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Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B. This journal is The Royal Society of Chemistry 2016 SUPPLEMENTARY INFORMATION Nanoengineered biomimetic hydrogels for guiding
Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B. This journal is The Royal Society of Chemistry 2016 SUPPLEMENTARY INFORMATION Nanoengineered biomimetic hydrogels for guiding human stem cell osteogenesis in three dimensional microenvironments Arghya Paul a,b,c,d*, Vijayan Manoharan a,b,d, Dorothee Krafft a,b, Alexander Assmann a,b,c,e, Jorge Alfredo Uquillas a,b,f, Su Ryon Shin a,b,c, Anwarul Hasan a,b,g, Mohammad Hussain h, Adnan Memic i, Akhilesh K. Gaharwar j, Ali Khademhosseini a,b,c,k,l,m* a Biomaterials Innovation Research Center, Division of Biomedical Egnineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA b Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA c Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA d Department of Chemical and Petroleum Engineering, Bioengineering Graduate Program, School of Engineering, University of Kansas, Lawrence, KS, USA e Department of Cardiovascular Surgery, Heinrich Heine University, Medical Faculty, Duesseldorf, Germany f Department of Orthopaedic and Trauma Surgery, Pontificia Universidad Católica del Ecuador, Quito-Ecuador g Department of Mechanical and Industrial Engineering, College of Engineering, Qatar University, Doha, Qatar h Department of Electrical and Computer Engineering, King Abdulaziz University, Jeddah 21589, Saudi Arabia i Center of Nanotechnology, King Abdulaziz University, Jeddah 21589, Saudi Arabia j Department of Biomedical Engineering and Department of Materials Science and Engineering, Texas A&M University, 5024 Emerging Technology Building, College Station, TX k Department of Bioindustrial Technologies, College of Animal Bioscience and Technology, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul , Republic of Korea. l Department of Physics, King Abdulaziz University, Jeddah 21569, Saudi Arabia m WPI-Advanced Institute for Materials Research, Tohoku University, Sendai, Japan [*] Corresponding author: Arghya Paul and Ali Khademhosseini ; Running title: Nanoengineered osteoinductive hydrogel 1 GelMA-NS GelMA SUPPLEMENTARY FIGURES AND TABLE (A) NS Porous hydrogel (B) Alizarin Red Staining (Lower Mag) Growth media Osteogenic Figure S1: Fabrication of hmsc encapsulated GelMA-nSi hydrogels for osteogenic differentiation. (A) Schematic representation of in vitro hydrogel fabrication method using GelMA, nanosilicates and hmscs by covalent crosslinking under UV radiation. Transmission Electron Microscope (TEM) image of nsi particles dispersed in water (Scale: 100nm). Bright field picture of porous hydrogel (Scale bar: 200μm). (B) Confirmation of osteogenic differentiation of hmscs after 21 days of culture in growth media and osteogenic media (with no drugs) by alizarin red staining of cross-section of the hydrogels to trace calcium deposits. Scale bar: 200μm. 2 Normalized Fluoresnece intensity (ROS) pg/ml (A) (B) Ctrl (LPS) 0% NS 0.01% NS 0.05% NS 0.5% NS Ctrl (0) NS Concentration (%) 0 IL6 TNF alpha Figure S2: (A) Effect of the GelMA-nSi hydrogel on production of reactive oxide species (ROS) in encapsulated hmscs. The formation of radicals as a measure of intracellular stress that generates a cytotoxic response was determined. The intracellular production of ROS was evaluated after hmscs incubation in the presence of different NSi concentrations in GelMA-nSi hydrogel. As the silicate concentration increased, no intracellular oxidative stress (ROS) was noticed until 0.05% NS. However, at higher silicate concentrations (0.5% NS), a significant increase in ROS was observed as quantified by ROS fluorescence assay in a plate reader and represented as fold change compared to 7% GelMA with 0% NS. Serum starved group with 7% GelMA hydrogel was used as the experimental control, Ctrl (0). (B) Secretion of pro-inflammatory cytokines, IL6 and TNFα, from RAW macrophages encapsulated in hydrogels with different formulations (7% GelMA hydrogels with different percentages of NS) after 24h of exposure represented in the bar graph with different colors [Black: Ctrl (LPS), White: 0% NS, Red: 0.01% NSi, Grey: 0.05% NS, Blue: 0.5% NS] as obtained by ELISA analysis. As positive control group, RAW cells were treated with LPS. Data represent Mean ± SD (n=3). *=p 0.05 & =p compared to control GelMA group (0% NS). 