Effect of boron on the differentiation of mesenchymal stem cells

EFFECT OF BORON ON THE DIFFERENTIATION OF MESENCHYMAL STEM CELLS Valencia, septiembre de 2016 Universitat Politècnica de València Mónica Victoria Gutiérrez Salazar Grado en Biotecnología, ETSIAMN 2015-2016 Tutor: José Luis Gómez Ribelles Cotutor: Patricia Rico Tortosa Effect of boron on the differentiation of mesenchymal stem cells Boron is an essential microelement in the metabolism of living organisms. However, its role is not well defined yet. It has been shown recently that boron has a positive effect on the differentiation on murine myoblasts and some reports describe boron to be involved on bone mineralization. For that reasons, it is done in this work a research using different concentrations of boron in a material system composed by PLLA (poly lactic-L-acid) as a substrate, in order to verify if boron has also an effect on the differentiation of mesenchymal stem cells to different lineages (myoblast, osteoblast, adipocyte or for the contrary stemness maintenance). With this, it could be favored the regeneration of both damaged muscle tissue and bone tissue, achieving in that way an important progress on tissue engineering, adding to the current techniques just a microelement, boron. Keywords: boron – differentiation – mesenchymal stem cells – PLLA Efecto del boro en la diferenciación de células madre mesenquimales El boro es un microelemento esencial en el metabolismo de los seres vivos, aunque su papel en este no está bien definido. Se ha demostrado recientemente que el boro tiene un efecto positivo en la diferenciación de mioblastos murinos, y algunos estudios describen que el boro puede intervenir en la mineralización del hueso. Por ello, se realiza aquí un estudio empleando diferentes concentraciones de boro y PLLA (ácido-L-poliláctico) como soporte, con el fin de comprobar si el boro tiene también un efecto en la diferenciación de células madre mesenquimales hacia varios linajes diferentes (mioblasto, osteoblasto, adipocito o por el contrario mantenimiento de las células en su estadío indiferenciado). Se podría con ello favorecer la regeneración tanto de tejido muscular como tejido óseo dañado, consiguiendo así un avance importante en la ingeniería de tejidos, añadiendo a las técnicas actuales tan solo un microelemento, el boro. Palabras clave: boro – diferenciación – células madre mesenquimales – PLLA Alumna: Dña. Mónica Victoria Gutiérrez Salazar Valencia, septiembre de 2016 Tutor Académico: Prof. José Luis Gómez Ribelles Cotutora: Dra. Patricia Rico Tortosa Acknowledgements The first and most important people to acknowledge in this work are my parents. Obtaining a bachelor’s degree in Biotechnology was my dream and I’m aware I couldn’t have reached it without their support, love, effort and sacrifice. They have been guiding me through the correct path, teaching me how to do things well in life and at the same time letting me make my mistakes and decide my direction. My professional and personal life begins here and I feel happy and brave knowing they will be by my side the following years. I would like to thank Dra. Patricia Rico, Pachi, my supervisor, for being so helpful when I was willing to make my bachelor thesis in the Center for Biomaterials, for offering me to research in this interesting and unique topic, and for teaching me proper cell biology. She has been patient and sweet. She is my first contact with the world of real science, the one that was able to tell things as they were, and one of the most passionate about science people I have met. Thanks also to Marina and Adrian, who were also under Pachi’s charge, for making those days in the laboratory fantastic. We know how awesome Team Pachi is. And all thanks to our director. Special thanks to the person that is always with me, to support me whatever is the situation. He has helped me to finish my bachelor’s degree without becoming crazy, he has always been trying to make sure that I was smiling, and made everything that can be imagined to make me happy. Thanks for your support, and thanks for reading a thousand times this work. Finally, I would like to thank all people that was there when I needed them, during the elaboration of this work and during my degree: my little brother, always there to talk, to laugh, to dream, to share. Miriam and Mabel, sorry for being disappeared during four years, and thanks for being there anyways. The iGEM team 2015 and 2016, for creating the two most incredible summers that can exist, and to the instructors, for giving us, the students, the opportunity to participate. Probably my future will be very different thanks to them. Thanks to Alberto Conejero, for believing in me and give me the chance to develop myself as a leader. Thanks also to my university, Polytechnic University of Valencia, for all the transversal activities, resources, facilities and education that it offers to the students. I have learned a lot in this university and from here is where my future begins. Contents 1. Introduction .......................................................................................................... 1 1.1. Context: tissue engineering ....................................................................................... 1 1.2. Biochemical factor as bioactive ion: boron (B) ............................................................ 2 1.2.1. Role of boron in physiology ....................................................................................................... 