Retracted: Ascorbic Acid Improves Embryonic Cardiomyoblast Cell Survival and Promotes Vascularization in Potential Myocardial Grafts In Vivo

Abstract

The senior author of the article entitled, “Ascorbic Acid Improves Embryonic Cardiomyoblast Cell Survival and Promotes Vascularization in Potential Myocardial Grafts In Vivo,” published in the April 2010 issue of Tissue Engineering Part A (vol. 16, no. 4, pages 1349–1361), contacted the editorial leadership of the Tissue Engineering journal to identify unintentional but significant duplication in figure 2 as well as magnification flaws in figure 7 within the published article.

Based on their reviews of the inaccuracies, the editors have decided to officially retract the paper from the literature.

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Introduction

The fabrication of sufficiently thick three-dimensional (3D) myocardial grafts by means of tissue engineering is limited by impaired cell viability in unperfused in vitro systems.¹ Further, cell death constitutes a major drawback when attempts are made to restore heart muscle via cell or bioartificial tissue implantation.^2–4 Following transplantation, cells in 3D grafts will be challenged by proapoptotic stimuli and an intense inflammatory response arising from the surrounding ischemic tissue,^5,6 as well as by hypoxia, particularly in the core of the bioengineered graft.⁷ Besides donor cell survival, functional integration and engraftment of bioengineered tissues in the recipient organism are equally critical for a successful myocardial restoration. It has been reported that neovascularization within myocardial grafts plays a key role for both cardiomyocyte survival and organization.⁸ All together highlights the paramount importance of blood supply in myocardial grafts for heart repair.

Given the relevance of hypoxia and oxidative stress for the fate of cardiac cells after ischemic injury as well as within thick bioengineered tissues, we postulate that ascorbic acid (AA) is a factor that might reduce cell death in myocardial grafts both in vitro and in vivo. This hydrophilic antioxidant scavenges toxic free radicals and other reactive oxygen species efficiently.⁹ Further, attenuation of hypoxia-induced apoptosis after AA treatment in vitro has been demonstrated in HL-1 cardiomyocytes¹⁰ and endothelial progenitor cells.¹¹ AA also exerts a stimulatory effect on angiogenesis through increase of collagen type IV synthesis by endothelial cells.¹² It has been shown that human umbilical vein endothelial cell type IV collagen production is enhanced when AA is added in vitro. At physiological concentrations (up to 100 μmol/L), AA induces tube formation by human umbilical vein endothelial cells in cultured extracellular matrix.¹² Further, large doses of AA induce superior mesenchymal tissue healing in rats, because of early angiogenesis induction and increased collagen synthesis.¹³ Finally, AA is available ubiquitously, is an essential part of our diet at a global scale, and can be easily administered in large doses as a therapeutic supplement in various formulations. However, the potential of AA for cell therapy and tissue repair has not been exploited yet. Experimental studies to assess the effect of AA on cell survival in 3D compounds destined for myocardial restoration have yet to be conducted.

Accordingly, the aim of our study was to investigate whether bioartificial grafts (scaffold–cell compounds) for myocardial repair, which have been enriched with AA, display improved donor cell viability, both in vitro and in vivo. In addition, we assess whether such enrichment would have any effect on angiogenesis and remodeling of thick bioartificial tissues, suitable for implantation and myocardial repair. To examine our hypothesis, a novel renal pouch model for in vivo prevascularization of 3D tissue grafts was developed in healthy rats.

Materials and Methods

Cell culture

H9C2(2-1) cardiomyoblasts derived from embryonic rat hearts were obtained from the American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100,000 U/L of penicillin, and 100 mg/L of streptomycin (GIBCO^®, Invitrogen Corporation, Carlsbad, CA), under 5% CO₂ at 37°C. Cells were subcultured on reaching subconfluency to maintain the myoblast phenotype.

