Aus der Universitätsklinik für Zahn- Mund- und Kieferheilkunde der Albert-Ludwigs-Universität Freiburg i. Br. Klinik für Mund-, Kiefer- und Gesichtschirurgie Ärztlicher Direktor: Prof. Dr. Dr. R. Schmelzeisen
Effects of Oleic Acid and Gabapentin-Lactam on Ovine Bone Marrow-Derived Mesenchymal Stem Cells
INAUGURAL - DISSERTATION zur Erlangung des Zahnmedizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg i. Br.
vorgelegt 2012 von Eva-Marie Jablonka geboren in Freiburg im Breisgau I
Dekan Prof. Dr. Dr. h.c. mult. Hubert E. Blum
1.Gutachter PD Dr. Dr. Sebastian Sauerbier
2.Gutachter Prof. Dr. Olga Polydorou
Jahr der Promotion 2013 II
Meinen Eltern und Großeltern gewidmet. III Contents
1 Introduction 1
1.1 Mesenchymal stem cells 1
1.1.1 Discovery 1
1.1.2 Defining criteria 3
1.1.3 Harvesting 4
1.1.4 Clinical relevance 4
1.1.5 Growth factors 6
1.2 Gabapentin-lactam 8
1.3 Oleic acid 10
1.4 Aim of this thesis 12
2 Materials and Methods 13
2.1 Ovine MSC 13
2.1.1 Cryopreservation 13
2.1.2 Resuscitation 14
2.1.3 Passaging 14
2.2 Differentiation of ovine MSC 16
2.2.1 Adipogenesis assay 16
2.2.2 Osteogenesis assay 16
2.2.3 Chondrogenesis assay 18
2.3 Proliferation of ovine MSC 20
2.3.1 Oleic acid and GBP-L media 20 IV
2.3.2 EZ4U cell proliferation assay 21
2.3.3 Statistical analysis 22
2.4 Human MSC 23
2.4.1 Cell culture 23
2.4.3 CyQuant cell proliferation assay 24
2.4.4 EZ4U cell proliferation assay 25
3 Results 26
3.1 Oleic acid induces morphological change in ovine MSC 26
3.2 Effect of oleic acid and GBP-L on ovine MSC proliferation (EZ4U assay) 27
3.3 Effect of GBP-L on human MSC proliferation (CyQuant assay) 33
3.4 MSC culture 35
3.4.1 Ovine cells 35
3.4.2 Human cells 35
3.5 Multipotency 36
4 Discussion 37
4.1 Results: Oleic acid 37
4.2 Results: Gabapentin-lactam 42
4.3 Methods: Cell proliferation assays 44
5 Conclusion 47 6 Summary 48 6 Zusammenfassung 49 7 References 50 8 Appendix 65 V 1 Introduction 1 Introduction
1.1 Mesenchymal stem cells
1.1.1 Discovery Mesenchymal stem cells (MSC) can be isolated from adult bone marrow stroma (Caplan 1991). They were first discovered in 1966 by A. Friedenstein and colleagues, who described them as a population of adherent, colony building, fibroblast-like cells with an osteogenic differentiation ability. Since then, MSC have shown their ability to differentiate into multiple other cell types of the mesodermal lineage, including cells of cartilaginous, osseous, adipose, muscle and tendon tissues, as well as bone marrow stroma (Caplan 1991; Prockop 1997; Pittenger et al. 1999). As non-hematopoetic adult stem cells, they are also characterized by a substantial proliferative potential (Pittenger et al. 1999).
EMBRYONIC TISSUES Embryonic ectoderm
Embryonic Embryonic epiblast endoderm
Primitive Epiblast streak
Inner cell Amnionic Embryonic mass ectoderm mesoderm
Extraembryonic Extraembryonic Blastocyst Hypoblast Yolk sac endoderm mesoderm
EXTRAEMBRYONIC Trophoblast Cytotrophoblast Syncytiotrophoblast TISSUES
FIG. 1: Development of the embryonic germ layers (modified after Gilbert 2010).
Adult stem cells - undifferentiated progenitor cells that are present in a variety of adult tissues including bone marrow - provide a viable alternative to embryonic stem cells for in vitro research and in vivo therapeutic application, especially since the latter have been subject to ethical concerns (Weissman 2002; Chin 2003). Unlike the pluripotent embryonal stem cells derived from the human blastocyst with their ability to differentiate 2 Introduction into cells of all three embryonic germ layers (Fig. 1; Thomson et al. 1998; Trounson 2001), the adult stem cell‘s potential for differentiation is usually limited to the lineage of its tissue of origin (multipotency, Fig. 2). However, a number of studies have shown a potential for transdifferentiation into other cell types such as hepatocytes and neuronal tissue (Jiang et al. 2002; Schwartz et al. 2002; Suon et al. 2004).
FIG. 2: Mesengenesis - MSC differentiation potential (Singer and Caplan 2011).
The presence of adult stem cells in each living individual opens up new perspectives for therapeutical use in autologous cell grafts and tissue engineering. The main advantage of autologous grafts is the absence of a host immune response, as the HLA (human leucocyte antigen) surface markers on the grafted cells are identical with those of the host. 3 Introduction
1.1.2 Defining criteria MSC are defined by certain criteria: They must show plastic-adherence in standard culture conditions. They must have the capacity to differentiate into osteogenic, adipogenic and chondrogenic progenitor cells in vitro. Finally, they should express the surface markers CD73, CD90 and CD105 and lack the expression of CD11b, CD14, CD19, CD34, CD45 and CD79α (Dominici et al. 2006). A combination of surface markers is used to identify MSC by flow cytometry, since there is no single characteristic surface marker known to isolate them from a mixed population (Pittenger et al. 1999). MSC have been shown to express CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, whilst testing negative for CD11b, CD31, CD34 and CD45 (Cheng et al. 2012). However, these surface markers can also be found on a variety of other cells. They are not specific for MSC and hence criticized as a means of identification (Wulf et al. 2006). The most reliable way to identify mesenchymal stem cells therefore remains the isolation by their ability to adhere to plastic surfaces and the ensuing differentiation of the cultured MSC into three mesenchymal lineages: osteogenic, adipogenic and chondrogenic (Fig. 3).
FIG. 3: Cell morphology of osteogenic, chondrogenic and adipogenic differentiation (Barry et al. 2001). 4 Introduction
1.1.3 Harvesting Mesenchymal stem cells have been successfully isolated from various tissues such as bone marrow, skeletal muscle, skin and fat, as well as dental pulp and periodontal tissue (Gronthos et al. 2000; Lee et al. 2000; Deasy et al. 2001; Zuk et al. 2002; Miura et al. 2003; Seo et al. 2004). Bone marrow aspiration from the pelvic bone is a relatively easy method to obtain an adequate quantity of MSC (Healey et al. 1990; Connolly et al. 1991; Tiedeman et al. 1991). Being less invasive and more cost-efficient, it is a viable alternative to the explantation of bone marrow and the method of choice for harvesting mesenchymal stem cells for therapeutic application.
1.1.4 Clinical relevance Craniofacial surgical reconstruction of defects caused by ablative tumor surgery, congenital malformation such as cleft lip and palate, trauma, and other deforming skeletal conditions often involves the repair of a variety of tissues including bone, cartilage, fat, and ligaments. The multipotent mesenchymal stem cells appear to be an advantageous source for craniofacial tissue-engineering applications since they possess the ability to differentiate into progenitor cells that produce all of the tissues mentioned above (Salinas and Anseth 2009). The possibilities of a clinical application of MSC in craniofacial and skeletal tissue reconstruction have been reviewed by several authors (Arthur et al. 2009; Rodrigues et al. 2010). In orthopedic surgery, MSC in composite grafts have been used successfully in the repair of thoracolumbar and non-union long bone fractures (Quarto et al. 2001; Faundez et al. 2006). It has also been shown that the addition of cultured allogeneic MSC to bone marrow transplants in the treatment of osteogenesis imperfecta enhances the effect of the graft (Horwitz et al. 2002). A comparison study of adolescent idiopathic scoliosis confirmed better results for aspirated autologous bone marrow combined with a mineralized collagen matrix grafts than for a freeze-dried corticocancellous allograft regarding posterior spinal fusion (Price et al. 2003). Another possible therapeutic application is cartilage regeneration (Jorgensen et al. 2008; Mrugala et al. 2008). A hyaline-like cartilage formation was observed in a pre-clinical ovine model 9 weeks after the transplantation of MSC in a composite graft (Mrugala et al. 2008). 5 Introduction
K. Esato and colleagues found neovascularization at the location of injection of bone marrow-derived mononuclear cells in patients with peripheral occlusive arterial disease (PAD) (2002) . In the therapy of myocardial infarction, improvements of ventricular function have been observed after the application of MSC (Grauss et al. 2007; Vassalli et al. 2007). The apparent advantage of MSC combined with biodegradable materials over conventional treatment alternatives for craniofacial defects has been stated by several reviewers (Miura et al. 2006; Howard et al. 2008). There have also been reports of significant cranial bone formation with MSC that were expanded in culture and various scaffolds with or without the addition of growth factors (Krebsbach et al. 1998; Dean et al. 1999; Shang et al. 2001; Rohner et al. 2003; Akita et al. 2004; Chang et al. 2004; Mankani et al. 2006; Velardi et al. 2006; Gimbel et al. 2007; Umeda et al. 2007) . M. Soltan and colleagues proposed the use of MSC as a possible new „platinum“ standard for the acquisition of osteoinductive bone grafting material (2005). In the Department of Maxillofacial Surgery of the Freiburg University Hospital, the transplantation of human autologous mesenchymal stem cells was first performed in 2005 in a maxillary sinus augmentation after several promising pre-clinical studies employing large animal models (Gutwald et al. 2009; Sauerbier et al. 2010; Wongchuensoontorn et al. 2009). The histomorphometric analysis of biopsies obtained during the placement of dental implants 5 months later showed satisfactory bone regeneration for the combination of bone substitute material (BioOss, Geistlich, Wolhusen, Switzerland) and autologous MSC (Scherfler 2010). A chair-side method using the BMAC procedure pack (Harvest Technologies, Plymouth, MA, USA) to concentrate mononuclear cells (including MSC) in the bone marrow aspirate for transplantion was established successfully for routine procedures in the department (Fig. 4; Sauerbier 2008; Sauerbier et al. 2010). 6 Introduction
FIG. 4: BMAC procedure: aspirated bone marrow is centrifuged to concentrate mononuclear cells (left) and added to bone substitute material (right) (Pictures: Department of Maxillofacial Surgery, Freiburg University Hospital).
1.1.5 Growth factors In the human body, all cells reside in the extracellular matrix, a microenvironment providing them with mechanical stimulation as well as with signaling molecules such as growth factors, cytokines and other molecules. Through signal transduction which involves the binding of a specific signaling molecule to a receptor on the cell‘s surface and an intracellular answer often mediated by a second messenger, a cellular response such as cell proliferation, differentiation, gene activations and metabolism changes is created (Rodbell 1980). Lipophilic signaling molecules like steroid hormones usually pass through the cell membrane, binding to intracellular receptors with a direct influence on nuclear gene expression. This extracellular niche is imitated in the therapeutical application of MSC by the use of a scaffold for adhesion and migration, and supplementation of growth factors to optimize cell proliferation and differentiation (Langer and Vacanti 1993; Arnaud et al. 1999; Donahue 2004). Since the immediate application of mesenchymal stem cells to a site of defect has shown success in the regeneration of tissue, it is now of great interest to find a method of 7 Introduction application that closely mimics the cells‘ natural environment to promote even further cell proliferation and ultimately differentiation of the grafted cells.
Several growth factors have been identified to be relevant for MSC proliferation and differentiation. Scaffold-tethered epidermal growth factor (EGF) has been observed to facilitate MSC adhesion and to improve the survival of cells (Fan et al. 2007). Platelet-derived growth factor (PDGF), transforming growth facor beta (TGF-β) and fibroblast growth factor (FGF) have been identified as signal molecules for key pathways in MSC proliferation in a serum-free environment as well in differentiation (Ng et al. 2008). PDGF, though, does not seem to be involved in the osteogenic differentiation of MSC (Kumar et al. 2009). However, mesenchymal stem cells exposed to the cytokines basic fibroblast growth factor (bFGF) and bone morphogenetic protein (BMP) have been observed to differentiate into an osteogenic lineage in vitro and to accelerate cranial bone healing in a nude rat model (Akita et al. 2004). The addition of TGF-β-1 in a biodegradable hydrogel composite matrix seems to promote chondrogenic differentiation of MSC and the cells‘ production of cartilage matrix (Park et al. 2007). Various studies have indicated an increase in cell proliferation involving BMP, TGF-β, FGF, PDGF and insulin-like growth factor (IGF) (Xiang et al. 1993; Kawaguchi et al. 1994; Talley-Ronsholdt et al. 1995; Hanisch et al. 1997; Shen et al. 2002; Pountos et al. 2010), with most studies focusing on BMP (Ripamonto et al. 1994; Terheyden et al. 1999; Jung et al. 2003) which has since been applied clinically to promote bone regeneration. BMP has been shown to be more effective compared to conventional treatment in acute open tibial fractures and spinal fusion surgery (spondylodesis) (Garrison et al. 2007). The clinical use of BMP is expensive (Cahill et al. 2009), hence alternative agents that provide better cost-efficiency and availability are in the focus of research. An example are statins, a class of drugs used to lower blood cholesterol levels that have been shown to increase the production of BMP-2 in vitro and in vivo (Mundy et al. 1999; Benoit et al. 2006). 8 Introduction
1.2 Gabapentin-lactam
FIG. 5: Gabapentin (left) and gabapentin-lactam (GBP-L, right).
