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Extracellular matrix-coated composite scaffolds promote mesenchymal stem cell persistence and osteogenesis Jenna N. Harvestine, Nina L. Vollmer, Steve S. Ho, Christopher Andrew Zikry, Mark A. Lee, and J. Kent Leach Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01005 • Publication Date (Web): 16 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016
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Extracellular matrix-coated composite scaffolds promote mesenchymal stem cell persistence and osteogenesis Jenna N. Harvestinea; Nina L. Vollmera; Steve S. Hoa: Christopher A. Zikrya; Mark A. Leeb, J. Kent Leachab* a
Department of Biomedical Engineering, University of California, Davis, Davis, CA 95616
b
Department of Orthopaedic Surgery, School of Medicine, UC Davis Medical Center, Sacramento, CA
95817 Key Words: extracellular matrix, bone, mesenchymal stem cell, bioactive glass, composite
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ABSTRACT
Composite scaffolds of bioactive glass and poly(lactide-co-glycolide) provide advantages over homogenous scaffolds, yet their therapeutic potential can be improved by strategies that promote adhesion and present instructive cues to associated cells. Mesenchymal stem cell (MSC)-secreted extracellular matrix (ECM) enhances survival and function of associated cells. To synergize the benefits of an instructive ECM with composite scaffolds, we tested the capacity of ECM-coated composite scaffolds to promote cell persistence and resultant osteogenesis. Human MSCs cultured on ECM-coated scaffolds exhibited increased metabolic activity and decreased apoptosis compared to uncoated scaffolds. Additionally, MSCs on ECM-coated substrates in short-term culture secreted more proangiogenic factors while maintaining markers of osteogenic differentiation. Upon implantation, we detected improved survival of MSCs on ECM-coated scaffolds over three weeks. Histological evaluation revealed enhanced cellularization and osteogenic differentiation in ECM-coated scaffolds compared to controls. These findings demonstrate the promise of blending synthetic and natural ECMs and their potential in tissue regeneration.
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INTRODUCTION The extracellular matrix (ECM) is comprised of soluble and insoluble signals unique to tissuespecific microenvironments.1 The composition of these matrices provides the niche responsible for directing cell fates necessary for tissue homeostasis such as adhesion, proliferation, migration, and differentiation.2 The instructive nature of the ECM lends itself as a powerful tool for tissue engineering and regenerative medicine as more effort is placed on controlling persistence and cell fate in vivo. The effect of individual ECM proteins such as fibronectin, collagen type I, and laminin on MSC adhesion and differentiation has been widely examined.3-5 However, individual molecules do not recapitulate the composition and complex interactions between the networked macromolecules of the native ECM. While all cells secrete an ECM, mesenchymal stem cells (MSCs) are particularly relevant because they deposit substantial quantities of ECM during in vitro culture, which can subsequently be harvested and deposited on other substrates.6 The composition of these matrices can be controlled through manipulation of the biochemical environment during culture (i.e. oxygen tension, media supplementation, etc.)7 and decellularized for use as an instructive biomaterial. Many studies using decellularized ECM in both 2 and 3 dimensions are performed without removal of ECM from its location of deposition.8-10 For clinical application of ECM-coated materials, it is advantageous to generate ECM in 2D and transfer it to the desired material after synthesis to eliminate heterogeneous ECM composition resulting from nutrient availability and cellular response in 3D. We previously demonstrated the potential of MSC-secreted decellularized matrices to serve as instructive biomaterials for osteogenic differentiation of undifferentiated MSCs in both the presence and absence of soluble factors.11-12 Moreover, we and others reported enhanced cell persistence and implant vascularization in vivo when cells were delivered on ECM-coated biomaterials compared to uncoated materials.11, 13 Polymeric biomaterials are widely explored for tissue engineering applications. Despite the advantages of tailorability and reproducibility, these materials lack the robust mechanical properties
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desirable for many orthopedic interventions. Bioceramic-polymer composite materials address many of the drawbacks to homogenous scaffolds. Bioactive glass (BG) is one bioceramic under investigation due to the proliferative and proangiogenic effect of its dissolution constituents on associated cells.14-16 BG is of particular interest for bone tissue engineering because the material’s quick dissolution and generation of Ca2+ reacting with (PO4)3- lead to the formation of an amorphous calcium phosphate layer on the surface, which continues to incorporate (OH)- and (CO3)2- from solution and crystalize as a carbonate-substituted hydroxyapatite-like layer.17 Although osteoconductive, composite substrates are relatively inert and lack vital instructional cues for associated cells which jumpstart the regenerative process, providing an opportunity to advance the therapeutic potential of these materials. In an effort to synergize the promising effects of ECM with improved physical properties of composite scaffolds, we investigated the potential of cell-secreted ECM deposited on composite, macroporous scaffolds to enhance MSC persistence and engraftment in vivo for bone tissue engineering. BG-PLG composite scaffolds were coated with MSC-secreted ECM (Scheme 1). The proangiogenic potential and osteogenic differentiation of associated MSCs were assessed in vitro. Cell persistence and proliferation was evaluated in vivo using bioluminescent imaging, and explants were analyzed histologically for tissue morphology and osteogenesis.
