where they play a critical role in human physiology and skeletal mineralization (Evenepoel & Wolf, 2013; Gharibzahedi & Jafari, 2017; Schäfer et al., 2003). For example, the bones contain around 99% and 80% of the body's calcium and phosphate concentration, respectively (Bonjour, 2011). The balance of these ions regulated at renal and intestinal levels leads to the maintenance of bone health (osteoporosis management) and influences the bone-forming and bone-resorbing cells (Bonjour, 2011). Hard tissue biomineralization can be described as the process where ions, like calcium and phosphate, accumulate in a coordinated way to form bone and teeth tissue. This biological process is reported to be a correlation between extracellular matrix suitable for mineralization, high concentration of extracellular inorganic phosphate and calcium and levels of mineralization inhibitors that may be expressed locally or systemically (Brylka & Jahnen-Dechent, 2013). Calcium and phosphate deposits in and outside the collagenous fibrous network, a significant component of the extracellular matrix, in the cells, and form hard tissue structures. In the process of biomineralization, one of the main purposes is the formation of hydroxyapatite – a mixture of inorganic phosphate and calcium ions – one of the principal components of fully developed hard tissues. For example, particularly in bones, osteoblast secret an extracellular matrix and its subsequent mineralization by crystalline hydroxyapatite formation that forms in a coordinated way along with the organic protein molecules, strengthening the organic matrix due to Ca and Pi influx in the cells in hard tissues (Bonjour, 2011; Sharma et al., 2021; Snijders et al., 2023). This mineral homeostasis is one of the most important prerequisites in the biomineralization process since maintaining the correct concentration of calcium and phosphate ions allows physiologic mineralization.
Real-Time Imaging of Cell Mineralization 20 Also, this dynamic equilibrium of intake and uptake of ions is reported to be highly controlled by several regulatory processes – such as feedback inhibitions - and to be responsible for preventing the formation of calcific deposits in soft tissues (Sharma et al., 2021; Snijders et al., 2023). However, a disturbance in these biologic processes influencing the Ca and P concentration balance and its regulatory processes may lead to calcification of soft tissues, also called pathological ectopic calcification. This study aims to comprehend the mechanisms behind the process of biomineralization and furthermore analyze the biological differences, cellular behaviour and responses associated with this process and calcification. 2.2 Vascular Calcification – Ectopic Calcification Cellular mineralization is one of the most vital physiologic processes in the human body, as it is integral in the development and maintenance of bone and teeth tissues. However, not all forms of mineralization are beneficial for the organism, such as in the case of “soft” tissue mineralization entitled vascular calcification (Kempf et al., 2021; Schäfer et al., 2003). Vascular calcification is a pathological mineralization widely described as the inappropriate deposition of calcium and phosphate in soft tissues such as blood vessels, joints, or tumours (Giachelli, 1999; T. Li et al., 2022; Persy & D’Haese, 2009; Snijders et al., 2023). The mineral deposits within the soft tissues tend to disrupt the biomechanical function of these tissues, restricting the patient's mobility and possibly contributing to mortality (Hutcheson et al., 2015). This complex biological process involves elaborate pathophysiological mechanisms, that can induce metabolic alterations due to calcium and phosphate dysfunction, absence of calcification and mineralization inhibitors, osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) to calcifying vascular cells, the release of apoptotic bodies or necrotic debris, deposits calcium phosphate hydroxyapatite due to modifications in mineral homeostasis or establishment and development of ectopic mineralization and nucleation complexes due to bone remodelling (Pan et al., 2023). Also, local and systemic changes within the human body can trigger or favor ectopic calcification by modifying the supply of calcium and phosphate, ectopic activation of osteogenic signalling, or by downregulating inhibitory mechanisms (Schäfer et al., 2003). Hence, a continuous, in-depth study of the available inhibitors in these biological processes, along with an understanding of their role and contribution, could be the basis for developing and establishing therapy to prevent disease-associated calcification.
Real-Time Imaging of Cell Mineralization 21 This pathology is classified broadly, according to the part of the body and soft tissue it affects, such as kidney, skin, or tendons, as illustrated in Figure 1 (Giachelli, 1999). Some literature even describes the existence of a genetic component associated with ectopic calcification correlated with a main disease like atherosclerosis, cancer or chronic renal failure. In this case, the inappropriate deposition of minerals can be broadly distributed and affect multiple organs (Schäfer et al., 2003). Vascular calcification can be correlated to cardiovascular disease, by affecting arteries and heart valves. Although valves are predisposed to calcify with apatitic mineral deposits, calcification in the arteries is followed by atherosclerotic plaque burden, increased risk of myocardial infarction, increased ischemic episodes in peripheral vascular disease, and increased risk of dissection, following angioplasty. All these repercussions are the basis of some of the most common cardiovascular diseases: hypertension, atherosclerosis, coronary artery disease, aortic valve stenosis and atherothrombotic cardiovascular disease as outlined in Figure 1 (Leopold, 2015; Pan et al., 2023; Snijders et al., 2023). Due to the deposits, these tissues become stiffer, harder and more susceptible to general failure, which can lead to a significant disease burden. On the other hand, ectopic calcification is strongly associated with the pathological mechanism of conditions like chronic diseases, such as atherosclerosis, hypercholesterolemia, diabetes mellitus (DM), hypertension, chronic kidney disease (CKD) and coronary artery disease (Figure 1). It may also manifest in the skin, as seen in rare conditions like Pseudoxanthoma Elasricum (PXE), systemic calcification, where skin plaque might arise or develop in the basal ganglia like the Primary Familial Brain Calcification (PFBC) condition (Snijders et al., 2023).
Real-Time Imaging of Cell Mineralization 22 Vascular calcification can be classified into different types based on the layer/area of the blood vessels where the calcification begins to appear: intimal calcification, media calcification, or cardiac valve calcification as presented in Figure 2. Intimal calcification is typically associated with atherosclerotic diseases and the development of atherosclerotic plaques, which may eventually lead to microcalcifications. These microcalcifications tend to scatter along the inner wall of the blood vessels in the form of small spots (Figure 2, Li et al., 2022; Nakahara et al., 2017). On the other hand, medial calcification, as the name suggests, occurs in the media layer of blood vessels and is more common in small and medium arteries. This type of calcification is associated with aging, diabetes, and chronic kidney disease (Zazzeroni et al., 2018). Cardiac valve calcification is normally correlated to metabolic diseases like diabetes or uremia, resulting from mechanical stress and inflammation (Shekar & Budoff, 2018). Figure 1:Example of different diseases correlated to vascular calcification (VC). VC plays a key role in the pathological process of some diseases, like cerebrovascular diseases, peripheral arterial diseases, and coronary artery disease (Pan et al., 2023).
Real-Time Imaging of Cell Mineralization 23 Moreover, vascular calcification is followed by a complex biological process involving the formation of precipitates (Johnson et al., 2006; Persy & D’Haese, 2009; Shekar & Budoff, 2018; Zazzeroni et al., 2018). Those precipitates are typically composed of calcium-phosphate complexes, specifically hydroxyapatite (HAP) crystals. In the early stages of calcification, these deposits are described as consisting of amorphous calcium phosphate (ACP), which gradually transforms into HAP crystals over time (Figure 3, Millán et al., 2021; Persy & D’Haese, 2009). Therefore, when the calcium and phosphate ions level rise and exceed the solubility product, it tends to create a supersaturated environment that initiates the calcification process and promotes the nucleation of calcium-phosphate deposits. As illustrated in Figure 3, initially the small ACP clusters are unstable and disordered, but with the constant aggregation and organization of these, more stable and crystalline structures start to appear, such as HAP crystals. As depicted in Figure 3, the transformation of small clusters into HAP crystals tends to be an irreversible process, even with the normalization of the calcium and phosphate concentration levels in the vascular layers (Millán et al., 2021). Figure 2:Schematic representation of the different calcification and constitution of ectopic calcification in the blood vessels and valves within the human body (Millán et al., 2021).