Gabapentin is a neuroleptic drug commonly used in the treatment of epilepsy and neuropathic pain. It is an analog of the amino acid GABA (γ-aminobutyric acid), the main inhibitory neurotransmitter of the central nervous system. Several derivates of gabapentin including gabapentin-lactam (GBP-L) and GABA-lactam analogs have been isolated and modified by T. Feuerstein and colleagues of the Department of Neuropharmacology, Freiburg University Hospital, Freiburg, Germany (PCT/EP 98/07383; US patent number: 09/554,587). The new substance GBP-L and the anti-epileptic drug gabapentin were tested on rats in an in vivo model of acute retinal ischemia, during which intraocular pressure was elevated to values above systolic blood pressure for 60 minutes. While a protective effect could be observed for gabapentin, GBP-L was shown to double the number of surviving retinal ganglion cells (Jehle et al. 2001; Lagreze et al. 2001). One possible explanation for the neuroprotective effect could be the inhibition of the excitatory and potentially toxic neurotransmitter glutamate, since a significant lower release of ischemia-induced glutamate could be detected for both gabapentin and GBP-L in vitro. The blockage of ATP- sensitive potassium channels of the cell membrane and mitochondria with glibenclamide was observed to completely antagonize the effects of GBP-L, suggesting that the opening of these channels might play a role in mediating the effects of GBP-L (Jehle et al. 2001; Pielen et al. 2004). A potential neurotrophic effect of GBP-L with an enhancement of dendritic filopodia essential for synapse formation in cultured hippocampal neurons has also been reported (Henle et al. 2006). 9 Introduction
A positive effect of GBP-L and GABA-lactam analogs on the proliferation of cultured osteoblasts has been observed by Feuerstein (2011). Mitochondrial production of reactive oxygen species (ROS) as an endogenous defense mechanism can promote cell growth (Irani 2000) and therefore mediate cell survival (Dzeja et al. 2001). GBP-L and GABA- lactam analogs have been shown to result in a significant increase in mitochondrial ROS formation. A causal link of the observed increase in cell proliferation and elevated ROS synthesis may be assumed (Feuerstein 2011). These findings suggest a growth factor-like effect of GBP-L and GABA-lactam analogs on cell proliferation. The same effect has also been studied with mesenchymal stem cells. A significant raise in growth rate could be observed in studies with ovine MSC cultured in proliferation media with GBP-L and GABA-lactam analogs as additives. MSC gained up to 26,2% in proliferation depending on the dosage of the additive (Wolter 2009; Obermeyer 2010; Sauerbier et al. 2011). Since GABA-lactam analogs are not water-soluble, the addition of dimethyl sulfoxide (DMSO) as a solvent was required. DMSO showed a suppressive effect on cell proliferation in a control group, and GBP-L and GABA-lactam analogs seemed to be able to antagonize this effect (Wolter 2009; Obermeyer 2010). For a systemic application in mice, the threshold dose of GBP-L that caused toxic reactions such as seizures was 10 µM (Feuerstein, unpublished data). The proliferative effect of GBP-L and GABA-lactam analogs does not seem to interfere with the differentiation potential of MSC, as cells could be differentiated into osteogenic, chondrogenic and adipogenic progenitors after the trials (Wolter 2009; Obermeyer 2010; Degenhardt 2010). 10 Introduction
1.3 Oleic acid
FIG. 6: Oleic acid.
Oleic acid (18:1) is a monounsaturated 18-carbon fatty acid that is ubiquitously present in plant and animal tissues. It is classified as a omega-9 fatty acid with one double bond (cis-9). The name ,oleic‘ is derived from the high content of oleic acid in oil and olives. Fatty acids are carboxylic acids with long unbranched carbon chains. They are a source of energy, and a structural membrane component of cells. Fatty acids are the major component of biological lipids necessary for energy storage such as triglycerides - and they constitue a basic element of the phospholipids that make up all cell membranes. They also act as membrane-associated signaling molecules (Sweeney et al. 2005). The phospholipids in cell membranes form a bilayer with the hydrophilic ,heads‘ facing the intra- and extracellular spaces and the hydrophobic ,tails‘ aggregating to establish a hydrophobic barrier inside the membrane. While researching the possibility of supplementing cell growth media with the fatty acids oleic acid (18:1) and linoleic acid (18:2), D. Grimm discovered a positive effect of oleic acid on the proliferation of cultured gingival keratinocytes (Grimm 2009). The analysis of the fatty acid composition of cells cultured with oleic acid showed a significant increase in oleic acid and palmitic acid (16:0) as well as a significant decrease in linoleic acid. This proliferation-enhancing effect of oleic acid as a media additive on cultured keratinocytes has also been described by Marcelo and colleagues (1992; 1994). Linoleic acid on the other hand seems to substantially decrease cell proliferation (Marcelo et al. 1994; Schurer et al. 1999; Grimm 2009). Oleic acid has also been shown to increase membrane fluidity (Heron et al. 1980; Ismaili et al. 1999). K. Pelz and colleagues showed the influence of different culture medium additives on the fatty acid composition of KB cells and gingival keratinocytes, observing an 11 Introduction increase in membrane fluidity depending on the additive (2006). In 1972, Singer and Nicholson described cell membranes to possess a fluid mosaic structure. Fatty acid composition in this structure might vary depending on the extracellular environment. Cells cultured in vitro are subject to changes in their membranes‘ fatty acid composition, determined by which type of serum the growth medium is supplemented with (Stoll and Spector 1984). The membrane viscosity of human keratinocytes has been shown to differ significantly with the addition of certain fatty acids to the growth medium (Dunham et al. 1996). The fatty acid-induced alterations in membrane fluidity and permeability have also been confirmed by A. Cader and colleagues (1995). Membrane fluidity is one of the factors considered to be relevant to the modulation of the immune response (de Pablo and Alvarez de Cienfuegos 2000), moderating changes in cytokine receptor affinity as well as macrophage functions (Stubbs and Smith 1984; Calder et al. 1990; Grimble and Tappia 1995). A higher membrane fluidity correlates with an increased receptor affinity and chemotaxis in neutrophilic granulocytes (Yuli et al. 1982). Oleic acid has been shown to stimulate the production of cytokine interleukin-8 (IL-8) (Andoh et al. 2000; Tanaka et al. 2001; Grimm 2009), as well as IL-1 and IL-2 (Tappia and Grimble 1994; Yaqoob and Calder 1995; de Pablo et al. 1998). Interleukins are important signaling molecules in the activation of the immune response such as the chemotaxis of leukocytes. Since an increased membrane fluidity and an interleukin production has been observed in previous trials, oleic acid is likely to have an immunomodulatory effect and to improve the cells‘ resistance to infection. It is known that a diet supplemented with oleic acid- containing olive oil has a positive effect on the immune system (Jeffery et al. 1996; Yaqoob et al. 1998; de Pablo and Alvarez de Cienfuegos 2000; Puertollano et al. 2007). An enhanced proliferation with additional immune protection would be beneficial for in vitro MSC expansion as well as in vivo MSC grafts. 12 Introduction
1.4 Aim of this thesis
The aim of this in vitro study was to examine the suggested growth factor-like effect of gabapentin-lactam and oleic acid on ovine mesenchymal stem cells.
The first objective was to verify the observed proliferative effect of GBP-L on ovine MSC (Wolter 2009; Obermeyer 2010) using the same proliferation assay (EZ4U, Biomedica, Vienna, Austria) as the previous studies. It was conducted as a time course assay with a baseline measurement at the beginning of the culturing period (day 1), as well as several measurements before cells reached confluency (day 3, 5, 10). Given that it showed the highest proliferation rate in preliminary studies, the concentration of 10 µM was selected for the GBP-L medium (Feuerstein, unpublished data).
A proliferation-enhancing effect of oleic acid has been observed in cultured keratinocytes (Grimm 2009). It was now of interest to find out whether this effect could be reproduced with ovine MSC. Six different concentrations (50 µM, 100 µM, 200 µM, 400 µM, 600 µM, 800 µM) were chosen for oleic acid media. Since the concentration of 600 µM has been shown to be lethal for chicken fibroblasts (Liu et al. 2009), it was hypothesized that the same could apply to ovine MSC.
It has previously been shown that GBP-L seems to be able to antagonize the toxic effect of the anti-freezing agent DMSO on ovine MSC proliferation (Wolter 2009; Obermeyer 2010). A combination of 10 µM GBP-L and 600 µM oleic acid was added to the tested culture media to find out whether GBP-L would offset the possible toxic effect of oleic acid at this concentration.
Furthermore, the 10 µM GBP-L medium was tested on human MSC (approved by the ethics committee EK Freiburg 26/05) in comparison to a control group. The effect was quantified with the CyQuant proliferation assay (Invitrogen/Life Technologies, Carlsbad, CA, USA) to establish this method as a possible alternative to the EZ4U assay. 13 Material and Methods 2 Materials and Methods
2.1 Ovine MSC Cryopreserved ovine mesenchymal stem cells from six different lineages were resuscitated to be used for the experiments of the present study. The bone marrow for the primary culture was harvested from the pelvic bones of six sheep (age: < one year) in a time period of less than 24 hours after slaughtering by U. Degenhardt with the aseptic explantation technique detailed in her thesis (2010). The primary culture was expanded in 75 cm2 cell culture flasks (Corning BV, Schiphol-Rijk, Netherlands) with the addition NH Expansion Medium (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) containing 1 % penicillin- streptomycin (Biochrom AG, Berlin, Germany). NH Expansion Medium is a ready-to-use proliferation medium for human MSC containing Dulbeco‘s modified Eagle‘s medium (DMEM), L-glutamine and fetal bovine serum (FBS). The cultured cells were kept in an incubator (Heracell incubator, Heraeus Medical, Hanau, Germany) at 37° C, 95 % humidity and 5 % CO2 at all times. MSC from each lineage were cryopreserved as described in the next paragraph.
2.1.1 Cryopreservation After being expanded for one or two passages, MSC from the six different lineages were harvested. The growth medium was removed from the culture flasks and a washing step with phosphate-buffered saline (PBS) (PAA Laboratories, Pasching, Austria) was performed. One mL trypsin / EDTA (0.05 % / 0.53 mM, PAA Laboratories, Pasching, Austria) was added to each culture flask to cover the cells. The flasks were incubated with the trypsin / EDTA at 37° C for 5 - 7 minutes. EDTA is a calcium chelator that loosens cell-cell-junctions; whereas trypsin, a protease isolated from the pancreas, breaks down cell-matrix-junctions, cleaving cell connections to the culture dish (detachment). Cells that are exposed to trypsin for too long can be damaged. Therefore the incubation period should not excess the necessary time frame for detachment. The complete dissociation was verified with the microscope (Axiovert 135, Zeiss, Göttingen, Germany). The detached cells were suspended in 10 mL medium and transferred to a 50 mL conical tube (Greiner-bio-one, Frickenhausen, Germany). After centrifuging the cell suspension at 300 x g for 10 minutes at room temperature (Biofuge Stratos, Heraeus Instruments, Osterode, Germany), the supernatant was removed by pipetting and the cell pellet was carefully resuspended. 14 Material and Methods
The pellet was suspended in NH Expansion Medium (Miltenyi Biotec, Bergisch Gladbach, Germany) with the addition of 10 % fetal bovine serum (FBS) and 10 % dimethyl sulfoxide (DMSO) at a concentration of 1 x 106 / mL. DMSO acts as a cryoprotectant by inhibiting the formation of crystals inside of and around the cells, and by preventing the partial dehydration of the cytoplasm. The cell suspension was then transferred to cryovials in 1.5 mL aliquots and frozen in a three-step process. The cryovials were first placed in the refrigerator at 8° C for 15 minutes to allow for the permeation of the cryopreservation medium. Afterwards, they were kept at -20° C for 2 - 4 hours and at -80° C for 10 hours. Finally, the vials were transferred to liquid nitrogen (-196° C).
2.1.2 Resuscitation To minimize cellular damage, thawing should be performed as quickly as possible. Hence, the cryovials with frozen MSC were placed in a 37° C water bath before their content was suspended in 10 mL NH Expansion Medium (Miltenyi Biotec, Bergisch Gladbach, Germany) in 50 mL conical tubes (Greiner-bio-one, Frickenhausen, Germany) and centrifuged at 1200 rpm for 10 minutes. The resulting pellet was then resuspended in 25 mL NH Expansion Medium and centrifuged one more time to allow for thorough removal of DMSO residue. After resuspension in 10 mL NH Expansion Medium, cells were plated in 75 cm2 cell culture flasks (Corning BV, Schiphol-Rijk, Netherlands). The medium was changed after 24 hours to remove the potentially cytotoxic residue of the cryoprotectant. Afterwards, the medium was refreshed every two days. When the MSC reached sub- confluency after approximately six days, they were passaged as described below.
2.1.3 Passaging After the removal of the growth medium and a washing step with phosphate-buffered saline (PBS) (PAA Laboratories, Pasching, Austria), 1 mL trypsin / EDTA (0.05 % / 0.53 mM, PAA Laboratories, Pasching, Austria) was added to each culture flask to cover the cells. The flasks were incubated at 37° C for 5 - 7 minutes thereafter. Again, the exposure to trypsin was kept as short as possible. The complete dissociation was verified with the microscope (Axiovert 135, Zeiss, Göttingen, Germany). The detached cells were suspended in 10 mL NH Expansion medium and transferred to a 50 mL conical tube (Greiner-bio- one, Frickenhausen, Germany). After centrifuging the cell suspension at 300 x g for 10 minutes at room temperature (Biofuge Stratos, Heraeus Instruments, Osterode, Germany), the supernatant was removed by pipetting and the cell pellet was carefully resuspended in 2 mL medium. 15 Material and Methods
To test MSC viability, cells were stained with trypan blue (Biochrom AG, Berlin, Germany). In this dye exclusion method, only irreversibly damaged cells are stained since the acidic dye is prevented from penetrating intact cell membranes due to its negative ion charge. The cell number was then determined by counting the unstained cells using a Neubauer counting chamber (haemocytometer) (B. Brand, Wertheim, Germany). The harvested MSC were resuspended in medium to be used in the experiments described in the following sections. 16 Material and Methods
2.2 Differentiation of ovine MSC To prove the multi-lineage potential of mesenchymal stem cells, the cultured cells were differentiated into adipogenic, osteogenic and chondrogenic phenotypes using specific MSC differentiation media (NH AdipoDiff / OsteoDiff / ChondroDiff Medium, Miltenyi Biotec, Bergisch-Gladbach, Germany).