MATERIALS AND METHODS Scaffold preparation Scaffolds were fabricated using a gas foaming/particulate leaching method as previously described.18 Poly(lactide-co-glycolide) (PLG) microspheres were formed from PLG pellets (85:15 DLG 7E; Lakeshore Biomaterials, Birmingham, AL) using a double-emulsion process and lyophilized to form a free-flowing powder. PLG microspheres (7.1 mg) were mixed with 17.8 mg 45S5 Bioglass® particulate
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(BG, 90-170 μm particle size, Novabone, Alachua, FL) and 135.1 mg NaCl particles (250-425 µm diameter, Fisher Scientific), creating a 2.5:1:19 mass ratio of ceramic:polymer:salt.18 The powder was compressed using a Carver Press (Carver, Wabash, IN) and stainless steel die for one minute under 2 metric tons of force to form solid disks of 8.5 mm diameter and 1.5 mm thickness. Compressed disks were pressurized in CO2 gas (5.5 MPa) for 16 h followed by a rapid pressure release over 1-2 min. NaCl particles were leached from scaffolds by submersion in distilled H2O for a minimum of 24 h. Scaffolds were sterilized in a sealed 50 mL Steriflip conical tube (Millipore, Billerica, MA) with 70% ethanol under gentle vacuum for 30 min. Scaffolds were rinsed twice (15 min) with sterile PBS under a gentle vacuum and transferred to a 24-well plate to dry in a biosafety cabinet overnight.
Cell culture Human bone marrow-derived MSCs (Lonza, Walkersville, MD) were used without further characterization. MSCs were expanded under standard conditions until use at passages 4-6 in growth medium (GM) containing minimum essential alpha medium (α-MEM; w/L-glutamine, w/o ribo/deoxyribonucleosides (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA) and 1% penicillin (10,000 U/mL) and streptomycin (10 mg/mL) (Mediatech, Manassas, VA). Human MSCs genetically modified to express luciferase (Luc-MSCs) were used during in vivo studies to observe cell persistence.11 Media was refreshed every 3 days.
Synthesis and collection of MSC-secreted extracellular matrix Cell-secreted ECMs were prepared as we described.6-7, 11-12 Briefly, MSCs were cultured in supplemented medium (SM) containing 50 mg/mL ascorbate-2-phosphate (A2P) one passage prior to experimental use to prime cells for enhanced matrix deposition. MSCs were then seeded at 50,000
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cells/cm2 and cultured in SM for 14 days under standard culture conditions with media changes performed every 3-4 days. Flasks were rinsed once with sterile PBS and treated with 0.5% Triton X-100 (Sigma, St. Louis, MO) in 20 mM NH4OH in PBS for 15 min at 37oC. Following an additional PBS rinse, wells were treated with DNAse (Sigma, 100-200 units/20 mL PBS supplemented with calcium) for 1 h at 37oC and rinsed with calcium-free PBS. ECM was collected with a cell scraper and transferred into sterile 0.02 N acetic acid. The solution was sonicated on ice with 2-3 second pulses to mechanically homogenize the contents. Total protein within the collected ECM was quantified using a bicinchoninic acid (BCA) protein assay (ThermoFisher, Rockford, IL). ECM solutions were frozen at -20oC until use.
Deposition of ECM on composite scaffolds Scaffolds were coated with ECM as described previously.12 Briefly, sterilized composite scaffolds were placed on a Steritop vacuum filter (Millipore) attached to a 300 mL glass bottle, and ECM solution was dispensed using a micropipette onto each scaffold. A gentle vacuum was applied to the bottom of the scaffolds until scaffolds appeared dry (approximately 30 min). Scaffolds were flipped, and the process was repeated to achieve the desired coating of 0, 50, or 100 µg of total protein per scaffold. Scaffolds were placed in sterile 24-well plates, dried overnight in a biosafety cabinet, and stored in the dark at room temperature until use.