Real-Time Imaging of Cell Mineralization 30 Since it was only necessary for the growth of E. coli cells it was crucial the use of antibiotics to create a selective bacterial medium . Therefore, a concentration of 100 g/mL of ampicillin was adjusted and spiked into the culture medium. The ampicillin used was provided from a previously prepared stock solution sterile filtered using a 0.22 m syringe filter (VWR, Radnor, Pennsylvania, USA) and stored at 20°C. Sterile Petri dishes with the selective bacteria conditions were stored at room temperature until further use (chapter 3.6.1). E.Coli suspension cultures were let to grow overnight in autoclaved Erlenmeyer flasks at 37°C and 250 rpm in a shaking incubator. For each suspension culture volume, 1/5 of the cell suspension was filled with LB medium to ensure adequate gas exchange. The transformation of E.Coli cells was afterward performed as detailed in chapters 3.6.1 and 3.6.2. 3.3 Chinese Hamster Ovary Cells – ExpiCHO-𝐒𝐓𝐌 Chinese Hamster Ovary cells (Gibco, Thermo Scientific, Waltham, USA) were used for protein expression due to their high-density suspension growth properties, resembling human post-translational modification patterns and their ability to produce satisfactory yields and high-quality protein expression (Z. M. Li et al., 2022). Firstly, ExpiCHO-STM cells were taken and defrosted from a liquid nitrogen tank (-196 C). The cryovial was warmed in the water bath (37C) and added to a previously prepared Erlenmeyer flask with 30 mL of prewarmed ExpiCHO Expression medium (Gibco, TermoFisher Scientific, USA). The cell culture rested in an incubator with a shaker (110 rpm) at 37C, 5% CO2. After 3.5 days the cell culture density was checked with the Neubauer Chamber (Attachment 7.3) to verify if the first cell splitting could be performed (density 4 × 106cells/mL). The cell counting process was possible with a Neubauer Chamber (Attachment 7.3). The next two subculture densities should be around 4 × 106 − 6 × 106cells/mL, and the new culture was adjusted to a cell density 0. 2 × 106cells/mL as well as fresh ExpiCHO Expression medium in an Erlenmeyer flask. Before the day of the transfection, a cell density of 3.5 × 106 viable cells/mL was prepared with a fresh ExpiCHO expression medium. The cells used for transfection were at least, split three times.
Real-Time Imaging of Cell Mineralization 31 3.4 Thawing of OCCM, SAOS, IM1 and IM3 Cells For the cell growth curves and calcification assays (chapters 3.7 and 3.8), professional mineralizing cells such as SaOs and OCCM and calcifying cells like IM1 and IM3 were thawed. SaOs-2 cell lines were derived from osteosarcoma, while the OCCM cell line originated from cementoblasts found in dental cementum. Additionally, the IM1 and IM3 cell lines were derived from vascular smooth muscle cells located in the blood vessels of two different patients. Thus, two cell lines of smooth muscle cells – IM1 and IM3, one cell line of osteosarcoma cells – SaOS-2 and one line of cementoblasts (OCCM) were taken from the liquid nitrogen at -196C. Low glucose DMEM medium with 10% FBS and 1% PSLG was prepared and pre-warmed in the water bath at 37C. The cryovials were placed in the water bath for some seconds to one minute to allow the cells to thaw but not completely as the presence of DMSO, a very toxic cryopreservant, decreases the cell viability. So, after the first layer of ice has melted, the cells are resuspended in 10mL of the medium previously prepared in T75 flasks. The flasks were placed in an incubator at 37C, 5% CO2. Since the medium had a red-color pH indicator, the cell culture was carefully checked daily to verify any color changes on the plates, indicating cell activity and proliferation of the cell lines in the study. The shift to an orange color indicated the production of cell activity residues and oxygen consumption inside the flask. Therefore, the medium was changed after around 2-3 days. 3.5 Cryopreservation of Eukaryotic cell lines Cryopreservation was performed for all the cell lines used during the present study at the end of each assay (Chapters 3.7 and 3.8), ensuring sufficient cell viability for future use. Cells were distributed in Falcon tubes and centrifuged at 300 × g for five minutes, at room temperature. The supernatant was discarded, and the cell pellet was resuspended in a culture medium previously described, containing 10% DMSO. The cell concentration was adjusted to 1×107cells/mL, in 1mL volume cryovial. The cryovials were put in a freezing container (Mr. Frosty, NALGENE, Thermo Scientific, Waltham, USA) filled with isopropanol. The freezing container was stored for one day at -80°C and afterward the cells were transferred into the liquid nitrogen tank, at -196°C.
Real-Time Imaging of Cell Mineralization 32 3.6 Protein Production and Purification 3.6.1 Transformation of NEBTurbo Competent E. coli cells The fetuin-A expression vector (Chapter 3.1) was received lyophilized, so it was necessary the addition of a liquid for use. The plasmid powder was first centrifuged for 10 seconds to allow the powder to settle down. 50L of highly pure nuclease-free water was added to prevent DNA sample loss, since this type of water is free of any kind of nucleases (DNase and RNase). The plasmid was incubated for 10 minutes at 37C and 800 rpm in the ThermoMixer. The introduction of Fetuin-A plasmid into Escherichia Coli cells (transformation) was based on a heat shock protocol. A NEBTurbo Competent E. Coli cells cryovial was taken from a -80C freezer and thawed, until all ice crystals defrosted. 50L of cells suspension was removed into a new transformation eppendorf, previously resting in an ice bath, and 1.5L containing 0.9ng of fetuin-A plasmid was added to the cell mixture. The tube was flicked carefully 4-5 times and put in ice for 2 minutes, so the cells and the DNA could mix without using the vortex. The heat shock was processed by prewarming the ThermoMixer at 42C, without any type of shaking, quickly putting the tube in for precisely 30 seconds, and putting it back on ice. Lastly, 600 L of room temperature SOC medium (Table 1) previously prepared was pipetted into the tube and left to incubate for one hour at 37C and 300 rpm in the ThermoMixer. With a pre-prepared sterile LB agar plate with ampicillin (Table 1 and chapter 3.2), 620 L of cell mixture was added and spread evenly on the plate and incubated for 8-12 hours at 37C. 3.6.2 Amplification of fetuin-A plasmid in Escherichia Coli cells For the amplification of the plasmid in bacteria, a previously prepared Petri dish with a selective medium was used (chapter 3.2). Therefore, the transformated E.Coli cells with the fetuin-A plasmid were added to the Petri dish for plasmid amplification. A colony in the incubated plate (Chapter 3.6.1) was taken with a sterile pipette and dropped in a 100 mL sterile Erlenmeyer filled with 20 mL of sterile LB medium. Ampicillin was added to adjust a final concentration of 100L/mL of the antibiotic. The culture was incubated overnight at 37C and 250 rpm. On the next day, the plasmid was amplified and purified with the kit “Pure Yield Plasmid Midiprep System” from Promega (Madison,USA) as described in attachment 7.4.
Real-Time Imaging of Cell Mineralization 33 For storage, 660 L of transfected E.Coli cell culture was added to 660 L of sterile glycerol to stabilize the cells during the frozen state. Therefore, the cells were stored for future use in the freezer at -80°C. 3.6.3 Transfection of ExpiCHO-STcells Transfection is the process that requires the intentional introduction of genetic material like DNA in cells to overexpress a gene of interest in CHO cell line. So, for the study, it was necessary to transfect ExpiCHO-STcells (Gibco, Thermo Scientific, Waltham, USA) using fetuin-A plasmid. The transfection protocol requires two days to achieve the cell density required for an efficient process. Thus, a density of ExpiCHO-STcell culture (chapter 3.3) of 3.5 × 106 viable cells/mL was prepared on day -1 from an ExpiCHO-STcell culture, with a range of 4 × 106− 6 × 106 viable cells/mL. On day 0 (transfection day), the cell density and percent viability were determined. Subsequently, a final density of 6 × 106 viable cells/mL was prepared with fresh ExpiCHOExpression medium pre-warmed to 37C, until a cell culture volume of 25 mL was reached. Since the fetuin-A plasmid concentration in stock was 423g/L and the goal was to have a concentration of 0.9 g/L for the transfection, a volume of 53 L of the stock concentration was used. The transfection required a kit constituted by ExpiFectamine CHO reagent, OptiPRO medium, ExpiCHO Feed and ExpiCHO Enhancer. So, 80L of ExpiFectamine CHO reagent was added to 920L of OptiPRO medium in a 1mL Eppendorf and inverted 4-5 times to mix the compounds. The 53 L of fetuin-A plasmid was added to 947L of OptiPRO medium in a 2mL Eppendorf. The Eppendorf was added to the 25mL Erlenmeyer with the CHO cell suspension cell culture, and the flask was left to rest in an orbital shaker in the incubator, at 125 rpm 37C and 5% CO2. During cell division of CHO cells, the plasmid present on the medium reaches the nucleus and the gene of interest is transcribed. A time of 18-22 hours posteriori to transfection is required for optimal expression of the transfected genes. On day 1, 150L of ExpiCHO Enhancer and 6mL of ExpiCHO Feed were added to the suspension cell culture, and the incubator temperature was shifted to 32C to slow down the growth of the cells and thereby enhance the expression of the protein of interest. In the next days, a 1mL sample of the CHO cell culture was taken, to verify and determine the cell percentage viability of the cell culture suspension by using the Neubauer chamber method (Attachment 7.3). The harvesting of the protein was only performed when the cell viability was above 60%, after a couple of days.