2.2.1 Adipogenesis assay The harvested MSC were suspended at 5 x 104 cells / mL in NH AdipoDiff Medium (Miltenyi Biotec, Bergisch Gladbach, Germany) with 1 % penicillin-streptomycin (Biochrom AG, Berlin, Germany) and seeded onto 6-well-plates (Corning BV, Schiphol-
Rijk, Netherlands) in 1.5 ml aliquots. The plates were then incubated at 37° C with 5 % CO2 and 95 % humidity with a medium change every 2 - 3 days. Large fatty vacuoles could be detected in the cytoplasm by microscopic control after 2 - 3 weeks.
Oil Red O staining was used to visualize lipid droplet formation. A working solution was prepared from 0.5 % Oil Red O (Sigma-Alldrich Chemie GmbH, Steinheim, Germany) stock solution in isopropanolol by diluting 6 mL stock solution with 4 mL distilled water. Immediately before staining, the working solution was filtered through a 0.22 µm filter (Schleicher & Schuell, Dassel, Germany). After removing the medium from wells and washing cells twice with PBS (PAA Laboratories, Pasching, Austria), 2 mL methanol (cooled at -20° C) were added and incubated for 5 minutes. The methanol was removed by pipetting and the wells were washed twice with 2 mL distilled water each. 2 mL Oil Red O working solution was then added to each well and mixed slowly on a plate shaker (IKA Basic 260 B, IKA-Werke GmbH & Co KG, Staufen, Germany) at room temperature. After 20 minutes, the staining reagent was removed and each well was washed with 2 mL distilled water. Immediately after staining, the cells were examined under the microscope.
2.2.2 Osteogenesis assay The passaged MSC were suspended at 5 x 104 cells / mL in NH OsteoDiff Medium (Miltenyi Biotec, Bergisch Gladbach, Germany) with 1 % penicillin-streptomycin (Biochrom AG, Berlin, Germany) and plated onto 6-well-plates (Corning BV, Schiphol- Rijk, Netherlands) in 1.5 mL aliquots. The plates were then incubated at 37° C with 5 %
CO2 and 95 % humidity with a medium change of every 2 - 3 days. After 10 days, osteogenic progenitor cells, characterized by their cuboid appearance and nearby synthesized bone matrix, could be detected by microscopic control. 17 Material and Methods
Alkaline phosphatase (ALP) is an isoenzyme present in all tissues of the human body but especially concentrated in the bone matrix. The concentration of ALP is a sensitive marker for osteoblastic activity. An ALP test kit (Sigma, Deisenhofen, Germany) was used to detect alkaline phosphatase activity. SIGMA FAST BCIP/NBT substrate (mixture 1) was prepared by combining 0,25 mL FBB solution (kit solution 1) with 0,25 mL sodium nitrite solution (kit solution 2) and incubating the mixture for 2 minutes. Meanwhile, 0,25 mL alkaline naphtol AS BI solution (kit solution 3) were added to 11,25 mL distilled water (mixture 2). Mixtures 1 and 2 were then combined for immediate usage. All medium was removed from the 6-well-plates and plates were left to air dry. Cells were then fixed by the application of a solution containing 5 mL citrate solution, 6,5 mL acetone and 0,8 mL formaldehyde for 30 seconds. After fixation, the plates were washed with distilled water. 2 mL substrate solution were added to each well and incubated for 30 minutes in darkness. Another washing step with distilled water followed after the removal of the supernatant solution. Afterwards, cells were counterstained with neutral red (toluylene red) and washed again with distilled water. Alkaline phosphatase activity can be detected by a diffuse blue precipitate in the cytoplasm whereas cell nuclei should appear red.
Type I collagen was detected by immunohistochemical staining. For the fixation of cells, the medium was removed from the 6-well-plates and the cells were washed with phosphate-buffered saline (PBS) (PAA Laboratories GmbH, Pasching, Austria) for 5 minutes. The cells were then covered with 70% ethanol for one hour. After the removal of the supernatant, the cells were again washed with PBS for 5 minutes and air dried over night.
The fixed plates were again covered with PBS for 5 minutes and incubated with 0.3 % H2O2 in methanol for 10 minutes, followed by another three washing steps with PBS for 5 minutes each. To inhibit unspecific immune reactions, 10 % bovine albumin was added for 10 minutes. The first antibody (Anti-Kollagen-Typ-I, Sigma, Deisenhofen, Germany) was added at a 1:100 dilution ratio and incubated for one hour. The plates were then incubated with the diluted biotinized second antibody (Vectastain Elite Kit, Vector Laboratories, Burlingame, USA) and an avidin-biotin-peroxidase complex (Vectastain Elite Kit, Vector Laboratories, Burlingame, USA). After each incubation step, the cells were washed several times with PBS. Peroxidase substrate solution (4 mg diaminobenzidine, 10 mL 0.05 M Tris 18 Material and Methods buffer, 17 µL 30 % H2O2) was added to start the immune reaction. Plates were again washed three times with PBS and counterstained with haemalaun (Merck, Darmstadt, Germany). After another washing step with water, the cells were examined by light microscope (Zeiss, Göttingen, Germany) at the 100 x magnification. The cytoplasm of positive cells should appear brown and nuclei blue.
Mineralization can be visualized by von Kossa staining. In this method, deposited calcium is replaced by silver ions which appear black under light microscopy. After the removal of the medium, the cells are fixed with 70 % ethanol and incubated in darkness with 5 % silver nitrate solution (Carl Roth, Karlsruhe, Germany) for one hour. The plates were then washed with distilled water. 1 % pyrogallic acid was added for 3 minutes to reduce the silver ions to metallic silver and the plates were again washed with distilled water. The cells were counterstained with nuclear fast red solution (0,1 g nuclear fast red, 5 % aluminum sulfate, 100 mL distilled water) for 8 minutes and washed with distilled water. An existing calcification in the cytoplasm and extracellular matrix should appear black in microscopy. The cell nuclei should be red.
2.2.3 Chondrogenesis assay The harvested MSC were suspended at 5 x 104 cells / mL in NH ChondroDiff Medium (Miltenyi Biotec, Bergisch Gladbach, Germany) with 1 % penicillin-streptomycin (Biochrom AG, Berlin, Germany) and transferred to 1.5 mL Eppendorf tubes (Eppendorf, Hamburg, Germany) in 1 mL aliquots. The cells were cultured in suspension rather than plated to prevent them from attaching to the polystyrene surface and to encourage the formation of cell nodules. The lids of the tubes were perforated with a sterile needle (B. Braun, Melsungen, Germany) to allow for air circulation. The tubes were centrifuged for 5 minutes at 150 x g at room temperature and placed upright in the incubator at 37° C with 5
% CO2 and 95 % humidity. The cells were cultured for 3 - 4 weeks with a medium change every 2 - 3 days.
The supernatant NH ChondroDiff Medium (Miltenyi Biotec, Bergisch Gladbach, Germany) was carefully removed by pipetting and the cell nodules were washed with phosphate- buffered saline (PBS) (PAA Laboratories, Pasching, Austria). Cells were then fixed with formalin on a plate shaker (IKA Basic 260B, IKA, Staufen, Germany) at room temperature and dehydrated with increasing ethanol concentrations. After the cell nodules were bathed 19 Material and Methods twice in Roti-Histol (Carl Roth, Karlsruhe, Germany) for 30 minutes at a time, they were imbedded in paraffin (pre-warmed to 58° C) and cooled down to -20° C over the next 8 hours. The embedded cell samples were then cut to 5 µm slices in a microtome (Leica, Nussloch, Germany) and mounted on microscope slides. The sections were deparaffinized in Roti-Histol. After rehydration with a descending ethanol dilution series (Allchem, Breisach, Germany), the sections were washed twice, first with distilled water and then with PBS for 5 minutes. It is important for the tissue samples to stay hydrated throughout the whole staining process, to guarantee reproducible specific staining results. A washing buffer was prepared by adding 1 % bovine serum albumin (BSA) (Sigma- Alldrich, Taufenkirchen, Germany) to PBS. To prepare for a blocking buffer, 10 % donkey serum (Dianova, Hamburg, Germany) was added to the washing buffer. While the sections were incubated with a permeabilization buffer (blocking buffer, with 0.3 % Triton X-100 (Fluka, Sigma Alldrich, Buchs, Germany)) at room temperature for 45 minutes, the primary antibody (mouse anti-human aggrecan) (Milipore, Schwalbach, Germany) was diluted with blocking buffer to a final concentration of 10 µL / mL. After permeabilization, the slides were dabbed dry and a circle was drawn around sections with a waterproof pen. The primary antibody was applied and the slides were incubated in a humidified chamber over night at 2 - 8° C. After the incubation, the sections were washed thrice with the washing buffer for 5 minutes at a time and incubated with the secondary antibody (donkey anti-mouse IgG rhodamine (Dianova, Hamburg Germany) diluted at a 1:50 ratio in washing buffer) in the dark for 60 minutes at room temperature. The fluoroscent stain DAPI (4',6-diamidino-2-phenylindole) (Sigma-Aldrich, Taufkirchen, Germany) was diluted with washing buffer at a 1:1,000 ratio and incubated in the dark for 15 minutes. The sections were washed twice for 5 minutes with the washing buffer, followed by a washing step with distilled water. The slides were then dabbed dry and cover slips were mounted with Fluoromount-G (Southern Biotechnology Association, Birmingham, USA) to protect the stained sections from light until they were analyzed by fluorescent microscopy (Axiovert 135, Zeiss, Oberkochen, Germany). 20 Material and Methods
2.3 Proliferation of ovine MSC To evaluate the effects of oleic acid and gabapentin-lactam on ovine MSC proliferation, cryopreserved cells were resuscitated and seeded in 24-well-plates (Corning BV, Schiphol- Rijk, Netherlands) at a density of 1 x 104 cells / well with NH Expansion Medium (Miltenyi Biotec, Bergisch Gladbach, Germany) in the presence or absence of oleic acid and gabapentin-lactam respectively. Five separate experiments were performed in three replicates (three wells for each medium). The medium was refreshed every 2 - 3 days.
2.3.1 Oleic acid and GBP-L media
Proliferation media
Gabapentin-lactam (GBP-L) Oleic acid (OA)
NH (control group) -- --
GBPL10 10 µM --
GBPL10_OA600 10 µM 600 µM
OA50 -- 50 µM
OA100 -- 100 µM
OA200 -- 200 µM
OA400 -- 400 µM
OA600 -- 600 µM
OA800 -- 800 µM
TABLE 1: Tested concentrations of media additives gabapentin-lactam (GBP-L) and oleic acid (OA).
For the preparation of the tested media, various concentrations of oleic acid (O1383-1G, Sigma München, Germany) as well as gabapentin-lactam (provided by the Department of Neuropharmacology, Freiburg University Hospital, Freiburg, Germany) were added to NH Expansion medium (Miltenyi Biotec, Bergisch Gladbach, Germany) under laminar flow in sterile conditions (Table 1). An oleic acid stock solution was prepared by the addition of 1 g oleic acid (O1383-1G, Sigma München, Germany), 850 µL 100 % undenatured ethanol (J.T. Baker, Deventer, Netherlands) and 9,15 mL culture medium in a 50 mL tube (Greiner Bio-One GmbH, 21 Material and Methods
Frickenhausen, Germany). This stock solution was then diluted with NH Expansion Medium (Miltenyi Biotec, Bergisch Gladbach, Germany) to achieve the desired molarity. A GBP-L working solution was prepared by diluting a GBP-L stock solution (50 mg / mL) with lactated Ringer‘s solution at a 1:10 ratio. To achieve a final concentration of 10 µM, 153 µL of the working solution were then added to 500 mL NH Expansion medium (Miltenyi Biotec, Bergisch Gladbach, Germany). The prepared media were sterile filtered before further usage.
2.3.2 EZ4U cell proliferation assay After 1, 3, 5 and 10 days of culture, the cell number was analyzed using the EZ4U assay (Biomedica, Vienna, Austria). This non-radioactive assay is based on the reduction of tetrazolium salts as an indicator of cell viability (Fig. 7). In living cells, the slightly colored tetrazolium salts are transformed to intensely colored formazan derivatives by the mitochondrial membrane-bound enzyme complex succinate dehydrogenase and ubiquitary cytosolic processes involving NADH and NADPH as co-factors (Berridge and Tan 1993). This metabolic activity can be measured by spectrometry and used as an indirect correlate to the number of living cells.
FIG. 7: Reduction of a tetrazolium dye to a formazan dye.
The EZ4U substrate solution was prepared according to the manufacturer‘s protocol by dissolving one vial of the substrate in 2.5 ml activator solution (pre-warmed at 37° C) immediately before performing the assay. After removing all culture medium from the 24- well-plates (Corning BV, Schiphol-Rijk, Netherlands), 500 µL of the respective medium and 50 µL EZ4U substrate were pipetted to each well and swirled gently. The plates were incubated for 3 hours at 37° C with 5 % CO2 and 95 % humidity (Heracell Inkubator, Heraeus Medical, Hanau, Germany). After the incubation period, the supernatant medium of each well was pipetted into three wells of a 96-well-microplate (Corning BV, Schiphol- Rijk, Netherlands). The absorbance was measured with a microplate reader (ELISA- 22 Material and Methods
Reader, Anthos Labtech, Salzburg, Austria) set at 450 nm, with 620 nm as a reference wavelength.