2 1.2.2. Drugs and boron ........................................................................................................................ 4 1.2.3. Boron in bone regeneration and maintenance ......................................................................... 5 1.2.4. Boron in myogenic differentiation ............................................................................................. 8 1.3. Biocompatible material: poly-L-lactic acid ................................................................ 10 1.4. Living cells: mesenchymal stem cells ........................................................................ 11 2. Objective ............................................................................................................ 12 3. Materials and methods ....................................................................................... 13 3.1. Experimental design ................................................................................................ 13 3.2. Polymeric substrate ................................................................................................. 14 3.3. Biological material and culture media ...................................................................... 14 3.4. Immunofluorescence assay ...................................................................................... 16 3.5. Cell staining............................................................................................................. 17 3.6. Image treatment ..................................................................................................... 17 4. Results and discussion......................................................................................... 19 4.1. Stemness ................................................................................................................ 19 4.2. Osteogenesis ........................................................................................................... 20 4.2.1. RunX2 ....................................................................................................................................... 21 4.2.2. OPN .......................................................................................................................................... 24 4.3. Myogenesis ............................................................................................................. 28 4.4. Adipogenesis ........................................................................................................... 32 5. Conclusions ......................................................................................................... 37 References ............................................................................................................. 39 Figures Figure 1. Immunofluorescence of Sca 1. Cell culture of MSC with basal medium. A and B) Culture over crystal slide cover, at 10X and 20X magnification respectively. C and D) Culture over PLLA, at 10X and 20X magnification respectively. .............................................................. 19 Figure 2. Immunofluorescence of Sca 1. Cell culture of MSC with basal medium over PLLA. A and B) Culture in medium with 2% boron added, at 10X and 20X magnification respectively. C and D) Culture in medium with 5% boron added, at 10X and 20X magnification respectively. ................................................................................................................................ 20 Figure 3. Immunofluorescence of RunX2. Cell culture of MSC over crystal slide cover. A and B) Culture with basal medium, at 20X and 40X magnification respectively. C and D) Culture with osteogenic medium, 20X and 40X magnification respectively. .......................................... 21 Figure 4. Immunofluorescence of RunX2. Cell culture of MSC over PLLA. A and B) Culture with basal medium, at 20X and 40X magnification respectively. C and D) Culture with osteogenic medium, 20X and 40X magnification respectively. .................................................. 22 Figure 5. Immunofluorescence of RunX2. Cell culture of MSC over PLLA with 2% boron in medium. A and B) Culture with basal medium, at 20X and 40X magnification respectively. C and D) Culture with osteogenic medium, 20X and 40X magnification respectively. .................. 23 Figure 6. Immunofluorescence of RunX2. Cell culture of MSC over PLLA with 5% boron in medium. A and B) Culture with basal medium, at 20X and 40X magnification respectively. C and D) Culture with osteogenic medium, 20X and 40X magnification respectively. .................. 24 Figure 7. Immunofluorescence of OPN. Cell culture of MSC over crystal slide cover. A and B) Culture with basal medium, at 10X and 40X magnification respectively. C and D) Culture with osteogenic medium, 10X and 40X magnification respectively. .......................................... 25 Figure 8. Immunofluorescence of OPN. Cell culture of MSC over PLLA. A and B) Culture with basal medium, at 10X and 40X magnification respectively. C and D) Culture with osteogenic medium, 10X and 40X magnification respectively. .................................................. 26 Figure 9. Immunofluorescence of OPN. Cell culture of MSC over PLLA with 2% boron in medium. A and B) Culture with basal medium, at 10X and 40X magnification respectively. C and D) Culture with osteogenic medium, 10X and 40X magnification respectively. .................. 