Real-Time Imaging of Cell Mineralization 34 All the cell culture was divided into 50mL Falcon and centrifuged at 300 rpm and 4C for 15 minutes. The pellet and the supernatants were separated on different Falcons and protease inhibitor was added to the supernatant. To preserve the samples, the Falcons were dropped into a liquid nitrogen bucket to shock freeze them. Afterward, the samples were put in the -20°C freezer until purification (chapter 3.6.5). 3.6.4 Western Dot Blot The Western Dot Blot protocol was used for the immunodetection of protein, utilizing a specific antibody, as described below, for the detection of protein in the nitrocellulose membrane. During protein production (chapter 3.6.3) 1 mL samples were taken every day to check the efficiency of expression. Each sample was vortexed and slightly centrifuged (15 minutes, 4°C, 300g) to allow the separation of the cells and cell debris (pellet) from the supernatant. The supernatant was removed and transferred to a new eppendorf. Both components were then rapidly frozen using liquid nitrogen (-196C) and stored in the freezer. Since cell viability on day 5 was around 67.9%, the cell culture was harvested, and all the suspension culture was distributed in 50 mL volume falcons. Each falcon was then vortexed and centrifuged (15 minutes, 4°, 300g) to separate the cell pallet from the supernatant. Both compounds were separated, shock freeze with liquid nitrogen (-196C), and stored in the freezer. After the end of the cell culture, each supernatant eppendorf of the five days samples were taken from the freezer and pre-warmed in the water bath at 37C. A droplet of around 3L of supernatant each day was pipetted onto a nitrocellulose membrane and let to air dry. In a 50 mL volume falcon a 20mL blocking buffer was prepared (Table 2). The membrane was then rolled and incubated for one hour inside a falcon with 10mL of the blocking solution prepared. Then, the membrane was removed from the falcon, put into the 50mL-falcon with the remaining blocking buffer with 10µL of a His-blot (antibody used for immunodetection of full-length murine FetuinA), and incubated for another hour at 37°C. The membrane was washed three times with PBST (0.05% Tween) at intervals of five minutes transferred into the chemiluminescent buffer (Table 2) and incubated for one minute. Immunodetection fundamentally relies on the principle of antigen-antibody binding. Primarily the His-blot (antibody) binds to His-tag attached to the protein intended for detection. The antibody used is directly coupled with HRP (horseradish peroxidase). This arrangement allows the Histag to associate with a single primary antibody, thereby serving a dual function as both a signal amplifier and a visualizer.
Real-Time Imaging of Cell Mineralization 35 So, the reaction of luminol and hydrogen peroxide, H2O2 (both from the chemiluminescent buffer) with the HRP (from His-tag) emits light and its intensity is proportional to the concentration of the target protein in the samples (chapter 4.2). After the washing steps, the membrane was taken to the chemiluminescence signal detection, named fluorescence imager (Fuji LAS Mini 4000, GE Healthcare,Boston,USA), to verify the presence of protein in the membrane. Table 2: Reagents required for the western Dot Blot protocol. Blocking Buffer Chemiluminescent Buffer Reagent Volume Reagent Volume PBST (0.05% Tween) 20 mL 0.1M TRIS/HCl pH 8.5 20mL Albumin 0.5g 90 mM p-Coumaric acid in DMSO 100L Block buffer 25% 250 mM Luminol IN DMSO 100L - - 30% 𝐇𝟐𝐎𝟐 10L 3.6.5 Immobilized Metal Chelate Affinity Chromatography (IMAC) The purification of Fetuin-A was crucial to extract all the desired protein from the remaining supernatant of the CHO cell suspension culture (Chapter 3.6.3). Therefore, since all proteins produced were expressed with a 6x-Histidine-tag, purification was performed using immobilized metal chelate chromatography (IMAC). The column material of Ni2+-nitrilo-acetic acid (NTA) column (HisTrap HP 5 mL, GE Healthcare, Boston, USA) consists of Ni2+ions that are bound to sepharose. Then, the his-tagged proteins specifically bind to the Ni2+ ions and form high-affinity complexes (Figure 6). Thus, IMAC has continued to be a widely used protein purification protocol since its foundation in the mid-1970s (Moore et al., 2021). Firstly, the frozen falcons from chapter 3.6.3 containing the supernatant were defrosted in the 37.5 C water bath and the centrifuge at 20.000 g, 10 minutes, 4C. During centrifugation, a column was attached to the pump with flow rate of 1 mL/min. All the buffers and solutions required (Table 3) were filtered and degassed to prevent the entrance of large particles or the formation of air bubbles during the process.
Real-Time Imaging of Cell Mineralization 36 The column was attached on the end of the tubing using a wet-to-wet method with 20% ethanol (Table 3). The ethanol was left to rinse tubing and His-trap column for around 10 minutes and a waste goblet was placed under the exit structure to collect the reagents. After the period of centrifugation, the supernatant was poured into a 500 mL bottle built with a 0.22 µm filter (Corning Inc., Corning, USA) that was previously placed inside an ice recipient. As the supernatant was being poured and sterile filtered, a volume of equal parts of low-imidazole buffer was added simultaneously (Table 3). All procedures were done on ice, to avoid protein denaturation. After the ethanol wash, MilliQ and the low imidazole buffer (at a flowrate of 2mL/min) were used for washing the tubing, with each wash lasting for 10 minutes. After the supernatant was connected to the affinity chromatography tube, the waste goblet was exchanged for another recipient to collect the flowthrough during the process. The supernatant was then filtered through the affinity chromatography tube at a flowrate of 4mL/min until only around 5mL of solution remained, as the tube cannot be completely dry. The supernatant goblet was exchanged for the low imidazole buffer and a falcon with a volume of 15mL was taken – wash elution (Figure 6). So, the unbound proteins were flushed from the column by this washing step, while the His-tagged proteins were still bound specifically to the Ni2+ions of the column. To extract the protein of interest that was bound to the affinity chromatography column, the supernatant goblet was exchanged for the high imidazole buffer with a flowrate of 3mL/min. Since the chemical structures of the imidazole on the buffer and the histidine (on the His-tag) are similar, the imidazole binds to the Ni2+ions in the column allowing the protein with the His-tag to detach and be collect in a first Eppendorf, with a volume of 5mL (dead volume elution), followed by 10 consecutive eppendorfs, with a volume of 1mL each (10 elution fractions) (Figure 6). Finally, the supernatant, flow through, wash elution, dead volume and the 10 elution fractions were analyzed spectrophotometrically to verify the presence of fetuin-A and its concentration (chapter 3.6.6). To verify the presence of Fetuin-A and other proteins in the previously mentioned solutions, an SDS-Page gel was conducted (chapter 3.6.7). To preserve the column for future use, it was firstly buffered in Milli-Q and stored in 20% ethanol.
Real-Time Imaging of Cell Mineralization 37 Table 3:List of buffers and solutions used in the affinity chromatography process as well as each reagent concentration needed. Reagent Volume 20% Ethanol 20 mL 100% Ethanol 80 mL MiliQ Milli-Q 50 mL Low Imidazole Buffer 20mM Na3PO4 500mM NaCl 40mM Imidazole MilliQ High Imidazole Buffer 20mM Na3PO4 500mM NaCl 500mM Imidazole MilliQ Figure 5: Scheme of the overall purification steps required on the IMAC chromatography process to purify a His-tagged protein sample.