2.3.3 Statistical analysis The cell populations were cultured in three replicates (three wells on a 24-well-plate) for each of the tested proliferation media. The absorbance was measured in three samples from each replicate. For the statistical analysis, arithmetric means were calculated for the measured absorbances. The statistical analysis was performed by Dieter Menne of Menne Biomed Consulting, Tübingen, Germany using the software R (R 2005). 23 Material and Methods
2.4 Human MSC Three patients who presented for oral bone grafts with autologous mesenchymal stem cells were asked to donate a portion of the aspirated bone marrow for research purposes. Two patients were healthy donors that underwent maxillary sinus augmentations prior to the placement of dental implants. One patient received reconstructive surgery for bisphosphonate-related osteonecrosis of the jaw. The procedures were performed under general anesthesia. Informed consent was retrieved from all patients and the protocol was approved by the local ethics committee (EK Freiburg 26/05).
2.4.1 Cell culture Bone marrow was aspirated from the posterior iliac crest. Mononuclear cells were isolated using the BMAC Procedure Pack (Harvest Technologies Corporation, Plymouth, MA, USA) with the method previously described by S. Sauerbier (2008). The cell number in both bone marrow aspirate and concentrate was determined by cell counting (Sysmex KX-21N, Norderstedt, Germany). The bone marrow concentrate was placed in 25 cm2 cell culture flasks containing 5 mL NH Expansion Medium (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) with the addition of 1 % penicillin-streptomycin (Biochrom AG, Berlin, Germany). The cell cultures were stored in an incubator (Heracell incubator, Heraeus Medical,
Hanau, Germany) at 37° C with 95 % humidity and 5 % CO2. The first medium change was performed after 24 hours. At that time, plastic-adherent cells were detected by inverted light microscopy (Axiovert 135, Zeiss, Göttingen, Germany). The medium was refreshed every 2-3 days and cell growth was observed regularly with the inverted microscope. When the cells reached a confluency of 80 %, they were passaged as described in section 2.1.3. After the first passage, the cells were used for the experiments described in the following sections. 24 Material and Methods
2.4.3 CyQuant cell proliferation assay To determinate the effect of GBP-L on the proliferation of human MSC, the passaged cells were cultured in medium with the addition of 10 µM GBP-L. The GBP-L medium was prepared as described in 2.3.1. A controlProtokoll: group CyQuant was expanded Cell in Proliferation NH Expansion Medium Assay 1 (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Five separate experiments were conducted with the cellsMaterialien from passages 1 and 2. • Zellsuspension • drei 96-Well-Platten (eine pro Messzeitpunkt) The CyQuant cell proliferation assay (Invitrogen/Life Technologies, Carlsbad, CA, USA) • Zellkulturmedien (NH Expansion Medium, Medium mit 10 µM GBP-L) uses DNA quantification as a measure to determine the number of cells.
Versuchsansatz (Tag 0)
• Inkubationszeiten 24 h, 3 d, 6 d = eine Platte pro Inkubationszeit • Dreifach-Ansatz (siehe Skizze) • 1000 Zellen / Well
Mediumwechsel 3 x /Woche • 200 µl Medium / Well
nonur cells Medium nonur cells Medium cellsmit Zellen cellsmit Zellen GBP-L Medium NH Medium NH Expansion 10 µM GBP-L Medium Medium FIG. 8: 96-well-microplate for CyQuant assay.
The cells were seededEinfrieren in 96-well-plates (Corning BV, Schiphol-Rijk, Netherlands) at a density of 1,000 cells! / nachwell withjeweiliger 200 µ InkubationszeitL medium with (24or without h, 3 d, 6 GBP-L d) Platten (Fig. aus 8). Onedem plateBrutschrank nehmen was prepared for eachund incubation Medium verwerfen period with three wells per medium. Three wells without cells were added for !each mit medium PBS waschen as a control of each medium‘s specific fluorescence. The cells were cultured for! 1,Platten 3 and bei6 days, -80 °Cwith einfrieren medium (wichtig changes für three Lyse times der Zellen!)a week. After the respective incubation! Platten können period, beithe -80medium °C bis was zu 4removed Wochen from gelagert the plates. werden The wells were washed with phosphate-buffered saline (PBS) (PAA Laboratories GmbH, Pasching, Austria). The plates were then frozen at -80° C. The freezing step is critical for cell lysis. The plates can be stored at -80° C for up to 4 weeks. For the assay, a working solution was prepared by adding 50 µL CyQuant GR stock solution (Component A) to a mixture of 1 mL cell-lysis buffer stock solution (Component B) and 19 mL nuclease-free water (Qiagen, Venlo, Netherlands) in a 50 mL conical tube (Greiner-bio-one, Frickenhausen, Germany). After the microplates were thawed at room 25 Material and Methods temperature, 200 mL of the working solution were added to each well by pipetting (Multipette Plus, Eppendorf, Norderstedt, Germany) and the plates were incubated for 3 minutes. The fluorescence was determinedProtokoll: by a microplate EZ4U Cell reader Proliferation (ELISA-Reader, Anthos Assay Labtech, Salzburg, Austria) at an excitation wavelength of 480 nm and an emission Materialien wavelength of 520 nm. • Zellsuspension • drei 24-Well-Platten (eine pro Inkubationszeit) 2.4.4 EZ4U cell proliferation• Zellkulturmedien assay (NH Expansion Medium, Medium mit 10 µM GBP-L) The cells were seeded in 24-well-plates at a density of 1 x 104 cells / well in 1 mL medium • EZ4U Assay Kit with or without GBP-L (Fig. 9). They were cultured for 1 and 6 days with medium changes • drei 96-Well-Platten (eine pro Messzeitpunkt) three times a week. After the respective incubation period, EZ4U assays (Biomedica, Vienna, Austria) were performed as detailed in 2.3.2.
Versuchsansatz (Tag 0) • Inkubationszeiten 24 h, 3 d, 6 d = eine Platte pro Inkubationszeit • Dreifach-Ansatz (siehe Skizze) • 1 x 104 Zellen / Well
Mediumwechsel 3 x /Woche • 1 ml Medium / Well
NH Expansion Medium
10 µM GBP-L Medium
FIG. 9: 24-well-plate for EZ4U assay. Messtag
! EZ4U ansetzen ! im Brutschrank für 3 Stunden inkubieren ! in 96-Well-Platte pipettieren
nur Medium
Verdünnung 1:2
unverdünnt
! Messung (Messprotokoll „EZ4U_neu“ , Matrix-Darstellung!) ! speichern 26 Results 3 Results
3.1 Oleic acid induces morphological change in ovine MSC Ovine bone marrow-derived mesenchymal stem cells (MSC) exposed to oleic acid in various concentrations (50 - 800 µM) showed distinctive changes in their morphological appearance after approximately one week of culture. Lipid droplets could be detected in the cytoplasm and cells appeared to differentiate into an preadipocyte-like phenotype with a maintained fusiform shape (Fig. 10). These morphological changes could be found in MSC treated with all concentrations of oleic acid, as well as in a combination of oleic acid and gabapentin-lactam (GBP-L).
To visualize lipid droplet formation, the cells were stained with Oil Red O as described in 2.2.1 after 9 days of culture in 600 µM oleic acid medium with or without GBP-L.
A B
C D
FIG. 10: Morphological changes of ovine MSC treated with medium containing oleic acid, stained with Oil Red O. (A) Cells cultured in OA 600 µM, counterstained with methylen blue (B), (C) Cells cultured in OA 600 µM. (D) Cells cultured in OA 600 µM and GBP-L 10 µM. 27 Results
3.2 Effect of oleic acid and GBP-L on ovine MSC proliferation (EZ4U assay) Proliferation rate compared to control
Time (days)
FIG. 11: Proliferation rate (dots) and 95% confidence interval for the different proliferation media compared to the reference group (medium NH).
A significant increase in cell proliferation (p < 0.05) was found for all concentrations of oleic acid except 800 µM (Fig. 11). The highest proliferative effect could be detected for concentrations of 400 µM and 600 µM with an increase in cell growth rates of 3.5 %. A dose-effect relationship in favor of medium-high concentrations of oleic acid, as suggested by the data, could not be proven. It is noticeable, however, that the combination of both gabapentin-lactam (GBP-L) and oleic acid seems to cause a suppression of cell growth. No proliferative effect could be detected for gabapentin-lactam itself compared to the control group. In Figure 11, the black dots show the proliferation rate of the different proliferation media compared to the reference group with NH medium, whereas the black horizontal lines indicate the 95% confidence intervals. The value of 1.00, implied by a grey vertical line, was equated with the proliferation rate of the reference group. A significant difference in log (absorption) absoption ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! 2 ! ! ! 2 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !! ! ! ! ! ! !! ! ! ! !! ! !! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! !! 1 ! ! !!! ! !!! ! !!!! 1 ! ! !! ! !!! ! !! !! !!! ! ! !!! !! ! !!! !! ! !! ! !! ! ! ! !! ! ! !!! !!!! ! !!! ! ! ! ! ! ! ! !!!!! ! !! !! !!! !!! ! !! !! !! !! !!!! !! !! !! !!!! ! !! ! !!!! !!!!!!!!! ! !!!! !! !!! ! ! !! ! !!! ! ! !! !! ! !!!! !!!!! !!!!!! !!! !!!!! ! !!!!! !!!! !! !! !! !! ! ! ! ! !!! !!!!!!! !! ! !! ! !!! ! !!!!!!! !! !!! !!! !!!! !!! ! !!!! !!!! !!!!!!! !! ! ! !!!!!!!!!!! !!! ! !! ! !!!!!!!! !!!!! !! !! !! ! !!!!!!!!!!!! !!!!!! ! ! 0 !!!!!!!!!!!!!!!!!!! ! ! ! 0 !!!!!!!!!!!! !!!!!!!! !! !! !! !!!!!!!!!!!!!!!!!!! !!! !! !!! !!!!!!!!!!!!!!!! ! !! !!!!!!!!! ! !!!!!!! ! !!!!!!!!!!!!!!!!!!! ! !! !!! ! !!! !!! !!!!!!!! ! !!!!!!!!!!!!!!!!!!! ! ! !!! ! !!!!!!!! ! ! ! !! !!! ! !! !!!!!!!!!!!! ! ! ! ! !! !! !! ! ! ! !!!!!!!!!!!! !! ! !! !! !! !!!!!!! !! !!! ! !! !!! !! !! ! !!!! !!!!!!! !!! ! ! ! !! !! ! ! ! !!!!! ! ! !! ! ! ! !! !! ! !! !1 ! ! ! ! ! ! ! !1 ! !!! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! !! ! !! ! ! ! !!!!!! ! ! !!!! !! ! ! ! !! !! ! Standardized residuals Standardized !2 residuals Standardized !2 ! !! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !2.0 !1.5 !1.0 0.1 0.2 0.3 ! 0.4 ! Fitted values 28 Fitted values Results proliferation can be derived from the grey line (id est the value 1.00) not being part of the confidence interval. Abbildung 4: ResidualplotTable bei2 shows einer Auswertungthe differences des Logaritmus in proliferation der Absorption rates (links)compared und der to Absorp- the reference group, the tion. Diese Graphiken zeigen, wie weit der Messwert von der gesch¨atzten Ausgleichsgeraden abweicht.confidence Im rechten intervals Bild zeigt and sich derthe Trend,p-values. dass kleine Werte zu niedrig gesch¨atzt werden, große zu hoch. Das linke Bild ¨ahnelt mehr einer gleichm¨aßigen Wolke, also einer Anpassung ohne systematischen Fehler.
Medium fak ciUpper ciLower p GPBL10 0.999 1.015 0.983 0.88 GBPL10 OA600 0.932 0.947 0.918 <.0001 OA50 1.023 1.040 1.007 0.0047 OA100 1.018 1.034 1.002 0.032 OA200 1.016 1.032 1.000 0.051 OA400 1.035 1.052 1.019 <.0001 OA600 1.035 1.051 1.019 <.0001 OA800 1.006 1.022 0.990 0.46
TABLE 2: fak: multiplier of the proliferation rates compared to medium NH. Tabelle 2: fak: Faktor der Wachstumsraten im Vergleich zu Medium NH; ciUpper, ciLower: ciUpper, ciLower: 95% confidence interval of the factor. 95% –Konfidenzintervall des Faktors; p.value: Fehlerwahrscheinlichkeit des Tests gegen die Hy- p-value: probability of the null hypothesis being true (fak = 1.00). pothese, dassThe der null Faktor hypothesis gleich is rejected 1 ist, alsoif the keinp-value Unterschied is less than 0.05 besteht. (significance level).
In Tabelle 2 und Abbildung 5 sind die Unterschiede im Wachstum im Vergleich zur Referengrup- pe FH dargestellt. Am auff¨alligsten ist, dass die Kombination GBPL mit OA eine sehr deutliche Suppression des Wachstums hervorruft, w¨ahrend fur¨ GPBL selbst kein Nachweis fur¨ eine Wachs-
Menne Biomed Consulting Tubingen¨ 6 29 Results Absorbance
Time (Days)
FIG. 12: Logarithmic representation of cell growth curves. The top number of each panel is the subject ID of the sheep from which the tested cell population came from. Abbreviated medium names stand below.
The cell proliferation rate was estimated with a linear mixed model (Pinheiro and Bates 2000). This analysis allows for a statistically correct consideration of the correlations between the data of a subject and the replicates. The shown slope values represent the maximum likelihood estimates. This provides for a more reliable output than using mean values of the slope as approximation estimates. 30 Results
Statistical exclusion of systematic error
log (absorbance) absorbance
FIG. 13: Residual plot of the analysis of the logarithm of absorption values (left) and the absorption. The graphs show how far measured values deviate from the estimated best fit line. horizontal axis = independent variable
The analysis of the original absorbance values did not deliver a reliable estimate of the slope values (proliferation rate) (Fig. 13, right panel). The plot on the right indicates the trend towards too low estimates for low numbers and too high estimates for high numbers. A linear regression model was therefore not appropriate for the analysis of this data.
