Annals of Biomedical Engineering ( 2014) DOI: 10.1007/s10439-014-1023-7
Comparison of Chondroitin Sulfate and Hyaluronic Acid Doped Conductive Polypyrrole Films for Adipose Stem Cells MIINA BJO¨RNINEN,1,2,3 ALIISA SILJANDER,1,2,3 JANI PELTO,4 JARI HYTTINEN,3,5 MINNA KELLOMA¨KI,3,5 SUSANNA MIETTINEN,1,2,3 RIITTA SEPPA¨NEN,1,3,5,6 and SUVI HAIMI1,2,3,7 1
Adult Stem Cells, Institute of Biomedical Technology, University of Tampere, Biokatu 8, 33014 Tampere, Finland; 2Science Centre, Pirkanmaa Hospital District, P.O. BOX 2000, 33521 Tampere, Finland; 3BioMediTech, Biokatu 10, 33520 Tampere, Finland; 4VTT Technical Research Centre of Finland, Sinitaival 6, P.O. Box 1300, 33101 Tampere, Finland; 5Department of Biomedical Engineering, Department of Electronics and Communications Engineering, Tampere University of Technology, P.O. 692, 33101 Tampere, Finland; 6Department of Eye, Ear, and Oral Diseases, Tampere University Hospital, Division 3, P.O. Box 2000, 33521 Tampere, Finland; and 7Department of Biomaterials Science and Technology, University of Twente, P.O. Box 217, 7500, AEEnschede, The Netherlands (Received 7 January 2014; accepted 29 April 2014) Associate Editor Kent Leach oversaw the review of this article.
Abstract—Polypyrrole (PPy) is a conductive polymer that has aroused interest due to its biocompatibility with several cell types and high tailorability as an electroconductive scaffold coating. This study compares the effect of hyaluronic acid (HA) and chondroitin sulfate (CS) doped PPy films on human adipose stem cells (hASCs) under electrical stimulation. The PPy films were synthetized electrochemically. The surface morphology of PPy–HA and PPy–CS was characterized by an atomic force microscope. A pulsed biphasic electric current (BEC) was applied via PPy films non-stimulated samples acting as controls. Viability, attachment, proliferation and osteogenic differentiation of hASCs were evaluated by live/dead staining, DNA content, Alkaline phosphatase activity and mineralization assays. Human ASCs grew as a homogenous cell sheet on PPy–CS surfaces, whereas on PPy–HA cells clustered into small spherical structures. PPy–CS supported hASC proliferation significantly better than PPy–HA at the 7 day time point. Both substrates equally triggered early osteogenic differentiation of hASCs, although mineralization was significantly induced on PPy–CS compared to PPy–HA under BEC. These differences may be due to different surface morphologies originating from the CS and HA dopants. Our results suggest that PPy–CS in particular is a potential osteogenic scaffold coating for bone tissue engineering. Keywords—Mesenchymal stem cells, Osteogenic, Electrical stimulation, Polysaccharide.
Address correspondence to Suvi Haimi, Adult Stem Cells, Institute of Biomedical Technology, University of Tampere, Biokatu 8, 33014 Tampere, Finland. Electronic mail: suvi.haimi@uta.fi
ABBREVIATIONS BEC ES hASC PPy–CS PPy–HA PS
Biphasic electric current Electrical stimulation Human adipose stem cells Chondroitin sulfate doped polypyrrole Hyaluronic acid doped polypyrrole Polystyrene cell culture plate
INTRODUCTION Conducting polymers are an arising interest in the field of tissue engineering as they can deliver electrochemical as well as electromechanical stimulation to cells. From those polypyrrole (PPy) and poly(3,4-ethylenedioxythiophene) (PEDOT) are the most investigated for biomedical applications owing to their good biocompatibility in vivo and in vitro.8 PPy is intensively investigated for bone9,37,38,46 and neural applications41,50 due to its easy modification with bioactive agents in ambient conditions and highly adjustable properties, such as surface charge and topography17,18,44,46 whereas PEDOT studies concentrate more on neural electrodes and nerve grafts1–5,13,20,21,35,42 mostly owing to PEDOT’s higher electrical conductivity and stability compared to PPy.8 In addition to bone and neural tissue engineering, PPy has so far been studied as bioactive coatings to improve osseointegration,11 in biosensors,7 drug delivery systems50 and actuators.34 Regards to the comprehensive research supporting
2014 Biomedical Engineering Society
BJO¨RNINEN et al.