3 Proliferation (fold increase) Osteocalcin ALP stain (A) (i) PEGDA coated glass (ii) PEGDA coated glass Spacer MSC encapsulated in hydrogel hmscs micropatterned in GelMA-nSi hydrogel (iii) UV Photo-Mask (v) (iv) (B) GelMA (0h) GelMA (72h) 0.05% NS (0h) 0.05% NS (72h) 0.05% NS (72h) (C) (D) GelMA 0.05% NS Magnified 1.4 0% NS 0.001% NS 0.01% NS % NS 1.2 Magnified hours Figure S3: (A) Schematic to step-wise microfabricate hmscs encapsulated in GelMA-nSi hydrogels on PEGDA coated glass slides by UV photo-crosslinking. Inset shows fluorescence image of the micropatterned cells stained with F-actin (green) and nuclei (blue). (B) Biocompatibility of micropatterned GelMA-nSi hydrogel was confirmed by fluorescence microscope images of calcein stained hmscs at 0h and 72h encapsulated in GelMA-nSi (0.05% NS) and control GelMA group (0%NS) in normal media. Additionally, (C) cell proliferation study by MTS assay demonstrated that all the micropatterned hydrogel groups had similar growth kinetics with no significant differences (p 0.05). (D) Increase in osteogenic differentiation potential of hmscs in micropatterned GelMA-nSi hydrogel compared to GelMA group was confirmed by ALP staining (upper panel, in purple) and osteocalcin immunostaining (lower panel, in red) after 21 days of culture in osteoconductive media. Scale bar: 100μm. 4 Table S1: Synthesis and Biological activities of Encapsulated Stem Cells within GelMA-nSi and other Nanocomposite Hydrogels reported for 2D and bone regeneration applications. Nanocomposite Materials Cell Type Mode of Cell Culture Growth Factors (Y/N) Remarks/Characteristics Photo-crosslinked gelatin and synthetic silicate nanoclay (GelMA-nSi) Chitosan + nanocrystalline hydroxyapatite + single-walled carbon nanotubes [1] Gelatin + chitosan nanoparticles + recombinant human bone morphogenic proteins (rhbmps) [2] GelMA + Gold nanoparticles [3] Hyaluronic acid methacrylate + growth & differentiation factor 5 [4] β-tricalcium phosphate scaffolds [5] Alginate [6] Alginate +anti-bmp2 monoclonal antibody [7] GelMA + fibronectin + laminin + osteocalcin [8] fetal osteoblasts MC3T3-E1 preosteoblasts & endothelial cells N Simple one-step fabrication method Biomimetic behavior GelMA-nSi hydrogel has strong osteoinductive properties Hydrogels can be micropatterned to design diverse osteogenic structures with hmscs in Biocompatible (tested in vitro and in vivo) 2D Seeding N Complex three step fabrication method Bioactivity of nanocrystalline hydroxyapatite Nanofibrous matrix using carbon nanotube reinforced substrate 2D Seeding Y Two step fabrication Multiple components needed rhbmp-2 promoted differentiation Chitosan was used to control delivery of rhbmp 2D Seeding N One step fabrication Bioactive gold nanoparticles Cells were not encapsulated 2D Seeding Y One step fabrication Application with human cells is not demonstrated 2D Seeding N Cells seeded on prefabricated scaffold Co-culture promoted osteogenesis N One step fabrication Result depends on cell density Y One step fabrication Expensive Large scale production N Rapid prototyping Need for multiple and costly biomolecules 5 Reference List [1] Im O, Li J, Wang M, Zhang LG, Keidar M. Biomimetic three-dimensional nanocrystalline hydroxyapatite and magnetically synthesized single-walled carbon nanotube chitosan nanocomposite for bone regeneration. International journal of nanomedicine. 2012;7: [2] Cao L, Werkmeister JA, Wang J, Glattauer V, McLean KM, Liu C. Bone regeneration using photocrosslinked hydrogel incorporating rhbmp-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles. Biomaterials. 2014;35: [3] Heo DN, Ko W-K, Bae MS, Lee JB, Lee D-W, Byun W, et al. Enhanced bone regeneration with a gold nanoparticle-hydrogel complex. Journal of Materials Chemistry B. 2014;2: [4] Bae MS, Ohe JY, Lee JB, Heo DN, Byun W, Bae H, et al. Photo-cured hyaluronic acid-based hydrogels containing growth and differentiation factor 5 (GDF-5) for bone tissue regeneration. Bone. 2014;59: [5] Kang Y, Kim S, Fahrenholtz M, Khademhosseini A, Yang Y. Osteogenic and angiogenic potentials of monocultured and co-cultured human-bone-marrow-derived and humanumbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold. Acta Biomaterialia. 2013;9: [6] Maia FR, Lourenco AH, Granja PL, Goncalves RM, Barrias CC. Effect of cell density on aggregation in RGD-alginate matrices under osteoinductive conditions. Macromolecular bioscience. 2014;14: [7] Moshaverinia A, Ansari S, Chen C, Xu X, Akiyama K, Snead ML, et al. Co- of anti-bmp2 monoclonal antibody and in alginate microspheres for bone tissue engineering. Biomaterials. 2013;34: [8] Dolatshahi-Pirouz A, Nikkhah M, Gaharwar AK, Hashmi B, Guermani E, Aliabadi H, et al. A combinatorial cell-laden gel microarray for inducing osteogenic differentiation of human. Scientific reports. 2014;4:
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