A better estimate could be achieved by employing the logarithm of the absorbance (Fig. 13, left panel). The plot on the left shows a more random pattern, indicating an adaption without systematic error. 31 Results Absorbance (log scale) (log Absorbance
Time (days)
FIG. 14: EZ4U results: smoothed mean values and 95% confidence intervals (gray ribbons) on a logarithmic scale. 32 Results Absorbance
Time (days)
FIG. 15: EZ4U results: smoothed mean values and 95% confidence intervals (gray ribbons) on a linear scale. EZ4U Cell Proliferation Assay
Patient 79 - Passage 0 Patient 79 - Passage 1 0,2000 0,2000
0,1750 0,1750
0,1500 0,1500 Absorption Absorption
0,1250 0,1250
0,1000 0,1000 1 8 1 6 Time (Days) Time (Days) 33 Results NH 1 NH 2 NH 3 GBP-L 1 NH 1 NH 2 NH 3 GBP-L 1 3.3 EffectGBP-L 2 ofGBP-L GBP-L 3 on human MSC proliferationGBP-L 2 (CyQuantGBP-L 3 assay)
CyQuant Cell Proliferation Assay
Patient 79 - Passage 1 Patient 81 - Passage 1 und 2 40000 40000
30000 30000
20000 20000 Fluorescence Fluorescence 10000 10000
0 0 1 3 6 1 3 6 Time (Days) Time (Days) NH GBP-L NH GBP-L NH GBP-L Passage 1 Passage 2
Patient 82 - Passage 1 und 2 40000
30000
20000 Fluorescence 10000
0 1 3 6 Time (Days)
NH GBP-L NH GBP-L Passage 1 Passage 2
FIG. 16: Results of CyQuant cell proliferation assay.
A tendency for a proliferation-enhancing effect of GBP-L on human MSC could not be detected (Fig. 16 and 17). Five separate experiments with three wells per medium were conducted for each medium and the cell number was analyzed by fluorescent DNA staining (CyQuant proliferation assay). The number of MSC in most bone marrow samples proved to be too low to set up both proliferation assays (CyQuant and EZ4U) for a direct comparison. The CyQuant proliferation assay was successfully established as an alternative to the EZ4U method in our department. 34 Results EZ4U Cell Proliferation Assay
Patient 79 - Passage 0 Patient 79 - Passage 1 0,2000 0,2000
0,1750 0,1750
0,1500 0,1500 Absorption Absorption
0,1250 0,1250
0,1000 0,1000 1 8 1 6 Time (Days) Time (Days)
NH 1 NH 2 NH 3 GBP-L 1 NH 1 NH 2 NH 3 GBP-L 1 GBP-L 2 GBP-L 3 GBP-L 2 GBP-L 3
FIG. 17: Results of EZ4U cell proliferation assay.
CyQuant Cell Proliferation Assay
Patient 79 - Passage 1 Patient 81 - Passage 1 und 2 40000 40000
30000 30000
20000 20000 Fluorescence Fluorescence 10000 10000
0 0 1 3 6 1 3 6 Time (Days) Time (Days) NH GBP-L NH GBP-L NH GBP-L Passage 1 Passage 2
Patient 82 - Passage 1 und 2 40000
30000
20000 Fluorescence 10000
0 1 3 6 Time (Days)
NH GBP-L NH GBP-L Passage 1 Passage 2 35 Results
3.4 MSC culture
3.4.1 Ovine cells The primary ovine mesenchymal stem cell cultures were maintained by U. Degenhardt who detailed the results in her dissertation (2010).
The cryopreserved MSC were successfully resuscitated and cultured in NH Expansion Medium. The cells regained their typical spindle-like shape and plastic-adherence in culture. The populations reached sub-confluency after 5 - 7 days.
Five out of six thawed cell populations could be used in the experiments. One population was discarded due to bacterial contamination after one week of culture.
3.4.2 Human cells Bone marrow-derived mesenchymal stem cells were cultured successfully in NH Expansion Medium. Plastic-adherent, spindle-shaped cells could be detected by the time of the second medium change after 72 hours. A sub-confluent cell monolayer developed over the following 3 - 4 weeks.
Donor ID
79 81 82
Bone marrow aspirate cells* / mL 2.1 x 107 2.4 x 107 1.7 x 107
Bone marrow concentrate cells* / mL 9.4 x 107 9.7 x 107 1.8 x 107
Passage 1 cells / mL 2.4 x 105 7.5 x 105 1.9 x 105
Passage 2 cells / mL -- 13.7 x 105 2.2 x 105
TABLE 3: Total cell counts. *mononuclear cells
The cell population of one individual (79) was used for both cell proliferation assays (EZ4U and CyQuant) at passage 1, but did not provide sufficient expansion for further subcultivation (Table 3). The other populations (81 and 82) were used for the CyQuant assay from passages 1 - 2. 36 Results
3.5 Multipotency Ovine MSC were successfully differentiated to osteogenic (Fig. 18: A, B, C), adipogenic (Fig. 18: E, F) and chondrogenic (Fig. 18: D) progenitor cells. The ability to differentiate in vitro is one of the criteria for defining mesenchymal stem cells .
A B
C D
E F
FIG. 18: Differentiated cells (40 x). A: Alkaline phosphatase stain. B: Type I collagen stain. C: von Kossa stain. D: Aggrecan stain. E and F: Oil Red O stain. 37 Discussion 4 Discussion
4.1 Results: Oleic acid In the present study, ovine mesenchymal stem cells (MSC) cultured with medium containing oleic acid in concentrations (varying from 50 to 800 µM) showed distinctive preadipocyte-like changes in their appearance after being cultured for approximately one week. To my knowledge, the effect of oleic acid on ovine MSC has not been previously described. The differentiation potential of oleic acid on other cells, however, has been reported before. The cell line NIH-3T3 has been shown to transdifferentiate into adipocytes when exposed to oleic acid (El-Jack et al. 1999). Liu and colleagues observed the effect of oleic acid on cultured chicken fibroblasts and preadipocytes (2009). After four days, fibroblasts showed a deposition of lipid beads in the cytoplasm. During this apparent transdifferentiation, cells expressed the adipogenic transcription factors PPARγ, C/EBPα and SREBP-1 (Gregoire et al. 1998; Davies et al. 2005) as well as adipocyte marker protein (A-FABP). PPARγ alone is able to induce the adipogenic differentiation of fibroblasts (Tontonoz et al. 1994).
FIG. 19: Regulators of MSC differentiation (Takada et al. 2009). 38 Discussion
The transdifferentiation potential of oleic acid has further been observed in studies with hepatocytes and OP9 mouse stromal cells (Jump et al. 2005; Wolins et al. 2006). Porcine stromal-vascular cells have been differentiated into adipocytes with oleic acid as a medium additive (Ding and Mersmann 2001), showing a dose-dependent effect and an increased level of differentiation over time. Chicken preadipocytes exposed to oleic acid developed the typical round shape of adipocytes as well as an increased cytoplasmatic lipid-droplet formation (Liu et al. 2009). Other studies with preadipocytes confirmed the up-regulation of adipogenic genes during culture with oleic acid (Ericsson et al. 1997; Xie et al. 2006). These findings suggest that oleic acid has a modulatory effect on adipogenesis, acting as a signaling molecule (Amri et al. 1994; Azain 2004). The mechanism of action could involve the binding to the promotor regions of adipogenetic genes (Duplus et al. 2000). It has been shown that a high fat diet can increase bone marrow adiposity (Chen et al. 2010), possibly by stimulating adipogenesis in bone marrow-resident mesenchymal stem cells.
In the present study, mesenchymal stem cells exposed to oleic acid (18:1) as a medium additive showed a higher proliferation rate compared to the control group cultured without oleic acid. This effect was statistically significant (p < 0.05) for the majority of concentrations (50 - 600 µM) except for 800 µM. The proliferation-enhancing effect of oleic acid on mammalian cell culture has first been reported in 1970 by Jenkin and Anderson. A similar effect has been documented for various other cell types including keratinocytes (Marcelo et al. 1992; Grimm 2009). The proliferative effect has recently been of interest in the study of atherosclerosis. Since perivascular adipose tissue constantly releases free fatty acids including oleic acid, it has been hypothesized that this might be a cause for the proliferation and inflammation of the vascular walls. It has been shown that oleic acid induces the proliferation of rat and human vascular smooth muscle cells (Yun et al. 2006; Lamers et al. 2010), an effect that can be antagonized by estrogen and palmitic acid (16:0) (Zhang et al. 2007; Jiang et al. 2009). Since the proliferative effect in the present study seems to decrease at the concentration of 800 µM, it could be possible that higher concentrations might actually have an inhibitory effect on cell proliferation. Oleic acid at a concentration of 600 µM was shown to inhibit the proliferation of chicken fibroblasts (Liu et al. 2009). Ovine mesenchymal stem cells seem capable of tolerating substantially higher concentrations. Although a dose-dependent effect could not be proven statistically, concentrations of 400 and 600 µM showed the 39 Discussion highest increase in proliferation in the performed assays. Further studies with a higher number of individuals should be designed to explore the dose-effect-relationship of various concentrations and to determine the potential toxicity of oleic acid at higher concentrations. A recent study showed an inhibiting effect of oleic acid on human MSC viability (Smith et al. 2012). This was reported for concentrations of 40 µM and higher. MSC in the cited study were plated at a density of 60 cells / cm2, expanded with regular medium (α- minimum essential medium (α-MEM) with glutamine, penicillin-streptomycin, HEPES and fetal bovine serum (FBS)) for 7 days and then switched to a medium containing α- MEM, 2% FBS and oleic acid for another 7 days. It was documented by the authors that no sign of adipogenic or osteogenic differentiation could be detected (Smith et al. 2012). In the present study, ovine MSC were seeded at a density of 500 cells / cm2 and cultured with oleic acid for 10 days without prior expansion. The medium supplemented with oleic acid was NH Expansion medium (Miltenyi Biotec, Bergisch Gladbach, Germany) containing Dulbeco‘s modified Eagle‘s medium (DMEM), penicillin-streptomycin, L- glutamine and fetal bovine serum (FBS). An inhibitory effect at the concentrations mentioned above could not be confirmed. On the contrary, oleic acid seems to have a proliferation-enhancing effect in the present experiments. The conflicting results of the two studies should be subject to further analysis. The applied methods differ in the two studies regarding seeding density, medium composition and proliferation assay. The seeding density used in this study conforms to the recommendation of plating at a density of 104 - 105 cells / ml to guarantee for sufficient cell-cell contacts and the conditioning of the culture medium by the cells (Lindl and Gstraunthaler 2008). Since the blood plasma levels of free fatty acids in patient with type-2-diabetes or obesity have been reported to be constantly at a concentration of around 600 µM (Roden 2004; Mathew et al. 2010), it seems plausible for mammalian cells to be able to tolerate these concentrations. The application of different proliferation assays is discussed in chapter 4.3. Oleic acid does have a low solubility and relatively high toxicity, and is therefore usually added to medium in combination with bovine serum albumin (BSA) (Nilausen 1978; Okuda et al. 1983). For serum-free culture, oleic acid esters have been proposed as as alternative growth factors (Yamada et al. 1990). They have been observed to stimulate proliferation in human HMy-2 cells and hamster HL-1 cells, with the effect being attributed to the fatty acid part. Having the chemical properties of detergents, they are water-soluble. 40 Discussion
Besides its proliferation-inducing benefits, oleic acid seems to induce adipogenic differentiation in MSC. This makes it unfit as a growth factor for MSC expansion in cell culture. For this purpose, growth factors should promote proliferation of MSC over several passages without differentiation toward a certain lineage (Rodrigues et al. 2010). However, oleic acid might be an useful medium additive for adipogenic differentiation. Other medium additives are known to promote the differentiation of mesenchymal stem cells. Soluble factors that are commonly added to culture medium to induce differentiation are listed below.
Cell type Soluble Factors
Osteoblasts β-glycerol-phosphate, ascorbic acid-2-phosphate, dexamethasone, fetal bovine serum
Chondrocytes ο-transforming growth factor β 1 and 3, bone morphogenetic protein-2, insulin-like growth factor, dexamethasone
Adipocytes isobutylmethylxanthine
TABLE 4: Medium additives to induce differentiation of MSC (Salinas and Anseth 2009).
To confirm the observations of this study, RT-PCR (reverse transcription polymerase chain reaction) and Northern blotting could be used to analyze the expression of adipogenic transcription factors and other adipocyte markers during the exposure to oleic acid. Further studies should be carried out to determine if the observed morphological changes in MSC phenotype are in fact the result of adipogenic differentiation.
The present study examined cryopreserved ovine MSC. The cells were successfully thawed and expanded in culture. It has been shown that cryopreservation does not have a negative effect on cell proliferation and differentiation abilities (Liu et al. 2008; Li et al. 2009). It can be assumed that the freezing and thawing process lead to a more homogenous cell culture than the original primary culture that showed very heterogenous results in the experiments by Degenhardt (2010). Cells could be successfully differentiated into adipogenic, osteogenic, and chondrogenic lineages, proving their MSC character. The analysis of MSC-associated cell surface markers such as CD29, CD44 and CD166 has been demonstrated in ovine mesenchymal stem cells before (McCarty et al. 2009) and should be part of future studies employing this cell type. 41 Discussion
An ovine model was chosen for comparability of results with previous studies of our department. Sheep are suitable test subjects for bone tissue engineering studies because of the similarities in bone structure and metabolism (Rehman et al. 1995). The effect of oleic acid should nonetheless be tested on human MSC, since the results of animal models are not always transferable.
