Interpenetrating Alginate-Collagen Polymer Network Microspheres for

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Interpenetrating Alginate-Collagen Polymer Network Microspheres for Modular Tissue Engineering Redouan Mahou, Alexander E. Vlahos, Avital Shulman, and M. V. Sefton ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00356 • Publication Date (Web): 25 Jul 2017 Downloaded from http://pubs.acs.org on July 30, 2017

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ACS Biomaterials Science & Engineering

Interpenetrating Alginate-Collagen Polymer Network Microspheres for Modular Tissue Engineering Redouan Mahoua, Alexander E Vlahosa, Avital Shulmana, Michael V. Seftona,b,* a

Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON,

M5S 3G9, Canada b

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto,

ON, M5S 3E5, Canada *

Address correspondence to:

Michael V. Sefton, ScD Institute of Biomaterials and Biomedical Engineering, University of Toronto 164 College Street, Toronto M5S 3G9, Ontario, Canada Phone: (+1) 416-978-3088; Fax: (+1) 416-978-4317; E-mail: [email protected]

ACS Paragon Plus Environment

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Abstract

The lack of vascularization limits the creation of engineered tissue constructs with clinically relevant sizes. We pioneered a bottom-up process (modular tissue engineering), in which constructs with intrinsic vasculature were assembled from endothelialized building blocks. In this study, we prepared an interpenetrating polymer network (IPN) hydrogel from a collagenalginate blend and evaluated its use as microspheres in modular tissue engineering. Ionotropic gelation of alginate was combined with collagen fibrillogenesis and the resulting hydrogel was stiffer and had greater resistance to enzymatic degradation relative to collagen alone; the viability of embedded mesenchymal stromal cells (adMSC) was unaltered. IPN microspheres were fabricated by a coaxial air-flow technique and an additional step of collagen coating was required to have human umbilical vein endothelial cells (HUVEC) attach and proliferate. When implanted subcutaneously in SCID/bg mice, adMSC-HUVEC microspheres promoted more blood vessels at day 7 relative to microspheres without adMSC but coated with HUVEC. Perfusion studies confirmed that these vessels were connected to the host vasculature. Fewer vessels were detected in both groups at day 21, but in adMSC-HUVEC explants more smooth muscle cells had wrapped around vessels and CLARITY processing of whole explants revealed a restricted leakage of blood. The capacity for rapid gelation and high throughput production are promising features for the use of these microspheres in modular tissue engineering.

Keywords: Modular tissue engineering; interpenetrating polymer network; vascularization; alginate; collagen


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Introduction The past decades have witnessed the emergence of tissue engineering in which living cells and biomaterials are combined into tissue-like constructs in order to restore or replace damaged tissues and organs.1,2 However, the approach is still limited by the inability to supply cells with oxygen and nutrients. As a result, the adoption of engineered constructs in clinical practice remains uncertain3 and has generally been restricted to thin4 or relatively avascular engineered tissues.5,6 Our group pioneered modular tissue engineering7-9 as a means of building vascularized constructs from the bottom-up.10 In this approach functional cells and vascular support cells were embedded in collagen hydrogels, referred to as modules, and endothelial cells (EC) were seeded on the surface of these modules. Upon implantation, EC detached from the surface and formed vessels. The premise for this approach was the advantage of a ‘built-in’ vascular network, with the vascular component already included by design. Modular tissue engineering allowed for uniform cell seeding within the construct as well as for mixing modules embedding different functional cells in different ratios. The sub-millimeter size of the modules ensures that even at high densities the cells do not experience hypoxia,11 a typical issue encountered when fabricating large tissues. In addition, the modules are injectable at the implant site in a minimally invasive manner.12 In addition to being biocompatible and relatively easy to make, collagen hydrogels support the adhesion of EC. On the contrary, they are relatively soft (and weak) with a modulus in the range of ~ 100 Pa.13,14 This is significantly lower, for example, than the modulus of small-diameter vascular tissue.15,16 Reinforcing collagen hydrogels with chemical crosslinking17 has unwanted