PPy’s use in bone tissue engineering, we chose PPy and evaluated the effects of the two most potential bioactive dopants, hyaluronic acid (HA) and chondroitin sulfate (CS) in the PPy films. In electrochemical polymerization of PPy, charged biomolecules, such as negatively charged glycosaminoglycans (GAGs), can be incorporated into the structure by doping when PPy is electrochemically polymerized by oxidation. Dopants play an important role in mediating the electric charges between the PPy chains.23 In addition, the surface topography and mechanical properties of PPy can also widely be altered by the choice of dopant.17,18 CS and HA are GAGs commonly found in the extracellular matrices (ECMs) of most animal tissues. CS is a major proteoglycan component in organic matrix of the bone and is involved in the mineralization of the bone tissue whereas HA takes part in various cellular processes, such as ECM organization and metabolism.43 Both GAGs are reported to support osteogenic differentiation of mesenchymal stem cells (MSCs) in scaffold structures in vitro.28,53 As regards integrating HA and CS into PPy surfaces, HA doped PPy (PPy–HA) has been studied with mouse bone marrow derived MSCs resulting in promoted osteogenic differentiation46 and with MC3T3E1 osteoblasts confirming cell differentiation on the surface.45 In addition, we recently were the first to report an excellent attachment, proliferation and early osteogenic differentiation of hASCs on chemically synthetized PPy–CS coating in non-woven polylactide fiber scaffolds.38 As both biomolecules are potential dopants for PPy coating in osteogenic applications, a systematic comparison is required to understand their benefits and differences with human MSCs. Inherent electrical currents and fields are essential in terms of the growth and remodeling of bone tissue. This was first demonstrated by Fukada and Yasuda, who reported bone formation under tension when positive charge was dominating, and the opposite in case of a negative charge and compression.15 This remark led to the development of electrical stimulation (ES) devices for treating severe bone defects.22 Even though ES has been acknowledged as a bone treatment method for several decades, the exploitation of ES to MSCs in bone tissue engineering has been studied only recently.25,26,31,36 These studies have shown that various types of ES can be applied to improve osteogenic differentiation and proliferation of MSCs, yet no specific parameters for the efficient differentiation of MSC towards mature osteoblasts have so far been identified. In addition, most of the above mentioned studies exploited inert conductive substrates; hence a conductive coating with bioactive molecules and topographical cues may yield interesting
synergy mimicking the natural environment in the bone tissue more closely. We therefore wanted to evaluate hASC spreading, proliferation and osteogenic differentiation on PPy surfaces under ES with our novel ES device developed in-house. To the best to our knowledge, this is the first paper to systematically compare HA and CS doped PPy coatings for hASCs.
MATERIALS AND METHODS Polypyrrole Synthesis Pyrrole (Sigma-Aldrich, St. Louis, USA) of 0.07 mL and 1 mg of HA from Streptococcus equi (SigmaAldrich) or CS A from bovine trachea (Sigma-Aldrich) were added per 1 mL of water. PPy–HA and PPy–CS films were grown electrochemically on a sputter-coated polyethylene-naphthalate film (PEN)/Au films (125 lm Dupont Teonex), with 50 nm Au-coating (VTT Technical Research Center of Finland) as a working electrode, platinum mesh as a counter electrode and Ag/AgCl as a reference electrode. Constant potential of 1.0 V was applied to the films until 300 mC cm22 polymerization charge had passed the cell. The stimulation plates and plate covers were sterilized by gamma irradiation (BBF Sterilisationsservice GmbH, Kernen, Germany) with an irradiation dose of >25 kGy that has not been reported to significantly alter the conductivity of the films.12,54 Surface Characterization of Polypyrrole Film The surface morphology and roughness (Ra) values of the PPy films were characterized by an atomic force microscope (AFM; Park Systems XE-100, Korea) in both dry and wet conditions due to the significant water absorption and hence swelling phenomenon of wet PPy films in physiological conditions.39,48 PPy–HA and PPy–CS films were incubated for 4 days in osteogenic medium (OM) containing 250 mM ascorbic acid 2-phosphate (Sigma-Aldrich), 5 nM dexamethasone (Sigma-Aldrich) and 10 mM b-glycerofosfate (Sigma-Aldrich) supplemented to maintenance medium consisting of Modified Eagle Medium/Ham’s Nutrient mixture F-12 (DMEM/F-12 1:1 Invitogen), 10% fetal bovine serum (FBS; Invitrogen), 1% L-glutamine (GlutaMAX I; Invitrogen) and 1% antibiotics/ antimycotic (100 U mL21 penicillin, 0.1 mg mL21 streptomycin; Invitrogen). To distinguish the swelling effect from the typical polysaccharide doped PPy nodular morphology,17,18,39,45,47 and in order to image the nanoscopic details of the soft films,17,38 dried samples were analyzed using non-contact AFM (Park Systems XE-100)