Generation of fluorescent/bioluminescent cell lines

pWPT-GFP (kindly provided by Dr. D. Trono; University of Geneva, Geneva, Switzerland) and pWPT-Fluc (modified from pWPT-GFP) lentiviral vectors were used to infect H9C2 cells. pWPT-Fluc was cloned by removing green fluorescent protein (GFP) from pWPT-GFP and inserting the firefly luciferase (Fluc) gene PCRed from pGL3 (Promega, Madison, WI). Lentiviral vectors were produced by transient transfection of 293T cells. About 5 × 10⁶ cells/plate were seeded in 10-cm tissue culture plates at 24 h before transfection. The latter was performed using calcium phosphate precipitation method with 10 μg pWPT-GFP or pWPT-Fluc vector, 7.5 μg helper plasmid pCMV8.91, and 2.5 μg of MD2G envelope plasmids (gifts from Dr. D. Trono, University of Geneva, Geneva, Switzerland). Cells were replaced with fresh medium at 14–16 h after transfection. The supernatant was filtered through a 0.45-μm filter, and the titer of supernatant on 293T cells was determined using flow cytometry. H9C2 cells were shown to be highly infectable (>90%) with unconcentrated lentiviral supernatant (unpublished data). H9C2 cells to be used in vivo were initially infected with supernatant containing pWPT-GFP, sorted for GFP positivity, and subsequently infected with pWPT-Fluc (H9C2-GFP-Fluc). Further, cells destined to in vitro experiments were infected with pWPT-Fluc (H9C2-Fluc).

3D graft preparation for in vitro studies

Prior to these experiments, AA concentrations without cytotoxic effect on H9C2 cells were established in our laboratory (data not shown) and have also been previously described in other cell types.^12,14 Sterile, porous sponges prepared from purified porcine skin gelatin (Gelfoam™; Pharmacia & Upjohn Company, Kalamazoo, MI) were used as scaffold material to fabricate 3D myocardial artificial grafts (MAGs). Gelfoam squares (10 × 10 × 5 mm) were placed in eight-well chamber slides (Lab-Tek™II Chamber Slide™; NUNC A/S, Roskilde, Denmark) under sterile conditions. Subsequently, foams were hydrated with 100 μL growth medium supplemented with 5 or 50 μmol/L L-AA (Sigma, St. Louis, MO). Scaffolds with just medium, that is, without AA and/or without cells, were used as controls. A 100 μL medium solution containing 2.5 × 10⁵ H9C2-Fluc cells was added on top of the 3D scaffolds. Chamber slides were then placed in an incubator under 5% CO₂ at 37°C for 30 min to allow cell solution absorption into the sponge. Next, scaffolds were covered with 0.4 mL growth medium, placed back into the incubator, and the medium was exchanged daily. Assays were conducted in quadruplet in five separate experiments.

In vitro bioluminescence imaging

Bioluminescence imaging (BLI) was used to evaluate the effect of AA on H9C2-Fluc cells in vitro survival when seeded in 3D MAGs after 1, 3, and 5 days in culture (n = 20/treatment/time point). For BLI, 150 μg/mL working solution of d-luciferin (Caliper Life Sciences, Hopkinton, MA) in prewarmed cell culture medium was added to each well and the chamber slides were scanned after 5 min using a Xenogen IVIS^® Lumina System (Caliper Life Sciences). Bioluminescence was quantified in units of photons per second total flux (p/s), using Living Imaging Software version 2.6 (Caliper Life Sciences).

dUTP nick-end labeling assay and immunohistochemical staining for active caspase 3

Three-dimensional MAGs were fixed in 10% buffered formalin, embedded in optimal cutting temperature (OCT) compound (Tissue-Tek^®, Sakura Finetek, Tokyo, Japan), and stored at −80°C after in vitro experiments at days 3 and 5. In situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis was done using the ApopTag Red In Situ Apoptosis Detection Kit (Millipore, Billerica, MA), according to the manufacturer's instructions, on 5-μm cryosections. The sections from at least three different experiments were visualized using a Leica TCS SP5, DMI6000 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). The number of TUNEL-positive cells was counted in 10 different fields (400×), from three different grafts/treatment. Next, to quantify apoptosis and exclude necrosis, 10-μm cryosections were stained with active caspase 3 antibody (1:100, rabbit polyclonal antibody to active caspase 3, ab2302; Abcam, Cambridge, United Kingdom). The sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Molecular Probes^®, Invitrogen, Carlsbad, CA) and z-stacks in 0.5 μm steps were obtained using confocal microscopy. A single image of maximum projection was obtained from the z-stacks by using Leica Application Suite Advanced Fluorescence quantification software (version 1.8.2, build 1465). The sections from at least three different experiments were analyzed. Cells were counted using Image J 1.42q (National Institutes of Health, Bethesda, MD). The percentage of TUNEL-positive and active caspase 3-positive cells was calculated as follows: 100 × (number of positive cells counted/total number of nuclei counted).