27 Figure 10. Immunofluorescence of OPN. Cell culture of MSC over PLLA with 5% boron in medium. A and B) Culture with basal medium, at 10X and 40X magnification respectively. C and D) Culture with osteogenic medium, 10X and 40X magnification respectively. .................. 28 Figure 11. Immunofluorescence of α-actinin sarcomeric. Cell culture of MSC over crystal slide cover. A and B) Culture with basal medium, at 10X and 20X magnification respectively. C and D) Culture with myogenic medium, 10X and 20X magnification respectively. ..................................................................................................................................................... 29 Figure 12. Immunofluorescence of α-actinin sarcomeric. Cell culture of MSC over PLLA. A and B) Culture with basal medium, at 10X and 20X magnification respectively. C and D) Culture with myogenic medium, 10X and 20X magnification respectively. ............................... 30 Figure 13. Immunofluorescence of α-actinin sarcomeric. Cell culture of MSC over PLLA with boron 2% in medium. A and B) Culture with basal medium, at 10X and 20X magnification respectively. C and D) Culture with myogenic medium, 10X and 20X magnification respectively. ..................................................................................................................................................... 31 Figure 14. Immunofluorescence of α-actinin sarcomeric. Cell culture of MSC over PLLA with boron 5% in medium. A and B) Culture with basal medium, at 10X and 20X magnification respectively. C and D) Culture with myogenic medium, 10X and 20X magnification respectively. ..................................................................................................................................................... 32 Figure 15. Staining of adipocytes. Cell culture of MSC over crystal slide cover. A and B) Culture with basal medium, at 20X and 40X magnification respectively. C and D) Culture with adipogenic medium, 20X and 40X magnification respectively. .................................................. 33 Figure 16. Staining of adipocytes. Cell culture of MSC over PLLA. A and B) Culture with basal medium, at 20X and 40X magnification respectively. C and D) Culture with adipogenic medium, 20X and 40X magnification respectively. ..................................................................... 34 Figure 17. Staining of adipocytes. Cell culture of MSC over PLLA with 2% boron in medium. A and B) Culture with basal medium, at 20X and 40X magnification respectively. C and D) Culture with adipogenic medium, 20X and 40X magnification respectively. .................. 35 Figure 18. Staining of adipocytes. Cell culture of MSC over PLLA with 2% boron in medium. A and B) Culture with basal medium, at 20X and 40X magnification respectively. C and D) Culture with adipogenic medium, 20X and 40X magnification respectively. .................. 36 1 1. Introduction 1.1. Context: tissue engineering This project is framed on the relatively new field of Tissue Engineering. This field applies the principles of engineering and biology in order to develop biological substitutes that restore, maintain or improve tissue function mainly when a tissue has been damaged or an organ has failed (Berthiaume, Maguire, and Yarmush 2011; Langer and Vacanti 1993). The strategy followed to create a new tissue structure combines three main components: a) Living cells: these cells can be obtained either from a donor or from a patient. When they are obtained from the patient, they can be mesenchymal stem cells, cells from the tissue that wants to be repaired or induced stem cells. b) Biocompatible materials: polymers that serve as support and protection for the cells, so they provide an environment similar to the extracellular matrix of their native tissue. c) Biochemical factors: substances and biomolecules that favor the proliferation and/or differentiation of the cells. They can be used in vitro to prepare the cells before implanting them, or they can be loaded into the biocompatible material to be released inside the body. These three strategies can be used to create a tissue-like structure with the objective of regenerating a damaged tissue or organ. In this project there is a special focus on a biochemical factor acting as a bioactive ion: boron. We performed a novel approach to test the effects of boron on mesenchymal stem cell differentiation. A biocompatible material, poly-lactic acid, is going to be used. This will allow assessing the potential of boron for applications in tissue engineering. In the following sections they will be developed the main reasons for choosing boron, poly-lactic acid and mesenchymal stem cells on this project. 8 The effect of boron in pre-osteoblastic cells (MC3T3) (Hakki, Bozkurt, and Hakki 2010) and bone marrow stromal cells (BMSC) (Ying et al. 2011) was tested. The metalloid remarkably enhanced cell viability, proliferation, mineralization nodules and expression of bone mineralized tissue-associated proteins, particularly collagen type I, osteopontin, bone sialoprotein, osteocalcin, alkaline phosphatase and RunX2, compared to untreated cells. It also increased mRNA expression of bone morphogenetic protein (BMP), specifically BMP4, BMP5 and BMP7. BMP are growth factors that belong to the TGF-β family which induce formation of new cartilage and bone. RunX2 is a transcription factor that stimulates osteoblastic differentiation from mesenchymal stem cells, as well as bone formation and bone maintenance (Franceschi et al. 2008). When its levels are reduced, other genes related to