Comparison of CS and HA Doped Conductive PPy Films
in air using silicon probe ACTA-905M (Applied NanoStructures, Inc.) with a nominal resonance frequency of 300 kHz, spring constant 40 N m21 and tip radius
Comparison of Chondroitin Sulfate and Hyaluronic Acid Doped Conductive Polypyrrole Films for Adipose Stem Cells MIINA BJO¨RNINEN,1,2,3 ALIISA SILJANDER,1,2,3 JANI PELTO,4 JARI HYTTINEN,3,5 MINNA KELLOMA¨KI,3,5 SUSANNA MIETTINEN,1,2,3 RIITTA SEPPA¨NEN,1,3,5,6 and SUVI HAIMI1,2,3,7 1
Adult Stem Cells, Institute of Biomedical Technology, University of Tampere, Biokatu 8, 33014 Tampere, Finland; 2Science Centre, Pirkanmaa Hospital District, P.O. BOX 2000, 33521 Tampere, Finland; 3BioMediTech, Biokatu 10, 33520 Tampere, Finland; 4VTT Technical Research Centre of Finland, Sinitaival 6, P.O. Box 1300, 33101 Tampere, Finland; 5Department of Biomedical Engineering, Department of Electronics and Communications Engineering, Tampere University of Technology, P.O. 692, 33101 Tampere, Finland; 6Department of Eye, Ear, and Oral Diseases, Tampere University Hospital, Division 3, P.O. Box 2000, 33521 Tampere, Finland; and 7Department of Biomaterials Science and Technology, University of Twente, P.O. Box 217, 7500, AEEnschede, The Netherlands (Received 7 January 2014; accepted 29 April 2014) Associate Editor Kent Leach oversaw the review of this article.
Abstract—Polypyrrole (PPy) is a conductive polymer that has aroused interest due to its biocompatibility with several cell types and high tailorability as an electroconductive scaffold coating. This study compares the effect of hyaluronic acid (HA) and chondroitin sulfate (CS) doped PPy films on human adipose stem cells (hASCs) under electrical stimulation. The PPy films were synthetized electrochemically. The surface morphology of PPy–HA and PPy–CS was characterized by an atomic force microscope. A pulsed biphasic electric current (BEC) was applied via PPy films non-stimulated samples acting as controls. Viability, attachment, proliferation and osteogenic differentiation of hASCs were evaluated by live/dead staining, DNA content, Alkaline phosphatase activity and mineralization assays. Human ASCs grew as a homogenous cell sheet on PPy–CS surfaces, whereas on PPy–HA cells clustered into small spherical structures. PPy–CS supported hASC proliferation significantly better than PPy–HA at the 7 day time point. Both substrates equally triggered early osteogenic differentiation of hASCs, although mineralization was significantly induced on PPy–CS compared to PPy–HA under BEC. These differences may be due to different surface morphologies originating from the CS and HA dopants. Our results suggest that PPy–CS in particular is a potential osteogenic scaffold coating for bone tissue engineering. Keywords—Mesenchymal stem cells, Osteogenic, Electrical stimulation, Polysaccharide.
Address correspondence to Suvi Haimi, Adult Stem Cells, Institute of Biomedical Technology, University of Tampere, Biokatu 8, 33014 Tampere, Finland. Electronic mail: suvi.haimi@uta.fi
ABBREVIATIONS BEC ES hASC PPy–CS PPy–HA PS
Biphasic electric current Electrical stimulation Human adipose stem cells Chondroitin sulfate doped polypyrrole Hyaluronic acid doped polypyrrole Polystyrene cell culture plate
INTRODUCTION Conducting polymers are an arising interest in the field of tissue engineering as they can deliver electrochemical as well as electromechanical stimulation to cells. From those polypyrrole (PPy) and poly(3,4-ethylenedioxythiophene) (PEDOT) are the most investigated for biomedical applications owing to their good biocompatibility in vivo and in vitro.8 PPy is intensively investigated for bone9,37,38,46 and neural applications41,50 due to its easy modification with bioactive agents in ambient conditions and highly adjustable properties, such as surface charge and topography17,18,44,46 whereas PEDOT studies concentrate more on neural electrodes and nerve grafts1–5,13,20,21,35,42 mostly owing to PEDOT’s higher electrical conductivity and stability compared to PPy.8 In addition to bone and neural tissue engineering, PPy has so far been studied as bioactive coatings to improve osseointegration,11 in biosensors,7 drug delivery systems50 and actuators.34 Regards to the comprehensive research supporting
2014 Biomedical Engineering Society
BJO¨RNINEN et al.