Assessment of H9C2 phenotype in 3D culture

Immunohistochemical assays were carried out to evaluate the effect of AA on H9C2 cardiomyoblast differentiation. Five-micrometer cryosections obtained from in vitro experiments at days 3 and 5 were stained with an antibody specific to α-cardiac and skeletal (sarcomeric) muscle actins (1:100, monoclonal mouse anti-sarcomeric actin, clone Alpha-Sr-1; DakoCytomatation, Glostrup, Denmark). The sections were then incubated with a secondary antibody Alexa Fluor^®-568, counterstained with DAPI (Molecular Probes, Invitrogen, Carlsbad, CA), and visualized with an Olympus BX61 fluorescence microscope equipped with a DP72 12.8 megapixel cooled digital camera (Olympus, Tokyo, Japan). Photomicrographs were processed using the DP2-BSW 2.2 (build 6212) software (Olympus).

Animals and renal pouch model

This study conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication no. 85-23, revised 1996). The experimental protocol was approved by the National University of Singapore—Institutional Animal Care and Use Committee. Male SPF Wistar rats (300–350 g) were used for our experiments. All surgical procedures were performed using aseptic techniques.

Anesthesia was induced and maintained in animals by inhalational isoflurane (2%) and intraperitoneal injection of ketamine:xylazine (90 mg/kg:10 mg/kg). Carprofen (5 mg/kg, subcutaneous [SC]) was administered preoperatively for analgesia. A midlaparotomy was performed, followed by displacement of the bowel and mild retraction of the kidney. Blunt preparation of the retroperitoneal fossa was done and a pouch was created between the retrorenal fat and the psoas muscle. Subsequently, the MAG was implanted into the retro-renal pouch. Grafts containing cells were implanted in the right renal pouch, whereas acellular controls were implanted in the contralateral pouch. We did not use suture material in the implantation process. Finally, the bowel was repositioned and the abdomen was closed in two layers. The animals were allowed to recover in a small-animal ICU. Carprofen (5 mg/kg/day, SC) was administered postoperatively for analgesia.

3D graft preparation for in vivo studies

Eight rats were used to test several 3D matrices and to identify the one displaying the least degradation and low inflammatory reaction after 10 days of implantation using the renal pouch model. Four different collagenous foam-scaffolds from diverse origin (i.e., equine collagen and human fibrinogen [Tachotop^®; Nycomed, Zurich, Switzerland], bovine collagen [Lyostypt^®; B. Braun Melsungen AG, Melsungen, Germany], native equine collagen [TissuFleece E^®; Baxter, Vienna, Austria], and porcine skin gelatin [Gelfoam]) were implanted in the renal pouch without cells (data not shown). The porcine gelatin foam was recovered almost intact after 10 days of implantation, and hence, it was chosen for the cell-based animal experiments.

H9C2-GFP-Fluc cells were washed three times in cold phosphate-buffered saline (PBS) and centrifuged. Gelfoam squares (20 × 20 × 5 mm) were placed in 12-well plates and covered with 4 × 10⁶ H9C2-GFP-Fluc cells in 250 μL PBS under sterile conditions. Based on our in vitro studies, we chose 5 μmol/L L-AA in PBS enrichment for the AA animal group (group A, n = 10). Plain MAGs without AA (PBS and cells) were used as control group (group B, n = 10). Acellular grafts containing just PBS (group C, n = 10) or 5 μmol/L AA in PBS (group D, n = 10) were used as negative controls and implanted in the left renal pouch.