tissue mineralization drop its expression, as bone sialoprotein, osteocalcin and osteopontin. Finally, several tissue engineering studies have shown beneficial effects of boron for osteoblast regeneration with different biomaterials. In 2003, Gough et al. tested the biocompatibility of PCL (polycaprolactone) and borontrifluoride for craniofacial bone regeneration. They showed improved cell proliferation, differentiation and higher amount of mineralized collagen 1 matrix in PCL scaffolds synthetized with borontrifluoride. Gorustovich et al. (2006) implanted bioactive glass particles modified with boron oxide in rat tibia. Boron increased bone regeneration, thickness and calcium:phosphorous ratio at 15 days. At 30 days postimplantation, boron only improved thickness. They suggest that boron acts in an early stage of osteogenic differentiation. Wu et al. (2011) tested mesoporous bioactive glass scaffolds containing boron for the proliferation and differentiation of human osteoblasts. It was improved the expression of collagen I and RunX2 in the boron-containing scaffolds. These studies show and support the potential of boron in bone tissue engineering. 1.2.4. Boron in myogenic differentiation In spite of all the evidence that supports the important role of boron in bone tissue regeneration, little is known about its effect on muscle tissue. Boron nitride nanotubes are being used as nanomaterials for biomedicine. However, this is mainly due to its physical-chemical characteristics, tridimensional structure and biocompatibility, not by a special improvement on muscle differentiation (Ciofani et al. 2013). It has been described how tetraphenylboron changes the charge of the surface of the sarcoplasmic reticulum, allowing Ca2+ release in skeletal muscle (Liu and Oba 1990; Soler, Fernandez-Belda, and Gomez-Fernandez 1989). However, the release of 9 Ca2+ is responsible for contraction of fully differentiated muscle cells, and does not have a paper in the differentiation. Only two recent studies have described the effects of boron in muscle differentiation. Apdik et al. (2015) tested the effect of boric acid on myogenic differentiation of human adipose stem cells (hADSC). Just 5μg/mL of boron increased cell viability and were not toxic for the cells. It increased expression of mRNA specific of myocytes in early stages of differentiation, particularly myosin heavy chain, myogenin, desmine and MyoD, being the last one the one with higher fold change compared to the control. However, a higher dose of boron at day 21 decreased the expression of these proteins. In the field of tissue engineering, Rico et al. (2015) showed the effect of poly-L-lactic acid (PLLA) substrates loaded with borax on the differentiation of murine C2C12 myoblasts, a model for muscle differentiation. The substrates containing borax had a higher percentage of differentiated cells and higher surface of myotube, compared to the standard control with collagen I as substrate. However, the myogenic markers MyoD and myogenin showed no significant improvement between the control and the PLLA substrates loaded with borax. This can be explained by the multistep process of differentiation of muscular cells, being possible that boron acts at an earlier stage than the moment when the analysis of the presence myogenic markers was done. 10 1.3. Biocompatible material: poly-L-lactic acid In tissue engineering, it is common the use of polymeric scaffolds or surfaces that serve as support and protection for the cells, having an environment similar to extracellular matrix of their native tissue (Langer and Vacanti 1993). The polymers can be used to induce differentiation of the cells ex vivo or can be implanted in vivo for tissue regeneration, with or without cells inside it. Ideal polymers for tissue engineering should be biocompatible and resorbable at a controlled rate which matches cell or tissue growth, should have a suitable chemistry for cell attachment, proliferation and differentiation, as well as mechanical properties to match those of the tissue to regenerate (Hutmacher 2000). Many polymers, natural or synthetic, have been used in tissue engineering. In this study poly-L-lactic acid (PLLA) has been chosen, as it can be considered as an ideal polymer, according to the characteristics mentioned above. It is composed by the L isomer of hydrophobic aliphatic polyester. PLLA has been widely approved by the US Food and Drug Administration (FDA) for direct contact with biological fluids and it is generally recognized as safe. As Farah et al. (2016) review, PLLA has numerous advantages compared to other biopolymers. PLLA’s production is eco-friendly, as it requires less energy to be produced -making it cheaper-, it is obtained from renewable sources and it is biodegradable. It is biocompatible and reabsorbable, meaning that its degradation products are not toxic and do not interfere with the tissue healing. In human body it is hydrolyzed to its constituent α-hydroxy acid, which is incorporated into the tricarboxylic acid cycle and excreted. It has higher processability, so it can be synthetized and shaped using multiple techniques. On the other hand, it has several drawbacks, as a slow degradation rate, low toughness and high hydrophobicity, which worsen cell affinity. However, these problems have been solved by blending PLLA with other polymers. PLLA has been widely used in biomedical applications that show its versatility: nerve and spinal cord injury regeneration, bioabsorbable screws in ankle, knee and hand; meniscus repair, guided bone regeneration, cardiac regeneration, stents, surgical sutures, dentistry, space filler, plastic and reconstructive surgery, dermal fillers, oncology, drug delivery, etc. (Tyler et al. 2016). It has been used both for bone and muscle regeneration, including in the Rico et al. ( 2015) study with boron, were they prove that boron does not affect the properties of PLLA and analyzed its release from the polymer. It is then a suitable material to use as biocompatible substrate in this study. In this case it is not used as scaffold, because it has not been shaped as a tridimensional porous structure, but it is used as a surface easy to synthetize and that is simple to later analyze the cell differentiation, without requiring complex equipment. 