The addition of oleic acid to culture medium could be a cost-efficient way of differentiating MSC to adipocytes in vitro and possibly in vivo. Adipose tissue engineering has been an area of research for soft tissue reconstruction (Patrick 2000). Soft tissue defects are likely to be encountered following ablative surgery in breast and head and neck tumors. Autologous adipose tissue grafts has been used to correct these defects. They are however subject to substantial and unpredictable shrinkage (von Heimburg et al. 2004) and require a sufficient amount of fat tissue at the donor site. There have been promising results with aspirated preadipocytes and hydrogel scaffolds (Halberstadt et al. 2002). Oleic acid could be added to these grafts to provide increased cell proliferation and differentiation of adipose tissue. Another possibility is the in vitro expansion and differentiation for MSC in culture medium containing oleic acid before therapeutic application. Oleic acid seems to be a promising signaling molecule for creating a micro-environment for adipogenic differentiation of MSC. 42 Discussion
4.2 Results: Gabapentin-lactam In this study, a proliferative effect on ovine and human mesenchymal stem cells (MSC) could not be detected for gabapentin-lactam (GBP-L) compared to the control group (Fig. 11). The effect of GBP-L on mesenchymal stem cells has been the subject of several in vitro studies in our department. After a proliferative effect had been observed on cultured osteoblasts in preliminary experiments (Feuerstein, unpublished data), subsequent studies suggested similar results for ovine mesenchymal stem cells (Wolter 2009; Obermeyer 2010). These studies involved testing of not just GBP-L, but also of other GABA-lactam analogs that were insoluble in water. All substances including the water-soluble GBP-L were added to medium with the addition of DMSO to create similar experimental conditions. DMSO is highly cytotoxic (Sexton 1980) and showed a suppressive effect on cell growth in the control group. Despite the addition of DMSO to culture media, all tested substances showed a significant enhancement in MSC proliferation compared to the control sample (Wolter 2009; Obermeyer 2010). A dose-dependent effect was suggested as well. It was hypothesized that the proliferation-enhancing effect would be even greater without DMSO added to the medium. Since GBP-L was the only water-soluble substance, it was chosen for testing on cultured ovine and human MSC in various concentrations (3,2 nM - 1 mM) in a consequent study (Degenhardt 2010). This study showed highly heterogenous results between all subjects. Enhancing and inhibiting effects on MSC proliferation could be observed in both groups, ovine and human. A dose-effect- relationship was not detected. The present study used cryopreserved MSC from 5 ovine subjects (passages 2 - 3) whose primary culture had been part of the previously conducted experiments. For the analysis of cell proliferation, the same method (EZ4U assay) as in the above-mentioned studies was employed. The concentration of 10 µM was chosen for the GBP-L medium since it seemed to promote the greatest growth rate in previous experiments (Degenhardt 2010). In contrast to prior experiments, the present study was designed as a time course assay performed on days 1, 3, 5, and 10.
In the present study, the effect of 10 µM GBP-L was also tested on human mesenchymal stem cells compared to NH expansion medium. Five independent experiments with MSC from three donors (passages 1 - 2) were conducted, with three wells per medium. The CyQuant proliferation assay was used to quantify cell proliferation. A statistical analysis 43 Discussion was not performed for this data, but the graphs did not show an apparent advantage in growth rate of the MSC exposed to GBP-L over the control group (Fig. 17). A potential toxic effect of GBP-L at 10 µM has been observed in systemic intravenous application (Feuerstein, unpublished). This observation, though, can not be supported by the data of the present and previous in vitro studies (Degenhardt 2010). Mitochondrial production of reactive oxygen species (ROS) has been suggested as a potential mechanism of action for GBP-L (Feuerstein 2011). However, recent studies imply that MSC proliferation is likely harmed by cytokines and ROS (Rodrigues et al. 2010). ROS have been shown to cause a decline in proliferation of hematopoetic progenitor cells (Meagher et al. 1988). Instead, differentiation seems to be stimulated (Reykdal et al. 1999; Carriere et al. 2003). ROS have also been shown to promote osteogenic differentiation in umbilical cord-derived mesenchymal stem cells by stimulation of TGF-beta 1 (Wang et al. 2004).
The combination of 10 µM GBP-L and 600 µM OA seems to cause a suppression of cell growth in ovine MSC at day 10 (Fig. 14). The possibility of a systematic error could not be eliminated completely for this result. However, increased ROS production has been shown to inhibit cell growth in preadipocytes (Carriere et al. 2003). If oleic acid induced adipogenic differentiation and the underlying mechanism of action for GBP-L was in fact related to ROS production, it can be speculated that this might be the reason for the ceased proliferation at the day 10-assay.
Yet the effect of GBP-L on MSC remains unclear. A growth factor-like effect for GBP-L on cultured ovine MSC could not be proven by the present thesis.
44 Discussion
4.3 Methods: Cell proliferation assays There are several methods to determine the number of vital cells in culture by testing different properties of living cells (Table 5).
Cell property Assays What is measured
1. Permeability Trypan blue exclusion Cell membrane integrity Cr, I-UDR release Cell membrane integrity LDH release Cell membrane integrity
2. Function ATP, ADP, AMP levels Cellular energy capacity MTT assay Mitochondrial function DNA synthesis Macromolecular synthesis capacity Protein synthesis Macromolecular synthesis capacity Ion, amino acid gradients Maintenance of ionic and pH environm.
3. Morphology Membrane blebbing Disruptions of plasma membrane Volume Changes in cellular osmotic properties Cytoskeletal Changes in cellular support structures
4. Reproduction In vitro colony formation Infinite cell division capacity In vivo colony formation Infinite cell division capacity Growth rate determination Increase in cell number with time
TABLE 5: Cell viability assays (modified after Cook 1989).
In this study, the EZ4U assay (Biomedica, Vienna, Austria) was chosen for analyzing MSC proliferation due to the comparability with previous studies. The EZ4U assay is a metabolic activity assay that uses light yellow XTT (tetrazolium) dye that is reduced intracellularly to water-soluble formazan derivates with an intense red color (Fig. 20; Roehm et al. 1991). The absorption intensity is measured by spectrophotometry after an incubation period of three hours.
FIG. 20: MTT assay. Tetrazolium dye is reduced to formazan dye. 45 Discussion
It is an advancement over the classic MTT assays which result in the production of blue formazan (Mosmann 1983; Scudiero et al. 1988). The water-insolubility of formazan makes it necessary to lyse cells after incubation and to release the formazan by the aid of a solubilization solution (Mosmann 1983; Carmichael et al. 1987). Since this step is skipped in the EZ4U, the continuation of cell culture is possible after performing the assay. It is important to adhere strictly to an equal incubation period in all assays to assure for a comparability of results. The EZ4U assay measures the activity of mitochondrial and cytosolic reductase enzymes. It does not measure DNA content or synthesis, and is therefore independent from cell cycle and not a measure for the current proliferation activity of cells. The succinate and tetrazolium dehydrogenase system is a part of the mitochondrial electron transport chain (complex II). The larger number of MTT reduction, however, seems to take part in the cytosol involving NADH and NADPH as coenzymes (Berridge and Tan 1993). The assay measures momentary cell metabolism. Since the processes involved are ubiquitously present in all vital cells, the measurements can be interpreted as an indirect correlate to the number of living cells (Mosmann 1983; Uludaq and Senfton 1990). In the present study, EZ4U assays were performed as time course assays after a certain number of days in culture (1, 3, 5, 10). Since the absorption correlates with the current quantity of vital cells, a growth curve can then be plotted to analyze cell proliferation. The proliferation (growth rate) can be determined by the slope of the growth curve. However, the EZ4U assay might not be a adequate fit for tissue engineering research. Miscorrelating changes in cell number and activity have been reported especially for cultures with a high cell density and 3D cultures using scaffolds (Ng et al. 2005).
The growth rate of human mesenchymal stem cells exposed to gabapentin-lactam (GBP-L) was analyzed using the CyQuant cell proliferation assay (Invitrogen/Life Technologies, Carlsbad, CA, USA). The CyQuant assay is a fluorescent-based method (Blaketa et al. 1991) with a sensitivity for as low as 50 cells. The cells are lysed using a buffer solution containing a green cyanine dye that binds to all cellular nucleic acids. The signal rate is six times higher for DNA than for RNA (Jones et al. 2001). The binding results in a strong enhancement in fluorescence which is measured by a microplate reader. The fluorescence correlates with the number of cells (Jones et al. 2001). Since the cells are lysed, they can not be used for further testing. To prevent photobleaching, it is critical that all steps are performed at equal time periods. 46 Discussion
Prior to cell lysis, the microplates on which the cells are cultured must be frozen at - 70° C. They can be stored for up to four weeks at that temperature. A washing step is necessary before freezing since salts and bovine serum albumin (BSA) can interfere with the fluorescence intensity (manufacturer‘s instructions). In contrast to the EZ4U method, the CyQuant assay ist faster to perform. Storing the samples taken at various times for up to four weeks allows for a more efficient performance of assays. It does not depend on metabolic activity like tetrazolium-based assays (Scudiero et al. 1988, Vistica et al. 1990) which can change independently from cell proliferation rates and is affected by medium components (Jones et al. 2001). A recent study showed that a metabolic activity assay comparable to the EZ4U method was not as accurate in determining the actual cell number as an assay with a DNA-binding fluorescent dye like the CyQuant. The results of the metabolic activity assay tended to overestimate cell numbers by up to 64 % (Quent et al. 2010). A direct comparison of the two methods could not be performed in the present thesis. This was due to the total number of MSC in the donor material being too low for both assays. However, the time-efficient manageability and the results of the studies cited above suggest that the CyQuant assay might be a better fit for future studies analyzing MSC proliferation. 47 Conclusion 5 Conclusion
Besides its proliferation-enhancing benefits, oleic acid seems to induce adipogenic differentiation in ovine mesenchymal stem cells.
A growth factor-like effect for GBP-L on cultured ovine and human MSC could not be proven by the present thesis.
If oleic acid induced adipogenic differentiation and the underlying mechanism of action for GBP-L was in fact related to ROS production, it could be speculated that this was the reason for the ceased proliferation at the day 10-assay.
