Cell isolation & maintenance
Mesenchymal stem cells derived from human embryonic stem cells (WA-09, a gift from Dr Peiman Hematti) were expanded and cultured as previously described [52
]. Briefly, cells were cultured on a 0.1% gelatin (Sigma-Aldrich, St Louis, MO, USA) pretreated flask containing α-minimum essential media (MEM)-complete. α-MEM-complete consisted of α-MEM (Invitrogen, Carlsbad, CA, USA), 10% fetal bovine serum (Hyclone, Logan, UT, USA), 1% nonessential amino acids (Invitrogen) and 1% l
-glutamine (Invitrogen). Cultures were allowed to grow to 60–70% confluency and were replated at a concentration of 1500 cells/cm2
. MSCs cultured in this way express CD73, CD90 and CD105 (Supplementary Figure 1
) (see online at www.futuremedicine.com/doi/suppl/10.2217/rme.11.48
) and with proper stimulation are able to differentiate down the chondrogenic, osteogenic and adipogenic pathways. Experiments were performed using passages 7–10 and all cultures were maintained at 37°C in 5% CO2
Preparation of the TissueMend matrix
For in vitro studies, pieces of TissueMend matrix (2 × 2 × 0.8 mm) were placed in wells of 24-well plates and hydrated with α-MEM-complete culture medium. H9MSCs were seeded on the TissueMend sections at a concentration of 1 × 106 cells/ml. The medium was changed every 3–4 days and cultures were maintained up to 3 weeks. For in vivo studies, TissueMend matrix was prepared in the same way and cultured with MSCs for 2 days prior to implantation (see Delivery of MSCs to the murine myocardium section) at which point the matrix contained approximately 2.3 × 104 total cells (see In vitro optical analysis below).
In vitro optical analysis
To determine the number of cells delivered, the degree of cell attachment and cell spreading, MSC-seeded TissueMend matrices were cultured for 2 days before staining with CellTracker™ Red (15 µM; Invitrogen), according to the manufacturer’s instructions. Cells of the matrix were imaged using multiphoton laser scanning microscopy (MPLSM) [53
]. MPLSM allows for deep sectioning of 3D tissues, such as the TissueMend matrix, and affords noninvasive analysis of collagen fiber orientation via second harmonic generation (SHG) [54
]. SHG signal is generated when two photons of incident light interact with the noncentrosymmetric structure of collagen fibers, such that the resulting photons are half the wavelength of the incident photons. For all multiphoton and second harmonic imaging, a custom multiphoton workstation at the University of Wisconsin Laboratory for Optical and Computational Instrumentation (LOCI) was used [55
]. The tissue samples were imaged using a TE300 inverted microscope (Nikon, Tokyo, Japan) equipped with a Plan APO VC 20× (numerical aperture 0.75; Nikon Instruments, Tokyo, Japan) objective lens by using a mode-locked Ti:Sapphire laser (Spectra-Physics®
, Mountain View, CA, USA). Tuning the excitation wavelength to 890 nm, a 445/1 nm narrow band pass emission filter (Thin Film Imaging, Greenfield, MA, USA) was used to detect the SHG signal of collagen in the backscattered mode using a H7422P GaAsP photon counting photomultiplier tube (Hamamatsu Photonics KK, Shizuoka, Japan). For detection of CellTracker Red, a 580 nm long pass emission filter (Thin Film Imaging) was used. Images of 1024 × 1024 pixels were acquired using WiscScan under identical conditions. The power of the laser at the sample and gain were set to allow 5% or less saturation prior to data collection. The number of cells in the TissueMend matrix just prior to implantation was determined by first counting the number of cells in at least three fields of three different matrices at a 200 µm 3D volume (images taken at 5 µm intervals); the total number of cells were expressed per unit volume (total volume of each field was 0.08 mm3
). Cell number per volume (mm3
) was then multiplied by the total volume of the matrix (3.2 mm3
) to yield total cell number per matrix. Quantitative analysis of spreading was conducted by outlining at least 16 cells from at least two matrices (3D) and one culture (2D). The area of outlined cells was determined using ImageJ (Fiji distribution, open source software). 3D reconstructions were generated using Imaris 7.2.3 software (Bitplane AG, Zurich, Switzerland).