11 1.4. Living cells: mesenchymal stem cells The key elements in tissue engineering are the living cells that are going to regenerate the damaged tissue. These cells can be derived from donor tissue or progenitor cells (Berthiaume, Maguire, and Yarmush 2011). The donor tissue can be from the patient or from other person, being the last one dangerous for the receptor due to immunological incompatibilities. Stem or progenitor cells have several advantages that make them suitable for tissue engineering: they have a high proliferative capacity and they are pluripotent (they can differentiate into several lineages). Stem cells can be obtained from embryos, placenta, umbilical cord, bone marrow of adults, or they can be induced pluripotent stem cells (iPSC), which can be any type of differentiated cell converted in stem cell. The cells chosen in this work are the murine embryonic mesenchymal stem cells C3H10T1/2. They have been demonstrated to differentiate into osteoblast, chondrocyte, adipocyte and myoblast, being suitable for this study (Shin et al. 2000). Mesenchymal stem cells (MSCs) have been used in tissue engineering (Ringe et al. 2002) due to their extensive in vitro proliferation and their potential to differentiate to multiple non hematopoietic lineages. MSCs can be differentiated to different lineages by adding specific inductors to culture medium. In this study, the lineages that are induced are osteoblast, myoblast and adipocyte. This last is used as a control for osteoblast differentiation, as bone induction blocks adipogenesis, and vice-versa (Q Chen et al. 2016). 12 2. Objective Myogenesis and osteogenesis are vital processes in the life cycle, which can be disrupted due to disease, injuries or aging. Tissue engineering aims at improving muscle and bone regeneration by using biomaterials, cells and biochemical factors that enhance genesis of tissue, to repair damages. Research and progress in developing and discovering any of the strategies of tissue engineering helps to this purpose. This study is motivated by the research done by our group in the Center for Biomaterials and Tissue Engineering (CBIT), where it is demonstrated that borax loaded PLLA substrates are able to induce muscle cell differentiation and myotube formation. Boron is a promising biochemical inducer, as it has demonstrated extensively its beneficial effects in life, health and muscle and bone differentiation, and hence regeneration. A step forward in the knowledge of how and under which circumstances does boron act can help to improve regenerative medicine. This work aims at testing the effect of boron in murine mesenchymal stem cells, particularly to demonstrate if it is capable to induce differentiation to osteoblast and myoblast lineages. This will allow knowing if boron acts at early stages of differentiation, before the compromise of cells to any particular lineage. The substrate used is poly-L-lactic acid prepared with a method in which PLLA merely acts as a support for the cells, being boron directly added to the culture medium. The study is qualitative, using fluorescence microscopy to analyze markers and morphology of the cells. 13 3. Materials and methods 3.1. Experimental design This study is based on the qualitative analysis of the cells to check their differentiation state. The workflow is the following: 1. Preparation of the polymeric substrates 2. Preparation of cells and the different culture media 3. Cell culture over the polymeric substrates 4. Immunofluorescence essay and analysis with fluorescence microscopy OR staining and analysis with inverted optical microscope. 5. Image treatment with specialized software A total of 4 different conditions are tested for each experiment, and there are a total of 9 experiments. The four conditions are the following, each one with three samples: 1. Crystal slide cover (C+, positive control) 2. PLLA 3. PLLA + 2% borax 4. PLLA + 5% borax The nine experiments are defined by different molecular markers that define each type of cell (osteoblast, myoblast, adipocyte and stem cell undifferentiated). To each of the four conditions explained above, either basal medium (negative control) or differentiation medium are used. 1. RunX2 (osteoblast) + basal medium 2. RunX2 (osteoblast) + osteoblast medium 3. OPN (osteoblast) + basal medium 4. OPN (osteoblast) + osteoblast medium 5. α-actinin (myoblast) + basal medium 6. α-actinin (myoblast) + myoblast medium 7. Liposome (adipocyte) + basal medium 8. Liposome (adipocyte) + adipocyte medium 9. Sca1 (stemness) + basal medium –> negative control 14 Given that, for each of the 9 experiments there are 4 conditions tested (positive control, PLLA, PLLA+2% borax, PLLA+5% borax), and for each condition there are 3 samples. 