Real-Time Imaging of Cell Mineralization 38 3.6.6 Spectrophotometer – Protein Concentration The concentration of protein was determined by spectrophotometry. This procedure is based on ultraviolet (UV) light absorbance, to determine the protein concentration in a sample. In general, proteins absorb UV light around the 280 nm wavelength, as amino acids like the tryptophan or tyrosine have aromatic side chains. Thus, a UV-Vis-Spectrophotometer (Eppendorf, Hamburg, Germany) with UV cuvettes were used in this work as each sample from chapter 3.6.5 was analyzed. The blank used was sodium chloride (NaCl) to minimize interference in the determination of protein concentration. 3.6.7 Sodium dodecyl-sulfate polyacrylamide gel electrophoresis – SDS-PAGE SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis) is a technique widely used to separate proteins of a sample according to their molecular weight in an electric field. The main procedure is the denaturation of the proteins with an anionic surfactant, that attributes the proteins a negative charge based on their molecular weight. The proteins are then separated by their weight with electrophoresis in an acrylamide gel. (Nowakowski et al., 2014). The protocol required the preparation of a separating and the stacking gel as described in Table 4 and briefly illustrated in Figure 6. Table 4:List of reagents for the production of the gels required for the SDS-PAGE method. This table represents the compounds necessary to produce 2 gels. Reagent Separating Gel (10%) Stacking Gel Milli-Q (mL) 4.2 3 Separating Gel Buffer (mL) 2.5 - Stacking Gel Buffer (mL) - 1.25 Acrylamide (30%, mL) 3.3 0.75 TEMED (L) 8 10 APS (10%, L) 50 50 End Volume (mL) 10 5 The buffers used were prepared in a 4-times stock solution: 1.8 M TRIS,0.4% w/v SDS, pH 8.8 as separating gel, and 0.5M TRIS, 0.4% w/v SDS, pH 6.8 as a stacking gel. The gels were prepared with the Mini-PROTEAN Tetra Cell Casting Stand from Bio Rad (Hercules, California, USA).
Real-Time Imaging of Cell Mineralization 39 First, the separating gel was poured between the glass plates and left to polymerize, and a thin layer of isopropanol was added on top of the gel for straight gel edges, for 30 minutes and 37 C. After the time established, the separating gel polymerized, and the layer of isopropanol was removed with the help of an absorbent paper. The stacking gel was added on top of the previous gel. A comb with the desired number of columns was placed inside the stacking gel and let to polymerize for 30 minutes at the same temperature. Laemmli buffer (25 mM TRIS pH 8.3, 192 mM glycine, 0.1% SDS) was added as the running buffer inside the gel caster until the guidelines were reached. The samples for the procedure were provided by the previous method: IMAC chromatography (chapter 3.6.5). The samples were prepared with a 1:2 dilution with protein solution and 2 SDS sample buffer (0.125 M TRIS/HCI, pH 6.8, 5% SDS, 10% glycerol, +/- 10% -mercaptoethanol, 0.01% bromphenolblue) and cooked on the ThermoMixer (Eppendorf) for 5 minutes at 95 C to allow the proteins to denaturize. The increase of temperature disrupts the hydrogen bonds of the proteins and as a result the amino acid-chains lose the secondary and tertiary structure. The proteins acquire a negative charge proportional to their length. Afterwards, the comb was removed and all volume of 15L of each sample was added to each column. 15L of low molecular weight marker (GE Healthcare, Boston, USA) was added to the first column to provide as a guideline of the molecular weight distribution. In each pocket created by the comb was added 15L of low molecular weight marker as well as of each sample. The gel was run at 33 mA for an hour until the samples reached a close point to the end of the casket. As the proteins within the sample are negatively charged, these migrate towards the positive electrode (anode, on the bottom of the casket). As smaller proteins migrate quicker through the gel matrix and stop closer to the end of the casket, larger proteins encounter more resistance, move slower, and tend to remain closer to the starting point. This differentiation in mobility allows for an effective protein separation based on molecular weight. The vertical gel apparatus Mini-PROTEAN Tetra Cell from Biorad (Hercules,California,USA) was used.
Real-Time Imaging of Cell Mineralization 46 The cells were cultured for seven days. After seven days, the cells were taken out of the CELLCYTE device, and parameters such as cell viability and calcification lesions were evaluated through Fetuin-A staining (chapter 3.8.2), FDA/PI staining (chapter 3.8.3), and Alizarin Red staining (chapter 3.8.4). The plate configuration incorporated duplicate samples. Table 7: List of reagents and each volume used during the preparation of both mediums necessary for the different phases of the calcification assay. The culture medium was prepared, stored on the fridge, and used whenever necessary during the assay. The calcification medium was prepared fresh and used on the same day. Reagent (mL) Culture Medium Calcification Medium Low-Glucose DMEM 500 10 Fetal Bovine Serum 50 1 Penicillin-Streptomycin (PS) 5 0.1 GlutaMAX 10 0.2 Calcium - 4.3 mM Phosphate - 3 mM 3.8.2 Fetuin-A Live Staining All the 24-well plates (chapter 3.8.1) with and without Fetuin-A on the media were stained with mFamRuby. This protein probe is a murine Fetuin-A probe with fluorescent properties, due to the Ruby chain, since it binds to mineral deposits allowing the visualization of early-stage cellular calcification. Therefore, the mFa-mRuby variant was produced as described in chapter 3.6.3 – with an initial concentration of 9.1mg/mL (based on the protocol in chapter 3.6.6), was adjusted to a final concentration of 100µg/mL and added to each well for 20 minutes at 37°C. The cells were washed with PBS three times for five minutes and representative pictures were taken at a LEICA 6000 microscope.
Real-Time Imaging of Cell Mineralization 47 3.8.3 Fluorescein/propidium iodide Staining Fluorescein diacetate/Propidium iodide staining or FDA/PI is used for live/dead staining. Therefore, fluorescence microscopy was required, as this staining protocol leans on the excitation of fluorophores by a specific wavelength of light and the subsequent emission of light at a longer wavelength(Shihan et al., 2021). By the selection of the appropriate fluorescence channel during microscopy, the cells take up the FDA (Fluorescein diacetate, Sigma, F7378) and convert the nonfluorescent FDA into a green fluorescent metabolite fluorescein, which can be observed in real-time simultaneously on both microscopy and the computer displaying the images. The intensity and signal measured provide an indicator of viable cells. On the other hand, PI (Propidium iodide, Sigma, 81845) is used for nuclei staining of cells with disordered areas on the membrane and binds to the cell deoxyribonucleic acid (DNA). Both PI and Fetuin-A (implemented in chapter 3.8.2) emit a red signal under fluorescence microscopy, posing difficulties in differentiation between them. Therefore, PI was not used during this staining protocol. The main objective of this assay was to visualize the cell outlines, hence FDA staining was employed for this purpose. A working solution was previously prepared by diluting the FDA stock solution (5 g/L in acetone) at a rate of 1:100 in sterile PBS. The cells on the 24-well plates (chapter 3.8.1) were washed with PBS and subsequently stained with the FDA working solution for 30 seconds. Cells were washed with PBS. 1mL of FluoroBrite DMEM (Thermo Fisher Scientific, A1896701) was added to each well, and representative pictures were taken with a LEICA 6000 microscope. The red and green fluorescence channels of the microscopy were used to detect signals emitted by the fetuin-A and FDA, respectively.
Real-Time Imaging of Cell Mineralization 48 3.8.4 Alizarin Red Staining The alizarin Red staining is used to colour calcium deposits red. Before the staining steps, it was necessary to fix the cells (from chapter 3.8.3) by causing their death by adding to each well a volume of 250 L of 4% PFA (paraformaldelhyde, ApppliChem, A3813,1000) for ten minutes at room temperature. After the time established, the cells were washed three times with 500 L PBS and stained with 750 L of filtered (10-20 m, VWR,516-0308) Alizarin Red working solution (40mM, pH4.1, Sigma, A5533) for ten minutes at room temperature. During this period the red colour stain binds to the calcification lesions. After the staining, the cells were washed around 5-6 times with PBS until the supernatant became clear. Representative pictures of each well and condition were taken with a Leica DMI 600 microscope with a bright field channel, that produces a dark image against a bright background.
Real-Time Imaging of Cell Mineralization 49 4. RESULTS 4.1 Construction of the Chicken Fetuin-A Plasmid The Chicken Fetuin-A plasmid was acquired from the Thermo Fisher Scientific website, but firstly it was necessary the construction of the desired plasmid, according to the needs of the study. For the design of the chicken Fetuin-A construct, pcDNA™3.4TOPO (A14697, Thermo Fisher Scientific) expression vector was used. This expression vector is a high-efficiency cloning plasmid for amplification protocols in Escherichia Coli cells (Chapter 3.6.2) and subsequently for production for recombinant protein in mammalian systems, such as CHO cells (Chapter 3.6.3). This vector enables a high yield of plasmid DNA as it contains an ampicillin resistance gene, which exclusively permits the growth of E. coli cells that have taken up the plasmid. This method enhances plasmid amplification and protein production ensuring reliable expression studies and high-quality protein variants. Therefore, for the design of the chicken Fetuin-A was crucial the initial addiction of a start codon (ATG), to initiate protein production, followed by the addition of a six-histidine chain (his-tag). Subsequently, the amino acid chain chosen (A0A8V0YUT8_CHICK, illustrated in Figure 8) was inserted into the vector. Ultimately, a stop codon TAG was added to signal the termination of protein synthesis. Figure 8: Sequence of amino acids of the A0A8V0YUT8_CHICK (chicken Fetuin-A) protein available in the UniProt Software.