For migration studies, H9MSCs were seeded at a concentration of 1 × 106
cells/ml on the TissueMend matrix and allowed to attach for 24 h. The matrix was then rinsed twice with fresh medium to remove nonadherent cells and transferred to new gelatin-coated wells (as above) containing fresh culture media. Over the course of 62 h, brightfield images at 10× magnification were taken using an Axiovert 40 CFL inverted microscope (Zeiss, Thornwood, NY, USA). The distance between matrix border and the leading edge of cells exiting the matrix was determined at 12, 40 and 62 h. Migration data was acquired from three matrices at each time point; at least three distance measures were made per matrix per time point. Migration data were plotted as the distance traveled (µm) as a function of time (h). Migration speed was defined as the distance traveled (µm) per unit time (h) [58
Proliferation was determined using a Click-it EdU assay (Invitrogen) according to the manufacturer’s instructions. Briefly, H9MSCs were seeded at a concentration of 1 × 106 cells/ml and allowed to adhere for 48 h on either 2D tissue culture substrate or 3D TissueMend matrix. Cell cultures (n = 3 each for 2D and 3D conditions) were incubated with 20 µM EdU for 24 h, followed by fixation with 4% paraformaldehyde (Acros Organics, Pittsburgh, PA, USA), permeabilization with 0.5% Triton-X (MP Biomedicals, Solon, OH, USA) and nuclear counter-stained with 10 µg/ml Hoechst 33342 (Invitrogen). Cell counts were taken from at least three fields per 2D culture or 3D matrix using an IX71 inverted deconvolution fluorescence microscope (Olympus, Center Valley, PA, USA).
Mechanical testing of TissueMend matrix
An ARES-LS2 rheometer (TA Instruments, New Castle, DE, USA) was used to measure the viscoelasticity of the hydrated TissueMend matrix. The matrix was cut with an 8 mm biopsy punch and submerged in phosphate-buffered saline (PBS) or 2 mg/ml collagenase II (Worthington Biochemical, Lakewood, NJ, USA) in PBS on a rocker for 24 h at room temperature. The upper plate (parallel plate, 8 mm diameter) was lowered until it was in conformal contact with the top surface of the matrix, corresponding to gap distances of 893 ± 80.22 mm and 453 ± 162.15 mm. Storage modulus and loss modulus were measured at 1.0% strain, the temperature was maintained in a chamber at 25°C and three independent matrices were measured for each condition.
Induction of myocardial infarction in mice
Myocardial infarction was induced in C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME, USA) by left coronary artery ligation as previously described [59
] and as is routinely performed in the University of Wisconsin Cardiovascular Physiology Core Facility. All animal procedures were performed in accordance with the guidelines of the American Association for Laboratory Animal Science and the University of Wisconsin-Madison Animal Care and Use Committee. Briefly, an intercostal thoracotomy was performed to visualize the heart. A 7–0 or 8–0 Prolene suture (Ethicon, Somerville, NJ, USA) was placed through the myocardium in the anterolateral wall. The suture was secured and coronary artery entrapment was confirmed by observing blanching of the distal circulation (ventricular apex). The ribs, muscle layers and skin were closed with 6–0 Prolene (Ethicon).
Delivery of MSCs to the murine myocardium
A 2 × 2 × 0.8 mm TissueMend matrix containing approximately 2.3 × 104 H9MSCs or unseeded (control) was tacked to the myocardium at each corner of the matrix using 7–0 Prolene suture 2 days after infarction. Human MSCs were selected as the donor cell population to most directly assess the clinical utility of human cell delivery with the TissueMend matrix. Tacking of the matrix to the myocardium utilized a stitch similar to a mattress stitch and the area covered by the matrix included a portion of the infarct and peri-infarct regions.
Optical analysis of heart/tissue explants
Murine hearts were harvested 3 weeks after matrix implantation to assess engraftment and cell state within the scaffold (). Hearts were bisected longitudinally through the scaffold. The tissues were immediately placed into 10% buffered formalin (pH = 7.2; Fisher Scientific, Forest Lawn, NJ, USA) for 24 h followed by 24 h of fresh 10% buffered formalin, and a final 24 h of 70% ethanol. Samples were further processed for histological analysis as previously described [60
Ventricular section and associated TissueMend® (TEI Biosciences, Boston, MA, USA) matrix 3 weeks after in vivo transplantation