3.2. Polymeric substrate The PLLA substrates were prepared from a solution of PLLA (Cargill Dow) dissolved in 100 mL of chloroform to obtain a final concentration of 2% (w/v). The materials were prepared using spin coating technique. Spin coating is a method that allows to deposit a thin layer of the biopolymer (PLLA 2%) over a surface. The surfaces used were crystal slide covers of 12 mm diameter, with a total surface of 2 cm2. The slide covers are vacuum dried at room temperature in order to evaporate the excess of solvent. Boron is not released from the polymer, but it is added to the culture medium, with the same concentrations as if it was released from a solvent casting prepared polymer, as described by Rico et al. (2015): major release is produced during the first 3 hours of culture, and the release of borax increases as the concentration of borax in the sample does. At 14 days there is still release of borax from the polymer. Two solutions of PLLA 2% containing 2% and 5% of borax (sodium tetraborate decahydrate Na2B4O7·10H2O) (Bórax España S.A) in relation to total mass of polymer were prepared, following the procedure of Rico et al. (2015). These will be added to the culture media depending on the experiment performed. The substrates were sterilized 30 minutes at UV light, before being used for cell culture. The two types of substrate were coated during 1h at room temperature with fibronectin (FN) from human plasma (Sigma-Aldrich), using a solution of 20 μg/mL in Dulbecco’s phosphatebuffered saline (DPBS). 3.3. Biological material and culture media The cells used were murine mesenchymal stem cells C3H10T1/2 (ATCC). They were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen), supplemented with 10% fetal bovine serum (FBS; Invitrogen), 1% Fungizone (Life Technology, Fisher) and 1% penicillin– streptomycin (Lonza) in humidified atmosphere at 37 ºC and 5% CO2. Cells were subcultured before reaching confluency, to obtain the needed concentration of cells before starting differentiation. 15 According to the nine experiments stated in section 3.1, it is necessary to use four different culture media. It was added either 2% or 5% borax to the culture media, depending on the experiment performed.  Basal medium: DMEM + 10% FBS + 1% fungizone + 1% penicillin-streptomycin. o Borax 2% o Borax 5%  Osteogenesis induction medium: DMEM + 10% FBS + 1% fungizone + 1% penicillin-streptomycin + 50 μg/mL ascorbic acid + 10mM β-glycerophosphate + 0.1 μM dexamethasone. o Borax 2% o Borax 5%  Myogenesis induction medium: DMEM + 10% horse serum (HS) + 1% fungizone + 1% penicillin-streptomycin. o Borax 2% o Borax 5%  Adipogenesis induction medium: DMEM + 10% FBS + 1% penicillin-streptomycin + 1% Fungizone + 0.5 mM, 3-isobutyl-1-methylxanthine + 60 μM Indometacine + 0.5 μM Hydrocortisone o Borax 2% o Borax 5% Once obtained the desired concentration of cells, the cultures were stimulated to differentiate. 20000 cells/cm2 were cultured over FN-coated crystal slide cover (C+), PLLA, PLLA2% borax and PLLA-5% borax, three samples per each one of the substrates. The substrates of 12mm Ø were placed in Nunclon plates of 24 wells. A total of 400 μL of culture media were added, according to the experiment (differentiation medium or basal medium; no borax, 2% borax or 5% borax). Every 3 days, the culture medium was changed. The cultures were kept in humidified atmosphere at 37 ºC and 5% CO2 until analysis of the samples was done. For OPN, α-actinin, adipogenesis and stemness experiments, the cultures were stopped at day 15. For RunX2, it was stopped at 3 days. For myoblast, osteoblast and stemness, the next step is an immunofluorescent essay. For adipocyte, it is a cell staining. 16 3.4. Immunofluorescence assay After the 3 or 15 days, depending on the experiment, the cultures were immunostained with specific markers of their lineage, in order to analyze their morphology and their stage of differentiation. The immunofluorescence assay is based in the binding of a primary antibody to the chosen molecular marker. Then, a fluorophore-conjugated secondary antibody is added. This binds to the primary antibody, and has fluorescence in a certain wavelength. Depending on the marker that wants to be seen, different primary and secondary antibodies are used. The general protocol for immunofluorescence begins fixing cells with formaline 4% and then permeabilizing the cells with DPBS/Triton X-100 0.5% during 5 minutes at room temperature. Next, cells are blocked during 1 hour at room temperature with DPBS and goat serum 5% (for RunX2, OPN and Sca1) or horse serum 5% for α-actinin sarcomeric. Cells are washed with DPBS/Triton X-100 0.1%. Cells are incubated with primary antibody 1 hour at 37 ºC or overnight at 4 ºC. The primary antibody is different depending on the marker:  Myoblast: mouse antibody anti mouse α-actinin sarcomeric (Abcam) dilution 1:200 in DPBS/HS 5%.  Osteoblast: o Rabbit antibody anti mouse RunX2 (Abcam) dilution 1:200 in DPBS/GS 5%. o Mouse antibody anti mouse OPN (Santa Cruz Biotechnology) dilution 1:200 in DPBS/GS 5%.  Stemness: rat antibody anti mouse Sca1 (Abcam) dilution 5μg/mL in DPBS/GS 5%. Unbound antibody is washed with DPBS/Triton X-100 0.1%. Cells are incubated with secondary antibody during 1 hour at 37 ºC. The secondary antibody is different depending on the primary antibody used. This is the antibody that gives the fluorescence, and depending on the fluorophore of the antibody, it will emit fluorescence in different wavelengths.  Myoblast: anti mouse Cy3 conjugated antibody (Jackson Immunoresearch) dilution 1:200 in DPBS/HS 5%.  