In vivo BLI

We performed optical in vivo BLI with the Xenogen-IVIS Lumina in vivo imaging system as previously described.^15–17

Briefly, rats were anesthetized with 2% isoflurane, and d-luciferin was administered intraperitoneally at a dose of 150 mg/kg of body weight. They were placed in the chamber in supine position and peak luciferase activity was detected by imaging the animals for 40 min with 2-min acquisition separated by 2-min intervals.¹⁷ The same rats were scanned repeatedly postimplantation at days 1, 3, and 6. Regions of interest indicating the locations of most intense signals were created using Living Imaging Software version 2.6. Bioluminescence was quantified in units of photons per second total flux (p/s).^15,18

Immunohistochemistry—Assessment of GFP and rat endothelial cell antibody expression

At 7 days postimplantation, MAGs were explanted and fixed in 10% formalin. The grafts were subsequently optimal cutting temperature (OCT) compound embedded and stored at −80°C. Immunohistochemical staining was performed in 5-μm cryosections using the primary antibody rat endothelial cell antibody-1 (RECA-1), a ubiquitous marker of rat endothelial cells (1:50 monoclonal mouse anti-RECA-1; HyCult Biotechnology b.v., The Netherlands). The sections were then incubated with secondary antibodies Alexa Fluor^®-647 and Anti GFP-Alexa Fluor^®-488, and nuclei were stained with DAPI (Molecular Probes).

Images were acquired using a Leica TCS SP5, DMI6000 confocal laser scanning microscope (Leica Microsystems), with 40 × and 20 × magnifications. They were subsequently processed using Leica Application Suite Advanced Fluorescence quantification software (version 1.8.2, build 1465).

The mean pixel intensity, a semiquantitative analysis of fluorescence, was used to evaluate GFP and RECA expression within the explanted 3D grafts. Predefined settings for laser power and detector gain were used for all experiments.

Histological analysis

We performed Masson's trichrome and hematoxylin–eosin staining on 5-μm sections of formalin-fixed and paraffin-embedded explanted MAGs. Histological assessment was performed by an experienced pathologist in a blinded fashion. To assess angiogenesis within the explanted graft, the number of blood vessels per high-power field (hpf ) (400 ×) was quantified. The degree of cell infiltration and fibrosis was evaluated as the percentage of area of the scaffold infiltrated by cellular reaction and collagen deposition covering the total cellular reaction, using a manual semiquantitative method. Ten random fields were chosen in each section for all the quantifications.

Statistical analysis

Data are presented as mean ± standard deviation. To test for statistically significant differences, we used two-way analysis of variance and the unpaired Student's t-test when appropriate. Differences were considered significant if p < 0.05. Regression plots were used to describe the relationship between bioluminescence and cell number in vitro; r²-values were reported to assess the quality of the linear regression model. All the statistical analyses were performed using GraphPad Prism^® software version 5.01 for Windows (GraphPad Software, San Diego, CA).

Results

In vitro BLI/effect of AA on 3D H9C2 cell graft survival in vitro

BLI showed that there was a significant increase in cell bioluminescent baseline signals from day 1 to 5 in grafts supplemented with both 5 and 50 μmol/L AA (55.23% ± 0.45%, p < 0.001 and 49.83% ± 0.45%, p < 0.01, respectively; Fig. 1A–D). H9C2-Fluc cells seeded within the 3D MAGs receiving 5 μmol/L AA-supplemented medium showed significantly larger bioluminescent signals after 3 days (2.1 × 10⁸ ± 9.5 × 10⁷ p/s vs. 1.5 × 10⁸ ± 7.4 × 10⁷ p/s, p < 0.05 vs. control) and 5 days in culture (2.3 × 10⁸ ± 7.9 × 10⁷ vs. 1.7 × 10⁸ ± 4.8 × 10⁷, p < 0.05 vs. control). Likewise, 50 μmol/L AA-supplemented grafts displayed significantly increased cell signals at day 5 when compared with control grafts receiving plain culture medium (2.0 × 10⁸ ± 8.0 × 10⁷, p < 0.05 vs. control; Fig. 1D). There were no large differences in photon emission between the two dosages of AA.

FIG. 1.

