N-Cadherin: β-Catenin Signaling Leading to Maintenance or Differentiation of Cell
Populations in Response to Different Dia...
the many ways in which cells can be affected when designing
bone scaffolds. Substrate geometry and stiffness can be
The results for the immunofluorescence staining showed
what was expected for the control coverslips which is that the
This REU has served as an excellent introduction to upper
level research. I intend to use my REU opportunit...
of 4


Published on: Mar 3, 2016
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Transcripts - Nagurney_Summer_2014_Final_Paper_OfficialDoc

  • 1. N-Cadherin: β-Catenin Signaling Leading to Maintenance or Differentiation of Cell Populations in Response to Different Diameter Electrospun Fibers Rebecca A. Nagurney Abstract--Non-union fractures, bone fusions, osteotomies and bone cancer are just some of the reasons that a person may need a bone graft. Bone grafts are procedures used to replace and help repair bone. Autogeneous bone grafts come from the patient’s bone, most commonly the hip bone. The problems with these grafts are limited supply in addition to pain and morbidity associated with the harvesting surgeries. Allogeneic bone grafts come from cadavers and these grafts can also cause complications. Allogeneic bone grafts have lost the mechanical integrity neededto grow bone, and they also have a higherrisk of disease transmission and immune rejection. Designing a scaffold for bone growth would reduce the need for autogeneous and allogeneic bone grafts which are not always the ideal option. β-Catenin is a protein that is involved in cell adhesion through cell-to-cell adherens junctions and is also a regulator of bone formation. Cells are influenced by chemical factors but can also be influenced by surface topography and geometry. Electrospun fibers mimic the extracellular matrix of bone. Growing cells on electrospun fiber surfaces can influence β- Catenin translocation to the nucleus and promote osteogenesis. Electrospinning uses a high voltage source to charge a solution, evaporate off the solvent, and stream fibers to a grounded source. The fibers being made can vary in diameterbased on the solution viscosity, the distance from the syringe to the grounded source, or the voltage applied. In this project, electrospun fibers were made out of Poly methyl methacralate (PMMA) Nitromethane solution and spun on glass coverslips spin coated with PolyHEMA (PHEMA) solution. Glass coverslips were also spin coated with a PMMA Nitromethane solution and not coated with fibers. MC3T3 cells were seeded and grown until confluent onto the fibers and onto the no-fiber surfaces. A β-Catenin assay using immunofluorescence was usedto show the locations and amounts of β-Catenin present on fiber and no-fiber surfaces. An increase in nuclear β-Catenin on fiber surfaces relative to the no-fiber surfaces would show that the fibers promote bone growth. (Abstract) Keywords—nanofibers, electrospinning, osteogenic I. INTRODUCTION Problems with bone can happen at any age and can include breaking a bone, bones that do not heal, bone cancer, and more. Each year there are over two million patients or medical procedures performed relating to bone replacement, reconstruction, or repair.1 Many procedures include using allogeneic or autogeneous bone grafts. Allogeneic bone grafts pose the rise of infection and immune rejection because they come from cadavers. Autogeneous bone grafts can come from the patient’s bone; however, these grafts can be painful and have negative effects on the site from which the bone was taken. Designing bone scaffolds would reduce the need for autogeneous and allogeneic bone grafts. While designing and testing scaffolds,scientists must keep in mind that they must be biocompatible with the body, provide adequate mechanical strength, and be porous.2 The biodegradability and the osteoinductive capabilities of the scaffold must also be examined. If the scaffolds degrade over time, patients would not need another surgical procedure to remove the scaffold once the area is healed.2 If the material is biodegradable over time, this can sacrifice the mechanical integrity that is needed for bone growth. Osteoinductive capability of the scaffold material is also important because growth factors may be needed to control cell differentiation. The purpose ofthis research is to determine the effects that different diameter electrospun fibers have on beta-catenin