Real-Time Imaging of Cell Mineralization 50 The addition of a histidine tag (His-tag) constituted by six histidine amino acids – HHHHHH – was crucial to facilitate some methods used for protein detection and purification. The addition of this polyhistidine affinity tag to the sequence of Chicken Fetuin-A makes an impactful difference, as it facilitates the detection of the target protein in the Western Dot Blot (Chapter 3.6.4), by using anti-His antibodies for an effective track of protein production, and to perform a high yield purification through IMAC chromatography (Chapter 3.6.5), as the his-tag has a high affinity to Ni ions present in the column of the purification system, allowing an optimal recovery of the desired protein. This specific affinity tag was chosen due to its versatility in several expression systems from bacteria to mammalian cells, a small size chain of around 0.84 kDa, which reduces any potential alterations on the proteins’ structure and function, enables one-step purification of the desired proteins from complex samples and the commercial availability of the anti-His-Tag antibodies. 4.2 Analysis of the Western Dot Blot The Western Dot Blot method is a protocol used to detect and analyze the presence of protein. This method allowed a quick verification of the quality of the expression recombinant proteins in CHO cells (chapter 3.6.3) and to perceive the presence of fetuin-A protein and its production during the fiveday suspension cell culture. The recombinant proteins (chicken Fetuin-A and mFa-Ruby) presence and expression were confirmed by chemiluminescence detection. The nitrocellulose membrane produced as described in chapter 3.6.4 was taken and processed to the fluorescence imager (Fuji LAS Mini 4000, GE Healthcare, Boston, USA) for the development of a signal to perform qualitative and quantitative analysis (Figure 10). This signal originated from the reaction of luminol, hydrogen peroxide and the HRP linked to the Hist-tag (chapter 3.6.4) and can provide crucial information about the concentration of the target protein. Each sample illustrates a droplet of supernatant collected daily during recombinant protein expression. The samples were labelled according to the harvest day, ranging from day 1 (D1) to day 5 (D5). So, if Fetuin-A was present in the supernatants, a positive signal would appear as a visible shade of grey with the shape of the pipetted droplet providing important insight into the recombinant protein expression and production. Figure 9 illustrates the nitrocellulose membrane after the chemiluminescence detection protocol. The visualization of the shape and colour of each droplet indicated the presence of Fetuin-A.
Real-Time Imaging of Cell Mineralization 51 Therefore, it is possible to say that the production of fetuin-A protein was constant and increased during the 5 days, as evidenced by the darkening of the droplets - therefore an increasing signal of the His Probe (six histidine residues in the Fetuin-A amino acid chains). Also, the droplets do not look perfectly round as it looks more like there are more than one droplet, indicating certain inaccuracies during the protocol. As the presence of fetuin-A was verified as desired, an IMAC chromatography was then performed to purify the supernatant obtained at the fifth day, stored in the freezer after the period of protein production and expression (chapter 3.6.6). Figure 9: Western dot blot membrane result of days 1, 2, 3, 4 and 5 of fetuin-A protein expression and production on a suspension of CHO cell culture. The droplets of each day are derived from the supernatant of the sample that was taken once a day. D1 D2 D3 D4 D5
Real-Time Imaging of Cell Mineralization 52 4.3 Expression and Purification of Recombinant Fetuin-A Probes and Determination of Protein Concentration Recombinant fetuin proteins – chicken and mRuby – were expressed in chinese ovary hamster cells, entitled ExpiCHO-STcells (Gibco, Thermo Scientific, Waltham, USA). The proteins were produced based on the protocol described in Chapter 3.6.3 and purified via IMAC chromatography (chapter 3.6.5). Thereafter, IMAC-purification fractions were analyzed by SDS-PAGE (chapter 3.6.7) pursued by Coomassie staining (chapter 3.6.8). Figure 11 displays the results of the purification of chicken fetuin-A and the mFA-mRuby. Thus, three rounds of purification were conducted two of them for chicken FetuinA and the last to produce m-Fetuin-A-mRuby for subsequent use in the calcification assay (chapter 3.8.1) and microscopy experiments (chapter 3.8.2). In all the SDS-PAGE gels in Figure 10, the first column on the left was used as a reference with a low molecular weight marker that indicates a range of molecular weights. This endorses the identification of Fetuin-A in the samples through its molecular weight, which is around 51-67 kDa. Since the ruby is a protein itself the mFetuin-A-mRuby molecular weight can go up to 90kDa. Therefore, in Figure 11A it was possible to verify the presence of fetuin-A in SDS-PAGE gel especially on elution 3 and strongly on elution 4 (from the IMAC – chapter 3.6.5), which indicated a high concentration of fetuin-A due to the thick band detected by Coomassie staining. Although elution 5 revealed a very narrow band on the fetuin-A molecular weight range, it was not preserved since the protein concentration was not significant. The absence of a band on the fetuin-A MW range on the flow-through, wash, and dead volume indicated that the purification method was well performed. Since the main goal of the method is to ensure that the protein appears solely in the elution fractions, the buffer is exchanged for one with a higher concentration of imidazole prior to dead volume eppendorf and the ten elutions. This is achieved because histidine binds the column through its imidazole ring, which enables imidazole to bind to the Ni2+ions in the column, releasing the His-tagged Fetuin-A and allowing its elution. Afterward, elution 3 and 4 were rebuffered and taken to the spectrophotometer to determine the protein concentration of each sample (chapter 3.6.6). Due to the low yield of chicken fetuin-A with a final concentration of 1,55 mg/mL after rebuffering (Figure 10A), a second round of production and purification was conducted (Figure 10B). On the second round of production of chicken Fetuin-A, elutions 1, 2, and 3 were subjected to spectrophotometric analysis to reveal the concentration of protein produced, due to a higher presence of the target protein, as indicated by the prominent bands in the Fetuin-A molecular weight.
Real-Time Imaging of Cell Mineralization 53 The protein concentration obtained was 2,56 mg/mL, indicating a higher yield compared to the previous round. This result was expected, as more prominent bands were observed in the second round (Figure 10B). A third round of protein production was carried out, during which the Fetuin-A mRuby supernatant was purified to assess the effectiveness of the production method and to evaluate the resulting production concentration. Thin bands with distinct molecular weights were identified in the supernatant, revealing unspecific protein on the sample and therefore a less effective purification. Also, dense layers in the elutions fractions 3, 4, and 5, spanning a high-spectrum molecular weight were spotted. However, these fractions displayed prominent bands on the Fetuin-A molecular weight, indicating a significant concentration of the target protein. So, the elutions 3,4 and 5 were subjected to spectrophotometric analysis, revealing a final concentration of 9.10 mg/mL of m-Fetuin-A-mRuby (Figure 10C). All the elution fractions previously analysed with the spectrophotometer were rebuffered, aliquoted, labelled and shock frozen in liquid nitrogen at -196C, and stored for future use in a -20˚C freezer. The necessary aliquots were subsequently used for the calcification assay (chapter 3.8.1) and fetuin-A staining (chapter 3.8.2). The chicken Fetuin-A produced initially was sent to Berlin BioGenes for Antibody production.
Real-Time Imaging of Cell Mineralization 54 Figure 10: SDS-Page of expression and purification of recombinant proteins. Two rounds of chicken Fetuin-A (10A and 10B) and one round of Fetuin-A-mRuby (Figure 10C) were purified by IMAC chromatography (chapter 3.6.5). From each purification process, 14 fractions were analyzed to verify the presence of the protein in the study: Fetuin-A. The culture supernatant (CM) was applied onto to the second column since the first one was used as a marker for the KDa scale. 15 L volume of culture supernatant (CM), flow-through (FT), wash fraction (W), dead volume (DV), and 10 consecutive elution fractions (1-10) solutions were prepared and were added to each column as described in chapter 3.5.8. On the second round of protein production (figure 7B) the 6 × histine sequence (his-tag) on the protein chain produced was digested and sent to Berlin BioGenes for antibody production. On the mFAmRuby production due to a high concentration of the protein of interest, the elution fractions 3, 4, and 5 were rebuffered, aliquoted, and stored. E=elution.