The CyQuant assay was successfully established as an alternative to the EZ4U assay for future studies in our department. It might be a better fit for analyzing MSC proliferation due to manageability and accuracy. 48 Summary 6 Summary
Mesenchymal stem cells (MSC) are a promising alternative to autologous bone grafts in craniofacial reconstructive surgery. One method for the clinical application of MSC is the combination of cells with scaffolds and growth factors. Gabapentin-lactam (GBP-L) as well as oleic acid have shown a growth factor-like effect in previous studies. Both substances could provide a possible alternative to the known growth factors. The aim of the present thesis was to examine the effects of GBP-L, oleic acid and a combination of both substances on ovine mesenchymal stem cells in vitro. Bone marrow-derived cryopreserved ovine MSC were resuscitated, plated and cultured with expansion medium with the addition of oleic acid in concentrations ranging from 50 - to 800 µM, GBP-L (10 µM) or a combination of both (600 µM oleic acid + 10 µM GBP-L). After 1, 3, 5, and 10 days of culture, the XTT-assay EZ4U was performed to determine the number of viable cells. To examine the multi-lineage potential of MSC, cells were differentiated into adipogenic, osteogenic and chondrogenic progenitors using standard differentiation media and stained specifically as a proof of multipotency. To establish a fluorescent-based assay as an alternative to the EZ4U assay, another experiment was performed with human bone marrow-derived MSC from iliac crest aspirate. Human MSC were cultured with expansion medium with or without 10 µM GBP-L. Cell proliferation was measured after 1, 3, and 6 days using the CyQuant cell proliferation assay. A significant proliferative effect on ovine MSC could be detected for oleic acid in concentrations from 50 to 600 µM. Interestingly, MSC exposed to oleic acid showed distinctive morphological changes typical for adipogenic differentiation. A proliferative effect for GBP-L on ovine and human MSC cultures could not be proven by these experiments. The combination of oleic acid and GBP-L seems to cause a suppression of cell growth after 10 days. This could be due to the production of reactive oxygen species (ROS) which has been postulated as a possible mechanism of action for GBP-L. The CyQuant proliferation assay was established as an alternative to the EZ4U assay for MSC culture with potential benefits regarding manageability and accuracy. 49 Summary 6 Zusammenfassung
Der Einsatz von mesenchymalen Stammzellen (MSCs) stellt eine vielversprechende Alternative zu autologen Knochentransplantaten in der kraniofazialen rekonstruktiven Chirurgie dar. Eine Möglichkeit der Anwendung besteht in der Kombination von Stammzellen mit Scaffolds und Wachstumsfaktoren. Die Substanzen Gabapentin-lactam (GBP-L) und Ölsäure zeigten in vorausgegangenen Studien einen wachstumsfaktor- ähnlichen Effekt auf Zellkulturen und wären dadurch als mögliche Alternative zu den bekannten Wachstumsfaktoren denkbar. In der vorliegenden Arbeit wurde die Wirkung von GBP-L, Ölsäure, sowie einer Kombination der beiden Substanzen auf mesenchymale Stammzellen vom Schaf in vitro getestet. Aus dem Beckenkamm von Schafen gewonnene, kryopreservierte MSCs wurden aufgetaut, ausgesät und mit Expansionsmedium kultiviert. Das Medium wurde mit unterschiedlichen Konzentrationen Ölsäure (50 - 800 µM), GBP-L (10 µM) oder einer Kombination (600 µM Ölsäure + 10 µM GBP-L) versetzt. Nach 1, 3, 5 und 10 Tagen Zellwachstum wurden mit dem XTT-Test EZ4U die Stoffwechselaktivität und somit näherungsweise die Zellzahl zum jeweiligen Zeitpunkt bestimmt. Um die Multipotenz der verwendeten Zellen zu beweisen, wurden diese mit Differenzierungsmedien zu adipogenen, osteogenen und chondrogenen Vorläufenzellen differenziert. Der Nachweis für die Differenzierung wurde mit spezifischen Färbungen erbracht. Um eine Alternative zum EZ4U Test zu etablieren, wurde ein weiterer Versuchsansatz mit humanen MSCs aus Knochenmarks-Aspirat vom Beckenkamm durchgeführt. Diese Zellen wurden entweder mit Expansionsmedium oder Medium mit 10 µM GBP-L kultiviert. Nach 1, 3 und 6 Tagen wurde die Zellproliferation mit dem Fluoreszenz-basierten Proliferationstest CyQuant gemessen. Bei Ölsäure-Konzentrationen von 50 bis 600 µM konnte ein signifikant wachstumssteigernder Effekt nachgewiesen werden. Interessanterweise zeigten alle mit Ölsäure kultivierte MSC nach etwa einer Woche distinktive morphologische Veränderung, die für eine Differenzierung in Adipozyten-Vorläufer sprechen. Ein proliferativer Effekt von GBP-L auf ovine und humane MSC ließ sich in dieser Arbeit nicht bestätigen. Die Kombination von GBP-L und Ölsäure scheint nach 10 Tagen eine wachstumshemmende Wirkung zu besitzen. Diese könnte auf der postulierten Bildung von reaktiven Sauerstoffspezies (ROS) durch GBP-L beruhen. Der CyQuant Proliferationstest wurde als geeignete Alternative zum EZ4U für die Stammzellkultur etabliert und weist potentielle Vorteile in Hinblick auf die Durchführung und Aussagekraft auf. 50 References 7 References
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Protokoll: EZ4U Cell Proliferation Assay
Materialien • Zellsuspension • drei 24-Well-Platten (eine pro Inkubationszeit) • Zellkulturmedien (NH Expansion Medium, Medium mit 10 µM GBP-L)
• EZ4U Assay Kit • drei 96-Well-Platten (eine pro Messzeitpunkt)
Versuchsansatz (Tag 0) • Inkubationszeiten 24 h, 3 d, 6 d = eine Platte pro Inkubationszeit • Dreifach-Ansatz (siehe Skizze) • 1 x 104 Zellen / Well
Mediumwechsel 3 x /Woche • 1 ml Medium / Well
NH Expansion Medium
10 µM GBP-L Medium
Messtag ! EZ4U ansetzen ! im Brutschrank für 3 Stunden inkubieren ! in 96-Well-Platte pipettieren
nur Medium
Verdünnung 1:2
unverdünnt
! Messung (Messprotokoll „EZ4U_neu“ , Matrix-Darstellung!) ! speichern 66 Appendix
Protokoll: CyQuant Cell Proliferation Assay 1
Materialien • Zellsuspension • drei 96-Well-Platten (eine pro Messzeitpunkt) • Zellkulturmedien (NH Expansion Medium, Medium mit 10 µM GBP-L)
Versuchsansatz (Tag 0)
• Inkubationszeiten 24 h, 3 d, 6 d = eine Platte pro Inkubationszeit • Dreifach-Ansatz (siehe Skizze) • 1000 Zellen / Well
Mediumwechsel 3 x /Woche • 200 µl Medium / Well
nur Medium nur Medium mit Zellen mit Zellen
NH Expansion 10 µM GBP-L Medium Medium
Einfrieren ! nach jeweiliger Inkubationszeit (24 h, 3 d, 6 d) Platten aus dem Brutschrank nehmen und Medium verwerfen ! mit PBS waschen ! Platten bei -80 °C einfrieren (wichtig für Lyse der Zellen!) ! Platten können bei -80 °C bis zu 4 Wochen gelagert werden 67 Appendix
Protokoll: CyQuant Cell Proliferation Assay 2
Materialien • eingefrorene 96-Well-Platten • CyQuant Kit • Nuclease-freies Wasser • BlueCup • Alu-Folie • Multipette
Vorbereitung der Reaktionslösung (für 8 Platten à 12 Wells) ! 1 ml cell-lysis buffer stock solution (Component B) und 19 ml Nuclease-freies Wasser in ein BlueCup pipettieren ! 50 µl CyQuant GR stock solution (Component A) dazu pipettieren
Messung ! Mikrotiterplatten bei Raumtemperatur auftauen lassen ! pro Well 200 µl Reaktionslösung pipettieren ! Messprotokoll CyQuant (Inkubationszeit 3 min, Exzitation 480 nm, Emission 520 nm) ! speichern unter C:\ Documents and Settings\ kons\ My Documents\ Assay\ Heike\ Eva\ CyQuant 68 Appendix
EZ4U data 0,150 0,150 0,149 0,115 0,116 0,151 0,160 0,155 0,157 0,150 0,146 0,154 0,153 0,152 0,156 0,128 0,135 0,140 0,130 0,142 0,143 0,188 0,175 0,167 0,232 0,210 0,209 0,185 0,188 0,208 0,153 0,217 0,150 0,197 0,187 0,195 0,304 0,253 0,249 0,286 0,296 0,255 0,214 0,257 0,253 0,151 0,141 0,142 0,133 0,135 0,123 0,142 0,139 0,135 0,135 0,137 0,130 GBPL+OA 0,135 0,126 0,130 0,125 0,129 0,131 0,144 0,140 0,136 0,117 0,132 0,131 0,175 0,157 0,151 0,130 0,135 0,132 0,136 0,140 0,142 0,154 0,169 0,160 0,148 0,163 0,158 0,201 0,180 0,211 0,180 0,186 0,169 0,176 0,166 0,164 0,208 0,194 0,199 0,188 0,205 0,190 0,184 0,238 0,232 0,263 0,235 0,202 0,269 0,203 0,200 0,283 0,295 0,287 0,331 0,301 0,313 GBPL 0,135 0,133 0,133 0,135 0,133 0,128 0,133 0,137 0,141 0,147 0,136 0,133 0,146 0,148 0,143 0,119 0,128 0,132 0,143 0,136 0,152 0,157 0,165 0,172 0,171 0,153 0,158 0,191 0,182 0,215 0,187 0,176 0,169 0,178 0,181 0,187 0,219 0,209 0,197 0,205 0,228 0,195 0,189 0,234 0,229 0,121 0,310 0,269 0,238 0,230 0,238 0,362 0,328 0,330 0,313 0,310 0,299 0,148 0,172 0,231 0,137 Kontrolle 2 GBPL+OA 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 0,137 0,157 0,192 0,265 GBPL 0,137 0,158 0,199 0,279 Kontrolle 2 Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx 0,122 0,122 0,128 0,134 0,130 0,128 0,124 0,119 0,118 0,124 0,122 0,118 0,145 0,150 0,148 0,199 0,191 0,213 0,227 0,200 0,230 0,160 0,167 0,163 0,162 0,225 0,225 0,172 0,191 0,153 0,157 0,156 0,157 0,197 0,210 0,205 0,145 0,147 0,176 0,156 0,184 0,186 0,249 0,204 0,184 0,194 0,198 0,198 0,320 0,276 0,276 0,279 0,344 0,420 0,321 0,298 0,311 0,129 0,192 0,181 0,264 Kontrolle 1 Kontrolle 1 0,132 0,123 0,129 0,141 0,138 0,140 0,132 0,134 0,135 0,130 0,129 0,127 0,139 0,143 0,128 0,162 0,168 0,158 0,182 0,185 0,182 0,198 0,206 0,210 0,171 0,177 0,175 0,181 0,173 0,149 0,196 0,183 0,184 0,248 0,240 0,229 0,366 0,313 0,318 0,218 0,225 0,203 0,280 0,298 0,288 0,188 0,255 0,246 0,349 0,359 0,338 0,381 0,183 0,208 0,408 0,421 0,440 0,133 0,179 0,253 0,315 800 µM 800 µM 0,119 0,119 0,121 0,124 0,125 0,127 0,121 0,122 0,122 0,121 0,126 0,122 0,150 0,151 0,151 0,165 0,159 0,161 0,182 0,183 0,179 0,188 0,190 0,172 0,174 0,173 0,171 0,182 0,217 0,206 0,183 0,193 0,177 0,253 0,249 0,241 0,354 0,313 0,341 0,231 0,223 0,220 0,229 0,192 0,231 0,302 0,361 0,319 0,437 0,421 0,401 0,409 0,415 0,412 0,475 0,366 0,423 0,128 0,180 0,242 0,365 600 µM 600 µM Mittelwerte 0,106 0,108 0,111 0,105 0,105 0,101 0,113 0,103 0,107 0,115 0,112 0,114 0,145 0,140 0,139 0,135 0,134 0,126 0,157 0,141 0,145 0,155 0,143 0,142 0,145 0,136 0,132 0,157 0,175 0,157 0,184 0,154 0,148 0,195 0,174 0,195 0,250 0,159 0,282 0,211 0,168 0,176 0,247 0,218 0,244 0,260 0,213 0,216 0,378 0,368 0,366 0,309 0,485 0,493 0,388 0,332 0,365 0,115 0,145 0,200 0,321 400 µM 400 µM 0,112 0,113 0,110 0,108 0,110 0,110 0,112 0,105 0,113 0,118 0,107 0,113 0,163 0,140 0,147 0,135 0,139 0,139 0,156 0,150 0,144 0,157 0,140 0,137 0,144 0,132 0,131 0,181 0,177 0,178 0,148 0,148 0,133 0,203 0,183 0,181 0,285 0,245 0,179 0,182 0,157 0,163 0,197 0,240 0,249 0,186 0,169 0,191 0,302 0,306 0,289 0,481 0,337 0,205 0,360 0,351 0,313 0,119 0,149 0,193 0,268 200 µM 200 µM 0,106 0,109 0,109 0,118 0,118 0,114 0,109 0,111 0,112 0,113 0,109 0,108 0,175 0,162 0,154 0,136 0,126 0,130 0,146 0,142 0,139 0,165 0,143 0,143 0,151 0,144 0,133 0,154 0,159 0,156 0,166 0,153 0,132 0,195 0,174 0,193 0,279 0,257 0,299 0,204 0,184 0,170 0,275 0,262 0,236 0,159 0,176 0,177 0,318 0,312 0,317 0,333 0,294 0,363 0,360 0,338 0,332 0,122 0,144 0,212 0,268 100 µM 100 µM 0,111 0,111 0,116 0,121 0,121 0,126 0,110 0,116 0,112 0,121 0,120 0,110 0,145 0,153 0,152 0,151 0,142 0,142 0,166 0,146 0,164 0,163 0,167 0,152 0,149 0,145 0,148 0,151 0,152 0,170 0,154 0,163 0,139 0,181 0,189 0,184 0,140 0,119 0,119 0,211 0,190 0,190 0,275 