Scaffold characterization For gross visual inspection of the ECM distribution, scaffolds were submerged in 0.1% (w/v) Coomassie Brilliant Blue (MP Biomedicals, Burlingame, CA) and placed on a shaker for 15 min at 250 rpm. Scaffolds were washed in PBS and then imaged. Scaffold porosity (void volume) was calculated as previously described.18 2 metric tons of force was applied for 30 sec using a Carver Press, and void
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volume was calculate from the difference in scaffold dimensions, measured with a caliper, before and after compression. Compressive moduli of acellular scaffolds were determined using an Instron 3345 testing device (Instron, Norwood, MA). Scaffolds were lyophilized for 24 hours prior to compression testing to remove moisture. Scaffolds were loaded between two flat platens and after an initial 1 N preload, scaffolds were compressed with constant deformation rate of 1 mm/min. Compressive moduli were calculated from the first 10% of strain.18 Pore architecture and protein adsorption to pore surface were visualized with scanning electron microscopy.12, 19 Scaffolds were fixed in Karnovsky’s fixative solution, flash frozen with liquid nitrogen, bisected with a razor, and dehydrated in increasing concentrations of ethanol. Following dehydration, samples were critical point dried (Supercritical AutoSamdri-931, Tousimis Research Corp, Rockville, MD), fixed to stubs with silver paste, sputter coated with gold (Pelco SC-7 Auto Sputter Coater), and imaged at 5 kV using a scanning electron microscope (Hitachi S3500-N, Hitachi Science Systems Ltd, Tokyo, Japan).
Characterization of MSC response to composite scaffolds MSCs (5x105 cells per scaffold) were suspended in α-MEM (35 µL) and applied dropwise to each scaffold. After 1 h to allow cell attachment, GM was added to each culture well, and plates were incubated on an XYZ shaker throughout the culture duration for up to 14 days or implanted. To analyze, cell-seeded scaffolds were rinsed in PBS, minced, and placed in 400 µL passive lysis buffer (PLB; Promega, Sunnyvale, CA). Following a freeze thaw cycle, the lysate was sonicated (10 s on ice) and separated from the scaffold material via centrifugation (10,000 rpm for 10 min at 4oC). Total DNA present in scaffolds was determined using the Quant-iT PicoGreen dsDNA kit (Invitrogen, Carlsbad, CA). Apoptosis was measured from lysates of scaffolds collected in PLB using the Caspase-Glo 3/7 luminescence assay (Promega) as we described.20 Luminescence was measured on a multifunctional plate reader (Synergy HTTR, Wisnooski, VT) and normalized to DNA content within each well. Cell
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metabolic activity was determined using an AlamarBlue assay per manufacturer’s instructions (Invitrogen). Osteogenic and proangiogenic potential were assessed at 1, 7, and 14 days by quantifying osteocalcin and vascular endothelial growth factor (VEGF) secretion, respectively, with protein-specific ELISA kits per manufacturer’s instructions (R&D Systems, Minneapolis, MN).
Murine ectopic model of bone formation Treatment of experimental animals was in accordance with the University of California, Davis animal care guidelines and all National Institutes of Health animal-handling procedures. Eight-week-old non-obese diabetic/severe combined immunodeficient gamma (NSG, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice (Jackson Laboratories, Bar Harbor, ME) were anesthetized and maintained under a 2% isoflurane/O2 mixture delivered through a nose cone. Using a scalpel, an incision was made down the back of the animal and 4 subcutaneous pockets were created for scaffold implantation. Every animal received four BG-PLG scaffold implants: acellular scaffold with 0 µg ECM (top left); 0 µg ECM with 5x105 MSCs (top right), 50 µg ECM with 5x105 MSCs (bottom left); and 100 µg ECM with 5x105 MSCs (bottom right). To track cell survival, animals were given an intraperitoneal injection of D-Luciferin Firefly (Caliper, Perkin Elmer, Waltham, MA) in sterile PBS (10 μg/g body weight).11 All animals were scanned with an IVIS Spectrum (Perkin Elmer), and cell persistence was measured using Living Image software (Perkin Elmer). Total photons per second per square centimeter were recorded from each bioluminescent region of interest. Animals were euthanized via CO2 inhalation at 2 (n = 3) and 6 weeks (n = 5) after scaffold implantation. Each scaffold was excised and fixed in 10% buffered formalin solution (Fisher Scientific). Scaffolds were soaked overnight and embedded in OCT, sectioned on a Leica CM1850 Cryostat (Leica Microsystems, Bannockburn, IL), and mounted on microscope slides for further histological processing.
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Slides were stained with a trichrome staining kit (AB150686, Abcam, Cambridge, MA) and hematoxylin and eosin (H&E) (Ricca Chemical, Arlington, TX). Images were converted to RGB stack in ImageJ and the thresholding tool was used to identify areas of section covered with tissue. The surface area above the identified threshold was divided by the total section area being evaluated. Immunohistochemistry for human osteocalcin (1:200, AB13420, Abcam), CD90 (1:250, AB133350, Abcam), and CD31 (1:500, AB28364, Abcam) was performed using HRP detection kit (AB64261, Abcam) per manufacturer’s instructions.
STATISTICAL ANALYSIS Data are presented as means ± standard deviation unless otherwise stated. Statistical analyses were performed with two-way ANOVA, followed by Tukey’s multiple comparison post hoc test (GraphPad Prism 7.0) to assess significance (p