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side effects, particularly if used in the presence of cells. Previously we combined collagen with poloxamine and achieved a 100-fold increase in stiffness relative to pure collagen gel,18 but the resulting hydrogels exhibited poor adhesive properties19-21 and had limited utility in modular tissue engineering. On the other hand, hydrogels made of sodium alginate (Na-alg) are commonly used as cellladen vehicles in various biomedical applications.22 Na-alg is a family of polysaccharides made of (1,4)-linked D-manuronic acid (M) and L-guluronic acid (G) residues, with various proportions and distributions. Na-alg form hydrogels through ionotropic gelation with divalent cations, referred to as egg-box model.23 As alginate hydrogels do not support cell attachment, one approach to improve their adhesive properties is the chemical modification with adhesion peptides; e.g. the sequence arginine-glycine-aspartic acid (RGD).24 Covalent cross-linking with gelatin,25 incorporation of magnesium ions,26 and enzyme-catalyzed gelation27 were also investigated for the purpose of enhancing the cell adhesion capacity of alginate hydrogels. Combining collagen and Na-alg yielded hydrogels with defined structures and biological features that are promising for multiple biomedical applications.28-34 In this study we blended collagen and Na-alg into an interpenetrating polymer network (IPN)35 in the form of submillimeter spheres and investigated their suitability as a biomaterial for modular tissue engineering. In order to render the resulting IPN microspheres suitable for seeding with EC, we used a two-step process as illustrated in Figure 1. IPN microspheres were prepared by extrusion of a collagen-alginate blend into calcium, followed by coating with a collagen/citrate solution and seeding with HUVEC. Specifically our study aimed to: (i) prepare a collagen-alginate IPN biomaterial and characterize its stiffness and degradation rate relative to collagen alone, (ii) process IPN into microspheres and confirm the feasibility of cell encapsulation and seeding with


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HUVEC and (iii) subcutaneously implant the IPN microspheres and investigate the formation, perfusion, and maturation of blood vessels. Tissue clearing (CLARITY) enabled us to visualize the intact explant vasculature in three dimensions.

Figure 1. Preparation of IPN microspheres: (A): A suspension of adMSC in collagen-alginate blend was extruded into a calcium solution. (B): Microspheres were further coated with collagen in sodium citrate and (C) seeded with HUVEC. IPN microspheres were (D) subcutaneously implanted into SCID/bg mice to investigate the formation of blood vessels. The photo on the bottom left shows a representative histology image of explants at day 7 stained with CD31. Arrows point to blood vessels with lumens.


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2. Experimental 2.1. Materials Na-alg (PRONOVA UP LVG, Batch no: BP-1111-32) was obtained from FMC BioPolymer, Norway. The apparent viscosity [η] and the molar guluronic acid fraction (FG) were 68 mPa.s and FG = 0.69, respectively. Endotoxin-free 3-(N-morpholino) propanesulfonic acid (MOPS) was purchased from Teknova (Hollister, CA). Bovine type-1 collagen solution (FibriCol®, ~ 10 mg/ml) was from Advanced BioMatrix (San Diego, CA). Phosphate-buffered saline (PBS), and Hank's balanced salt solution (HBSS 10x - without Ca2+ and Mg2+) were obtained from GIBCO (Burlington, Canada). Acrylamide, paraformaldehyde, bis-acrylamide, Dulbecco's Modified Eagle's Medium (DMEM), collagenase from clostridium histolyticum (C9263), calcium chloride, sodium chloride, sodium citrate, Tween-20, and sodium hydroxide were purchased from Sigma (Sigma-Aldrich, Oakville). All reagents were used without further purification. 2.2 Preparation of stock solutions A stock solution of Na-alg was prepared at 40 mg/mL in endotoxin-free water (Associates of Cape Cod, Inc. MA). The calcium solution consisted of 90 mM CaCl2 + 0.1% v/v Tween-20 in 10 mM MOPS, pH 7.2. A solution of 5 mM sodium citrate was prepared in DMEM, sterile filtered and stored at 4°C until further use. A solution of 0.2 mg/mL collagen in citrate was prepared immediately prior to use by mixing FibriCol® and sodium citrate solution in 1:50 volume ratio. 2.3. Cell culture