translocation to the nucleus by comparing beta-catenin levels in cells on fibers and not on fibers. This is important because an increase in beta-catenin in the nucleus would lead to an increase in osteogenic differentiation. Cells were grown on Poly methyl methacrylate (PMMA) electrospun fibers and on PMMA spin coated coverslips. PMMA is a biocompatible material that is used in bone cement and is not biodegradable. Because cells will be grown on the same material (PMMA), the only change will be in the surface on which the cells are grown. Therefore, the results recorded fromfibers and no fibers will be due to the change in the surface, not the material. II. LITERATURE REVIEW Surface topography has been studied and shown to affect cell differentiation. When developing a scaffold for bone tissue engineering,the effects that the surface geometry has on cells must be studied. Geometric cues can have strong effects on the fate of cells and is comparable to chemical factors. A study was conducted that tested the significance of scaffold geometry on cell differentiation. Mesenchymal stem cells (MSCs) were grown on flat surfaces and on straight and random nanofiber surfaces. The nanofiber surfaces directed the cell differentiation toward osteogeneis even though the medium in which the cells were grown had chemical factors that should have directed the cells into adipocytes.3 Surfaces that are convex tend to direct MSC differentiation toward adipogenesis while concave surfaces direct differentiation toward osteogenesis. Surfaces that force cell shape into rectangles also tend to direct MSC differentiation towards osteogenesis as aspect ratios increase.4 Electrospun fibers are long and thin like rectangles and therefore can influence cells to develop into bone cells. Along with shape, stiffness of a substrate can also impact the fate of cells. Stiff substrates tend to direct stem cells to differentiate into bone cells. The field of biomedical engineering is holistic. Because of this,scientists must think of
  • 2. the many ways in which cells can be affected when designing bone scaffolds. Substrate geometry and stiffness can be studied, but other factors such as porosity and topography must be recognized for the possible influences that they may exhibit on cell growth and differentiation.5 Bone scaffold designs must be examined because the slightest change in porosity, stiffness, size, topography, or geometry can direct cells to differentiate into a different lineage. Electrospinning is a technique that uses a high voltage source to charge a solution as it is being pushed through a syringe. The drip of solution at the tip of the syringe forms a Taylor cone. The solution streams out, the solvent evaporates off and the fibers travel to and are collected on a grounded source. Conditions such as temperature and humidity can affect the fibers that are being spun. High humidity results in straight fibers while low humidity tends to produce curly fibers. The temperature and humidity can also change the polymer to spray instead of stream to the grounded source. Fibers can be spun until the desired density is reached that would mimic the extracellular matrix. Fibers can be electrospun to have different diameters. Small diameter fibers allow more cell-to-cell adherens junctions to form. Larger diameter fibers allow cells to wrap around the fibers and create fewer adherens junctions. Adherens junctions are cell-to-cell contacts that connect transmembrane proteins known as cadherins with actin filaments. There are many different types of cadherins such as E (endothelial), N (neural), O (osteoblast), P (placental), and VE (vascular endothelial) Cadherin. The cadherins interact and connect with cytoplasmic proteins called catenins. An alpha-beta-catenin complex forms and those proteins then connect to the actin filaments.6 This creates a structural and biochemical connection between cells. This will in turn affect the organizational development of the cells.7 Beta-Catenin is one catenin that connects to the cadherins in adherens junctions. Beta-Catenin can be found at the plasma membrane, free in the cytoplasm, or in the cell nucleus. While beta-catenin is connected in the adherens junctions, the levels of free beta-catenin in the cytoplasmare kept low. When beta- catenin travels to the nucleus, it can affect transcription. There are many different sites where beta-catenin can be phosphorylated which would trigger its translocation to the cell nucleus. Phosphorylation controls the stability of the beta- catenin connection at the plasma membrane to cadherins in adherens junctions. For example, phosphorylation at tyrosine 142 increases transcription activity of beta-catenin by breaking the catenin-cadherin connection. This then leads to translocation to the cell nucleus and gene transcription. 