For phenotypic analyses, 5 µm thick paraffin histological sections were probed for MSCs, and cardiac and vascular proteins using immunofluorescent staining. To identify MSCs, sections were probed with goat anti-CD73 (polyclonal antibody, V-20; Santa Cruz Biotech, Santa Cruz, CA, USA). The primary antibody was used at a 1:25 dilution in diluting buffer (5% bovine serum albumin [Fisher Scientific], 0.02% NaN3− [Acros Organics] in PBS [Fisher Scientific]) and incubated overnight at 4°C. Detection of CD73 was achieved by incubating with a secondary antibody, donkey anti-goat Alexa Fluor (AF488; Invitrogen), at a 1:200 dilution in preadsorption solution (90% diluting buffer, 5% human serum [Pelfreez, Brown Deer, WI, USA] and 5% mouse serum [Equitech-Bio, Inc, Kerrville, TX, USA]) for 45 min at room temperature. To identify mature cell types, sections were probed with primary antibodies for cardiomyocytes (mouse anti-troponin T cardiac isoform Ab-1, cardiac troponin T [cTnT], clone 13–11 [Fisher Scientific]); for endothelial cells (rabbit anti-von Willebrand Factor [vWF]; Sigma-Aldrich); and for fibroblast-like cells (rabbit anti-discoidin domain receptor [DDR]-2 (H-108); Santa Cruz Biotechnology). Following deparaffinization and rehydration, sections were subjected to antigen retrieval as recommended by the manufacturer: anti-vWF–1 mM ethylenediaminetetraacetic acid (Fisher Scientific), pH 8.0 + 0.01% Triton-X 100 (MP Biomedicals) for 30 min at 97°C; anti-cTnT and anti-DDR2, 10 mM sodium citrate (Sigma-Aldrich), pH 6.0 + 0.01% Triton-X for 15 min to 30 at 97°C. Following incubation for 30 min at room temperature in blocking solution (either 5% bovine serum albumin, 1% glycine [Fisher Scientific], 2% donkey serum [Sigma-Aldrich], 0.1% Triton-X 100 for anti-cTnT or with 5% bovine serum albumin, 2% normal goat serum [Invitrogen], 0.1% Triton-X 100 for anti-vWF and anti-DDR2) the sections were incubated in the primary antibodies, diluted at 1:20 (anti-DDR2) or 1:50 (anti-cTnT and anti-vWF) in blocking solution overnight at 4°C. Sections were incubated in secondary antibodies (donkey anti-mouse AF 546, goat anti-rabbit AF 588 or goat anti-rabbit AF 647 [Invitrogen]) for 1 h at room temperature. All stained sections were mounted in 1,4-diazabicyclo[2.2.2]octane/4´,6-diamidino-2-phenylindole mounting medium (2.5% 1,4-diazabicyclo[2.2.2]octane [Sigma-Aldrich], 50% glycerol [Fisher Scientific], and 0.005% 4´,6-diamidino-2-phenylindole [Sigma-Aldrich] in PBS). Fluorescence emission was detected on an IX71 inverted deconvolution fluorescence microscope (Olympus). Images were acquired with 20× UPlanFluor objective (numerical aperture = 0.5) and analyzed with Slidebook software (Intelligent Imaging Innovations, Denver, CO, USA) and with ImageJA (Fiji; open source software). Images were normalized using a secondary only control for each labeling procedure in order to minimize background fluorescence attributable to nonspecific secondary antibody labeling and autofluorescence. Positive events were reported as a percentage of the total cell number obtained from analysis of at least eight optical fields from multiple histological sections of at least three matrices seeded with MSCs and at least one unseeded matrix. For DDR2-labeled tissue, a total of 11 optical fields of the border region from three sections from each of two hearts with seeded matrices or a total of six optical fields from three sections from one heart with unseeded matrix were counted. The total number of CD73+ in the matrix was determined by counting the number of positive events in at least three optical fields from multiple histological sections of at least three matrices. Based on the dimensions of the field of view and assuming a tissue depth of 5 µm, a cell number per mm3 was determined. Cell number per volume (mm3) was then multiplied by the total volume (3.2 mm3) per matrix to yield the total cell number per matrix. Cell retention was defined as the calculated number of CD73+ cells in the seeded matrices minus the calculated number of CD73+ cells in the unseeded matrices.
To determine the origin of cells in the border region, a fluorescent in situ hybridization tissue digestion kit targeting all human centromeres (Kreatech, Amsterdam, The Netherlands) was performed. Samples were processed by the Cytogenetics Laboratory (WiCell Research Institute, Madison, WI, USA) according to manufacturer’s protocol. Briefly, paraffin-embedded slides were baked for 4 h at 56°C. Specimens were incubated with pepsin for 20 min for tissue digestion prior to probe application.
For in vivo
remodeling studies, SHG imaging and analysis was conducted on histological sections of hearts containing the TissueMend matrix using MPLSM. Traditional Fourier-based analysis methods [61
] are not particularly effective at detecting edges in images with overlapping fibers or curves, which are a major feature in images of the ECM. As an alternative, the curvelet transform, developed by Candes and Donoho [63
], is specifically designed to determine a sparse representation of edges in images even in the presence of complex geometries such as those associated in the SHG images of the collagen. We used software from LOCI that utilizes the curvelet transform to interactively measure distribution of angles for a defined region of interest. Generated images were reconstructed using the LOCI curvelet software. The curvelet software was used to determine a ‘coefficient of alignment’ that corresponds to the relative distribution of angles of fibers in each sample. A coefficient that approaches 1 indicates that the orientation of fibers in the sample are grouped closely about the mean. Image analysis was conducted on at least three optical fields from one matrix seeded with MSCs and one unseeded matrix.
For comparison of collagen fiber alignment, MSC spreading and MSC proliferation in the TissueMend matrix versus controls, a normal distribution was assumed and one-way analyses of variance and Student’s t test were used. Data were analyzed with Microsoft Excel (Microsoft, Redmond, WA, USA).