PPy’s use in bone tissue engineering, we chose PPy and evaluated the effects of the two most potential bioactive dopants, hyaluronic acid (HA) and chondroitin sulfate (CS) in the PPy films. In electrochemical polymerization of PPy, charged biomolecules, such as negatively charged glycosaminoglycans (GAGs), can be incorporated into the structure by doping when PPy is electrochemically polymerized by oxidation. Dopants play an important role in mediating the electric charges between the PPy chains.23 In addition, the surface topography and mechanical properties of PPy can also widely be altered by the choice of dopant.17,18 CS and HA are GAGs commonly found in the extracellular matrices (ECMs) of most animal tissues. CS is a major proteoglycan component in organic matrix of the bone and is involved in the mineralization of the bone tissue whereas HA takes part in various cellular processes, such as ECM organization and metabolism.43 Both GAGs are reported to support osteogenic differentiation of mesenchymal stem cells (MSCs) in scaffold structures in vitro.28,53 As regards integrating HA and CS into PPy surfaces, HA doped PPy (PPy–HA) has been studied with mouse bone marrow derived MSCs resulting in promoted osteogenic differentiation46 and with MC3T3E1 osteoblasts confirming cell differentiation on the surface.45 In addition, we recently were the first to report an excellent attachment, proliferation and early osteogenic differentiation of hASCs on chemically synthetized PPy–CS coating in non-woven polylactide fiber scaffolds.38 As both biomolecules are potential dopants for PPy coating in osteogenic applications, a systematic comparison is required to understand their benefits and differences with human MSCs. Inherent electrical currents and fields are essential in terms of the growth and remodeling of bone tissue. This was first demonstrated by Fukada and Yasuda, who reported bone formation under tension when positive charge was dominating, and the opposite in case of a negative charge and compression.15 This remark led to the development of electrical stimulation (ES) devices for treating severe bone defects.22 Even though ES has been acknowledged as a bone treatment method for several decades, the exploitation of ES to MSCs in bone tissue engineering has been studied only recently.25,26,31,36 These studies have shown that various types of ES can be applied to improve osteogenic differentiation and proliferation of MSCs, yet no specific parameters for the efficient differentiation of MSC towards mature osteoblasts have so far been identified. In addition, most of the above mentioned studies exploited inert conductive substrates; hence a conductive coating with bioactive molecules and topographical cues may yield interesting
synergy mimicking the natural environment in the bone tissue more closely. We therefore wanted to evaluate hASC spreading, proliferation and osteogenic differentiation on PPy surfaces under ES with our novel ES device developed in-house. To the best to our knowledge, this is the first paper to systematically compare HA and CS doped PPy coatings for hASCs.
MATERIALS AND METHODS Polypyrrole Synthesis Pyrrole (Sigma-Aldrich, St. Louis, USA) of 0.07 mL and 1 mg of HA from Streptococcus equi (SigmaAldrich) or CS A from bovine trachea (Sigma-Aldrich) were added per 1 mL of water. PPy–HA and PPy–CS films were grown electrochemically on a sputter-coated polyethylene-naphthalate film (PEN)/Au films (125 lm Dupont Teonex), with 50 nm Au-coating (VTT Technical Research Center of Finland) as a working electrode, platinum mesh as a counter electrode and Ag/AgCl as a reference electrode. Constant potential of 1.0 V was applied to the films until 300 mC cm22 polymerization charge had passed the cell. The stimulation plates and plate covers were sterilized by gamma irradiation (BBF Sterilisationsservice GmbH, Kernen, Germany) with an irradiation dose of >25 kGy that has not been reported to significantly alter the conductivity of the films.12,54 Surface Characterization of Polypyrrole Film The surface morphology and roughness (Ra) values of the PPy films were characterized by an atomic force microscope (AFM; Park Systems XE-100, Korea) in both dry and wet conditions due to the significant water absorption and hence swelling phenomenon of wet PPy films in physiological conditions.39,48 PPy–HA and PPy–CS films were incubated for 4 days in osteogenic medium (OM) containing 250 mM ascorbic acid 2-phosphate (Sigma-Aldrich), 5 nM dexamethasone (Sigma-Aldrich) and 10 mM b-glycerofosfate (Sigma-Aldrich) supplemented to maintenance medium consisting of Modified Eagle Medium/Ham’s Nutrient mixture F-12 (DMEM/F-12 1:1 Invitogen), 10% fetal bovine serum (FBS; Invitrogen), 1% L-glutamine (GlutaMAX I; Invitrogen) and 1% antibiotics/ antimycotic (100 U mL21 penicillin, 0.1 mg mL21 streptomycin; Invitrogen). To distinguish the swelling effect from the typical polysaccharide doped PPy nodular morphology,17,18,39,45,47 and in order to image the nanoscopic details of the soft films,17,38 dried samples were analyzed using non-contact AFM (Park Systems XE-100)
Comparison of CS and HA Doped Conductive PPy Films
in air using silicon probe ACTA-905M (Applied NanoStructures, Inc.) with a nominal resonance frequency of 300 kHz, spring constant 40 N m21 and tip radius