8 III. METHODOLOGY Glass coverslips were spin coated with 100μL of 30% w/v PolyHEMA (PHEMA) 95:5 EtOH:H2O solution at 5000 rpm and a 2% w/v PMMA Nitromethane solution at 2500 rpm. Fibers were spun onto the PHEMA coverslips for 2 minutes at 5.5 mL/hr. while applying 13 kV. Fibers were not spun onto the 2% w/v PMMA coated coverslips because they were used as the control. Some problems that occurred were that the conditions such as temperature and humidity did not stay constant while electrospinning fibers on different days. The temperature and humidity affected the distance that the syringe needed to be placed in relation to the grounded source. The distance needed to be changed to prevent fluffy fibers. Fluffy fibers are not ideal because they disconnect from the coverslips during cell seeding and incubation. To tackle this, various heating methods were used. The coverslips with fibers were heated on a hot plate at 75°C for 1 minute in the center of the hot plate. The edges ofthe coverslips coated with fibers were also heated at 200°C. This created a border around the coverslip of melted fibers that would prevent them fromlifting off of the coverslip. Another method used to prevent the fibers from lifting off of the coverslips was UVgluing. The glue was placed on the four edges of the coverslips and quickly dried to prevent the glue from bleeding in to the remainder of the fibers on the cover slip. The method that produced the best results was heating the edges at 200°C. This method did not prevent all of the fibers from lifting off but it held down most of the fiber mat. This heating method was used to complete the experimentation. Another issue that must be addressed is the PHEMA solution not evenly coating the glass cover slips. PHEMA is spin coated onto the cover slips so that the cells adhere and grow on the fibers and not on the glass cover slips. When the cells are seeded on to the cover slips, pockets can be seen where the glass is exposed. The cells will settle into these pockets and grow in there instead of on the fibers. The PHEMA is spin coated on to the glass cover slips at 5000 rpm. To try and fix this PHEMA problem, the speed at which the cover slips were spin coated was reduced to 3000 rpm. Results have yet to be recorded for those upcoming trials using the 3000 rpm spin coated PHEMA solution. MC3T3-E1 S4 cells were grown and fed every 2 days using α MEM 1X media. They are an osteoblast precursor cell line that is derived from mouse calvarias (skulls). When the cells were confluent, they were seeded onto the fiber and no fiber cover slips in a 6 well plate. The cells were grown until confluent on the fiber and no fiber surfaces. Immunofluorescence was then performed to see the translocation of beta-catenin on the fiber and no fiber surfaces. Beta-Catenin (E-5) and cadherin primary antibodies were used to perform the beta-catenin assay using immunofluorescence. The secondary antibodies chosen for the immunofluorescence were Anti-Mouse IgG Dylight 488 and cy5 antibodies. Phalloidin and DAPI were used to stain the cell’s actin and DNA respectively. Phalloidin is a highly toxic small molecule that is taken from mushrooms. DAPI is also toxic and it binds to the adenine thymine (A-T) regions in DNA in the nucleus. Once the immunofluorescence was complete, the cover slips were mounted onto glass slides and viewed under the microscope. Expected results would show that beta-catenin would be highly concentrated at the plasma membrane in adherens junctions on the control surfaces. Beta- catenin would be expected to be more concentrated in the nucleus for the cells seeded onto the fiber surfaces. IV. RESULTS The MC3T3-E1 S4 cells were imaged using differential interference contrast (DIC) and fluorescence microscopy. The cell nuclei are in purple, actin is red, cadherins are blue and beta-catenin is green. Because there were many focal planes, the cells seeded and grown onto the PMMA fibers were difficult to image. Figure 1 shows the separate color channels for the cells. The four channels were overlapped to be viewed all at once when imaging the cells.