Osteoblast: o Alexa Fluor 488 goat anti rabbit (Invitrogen) dilution 1:100 in DPBS/GS 5%. 17 o Alexa Fluor 488 goat anti mouse (Invitrogen) dilution 1:200 in DPBS/GS 5%.  Stemness: Alexa Fluor 488 goat anti rat (Fisher) dilution 5μg/mL in DPBS/GS 5%. Unbound antibody is washed with DPBS/Triton X-100 0.1%. The samples are mounted in microscope slide covers, staining the nucleus with Vectashield containing DAPI (Atom) in the case of α-actinin sarcomeric, OPN and Sca1. For RunX2, it is mounted with FluorSave (Calbiochem WVR). The samples are observed under a fluorescence microscope (Nikon Eclipse 80i). 10 images for each condition (C+, PLLA, PLLA-B2%, PLLA-B5%) and experiment were taken. The channels were used according to the secondary antibody fluorophore:  α-actinin sarcomeric: DAPI channel (blue) and Cy3 channel (red). Images taken at 10X and 20X magnification.  RunX2: Alexa Fluor 488 channel (green). Images taken at 20X and 40X magnification.  OPN: DAPI channel (blue) and Alexa Fluor 488 channel (green). Images taken at 10X and 40X magnification.  Sca1: DAPI channel (blue) and Alexa Fluor 488 channel (green). Images taken at 10X and 20X magnification. 3.5. Cell staining After 15 days in adipocyte differentiation culture medium, the cultures are stained with Oil Red O, a fat soluble dye. Cells are equilibrated with isopropanol 60% during 2 minutes at room temperature. Oil Red O solution (1mg/mL Oil Red O (Sigma-Aldrich) + 60% isopropanol + distilled H2O) is added, 1mL per well, during 15 minutes at room temperature. Cells are washed twice with distilled water. The samples are mounted in microscope slide covers with glycerol. They are observed under inverted optical microscope. 10 images for each condition (C+, PLLA, PLLA-B2%, PLLA-B5%) and experiment were taken, at magnification 20X and 40X. 3.6. Image treatment The obtained images were treated using the software Fiji (Image J), specialized for scientific image analysis. Images with high background noise were readjusted. The brightness and 24 Figure 6 shows the cells cultured over PLLA substrate with boron 5% in the basal (A and B) and osteogenic (C and D) medium. These images show lower cell density comparing to PLLA+B2%, in basal and osteogenic medium. In both media the cells have a marked pre-osteoblast shape and defined nuclei. Additionally, in figures C and D the nuclei are clearly more intense, meaning that RunX2 is inside. When comparing these images to figure 3C and 3D (osteogenic medium in C+), and taking in account the nuclei intensity in figure 5, it is suggested that even if 3 days may be a short time for osteogenic differentiation, boron induces it, leading to the activation of RunX2. Figure 6. Immunofluorescence of RunX2. Cell culture of MSC over PLLA with 5% boron in medium. A and B) Culture with basal medium, at 20X and 40X magnification respectively. C and D) Culture with osteogenic medium, 20X and 40X magnification respectively. 4.2.2. OPN These cultures were left during 15 days. Taking in account those 3 days could be few time to differentiate MSC to pre-osteoblasts, it is expected that 15 days are enough for immature osteoblasts, which is the stage when OPN is expressed. 25 In figure 7 it is shown the culture of MSC over C+, with basal medium (A and B) and osteogenic medium (C and D). The most noticeable difference between basal medium and osteogenic medium is how the cytoplasm is more delimited and there is less background in osteogenic medium. OPN has been found in MSC, being excreted to the medium, because it is used as signaling molecule too (Chen et al. 2014). However, it is upregulated in osteoblasts. It is possible that it is being synthetized and accumulated in the cytoplasm, and its excretion is performed later in the osteogenesis process. Figure 7. Immunofluorescence of OPN. Cell culture of MSC over crystal slide cover. A and B) Culture with basal medium, at 10X and 40X magnification respectively. C and D) Culture with osteogenic medium, 10X and 40X magnification respectively. 26 In figure 8, cells cultured over PLLA substrate, with basal medium (A and B) and osteogenic medium (C and D) show similar behavior that those of the cells cultured over C+ (figure 7). Cell density is very similar. Figure 8. Immunofluorescence of OPN. Cell culture of MSC over PLLA. A and B) Culture with basal medium, at 10X and 40X magnification respectively. C and D) Culture with osteogenic medium, 10X and 40X magnification respectively. Figure 9 shows the cells cultured over PLLA substrate with boron 2% in the basal (A and B) and osteogenic (C and D) medium. It can be seen the same result as with the other substrates: with osteogenic medium, the cytoplasm is more defined. Cell density seems lower in osteogenic medium, meaning that there are more differentiated cells compared to C+ in osteogenic medium (figure 7C and D). 27 Figure 9. Immunofluorescence of OPN. Cell culture of MSC over PLLA with 2% boron in medium. A and B) Culture with basal medium, at 10X and 40X magnification respectively. C and D) Culture with osteogenic medium, 10X and 40X magnification respectively. Figure 10 shows the cells cultured over PLLA substrate with boron 5% in the basal (A and B) and osteogenic (C and D) medium. In basal medium, cell density is very low, and cells have an amorphous shape. Cytoplasm is almost no defined. In some cells there is an intense fluorescence next to the nucleus. It is possible that 5% boron concentration is too high for this stage of differentiation and it is being counterproductive, particularly in the case of basal medium, as there is no other osteogenic inductor. In pictures C and D (osteogenic medium), the cytoplasm is more defined than in pictures A and B, but less than the cells with 2% boron. Cell density is low, signaling differentiation. 