Real-Time Imaging of Cell Mineralization 55 4.4 Calcium-Phosphate Precipitation Inhibition Assay The Calcium-Phosphate Precipitation Inhibition Assay is a method used extensively to understand how a variety of substances stabilizes calcium and phosphate in a supernatant and consequently inhibit the precipitation of these minerals. Having expressed and produced Fetuin-A probes (chapter 3.6), it was imperative to evaluate the protein inhibition activity through this particular assay. As fetuin-A is widely reported in the literature as an inhibitor of calcification within the human body, ensuring and certifying the functionality of the protein probes was fundamental for accurate experimental outcomes in the forthcoming assays (Brylka & Jahnen-Dechent, 2013; Chekol Abebe et al., 2022; Jahnen-Dechent et al., 2008, 2011; Kovářová et al., 2021). The data obtained from the spectrophotometrically measured samples provided insight into an inhibitory potential of the Fetuin-A. The results obtained, with the establishment of the protocol described in chapter 3.6.9, were analysed in the GraphPad Software and the obtained graphs are shown in Figure 11. The commercial bovine Fetuin-A from Sigma was used as a control. The chicken Fetuin-A (Figure 11B) and m-Fetuin-A-mRuby (Figure 11C) were tested. The graph illustrates that the precipitate curve (red) in the bovine Fetuin-A (Figure 11A) started at 3.4 mM calcium and dropped close to zero after the turning point. The supernatant (blue) starting at 1.3 mM, moved up to around 4.0 mM calcium after the turning point. For the chicken Fetuin-A (Figure 11B) the precipitate curve (red) started at 3.2 mM calcium, remained relatively stable at this concentration, and dropped sharply until around 1.0 mM. The supernatant in blue starting at 1.9 mM, moved up to around 2.1 mM calcium. Additionally, in the murine Fetuin-A-mRuby (Figure 11C) the precipitate curve (red) started at 3.3 mM calcium and dropped close to 1 mM after the turning point. The supernatant (blue) started at 2.0 mM and moved up to around 4.9 mM calcium. In the three samples, the calcium content in the supernatant and the precipitate inverts their direction creating a turning point. The turning point is a parameter that marks the protein concentration at which its inhibition activity modifies the rate of precipitation. This parameter can also be entitled IC50(half maximal inhibitory concentration). The IC50 of each approach was determined and documented in Figure 11.
Real-Time Imaging of Cell Mineralization 62 Figure 13:Live staining of IM1, IM3, Saos-2, and OCCM cells over a period of seven days. FDA, Hoesht and Fetuin-A staining of the 4 cell lines incubated in culture medium (A-D, I-L) and calcification medium (E-H, M-P). Live cells were stained with the colour green (FDA), cell nuclei with blue (Hoechst), and red for fetuin-A signal, and both medium were studied using the fluorescence channels (overlap of the green, blue, and red channels) available on the microscope. Significant calcification lesions were highlighted with white arrows. Calcification lesions were observed in the alizarin red staining protocol, from the fetuin-A staining with the bright field. The fetuin-A staining was used as an end-point staining protocol.
Real-Time Imaging of Cell Mineralization 63 The last results (figure 14) portray the implementation of Fetuin-A as a continuous staining protocol during the seven days aligned with the FDA staining as an end-point protocol, to study the differential cellular responses of the professional mineralizers and the calcifying cells. So, in figure 14A – 14D, under DMEM culture conditions, all four cell lines revealed a similar number of FDA green positive cells as well as in Figures 14E – 14H, where the cells proliferated under calcification conditions. The bright field representative pictures illustrated the absence of Alizarin Red stain in vascular smooth muscle cells (Figure 14K, 14L, 14O and 14P) – IM1 and IM3 – and the professional mineralizers – SaOs-2 and OCCM (Figure 14I, 14J, 14M and 14N) under calcification and proliferation conditions. Figure 13 and 14 displayed notable differences around the SaOs-2 cells as well as the Alizarin Red staining results in both vascular smooth muscle cells used (IM1 and IM3).
Real-Time Imaging of Cell Mineralization 64 Figure 14: Live staining of IM1, IM3, Saos-2, and OCCM cells over seven days incubated with mFA-mRuby in the culture and calcification medium. FDA and Fetuin-A staining of the 4 cell lines incubated in culture medium (A-D, I-L) and calcification medium (E-H, M-P). Live cells were stained with green (FDA), and red for fetuin-A signal, and both mediums were studied using the fluorescence channels (overlap of the green and red channels) available on the microscope. Significant calcification lesions were not observed. The existence of calcification lesions was verified in the alizarin red staining protocol with the bright field. The fetuin-A staining was used during cell culture in the medium and as an end-point staining protocol.
Real-Time Imaging of Cell Mineralization 65 5. DISCUSSION 5.1 Production and Expression of Recombinant Fetuin-A Probes Fetuin-A/ ∝2-Heremans Schmid (HS) is a negatively charged glycoprotein produced in the liver and released into the bloodstream. Human Fetuin-A is widely found in literature due to its involvement and contribution to various biological processes such as a strong anti-inflammatory effect as it is reported to be a potent crystal-bound inhibitor preventing the development of hydroxyapatite crystals (JahnenDechent et al., 2011). The tertiary protein structure of Fetuin-A incorporates a calcium phosphate binding site that can bind calcium phosphate pre-nucleation clusters, amorphous calcium phosphate, and crystalline apatite (Brylka & Jahnen-Dechent, 2013). Therefore, Fetuin-A binds with high-affinity calcium phosphate and calcium carbonate, which inhibits the formation of new calcium-phosphate complexes without influencing the existent mineral deposits. These findings corroborated a deeper and thorough investigation into the correlation between this plasma protein, Fetuin-A, and the calcification of smooth muscle cells in the human body, culminating in health pathologies like atherosclerosis or CKD. The present dissertation aimed to study the expression and production of two Fetuin-A variants, namely i) the expression and amplification of the plasmid of Fetuin-A variants in Escherichia Coli cells; ii) transfection in Chinese Hamster Ovary (CHO) cells and iii) purification and quantification of Fetuin-A variants produced. Thus, chicken-Fetuin-A and m-Fetuin-A-mRuby were strategically chosen for the present work. The chicken-Fetuin-A was synthesized as requested by Berlin BioGenes and, therefore employed to gain insights into the methodologies for optimizing high-yield protein production, in the initial phases of the study. The m-Fetuin-A-mRuby was employed in the experimental protocol as it facilitates i) daily monitoring during protein production in CHO cells (chapter 3.6.3), due to the transition from a colourless to an orange-coloured medium, indicating the expression of the desired protein; ii) an easy tracking of the target protein localization, due to the fluorescence intensity, during image acquisition via microscopy, that provides important insights into the concentration and distribution of the protein along the cell culture matrix. Protein production required a deep understanding of each method employed and their fundamental role in achieving both high-yield expression of the desired proteins and a highly purified sample. Protocols like transfection in CHO cells, Western Dot Blot, IMAC chromatography, Spectrophotometer, SDS-Page, and Coomassie staining.