In vitro BLI of 3D MAGs. BLI of AA-enriched and plain MAGs after (A) 1 day, (B) after 3 days in culture, and (C) after 5 days in culture; n = 20. (D) MAGs receiving 50 and 5 μmol/L AA-supplemented medium showed significantly larger bioluminescent signals after 3 days in culture (p < 0.05 vs. control). About 5 μmol/L AA-enriched MAGs also displayed higher photon emission at day 3 in culture (p < 0.05 vs. control). An increase in cell bioluminescent baseline signals from day 1 to 5 was found in grafts supplemented with both 5 μmol/L AA (***p < 0.001) and 50 μmol/L AA (**p < 0.01). Correlation between mean bioluminescence (mean total photon flux) and histological mean cell number after (E) 3 days and (F) 5 days in culture. 3D, three-dimensional; AA, ascorbic acid; BLI, bioluminescence imaging; hpf, high-power field (400 ×); MAGs, myocardial artificial grafts; SD, standard deviation.

Histological cell counts (DAPI⁺ cells) indicated that the amount of DAPI⁺ cells per hpf (400 × ) in the 5 μmol/L AA-enriched grafts was significantly higher than in the plain grafts after 3 days in culture (53 ± 21.4 vs. 30 ± 7.18, p < 0.05). Likewise, mean cell number was higher in grafts that received 50 μmol/L AA after 5 days in static culture when compared with untreated MAGs (41.4 ± 14.3 vs. 19.6 ± 6.9, p < 0.05). There were differences in cell density between the AA-treated grafts. A good correlation of luciferase activity per graft to mean cell number (DAPI⁺ cells/graft) was found after 3 days (r² = 0.90) and 5 days (r² = 0.97) in culture (Fig. 1E, F).

The effect of AA on cell apoptosis in 3D myocardial bioartificial grafts in vitro

TUNEL assay and active caspase 3 staining were used to assess the effect of AA on H9C2-Fluc cells apoptosis when cultured in thick 3D MAGs (Fig. 2A–F). The percentage of TUNEL⁺ cells per hpf (400×) in plain grafts increased significantly from day 3 in culture (23.5% ± 12.6%) to day 5 (61.9% ± 30.1%, p < 0.001). The percentage of TUNEL⁺ cells was significantly inferior in the 5 and 50 μmol/L MAGs at day 5 in culture (4.8% ± 8.6%, p < 0.001 and 8.3% ± 9.2%, p < 0.001, respectively) when compared with plain control grafts (61.9% ± 30.1%) (Fig. 2G). Given that TUNEL assay tags apoptotic and necrotic cells, we used active caspase 3 staining as a more specific method to identify apoptotic cells. The percentage of active caspase 3⁺ cells was considerably higher in plain grafts after 3 days (41.6% ± 11.2%) and 5 days (44.8% ± 20.0%) in culture when compared with both dosages of AA (Fig. 2A–F, H). The 5 μmol/L AA grafts had 16.5% ± 6.17% (p < 0.01) apoptotic cells at day 3 and 15.7% ± 6.8% (p < 0.001) at day 5. Likewise, the 50 μmol/L AA MAGs had 11% ± 5.7% (p < 0.01) of apoptotic cells at day 3 and 18.8% ± 5.9% (p < 0.01) at day 5 in culture.

FIG. 2.

Apoptosis assessment in 3D MAGs. Confocal micrographs of active caspase 3 staining (red) performed after 3 days in culture in (A) plain MAGs, (B) 5 μmol/L AA MAGs, and (C) 50 μmol/L AA MAGS. Active caspase 3 staining after 5 days in culture in (D) plain MAGs, (E) 5 μmol/L AA MAGs, and (F) 50 μmol/L AA MAGS. The inset is a higher magnification showing cytoplasmic active caspase 3 expression. (G) In the plain MAGs, TUNEL assay showed an increase in the percentage of TUNEL-positive cells from day 3 to 5 in culture (***p < 0.001). (H) The percentage of apoptotic cells (active caspase 3⁺) was significantly inferior at days 3 and 5 in the 5 μmol/L AA (p < 0.01 and p < 0.001, respectively) and 50 μmol/L AA MAGs (p < 0.01). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scaffold autofluorescence appears in gray. Arrows pointing at some caspase 3-positive cells. Scale bars indicate 25 μm. TUNEL, dUTP nick-end labeling.