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Primary human umbilical vein endothelial cells (HUVEC) and human adipose derived mesenchymal stromal cells (adMSC) were obtained from Lonza. HUVEC were cultured in endothelial basal medium supplemented with the endothelial growth medium bullet-kit (EGM2, Lonza), which was changed every second day. adMSC were cultured in DMEM supplemented with 10% fetal bovine serum and 1% volume penicillin-streptomycin (Gibco). Cells were passaged upon reaching 80% confluence and were used for experiments at passages 3 to 6. 2.4. Preparation of IPN microspheres IPN microspheres were fabricated from a precursor solution containing 10 mg/mL Na-alg and 5 mg/mL collagen in a two-step process, as illustrated in Figure 1. The precursor solution was prepared as follows: 1 mL FibriCol®, 100 µL NaOH (0.3 N), 200 µL of DMEM, and 200 µL HBSS 10x were added to a 5 mL luer-lock syringe with sterile tip cap and gently mixed for 30 min at 4°C. Na-alg stock solution (500 µL) was then added into the syringe and the precursor solution homogenized at 4°C for an additional 30 min. The syringe was attached to a 25G blunt needle (SAI Infusion Technologies, IL, USA) and the solution was extruded into calcium solution using a homemade coaxial air-flow droplet generator.36 The speed of extrusion and the pressure of compressed air were 0.5 mL/min and 4 PSI, respectively. The resulting microspheres (~700 µm in diameter) were collected over a sieving device (pluriStrainer® 200 µm, pluriSelect, San Diego) and incubated for 1 h in DMEM at 37°C to enable fibrillogenesis.37 For the second step, microspheres were transferred to a 10 cm-diameter petri dish and cultured overnight at 37°C under mild shaking (20 rpm) with 20 mL of 0.2 mg/mL collagen in 5 mM sodium citrate solution. Microspheres were collected by filtration and were ready for seeding with HUVEC. For the preparation of adMSC-embedded microspheres, 1 mL FibriCol®, 100 µL NaOH (0.3 N), 200 µL HBSS 10x, and 500 µL Na-alg were added to the syringe and mixed at 4°C. adMSC (2


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million in 200 µL of DMEM) were added to the precursor solution and homogenized for 5 min. The suspension was then extruded into calcium and treated in a second step as above. 2.5. Rheological characterization The stiffness of hydrogels was monitored at 37°C by small-amplitude oscillatory shear (SAOS) on an AR-1000 rheometer (TA Instruments, New Castle, DE, USA). Disk-shaped hydrogels were fabricated as detailed in the supporting information SI1. The following protocol was applied: (1) a strain sweep at a frequency of 1 Hz to determine the linear viscoelastic region with respect to strain, and (2) a time sweep with values obtained from strain sweeps. All samples were analyzed in triplicate. 2.6. Enzymatic degradation Hydrogels were prepared in pre-weighted vials as described in the supporting information SI2 and their degradation by collagenase was monitored over time. Three vials of either IPN or collagen were washed with deionized water and lyophilized to determine the initial dry weight (m0). Collagenase (0.1 units/mL of PBS) was added and the vials were incubated at 37°C on an orbital shaker. The solution was withdrawn at periodic intervals and the hydrogels were washed with deionized water, freeze-dried and their dry weight (md) recorded. The percent mass loss was calculated as:

Percent mass loss =

( − ) × 100 ( )

Where xi is the weight fraction contributed by the collagen to the hydrogel formulation. xi corresponds to 1 and 0.33 for collagen-only and IPN, respectively.


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2.7. AlamarBlue assay The viability of adMSC in hydrogels was assessed by alamarBlue®. adMSC were embedded in IPN or collagen in a 24 well plate as described in the supporting information SI3. At days 1, 3, and 7, culture medium was replaced by 10% alamarBlue® (Invitrogen) in DMEM, and the plate was incubated for 6 hours at 37°C. The absorbance of the supernatant was monitored with a microplate absorbance reader (Tecan, Sunrise) at 570 nm, using 600 nm as a reference wavelength. The percent reduction was calculated and normalized to values of adMSC cultured in the absence of hydrogels, which were used as control. 2.8. Seeding IPN microspheres with HUVEC In a 10 cm-diameter petri dish, HUVEC (1 million in 15 mL of a mixture of DMEM:EGM-2, 50:50) were added to the IPN microspheres prepared in section 2.4 (with or without embedded adMSC) and placed on a low-speed uniaxial rocker for 60 minutes at 37°C. Microspheres were statically cultured for 3 days. HUVEC coated microspheres with or without embedded adMSC are referred to herein as adMSC-HUVEC or HUVEC microspheres, respectively. 2.9. Immunofluorescence and Confocal Imaging Confocal imaging microscopy with a Zeiss Axiovert fluorescence microscope was used to assess the viability of adMSC embedded in IPN microspheres, and to visualize the collagen at the surface of microspheres as well as VE-cadherin (for HUVEC adherens junctions). For viability studies, Calcein AM and ethidium homodimer-1 (live/dead™ kit, Molecular Probes, Eugene, OR) was used. Immunofluorescence of collagen was monitored using rabbit polyclonal antibody to collagen I (ab34710; Abcam) and goat anti-Rabbit IgG (H+L) secondary antibody, Alexa