Real-Time Imaging of Cell Mineralization 66 Both Fetuin-A plasmids were introduced into CHO cells, in the protocol known as transfection, to express the proteins desired and produce the recombinant proteins. CHO cells were the transfection system chosen due to their high-density suspension growth and their satisfactory yield and quality of recombinant protein expression (Z. M. Li et al., 2022). The Western Dot Blot (chapter 3.6.4) was undertaken after, revealing successful protein production during the five days. Given the laboratory´s prior experience with protein expression in CHO cells of m-Fetuin-A-mRuby, this method was only executed with chicken Fetuin-A. As this was the first instance of the use of chicken Fetuin-A plasmid in the laboratory it was essential to analyze and track the progression of protein expression and production every day. Therefore, the results demonstrated a consistent increase in the output of the target protein, as desired. In terms of the protein yield of each round of expression and protein production, the spectrophotometer results revealed that the two chicken Fetuin-A gels disclosed concentrations of 1,55 mg and 2,56mg per mL of elution, which indicated a total production of 3.1mg (Elutions 3 and 4, 1mL each) and 7,68 mg (Elutions 1,2 and 3, 1mL each) of chicken Fetuin-A, respectively. For the m-Fetuin-AmRuby a concentration of 9,1mg per mL of elution was obtained, which results in a total production of 27.3mg of protein as three elutions (E3, E4, E5) of 1mL each were aliquoted. These findings implied a significantly higher yield of the m-fetuin-A-mRuby compared to the two rounds of chicken Fetuin-A expression. As the protein expression occurred in a mammalian cell line (Chinese hamster ovary cells) and the murine plasmid originated from a mammal, it likely exhibited a higher affinity with the CHO cell system than the avian plasmid (chicken plasmid). Therefore, that might have resulted in a better optimization and efficient recombinant production system, due to the closer phylogenetic relationship between the mammalian expression cell line and the mammalian gene in the murine plasmid, resulting in more efficient post-translational modifications. As described in the literature, CHO cells are commonly used in mammalian protein expression, further explaining the observed yield differences (Z. M. Li et al., 2022). On the SDS-gel of recombinant m-fetuin-A-mRuby, theoretical molecular weight in the range of 70 to 90 kDa, given that ruby is also a protein, was set as a guiding reference, following the literature (Chekol Abebe et al., 2022). For the chicken Fetuin-A a smaller molecular weight in the range of 51 to 67 kDa was considered. Therefore, the three gels produced intended to separate the proteins in each sample based on their molecular weight, as evidenced by the satisfactory results illustrated in Figure 10. The Coomassie staining protocol subsequently employed permitted the visualization of the protein separation on the acrylamide gels from the previously performed SDS-PAGE.
Real-Time Imaging of Cell Mineralization 67 The intensity of the staining in each band correlated directly to the protein concentration allowing the assessment of the protein expression levels and the purity of each sample. In the three gels, thicker prominent bands were noted around the MW range corresponding to the reference size for mFA-mRuby and chicken Fetuin-A. These bands were notably clearer than other proteins present, detected at significantly lower concentrations, that appeared as faint bands in higher and lower molecular weight in all three gels. The m-Fetuin-A-mRuby exhibited a lower purification level due to the presence of additional proteins from elution 2 to 5, alongside the target protein. However, the two chicken Fetuin-A gels revealed similar top-notch purification levels. These gels contrary to that of the m-Fetuin-A-mRuby sample, showed higher purified samples, characterized by a prominent concentration of the target protein and a faint presence of other proteins with distinct molecular weights. The lower level of purification might result from some setbacks during the immobilized metal affinity (IMAC) chromatography. Inefficient elutions, where the non-specific proteins remained in the samples, could have been one of the complications. Furthermore, a high concentration of non-target proteins may have interfered with the effective binding of the desired protein, but also with the elution of the unwanted proteins during their respective extract phase. The outcomes of this study demonstrated a highly satisfactory alignment with the initial predictions, affirming the validity of the methodological framework employed. The level of congruency reflected the efficacy of each protocol implemented and paved the way for subsequent experimental procedures
Real-Time Imaging of Cell Mineralization 68 5.2 Calcium-Phosphate Precipitation Inhibition Assay – Fetuin-A The Calcium-Phosphate Precipitation Inhibition Assay is a method widely used to study how different substances affect delicate balances in biological mineralization. The assay uses conditions in which calcium and phosphate ions typically would precipitate to enable researchers to evaluate the efficacy of inhibitors such as proteins, polymers, or drugs in slowing or stopping crystal growth. The study of a substance’s inhibition activity is vital for understanding the mechanism underlying the development of mineral precipitates. This insight is fundamental to generate techniques to prevent soft tissue calcification in blood vessels, for example, and for establishing the balance for normal maturation and mineralization. This assay has been valuable in studies of bone and mineral metabolism as well as in research on pathological calcification such as ectopic calcification. The data obtained from spectrophotometric measuring was analysed using the GraphPad Software features (Figure 11) and provided information on the inhibitory potential of the Fetuin-A probes tested – Chicken Fetuin-A and the m-Fetuin-A-mRuby. The graph provided an important parameter to assess the protein effectiveness/affinity: the Turning Point also known as the IC50 value. This parameter gives a perception of the concentrations required for a prominent shift in the inhibition behaviour of the protein. The nomenclature IC50 translate to value of the half maximal inhibitory concentration and allows the determination of the inhibition potency of the probes tested, by indicating the concentration of protein needed to inhibit the growth of mineral crystals by 50%, based on the concentrations of calcium (5mM) and phosphate (3mM) added. The IC50 value endorsed the assessment of each protein efficacy, a lower IC50 value indicating that a smaller quantity of protein was required to achieve a half-maximal effect, thus suggesting an elevated potency. On the other hand, a higher IC50 value indicated a need for a higher amount of protein to achieve the same level of inhibition activity. The IC50 value was calculated with the GraphPad Software. According to the values acquired, the mFetuin-A-mRuby exhibited a IC50 value of 0.49 µM, significantly lower than that of the chicken Fetuin-A, 3.88 µM. Thus, the mFetuin-A-mRuby had a substantially higher inhibitory activity compared to the chicken Fetuin-A probe, further supporting the previously described hypothesis. Therefore, to achieve the same desired level of inhibition, a lower concentration of m-Fetuin-A-mRuby is necessary.
Real-Time Imaging of Cell Mineralization 69 5.3 Cell Culture-based Calcification Assay Following the production of m-Fetuin-A-mRuby, the next phase aimed to mimic the biological conditions present within the human body through a calcification assay. Four cell lines were chosen concretely for this assay. SaOs-2 (human osteosarcoma cell line) and OCCM (immortalized murine cementoblast cell line) were selected, due to their professional mineralizer activity. These cell lines are widely used as models for studying the bone formation, metabolism, and mineralization essential for bone tissue engineering (Declercq et al., 2004). Thus, they were ideal for mimicking the behaviour of professional mineralizing cells under the experimental conditions studied. Vascular smooth muscle cells were chosen due to their capacity to alter their phenotype from contractile cells to osteogenic, under pathological conditions, characterized by the development of calcifying vesicles and consequent mineral deposits (Durham et al., 2018). These calcifying cells are associated with pathological conditions and are widely associated with diseases such as hypertension, chronic kidney disease, diabetes mellitus , or atherosclerosis, therefore VSMCs are ideal to investigate in vitro pathological calcification and to comprehend the mechanism and factors behind these complex biological processes. The ability to mimic calcifying conditions and utilize the cells directly associated with these biological events within the human body offers a more accurate approximation of in vivo human conditions. Also, the employment of two VSMC lines (IM1 and IM3) from different patients was a crucial strategy to enhance the reliability and accuracy of the results, better modelling of the calcification conditions, compare if the calcification response alters or not between patients and have more consistency, precise and well-founded outcomes. Therefore, a cell culture-based assay was implemented, as this type of study is fundamental in biomedical research for in-depth study of biological processes, such as calcification, and bridging the gap between laboratory research and clinical applications. The opportunity to perform this assay with these four cell lines enables a more accurate and precise testing environment to assess therapeutic strategies to prevent, reverse, or eliminate calcifying deposits correlated to ectopic calcification. Figures 13 and 14 illustrate the representative microscope pictures, with the green and red fluorescence channels and the bright field, of the four cell lines – SaOs-2, OCCM, IM1, and IM3 – under culture and calcification medium. The fluorescence channels enabled the visualization of the green signal emitted by the FDA staining, the red signal corresponding to the mFetuin-A-mRuby in combination with the fluorescence ruby. The bright field imaging provided a more refined view of the Alizarin Red Staining, as this method provides an overview of the staining outcome to be observed with the naked eye. These mosaic images were distinguished by the protocol adopted.