AA effect on H9C2 cells phenotype in vitro

After 3 days in culture, H9C2 cardiomyoblasts were attached to the scaffold material and formed a primitive syncytium in all groups (Fig. 3A, C, E). Cells fused and formed elongated myotube-like structures that were evident in the AA-treated grafts (Fig. 3C, E). Immunohistochemical assays revealed that α-sarcomeric actin was expressed by untreated and AA-enriched cells after 3 and 5 days in 3D culture. However, the AA-treated groups displayed organized sarcomeric patterns at days 3 and 5 in culture, whereas z-lines were not apparent in the untreated cells after 5 days in culture (Fig. 3A–F).

FIG. 3.

AA effect on H9C2 cardiomyoblasts' phenotype in 3D culture. α-Sarcomeric actin staining (red) performed after 3 days in culture in (A) plain MAGs, (B) 5 μmol/L AA MAGs, and (C) 50 μmol/L AA; and after 5 days in culture in (D) plain MAGs, (E) 5 μmol/L AA MAGs, and (F) 50 μmol/L AA. Nuclei were counterstained with DAPI (blue). Right-side panels depict DIC images that were merged to the fluorescence micrographs to identify cell morphology and distribution in the scaffold. Scale bars indicate 10 μm. α-SA, alpha-sarcomeric actin; DIC, differential interference contrast.

In vivo BLI

Bioluminescent signals progressively decreased from day 1 to 6 in both the plain and AA-enriched MAGs (groups A and B; Fig. 4A, B). However, photon emission fell dramatically in plain MAGs (group B) from day 1 to 6 postimplantation (Fig. 4A). Cell photon emission by day 6 plunged in a 74% ± 0.9% of the baseline in the plain MAGs, whereas the AA-enriched graft signals decreased by just 36.42% ± 1.81% (p < 0.0001). There was no significant difference in the mean photon emission among groups at days 1 and 3 postgraft implantation. However, the AA MAGs (group A) displayed significantly higher cell signals at day 6 (6.1 × 10⁶ ± 1.7 × 10⁵ p/s vs. 1.8 × 10⁶ ± 2.8 × 10⁵ p/s, p < 0.05; Fig. 4C).

FIG. 4.

In vivo BLI after 1, 3, and 5 days of graft implantation in the renal pouch. (A) Plain MAGs. (B) AA-enriched MAGs. (C) The AA-enriched MAGs displayed significantly higher cell photon emission at day 6 after graft implantation (*p < 0.05).

Renal pouch model

None of the rats died before the scheduled euthanasia. There were no visible signs of infection, inflammation, adhesions, or foreign body reaction in any group after 1 week of MAG implantation in the renal pouch. Explanted grafts containing cells (groups A and B) displayed preserved size and shape. In contrast, the negative control grafts (groups C and D) were slightly smaller in size after 1 week of implantation, which could be due to scaffold's degradation. All the implanted grafts were surrounded by a thin layer of connective tissue adhered to the prerenal fat. Numerous blood vessels were visible by the naked eye in both plain (Fig. 5A) and AA-enriched MAGs (Fig. 5C), whereas fewer vascular networks could be seen in acellular negative control grafts (Fig. 5B, D).

FIG. 5.

Explanted MAGs. Abundant blood vessels infiltrating the (A) plain and (C) AA-enriched MAGs explanted from the right renal pouch. Fewer blood vessels could be observed by the naked eye in the (B) plain negative (acellular) control and (D) AA negative (acellular) control, explanted from the left renal pouch. Scale bars indicate 10 μm. Color images available online at www.liebertonline.com/ten.

Immunohistochemistry—Assessment of GFP and RECA expression

Fluorescence pixel intensity analysis of the confocal micrographs revealed that the amount of GFP-positive cells (donor cells) was significantly greater in group A (AA-enriched MAGs) when compared with the plain grafts from group B (2.09 ± 1.42 vs. 0.94 ± 0.24, p < 0.01; Fig. 6E).

FIG. 6.