28 Figure 10. Immunofluorescence of OPN. Cell culture of MSC over PLLA with 5% boron in medium. A and B) Culture with basal medium, at 10X and 40X magnification respectively. C and D) Culture with osteogenic medium, 10X and 40X magnification respectively. 4.3. Myogenesis The marker chosen for myogenesis was α-actinin sarcomeric. This is an actin-binding protein that is found in muscle. In skeletal muscle it is the major component of z-discs that define muscle sarcomeres, so it is expressed in developed myocytes. DAPI was used to stain the nucleus. In figure 11 it is shown the culture of MSC over C+, with basal medium (A and B) and myogenic medium (C and D). There is strong background and no definition of cytoplasm in A and B, showing the undifferentiated state of the cells. In myogenic medium (C and D), cells have an elongated shape, characteristic of myocytes and myoblasts. Cell density is much lower, meaning that there are differentiated cells. There are not, however, aligned cells that would indicate the formation of myotubes. It is possible that 15 days are not enough to obtain fully differentiated myoblast cells from mesenchymal stem cells. Rico et al. (2015) obtained myotubes in 4 days from the C2C12 myocyte cell line and Kubo (1991) obtained nucleated skeletal cells in two weeks from 29 C3H10T1/2 (the same cell line used in this work). However, both used different myogenic medium. It is possible that the chosen medium was not the suitable for this cell line. Another hypothesis is that the obtained cells are myocytes (an early stage of the myogenesis process) and not myoblasts, so α-actinin sarcomeric is less expressed and is not arranged in sarcomeres yet. Figure 11. Immunofluorescence of α-actinin sarcomeric. Cell culture of MSC over crystal slide cover. A and B) Culture with basal medium, at 10X and 20X magnification respectively. C and D) Culture with myogenic medium, 10X and 20X magnification respectively. Figure 12 depicts cells cultured over PLLA substrate, with basal medium (A and B) and myogenic medium (C and D). In basal medium, there is background and high density, same as in figure 11A and B. In C and D pictures, there is lower cell density and cells have lenticular shape, characteristic of myocytes. PLLA is possibly a substrate that enhances myogenesis, compared to slide covers. 30 Figure 12. Immunofluorescence of α-actinin sarcomeric. Cell culture of MSC over PLLA. A and B) Culture with basal medium, at 10X and 20X magnification respectively. C and D) Culture with myogenic medium, 10X and 20X magnification respectively. Figure 13 shows the cells cultured over PLLA substrate with boron 2% in the basal (A and B) and myogenic (C and D) medium. In basal medium there is still high cell density and strong background. However, cells are more defined than in basal PLLA (fig 12A-B) and basal C+ (fig 11AB) and have an elongated shape. Cells in myogenic medium have the same lenticular shape as depicted in figure 12C-D. Cell density is lower, showing less proliferation and more differentiation. 31 Figure 13. Immunofluorescence of α-actinin sarcomeric. Cell culture of MSC over PLLA with boron 2% in medium. A and B) Culture with basal medium, at 10X and 20X magnification respectively. C and D) Culture with myogenic medium, 10X and 20X magnification respectively. Figure 14 shows the cells cultured over PLLA substrate with boron 5% in the basal (A and B) and myogenic (C and D) medium. There is less background and less cell density than cultures in boron 2%. Cell cytoplasm is also more defined compared to PLLA and C+ substrates. This means that boron might induce differentiation in basal medium. However, it is not clear the cell morphology and fluorescence is less intense than in the cultures without boron. An explanation can be that spontaneously and without other biochemical factor, boron induces differentiation to another cell line different from myoblast, probably to osteoblast. Pictures C and D show very low cell density, as indicator of differentiation. The morphology of the cell supports the theory that they are myocytes, and 5% of boron improves myogenesis when using myogenic medium. 32 Figure 14. Immunofluorescence of α-actinin sarcomeric. Cell culture of MSC over PLLA with boron 5% in medium. A and B) Culture with basal medium, at 10X and 20X magnification respectively. C and D) Culture with myogenic medium, 10X and 20X magnification respectively. 4.4. Adipogenesis To test for adipogenic differentiation, an Oil Red O staining was performed. Oli Red O is a fat soluble dye which stains triglycerides and lipids in red. It makes visible the fats deposits in adipocytes. In figure 15 it is shown the culture of MSC over C+, with basal medium (A and B) and adipogenic medium (C and D). No red staining can be seen in basal medium, meaning there is not any adipocyte. By contrast, in adipogenic medium the stained fat vacuoles can be easily located. Cell density in the second case is low. 33 Figure 15. Staining of adipocytes. Cell culture of MSC over crystal slide cover. A and B) Culture with basal medium, at 20X and 40X magnification respectively. C and D) Culture with adipogenic medium, 20X and 40X magnification respectively. 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