0,233 0,274 0,199 0,200 0,171 0,337 0,332 0,327 0,469 0,277 0,471 0,354 0,340 0,355 0,123 0,154 0,184 0,295 50 µM 50 µM 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 1 3 5 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Tag Einfluss verschiedener Ölsäure-Konzentrationen auf die Proliferation von MSCs Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx 69 Appendix 0,150 0,150 0,149 0,115 0,116 0,151 0,160 0,155 0,157 0,150 0,146 0,154 0,153 0,152 0,156 0,128 0,135 0,140 0,130 0,142 0,143 0,188 0,175 0,167 0,232 0,210 0,209 0,185 0,188 0,208 0,153 0,217 0,150 0,197 0,187 0,195 0,304 0,253 0,249 0,286 0,296 0,255 0,214 0,257 0,253 0,151 0,141 0,142 0,133 0,135 0,123 0,142 0,139 0,135 0,135 0,137 0,130 0,397 0,380 0,406 GBPL+OA 0,135 0,126 0,130 0,125 0,129 0,131 0,144 0,140 0,136 0,117 0,132 0,131 0,175 0,157 0,151 0,130 0,135 0,132 0,136 0,140 0,142 0,154 0,169 0,160 0,148 0,163 0,158 0,201 0,180 0,211 0,180 0,186 0,169 0,176 0,166 0,164 0,208 0,194 0,199 0,188 0,205 0,190 0,184 0,238 0,232 0,263 0,235 0,202 0,269 0,203 0,200 0,283 0,295 0,287 0,331 0,301 0,313 0,388 0,380 0,401 GBPL 0,135 0,133 0,133 0,135 0,133 0,128 0,133 0,137 0,141 0,147 0,136 0,133 0,146 0,148 0,143 0,119 0,128 0,132 0,143 0,136 0,152 0,157 0,165 0,172 0,171 0,153 0,158 0,191 0,182 0,215 0,187 0,176 0,169 0,178 0,181 0,187 0,219 0,209 0,197 0,205 0,228 0,195 0,189 0,234 0,229 0,121 0,310 0,269 0,238 0,230 0,238 0,362 0,328 0,330 0,313 0,310 0,299 0,388 0,390 0,406 0,148 0,172 0,231 0,188 Kontrolle 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 GBPL+OA 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 0,137 0,157 0,192 0,290 GBPL 0,137 0,158 0,199 0,302 Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx Kontrolle 2 0,122 0,122 0,128 0,134 0,130 0,128 0,124 0,119 0,118 0,124 0,122 0,118 0,145 0,150 0,148 0,199 0,191 0,213 0,227 0,200 0,230 0,160 0,167 0,163 0,162 0,225 0,225 0,172 0,191 0,153 0,157 0,156 0,157 0,197 0,210 0,205 0,145 0,147 0,176 0,156 0,184 0,186 0,249 0,204 0,184 0,194 0,198 0,198 0,320 0,276 0,276 0,279 0,344 0,420 0,321 0,298 0,311 0,299 0,331 0,352 0,129 0,192 0,181 0,276 Kontrolle 1 Kontrolle 1 0,132 0,123 0,129 0,141 0,138 0,140 0,132 0,134 0,135 0,130 0,129 0,127 0,139 0,143 0,128 0,162 0,168 0,158 0,182 0,185 0,182 0,198 0,206 0,210 0,171 0,177 0,175 0,181 0,173 0,149 0,196 0,183 0,184 0,248 0,240 0,229 0,366 0,313 0,318 0,218 0,225 0,203 0,280 0,298 0,288 0,188 0,255 0,246 0,349 0,359 0,338 0,381 0,183 0,208 0,408 0,421 0,440 0,318 0,328 0,322 0,133 0,179 0,253 0,316 800 µM 800 µM 0,119 0,119 0,121 0,124 0,125 0,127 0,121 0,122 0,122 0,121 0,126 0,122 0,150 0,151 0,151 0,165 0,159 0,161 0,182 0,183 0,179 0,188 0,190 0,172 0,174 0,173 0,171 0,182 0,217 0,206 0,183 0,193 0,177 0,253 0,249 0,241 0,354 0,313 0,341 0,231 0,223 0,220 0,229 0,192 0,231 0,302 0,361 0,319 0,437 0,421 0,401 0,409 0,415 0,412 0,475 0,366 0,423 0,312 0,339 0,334 0,128 0,180 0,242 0,358 600 µM 600 µM Mittelwerte 0,106 0,108 0,111 0,105 0,105 0,101 0,113 0,103 0,107 0,115 0,112 0,114 0,145 0,140 0,139 0,135 0,134 0,126 0,157 0,141 0,145 0,155 0,143 0,142 0,145 0,136 0,132 0,157 0,175 0,157 0,184 0,154 0,148 0,195 0,174 0,195 0,250 0,159 0,282 0,211 0,168 0,176 0,247 0,218 0,244 0,260 0,213 0,216 0,378 0,368 0,366 0,309 0,485 0,493 0,388 0,332 0,365 0,291 0,303 0,307 0,115 0,145 0,200 0,317 400 µM 400 µM 0,112 0,113 0,110 0,108 0,110 0,110 0,112 0,105 0,113 0,118 0,107 0,113 0,163 0,140 0,147 0,135 0,139 0,139 0,156 0,150 0,144 0,157 0,140 0,137 0,144 0,132 0,131 0,181 0,177 0,178 0,148 0,148 0,133 0,203 0,183 0,181 0,285 0,245 0,179 0,182 0,157 0,163 0,197 0,240 0,249 0,186 0,169 0,191 0,302 0,306 0,289 0,481 0,337 0,205 0,360 0,351 0,313 0,342 0,332 0,360 0,119 0,149 0,193 0,283 200 µM 200 µM 0,106 0,109 0,109 0,118 0,118 0,114 0,109 0,111 0,112 0,113 0,109 0,108 0,175 0,162 0,154 0,136 0,126 0,130 0,146 0,142 0,139 0,165 0,143 0,143 0,151 0,144 0,133 0,154 0,159 0,156 0,166 0,153 0,132 0,195 0,174 0,193 0,279 0,257 0,299 0,204 0,184 0,170 0,275 0,262 0,236 0,159 0,176 0,177 0,318 0,312 0,317 0,333 0,294 0,363 0,360 0,338 0,332 0,355 0,362 0,372 0,122 0,144 0,212 0,286 100 µM 100 µM 0,111 0,111 0,116 0,121 0,121 0,126 0,110 0,116 0,112 0,121 0,120 0,110 0,145 0,153 0,152 0,151 0,142 0,142 0,166 0,146 0,164 0,163 0,167 0,152 0,149 0,145 0,148 0,151 0,152 0,170 0,154 0,163 0,139 0,181 0,189 0,184 0,140 0,119 0,119 0,211 0,190 0,190 0,275 0,233 0,274 0,199 0,200 0,171 0,337 0,332 0,327 0,469 0,277 0,471 0,354 0,340 0,355 0,408 0,440 0,342 0,123 0,154 0,184 0,314 50 µM 50 µM 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 1 3 5 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Tag Einfluss verschiedener Ölsäure-Konzentrationen auf die Proliferation von MSCs Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx Schaf 44 Schaf 45 Schaf 49 Schaf 50 Schaf 48 Schaf xx 70 Appendix 10 10 10 9 9 9 8 8 8 7 7 7 6 6 6 5 5 5 time (d) time (d) time (d) 4 4 4 OA 200 µM OA 800 µM GBPL 10 µM 3 3 3 2 2 2 1 1 1 0 0 0 0 0 0
0,500 0,375 0,250 0,125 0,500 0,375 0,250 0,125 0,500 0,375 0,250 0,125
OD 450 nm 450 OD nm 450 OD OD 450 nm 450 OD 10 10 10 10 9 9 9 9 8 8 8 8 7 7 7 7 6 6 6 6 5 5 5 5 time (d) time (d) time (d) time (d) 4 4 4 4 control 2 OA 100 µM OA 600 µM 3 3 3 3 2 2 2 2 GBPL 10 µM + OA 600 1 1 1 1 0 0 0 0 0 0 0 0
0,500 0,375 0,250 0,125 0,500 0,375 0,250 0,125 0,500 0,375 0,250 0,125 0,500 0,375 0,250 0,125
OD 450 nm 450 OD nm 450 OD OD 450 nm 450 OD nm 450 OD 10 10 10 9 9 9 8 8 8 7 7 7 6 6 6 5 5 5 time (d) time (d) time (d) 4 4 4 control 1 OA 50 µM OA 400 µM 3 3 3 2 2 2 1 1 1 0 0 0 0 0 0
0,500 0,375 0,250 0,125 0,500 0,375 0,250 0,125 0,500 0,375 0,250 0,125
OD 450 nm 450 OD OD 450 nm 450 OD OD 450 nm 450 OD 71 Appendix 10 10 10 9 9 9 8 8 8 7 7 7 6 6 6 5 5 5 time (d) time (d) time (d) 4 4 4 OA 200 µM OA 800 µM GBPL 10 µM 3 3 3 2 2 2 1 1 1 0 0 0 0 0 0
0,375 0,250 0,125 0,500 0,375 0,250 0,125 0,500 0,375 0,250 0,125 0,500
OD 450 nm 450 OD nm 450 OD nm 450 OD 10 10 10 10 9 9 9 9 8 8 8 8 7 7 7 7 6 6 6 6 5 5 5 5 time (d) time (d) time (d) time (d) 4 4 4 4 control 2 OA 100 µM OA 600 µM 3 3 3 3 2 2 2 2 GBPL 10 µM + OA 600 1 1 1 1 0 0 0 0 0 0 0 0
0,500 0,375 0,250 0,125 0,500 0,375 0,250 0,125 0,500 0,375 0,250 0,125 0,500 0,375 0,250 0,125
OD 450 nm 450 OD nm 450 OD nm 450 OD nm 450 OD 10 10 10 9 9 9 8 8 8 7 7 7 6 6 6 5 5 5 time (d) time (d) time (d) 4 4 4 control 1 OA 50 µM OA 400 µM 3 3 3 2 2 2 1 1 1 0 0 0 0 0 0
0,250 0,125 0,500 0,375 0,250 0,125 0,250 0,125 0,500 0,375 0,500 0,375
OD 450 nm 450 OD nm 450 OD OD 450 nm 450 OD 72 Appendix
CyQuant data
1 3 6 1 3 6 41470 27010 37676 Pat. 79 36319 31512 39873 Pat. 79 31138 30795 39731 P1 33445 34014 39718 P1 34713 40988 43170 NH 29119 24791 38825 GBP-L 27989 25651 22261 26237 26204 30607 30114 22338 23060 24750 25400 20181 43424 22755 32854 28578 25160 22154
Patient 79 - Passage 1 - NH Patient 79 - Passage 1 - GBP-L 50000 50000
37500 37500
25000 25000
12500 12500
0 0 1 3 6 1 3 6
Ansatz 1 Ansatz 2 Ansatz 3 Ansatz 1 Ansatz 2 Ansatz 3 Kontrolle 1 Kontrolle 2 Kontrolle 3 Kontrolle 1 Kontrolle 2 Kontrolle 3
1 3 6 1 3 6 35320 37842 44745 Pat. 81 44495 43483 26000 Pat. 81 40531 33942 33500 P1 37276 38351 25639 P1 36158 43204 26740 NH 42552 37142 26667 GBP-L 14013 10077 3674 13056 9221 3891 14284 9820 4914 20227 9061 3411 19389 10182 5586 13229 9249 3798
Patient 81 - Passage 1 - NH Patient 81 - Passage 1 - GBP-L 50000 50000
37500 37500
25000 25000
12500 12500
0 0 1 3 6 1 3 6
Ansatz 1 Ansatz 2 Ansatz 3 Ansatz 1 Ansatz 2 Ansatz 3 Kontrolle 1 Kontrolle 2 Kontrolle 3 Kontrolle 1 Kontrolle 2 Kontrolle 3
1 3 6 1 3 6 37279 45186 Pat. 81 23455 27741 Pat. 81 36609 40100 P2 28829 27165 P2 43421 43115 NH 27538 28376 GBP-L 22350 19461 8361 6229 22451 21824 8598 6774 22945 20346 8700 7110
Patient 81 - Passage 2 - NH Patient 81 - Passage 2 - GBP-L 50000 50000
37500 37500
25000 25000
12500 12500
0 0 1 3 6 1 3 6
Ansatz 1 Ansatz 2 Ansatz 2 Ansatz 1 Ansatz 2 Ansatz 3 Kontrolle 1 Kontrolle 2 Kontrolle 3 Kontrolle 1 Kontrolle 2 Kontrolle 3 73 Appendix
1 3 6 1 3 6 21274 37833 37815 Pat. 82 17610 41886 26667 Pat. 82 42672 43977 40377 P1 19303 38829 46336 P1 25783 37624 39728 NH 22277 38417 9958 GBP-L 11085 12581 6148 6471 11551 7567 7921 12478 9484 6060 11516 10717 8216 15617 8123 6677 12358 9958
Patient 82 - Passage 1 - NH Patient 82 - Passage 1 - GBP-L 50000 50000
37500 37500
25000 25000
12500 12500
0 0 1 3 6 1 3 6
Ansatz 1 Ansatz 2 Ansatz 3 Ansatz 1 Ansatz 2 Ansatz 3 Kontrolle 1 Kontrolle 2 Kontrolle 3 Kontrolle 1 Kontrolle 2 Kontrolle 3
1 3 6 1 3 6 31707 41545 37206 Pat. 82 39721 38490 36376 Pat. 82 43672 41108 39648 P2 36847 44451 38230 P2 36194 43276 31855 NH 36769 39405 44921 GBP-L 11399 12382 8507 14249 12057 8493 21366 12329 13536 14106 12941 7680 31558 16524 9091 14635 14054 7823
Patient 82 - Passage 2 - NH Patient 82 - Passage 2 - GBP-L 50000 50000
37500 37500
25000 25000
12500 12500
0 0 1 3 6 1 3 6
Ansatz 1 Ansatz 2 Ansatz 3 Ansatz 1 Ansatz 2 Ansatz 3 Kontrolle 1 Kontrolle 2 Kontrolle 3 Kontrolle 1 Kontrolle 2 Kontrolle 3 74 Appendix
Curriculum vitae
Zur Person Name: Eva-Marie Jablonka Geburtsdatum: 09.04.1984 Geburtsort: Freiburg im Breisgau
Kontakt: [email protected]
Ausbildung seit 2009 Studium der Humanmedizin, Albert-Ludwigs-Universität Freiburg und Ludwig-Maximilians-Universität München 03/2011 Physikum
2003-09 Studium der Zahnmedizin, Albert-Ludwigs-Universität Freiburg 08/2009 Zahnärztliche Approbation 07/2009 Zahnärztliche Prüfung (Note: 1,16 - Examensbeste) 04/2006 Zahnärztliche Vorprüfung
06/2003 Abitur, Albert-Schweitzer-Gymnasium Gundelfingen (Note: 1,7) 2000-01 Auslandsjahr, Waterford Mott High School, Michigan, USA 09/1990 Einschulung, Johann-Peter-Hebel-Grundschule Gundelfingen
Praktika 03/2013 Famulatur in der Abteilung für Mund-, Kiefer- und Gesichtschirurgie, Klinikum Rechts der Isar der Technischen Universität München 09/2012 Famulatur bei Dr. Dr. Günter Nahles, Praxis für Mund-, Kiefer- und Gesichtschirurgie, Berlin 03/2012 Famulatur in der Abteilung für Mund-, Kiefer- und Gesichtschirurgie, Virchow Klinikum der Charité Berlin 10/2010 Hospitation bei Dr. William McClure, Napa Valley Plastic Surgery, Napa, California, USA 10/2010 Hospitation bei Dr. John Pappas und Dr. Jeffrey Politz, Napa Valley Oral & Maxillofacial Surgery, Napa, California, USA 09-10/2010 Krankenpflegepraktikum am Queen of the Valley Medical Center, Napa, California, USA 04/2010 Krankenpflegepraktikum am Rehazentrum Leukerbad, Schweiz 08/2007 Famulatur am Institut d‘Odonto-Stomatologie Tropicale de Madagascar, Mahajanga, Madagaskar 75 Appendix
Danksagung
PD Dr. Dr. Sebastian Sauerbier danke ich herzlich für die Überlassung des hochinteressanten Themas, seine hervorragende Betreuung und die motivierende Zusammenarbeit.
PD Dr. Olga Polydorou danke ich für die schnelle und freundliche Übernahme des Zweitgutachtens.
Heike Jahnke danke ich für die intensive Einarbeitung in die Methoden der Zellkultur und ihre großartige Unterstützung bei der Durchführung.
Meinen Eltern und meinen Geschwistern Carolin und Felix danke ich von ganzem Herzen für die uneingeschränkte Unterstützung und ihren Rat bei der schriftlichen Ausarbeitung.