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Fluor® 568 conjugate (A11011, Invitrogen). Goat polyclonal IgG and donkey anti-goat IgGFITC (C-19 and sc-2024 respectively, both from Santa Cruz Biotechnology) were used as primary and secondary antibodies in staining for VE-cadherin. Detailed protocols are given in supporting information SI4. 2.10. In vivo vessel formation Six-week old male severe combined immunodeficiency/beige mice (SCID/bg) were purchased from Charles River Laboratories (Wilmington, MA) and used with the approval of the University of Toronto animal care committee. The SCID mutation results in severe combined immunodeficiency affecting both the B and T lymphocytes. The beige mutation results in defective natural killer (NK) cells. A packed volume of 0.2 mL of microspheres (adMSCHUVEC or HUVEC) were suspended in 0.5 mL PBS and subcutaneously injected using an 18G needle into the dorsum of mice. At days 2, 7, and 21, microspheres and the tissue surrounding were excised, fixed in 4% formalin and processed for histology in the pathology research laboratory at Toronto General Hospital. Explants were embedded in paraffin and 3 sections (6 micrometers thick, 100 micrometer apart) per implant were cut, mounted in a microscope slide, and stained for Hematoxylin and Eosin (H&E), Masson Trichrome, Ulex Europeus Agglutinin-1 (UEA-1, Vector, cat# B1201), CD31 (Santa Cruz, cat# sc1506), and alpha-smooth muscle actin (α-SMA) (Sigma #A5228). Each explant yielded three replicates (one per section for CD31, UEA-1, and α-SMA), which were averaged and presented as one biological replicate (n given in each figure caption corresponds to the number of animals). 2.11. Immunohistology quantification


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Histology slides were scanned at 20x magnification using ScanScope XT (Aperio technologies) in the Advanced Optical Microscopy Facility, UHN, and analyzed using ImageScope software. A region of interest (ROI; 600 x 600 µm) was identified as containing the most positively stained cells (hotspot method). A vessel was defined as a lumen-containing structure enclosed entirely by endothelial cells. Three ROI (at different levels) were manually drawn and the number of vessels within each ROI was recorded and divided by the area (vessels/mm2). The number (n) of animals is presented within each figure. 2.12. Perfusion study At day 7 post-implantation, 100 µg of fluorescein labeled UEA-1 (Vector Laboratories, Burlington, ON, Canada) in 150 µL PBS was injected via the tail vein 10 min prior to sacrifice. The microspheres and surrounding tissues were removed surgically, embedded in OCT compound (Tissue-Tek), snap frozen in liquid nitrogen, and cryo-sectioned. Slides were imaged using a Nikon A1 confocal microscope (Nikon, Melville, NY) at the Center for Microfluidics Systems (University of Toronto). 2.13. CLARITY At day 21 post-implantation, 150 µL of PBS containing 0.1 mg of Griffonia Simplicifolia Lectin (GSL-1; Vector Laboratories, Burlington; alexa fluor® 555 conjugated) was injected via the tail vein 10 min prior to sacrifice. The microspheres and the surrounding subcutaneous tissue were removed surgically and processed with CLARITY.37 Explants were fixed at 4°C in a solution containing 4% acrylamide, 4% paraformaldehyde, 0.05% bis-acrylamide and 0.25% (w/v) VA044 thermal initiator. After one week of incubation at 4°C, the acrylamide was polymerized at 37 °C for 3 h. Polyacrylamide-embedded explants were cleared for 14 days at 50 °C in the clearing


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solution (8% SDS in borate buffer, pH 8.5), which was changed every second day. The explants were counterstained with DAPI (ThermoFisher Scientific). Refractive index matching was performed by infusing explants with 67% of 2,2′-thiodiethanol. Explants were imaged using light-sheet microscopy (Zeiss Z1 light-sheet microscope, Sick Kids Imaging Facility). Images were digitally processed using Bitplane IMARIS (Version 8.1). 2.14. Statistical analysis Data are presented as average ± standard deviation. Statistical analysis was performed using GraphPad Prism (Version 6). Statistical comparison was performed using two-way ANOVA followed by Tukey’s post hoc test, or unpaired t-test if only two groups were compared. The level of statistical significance was set at p 90% from 1 to 7 days. Neither the shear associated with cell extrusion nor the presence of Tween 20 had a measurable impact.


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Figure 2. (A) Elastic modulus of hydrogels and (B) percent mass loss of collagen upon enzymatic degradation (***p

Interpenetrating Alginate-Collagen Polymer Network Microspheres for

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