GFP and rat endothelial cell antibody (RECA) expression in explanted MAGs. (A, C) Plain MAGs. (B, D) AA-enriched MAGs. Confocal micrographs show H9C2-Fluc-GFP cells in green; RECA⁺ cells in red (blood vessels), and nuclei stained with DAPI in blue. Vascular networks infiltrating the grafts could be observed in both (A) plain and (B) AA-enriched MAGs. The amount of GFP⁺ and RECA⁺ cells is more evident in the AA group. (C, D) Distribution of donor (GFP⁺) cells and blood vessels in the scaffold. GFP⁺ cells can be seen in white and nuclei in blue. Asterisks indicate the scaffold structure. Arrows point to blood vessels. (E) Semiquantitative fluorescence pixel intensity analysis showed that the number of GFP-positive cells was greater in the AA-enriched MAGs (**p < 0.01). Likewise, expression of RECA was higher in the explanted grafts from the AA group (*p < 0.05). GFP, green fluorescent protein.

Abundant vascular networks infiltrating the MAGs could be observed under the confocal microscope (Fig. 6A, B); angiogenesis was particularly abundant in the AA group (Fig. 6B, D). Further, blood vessel infiltration observed in group A (AA) was not limited to the outmost regions of the graft, as vessels in close contact with the gelatin scaffold were found toward the graft's core (Fig. 6D). Accordingly, pixel intensity analysis showed that expression of the RECA was significantly higher in the explanted grafts from group A (AA) than those from group B (plain) (4.28 ± 1.98 vs. 1.46 ± 1.21, p < 0.05). Fluorescence intensity quantification of DAPI⁺ cells did not reveal any difference among groups (Fig. 6E).

Histology

Histology analysis showed a higher amount of blood vessels per hpf (400×) in the AA-MAGs (group A) compared with plain MAGs (group B) (3.75 ± 0.5 vs. 2.75 ± 0.5, p < 0.05; Fig. 7A, B, E). These results are in agreement with the analysis of endothelial cell markers following confocal microscopy. In both groups, peripheral angiogenesis was higher. However, few capillaries could also be detected at the graft's core. Ingrowth of vessels was also observed in the periphery of the acellular negative controls (Fig. 7C, D), but no capillaries were detected toward the core. There was no difference in the number of blood vessels found in the negative control groups (Fig. 7E).

FIG. 7.

Masson's trichrome staining micrographs of (A) plain MAGs, (B) AA-enriched MAGs, (C) plain negative (acellular) control, and (E) AA negative (acellular) control. Mild collagen deposition was observed in all groups. Scaffold material is stained in blue and pointed out with asterisks, to differentiate it from extracellular matrix deposition. Arrows indicate blood vessels within the grafts. (D) Histological analysis of the explanted MAGs showed fewer amounts of blood vessels per high-power field (400 ×) in the plain MAGs when compared with the AA-enriched grafts (p < 0.05). Scale bars indicate 50 μm.

There was no significant difference in collagen deposition among groups. The mean collagen deposition score (% of area covered by collagen of the total cellular reaction) was mild (0–25%) for both AA-enriched and plain MAGs, as well as for negative controls (Table 1 and Fig. 7A–D).

Table 1.

Histological Semiquantitative Scoring of Explanted Myocardial Artificial Grafts

	Animal groups
Parameter	Group A AA MAGs	Group B Plain MAGs	Group C AA control	Group D Plain control
Cellular reaction (%)	0–25	0–25^a	0–25	0–25^a
No. of eosinophils/hpf	2.3 ± 0.9	2.0 ± 1.0	1.5 ± 0.0	1.5 ± 0.0
Collagen deposition (%)	0–25	0–25	0–25	0–25

Cellular reaction was defined as the percentage of scaffold area covered by cells, and collagen deposition reflects the percentage of collagen covered of the total cellular reaction.

Minimal cellular reaction.

MAGs, myocardial artificial grafts; AA, ascorbic acid; hpf, high-power field.

Cell infiltration was mild in all groups, with eosinophils being the most abundant cell type found within the grafts. Occasional lymphocytes and neutrophils were observed in all the graft sections (Table 1). Likewise, mild foreign body reaction was observed in all the groups. Macrophages and giant cell reaction were observed in all groups. Just one of the explanted grafts from the negative control (group D) displayed moderate necrosis, 26–50% collagen deposition, and abundant cell infiltration by neutrophils.