Real-Time Imaging of Cell Mineralization 70 Figure 14 employed on the incorporation of mFa-mRuby as a component of the mediums. Both figures 13 and 14, the Fetuin-A end-point staining protocol (Chapter 3.8.2) was implemented. In Figure 13, the fluorescence channels revealed a bright green signal in the culture medium, confirming FDA-positive cells in the four cell lines. This insight indicated the presence of viable cells within the wells of all four cell lines, thereby implying a healthy cell proliferation during the seven-day assay. However, the calcification medium appears to have a detrimental effect on SaOs-2, as evidenced by a lower number of FDA-positive cells and a faint green signal, highlighting a minimal cell density, in comparison to the outcome of the culture medium condition. A decrease in FDA-positive cells in the OOCM calcification medium culture was also detected, indicating possible cell apoptosis induced by the media composition or the hypothesis that the cells create shields connected to the extracellular matrix and are flushed away during the washing steps within the staining protocols. In the red fluorescence channel, SaOs-2 exhibited a very faint red signal in the form of specks only in the calcification medium, which may suggest the potential presence of calcifying lesions or calcified dead cells. On the other hand, the OCCM cells did not reveal any red mFa-mRuby signal, suggesting the nonexistence of microcalcifications in both conditions. Even though, SaOs-2, osteogenic cells and OCCM, highly differentiated mesenchymal cells of the periodontal ligament (PDL), are different cell lines, both contributing to biological processes related to mineralization, with the balanced deposition of calcium phosphate minerals in bone and cementum, respectively (Bao et al., 2013; Dvorakova et al., 2023). As cementum and bone have similar compositions of minerals, organic matrix, and water, that finding may justify their similar behaviour under calcification conditions and why diseases that affect the properties of bone often also affect the cementum. Therefore, for both cell lines physiologically higher concentrations of calcium and phosphate promote tissue mineralization, however, if these mineral levels are pathologically high the surrounding environment can become detrimental. As under pathophysiological conditions, phosphate and calcium plasma concentrations range from 2.85 – 2.95 mmol/L, those mineral levels can potentially initiate unbalanced mineralization, also known as calcification. Hence, the calcification assay (chapter 3.8) established a concentration of 3 mM phosphate and 4.3 mM calcium in the medium, significantly higher than pathophysiological conditions and approaching levels observed in disease states, which contemplate a phosphate concentration around 4.4mM (Bhargava et al., 2023). This excessive concentration of phosphate could have led to cell death and dystrophic calcification of dead tissues, as evidenced in ectopic calcification cases. Thus, as calcification and cell death are widely reported to have a strong correlation, the combination of 3 mM phosphate and 4.3 mM calcium may instead of triggering calcification, as expected, stimulate cell apoptosis.
Real-Time Imaging of Cell Mineralization 71 This finding may explain the lower number of FDA-positive cells observed in the fluorescence results in the OCCM cells under calcification medium, the absence of viable SaOs-2 cells, and the presence of small red specks, that may indicate calcified dead cells. For this reason, an in-depth investigation of the effective and optimal combination of the phosphate and calcium concentrations would represent a significant advancement, enabling a proper study of active calcification with significant cell survival, without inducing calcification through cell apoptosis. On the other hand, as expected, some prominent signs of the presence of possible calcifying lesions were observed in the vascular smooth muscle cells IM3. This hypothesis is based on the red signal observed in the representative image (Figure 13F), pointed with white arrows, that highlighted the m-Fetuin-A-mRuby potentially bonded to the calcification lesions, suggestive of its role in cell signalling and cellular interactions. Additionally, some red specks in the fluorescence image of the IM1 cells (Figure 13E) were detected, which may suggest the potential existence of microcalcifications. As the calcification medium has high levels of calcium and phosphate, these mineral concentrations exceed their solubility, mimicking pathological conditions. This high concentration of extracellular phosphate could lead to the inhibition of the production of calcification inhibitors and promote the production of calcifying extracellular vesicles, secreted by SMCs (smooth muscle cells), that aggregate and form microcalcifications. Bigger calcification lesions tend to arise from the continuous accumulation of microcalcifications and maturation of the minerals (Blaser & Aikawa, 2018; Goettsch et al., 2016; Hutcheson et al., 2016; Lee et al., 2020). Thus, the actual existence of calcifying lesions could only be confirmed by the Alizarin red staining, as evidenced by the bright field results. As expected, none of the four cell lines revealed any staining, under culture conditions, therefore confirming the absence of calcified lesions has been predicted previously based on the fluorescence images. Given that the culture medium supplied all the minerals and nutrients beneficial for cellular proliferation, the cells were maintained in an optimal growth environment, which effectively didn’t contribute to the development of microcalcification. Under calcification conditions, a clear distinction between the smooth muscle cells – IM1 and IM3 – and the professional mineralizers – SaOs-2 and OCCM - was witnessed. The Alizarin Red staining highlighted, with a red pigment, the calcified lesions present in the cell’s wells. Smooth muscle cells revealed a presence of red colouring on the cells on the bright field, confirming the development of calcified lesions over the seven days of the assay, supporting the initial hypothesis proposed based on the fluorescence channel. Conversely, the absence of red colouring on the representative images in the SaOs-2 and OCCM cells indicated the nonexistence of calcification in these cells’ lines.
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Real-Time Imaging of Cell Mineralization 80 7.1 Uniprot Protein Comparision 7.2 Chicken Fetuin-A Sequence 3D Image Figure 15:Blast Alignment of the amino acids of the protein chains of fetuin-A variants from mammals: Mouse, Cotja and two different protein chains lengths from chicken using the Uniprot Software. Figure 16:Percentage Identity Matrix from Uniprot Software of the four different fetuin-A protein variants from mammals: Mouse, Cotja and Chicken.
Real-Time Imaging of Cell Mineralization 81 7.3 Neubauer Chamber A cell culture sample was taken from the Erlenmeyer flask and diluted by a factor of 1:10 with PBS. Trypan blue was used to determine the cell culture viability as it only colours dead cells blue, so the live cells could be counted. So, a dilution of 1:20 with trypan blue (trypan blue in PBS, 0.5% w/v) was performed. A sample of 7 µL of cell culture + trypan blue was pipetted into the Neubauer chamber. The cells counted were inside the 4 squares represented (Figure 19). Subsequently, the number of cells in the Erlenmeyer flask was calculated with Equation 1. 𝑬𝒒𝒖𝒂𝒕𝒊𝒐𝒏 𝟏: 𝑐𝑒𝑙𝑙 𝑐𝑜𝑢𝑛𝑡 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 × 𝑁𝑒𝑢𝑏𝑎𝑢𝑒𝑟 𝑐ℎ𝑎𝑚𝑏𝑒𝑟 𝑓𝑎𝑐𝑡𝑜𝑟 × 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 Neubauer chamber factor = 10000 Dilution factor = 20,if trypan blue was used (1: 1) Figure 17: Potential representation of a 3D-Model of the protein structure of Fetuin-A derived from chicken using the ChimeraX software.
Real-Time Imaging of Cell Mineralization 82 7.4 PureYield Plasmid Midiprep System The PureYield™ Plasmid Midiprep System protocol was used to isolate and analyze plasmid DNA from Escherichia coli cells. In this study was important to quantify the production of chicken Fetuin-A plasmid by the E. coli cells. The following protocol was used. • A 100 mL transformed E. coli bacterial cell culture grow overnight at 37C and 250 rpm; • Two 50 mL falcon were filled with cell culture and centrifuge at 5000 g for 10 minutes; • The supernatant was discarded, and each pellet was resuspended in 3mL Cell Resuspension Solution; • 3mL of cell Lysis Solution was added to each falcon. The mixture was gently inverted 3-5 times. Incubation for 3 minutes at room temperature; • Centrifugation of the lysate at 15000 g for 15 minutes; • The PureYield clearing and binding columns were stacked and the lysate was added to the column; • Vacuum was added to allow the sample to go through the filter; • 5 mL of Endotoxin Removal wash and 20mL of column wash solution were added consequently and a vacuum was applied; • 1.5mL Eppendorf was placed in the base of the elution device with the cap open; • 600L of nuclease-free water was added to the DNA binding column and applied vacuum. Figure 18:Neubauer Chamber Layout for counting live/dead cells.
Real-Time Imaging of Cell Mineralization 83 • The DNA was collected and saved. 7.5 Cell Culture Growth Curve Assay Figure 19: General representation of a Cell Growth Curve
Real-Time Imaging of Cell Mineralization 84 7.6 Calcification Assay Figure 20: 24-well-plate display used for the calcification assay. The cell lines used: osteosarcoma cells (SaOs-2), cementoblast (OCCM) and two different cell lines from vascular smooth muscle cells from two different patients (IM1 and IM3). Before the addition of the calcification medium the culture medium was used in all the wells. The calcification medium was added to all the wells on column 2 and 4. At the end the culture medium on column 1 and 3 was compared to the calcification conditions. The impact of the presence of Fetuin-A was studied as well, so in one of the 24-well plate the target protein was in contact with the cells during the seven-day experiment; in the second 24-well plate the Fetuin-A was used as an end-point staining on the last day of the study. Each Fetuin-A study was conducted in duplicate (chapter 3.8.1 and 3.8.2).