  • 3. The results for the immunofluorescence staining showed what was expected for the control coverslips which is that the beta-catenin would be at the plasma membrane held in adherens junctions. Figure 2 shows the cells grown onto the control coverslips without fibers. The beta-catenin, stained green, is highly concentrated at the cell’s plasma membrane which can be seen by the large amount of green. This is because the cells are allowed adhere, spread out and make many cell-to-cell contacts on the flat PMMA surface. The cells seeded and grown on the PMMA fibers are imaged below in Figures 3 and 4. Some beta-catenin can be seen in the nucleus in Figure 3, but beta-catenin can also be seen outside nucleus oriented along the fiber in Figure 4. More trials will need to be performed to understand why the beta- catenin is found oriented along the fiber without any cell-to- cell contacts. Figure 2: MC3T3-E1 S4 cells grown on glass coverslips spin coated with 2% PMMA Nitromethane solution Figure 1: This figure shows the four separate color channels. Top Left: purple stained nuclei. Bottom Left: blue stained cadherin, Top Right: red stained actin, Bottom Right: green stained beta-catenin. Figure 4:MC3T3-E1 S4 cells grown on 35% w/v PMMA fibers. Beta-catenin (green) can be seen oriented along the fibers. Figure 3: MC3T3-E1 S4 cells grown on 35% w/v PMMA fibers. Beta-catenin (green) can be seen in and near the nucleus. Nuclei Actin Cadherin Beta-Catenin
  • 4. V. FUTURE WORK This REU has served as an excellent introduction to upper level research. I intend to use my REU opportunity as the start of my honors thesis work and I plan to continue this research through the fall semester and until I graduate. My future work will consist of electrospinning smaller diameter fibers. This will allow me to compare the translocation of beta-catenin for cells grown on large and cells grown on small diameter fibers. I have already begun to spin smaller diameter fibers and was confronted with beading issues on the fibers. The PMMA solution was switch to a 90:10 Nitromethane: DMF solvent and this reduced the amount of beading. Immunofluorescence will be performed again when cells are confluent on the different diameter fibers and control coverslips. One change for the immunofluorescence will be that an additional antibody (Y142) will be used to stain for nuclear beta-catenin. The beta-catenin antibody used in experimentation this summer only stained for total beta- catenin. A western blot will be performed to detect the intensity of nuclear beta-catenin using the Y142 antibody in the cells grown on fibers versus the cells grown on the flat PMMA control surface. Western blots separate proteins based on their molecular weight and the band intensity is shown by color. The results that will be obtained by this future work for the remainder of the summer will direct the pathway for my future research with beta-catenin and its influence on cell osteogenic differentiation. REFERENCES 1. Langer R, Vacanti JP. Tissue Engineering. 2014;260(5110):920-926. 2. Murphy CM, O’Brien FJ, Little DG, Schindeler A. Cell-scaffold interactions in the bone tissue engineering triad. Eur Cell Mater. 2013;26:120-32. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24052425. 3. Fujita S, Shimizu H, Suye S. Control of Differentiation of Human Mesenchymal Stem Cells by Altering the Geometry of Nanofibers. J Nanotechnol. 2012;2012:1-9. doi:10.1155/2012/429890. 4. Kilian K a, Bugarija B, Lahn BT, Mrksich M. Geometric cues for directing the differentiation of mesenchymal stemcells. Proc Natl Acad Sci U S A. 2010;107(11):4872-7. doi:10.1073/pnas.0903269107. 5. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126(4):677-89. doi:10.1016/j.cell.2006.06.044. 6. Niessen CM. Tight junctions/adherens junctions:basic structure and function. J Invest Dermatol. 2007;127(11):2525-32. doi:10.1038/sj.jid.5700865. 7. Meng W, Takeichi M. Adherens junction: molecular architecture and regulation. Cold Spring Harb Perspect Biol. 2009;1(6):a002899. doi:10.1101/cshperspect.a002899. 8. Couffinhal T, Dufourcq P, Duplàa C. Beta-catenin nuclear activation: common pathway between Wnt and growth factor signaling in vascular smooth muscle cell proliferation? Circ Res. 2006;99(12):1287-9. doi:10.1161/01.RES.0000253139.82251.31.

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