Discussion

The main findings in this study are twofold: first, AA enhances thick bioengineered myocardial tissue survival in vitro through inhibition of cell apoptosis; second, the graft's viability and angiogenic potential in vivo are superior with AA enrichment. Moreover, we introduced an in vivo model for myocardial graft prevascularization, which provides the implanted graft with blood vessels of autologous origin. We improved graft viability using a biocompatible and inexpensive compound that could be beneficial for the tissue engineering and the cell therapy field in general. Stem cell- and bioartificial tissue-based therapies for the heart remain supplementary measures. The unique architecture, terminal differentiation, and limited regenerative potential of the heart muscle restrict the capacity of random injections and implants to restore myocardium efficiently and permanently. The progress of such methods from random and supplementary to first-line therapeutic options will largely depend upon improvement on various levels: first, therapies have to be less traumatic, and second, the effect has to be stronger and more durable by enhancing graft viability and functionality in vivo. It is critical to sustain donor cell survival within bioengineered myocardial grafts through in vitro establishment of vascular networks for successful cardiac repair. Cell death constitutes a major limitation in cell and tissue implantation in the frame of restorative therapies. Some reports indicate that more than 70% of cells die during the first 2 days after injection into the myocardium,² whereas others point out a cell loss of ∼55% in just 10 min following implantation into the heart.³ Further, there is evidence that, in spite of capillary formation in vivo, donor cell survival after myocardial 3D graft implantation is still at risk.¹⁹

The addition of AA to the graft constitutes an uncomplicated method to counteract fulminant events that occur during acute ischemia and remodeling within the area of myocardial lesion. Such events are cytokine and radical oxygen species liberation, accumulation of purine derivates (danger signals), inflammation, and apoptosis. AA is a ubiquitous and essential substance with practically no side-effects even in high doses and can easily be integrated in a regenerative therapy protocol. AA participates in a variety of physiological functions in living organisms. In humans, its deficiency causes defective healing and disturbed blood vessel formation because of impaired collagen deposition.^12,20 Besides its role in angiogenesis, AA reduces hypoxia-induced apoptosis in vitro.^10,14 Accordingly, we hypothesized that AA could be used to reduce cell death in myocardial grafts both in vitro and in vivo.

We used BLI to assess H9C2-Fluc cell survival within thick, 3D MAGs in vitro. The high sensitivity of BLI to monitor viable cells^21,22 was effectively demonstrated in our in vitro model, as we found a robust correlation between luciferase activity and histological cell counts. We did not find significant difference in cell photon emission among groups after 1 day in culture, suggesting homogeneous delivery of cells within the grafts. However, an increase in cell photon emission was observed within 5 days in culture, indicating that cell survival and proliferation were enhanced¹⁸ when 3D myocardial artificial tissues are supplemented with AA. Our data suggest that AA enhances cell viability within bioengineered tissues in vitro in spite of the harsh conditions in culture (i.e., static 3D culture, hypoxia, and limited growth medium volume). Apoptosis is an active process associated with both the acute and chronic phases of myocardial infarction in response to oxidative insults.²³ The role of hypoxia on apoptosis via mitochondrial pathway activation of downstream effector caspases has already been demonstrated.²⁴ It has been shown that in hypoxic conditions, hypoxia inhibitor alpha (HIF-1α) promotes apoptosis in H9C2 cells and cardiomyocytes.^10,25,26 There is also evidence that depletion of AA interrupts HIF-1α proteosomal degradation, leading to increased expression of the latter.¹² Our in vitro experiments showed that graft viability improved via reduction of cell apoptosis, particularly when medium is supplemented with both 5 and 50 μmol/L AA. Further, the amount of cells within the AA grafts is preserved over time in static culture conditions, whereas it significantly decreases in the plain MAGS. These data correlate to our in vitro BLI results. Thus, our findings suggest that AA has a protective effect on donor cells when cultured in thick, 3D bioartificial constructs. The precise mechanism of such effect was not explored in this study. However, the enhancement in cell viability observed in the antioxidant-supplemented groups could be attributed to AA-driven reactive oxygen species scavenging, which preserves the integrity of the mitochondria and stabilizes the key regulator of hypoxia (HIF-1α).^10,12,25 We do not have direct proof that AA was in its active form during our study. However, previous studies have demonstrated that the oxidized form of AA—dehydroascorbic acid (DHAA)—is neuroprotective in ischemia models,^27,28 and it also prevents apoptosis and oxidative damage in macrophages,²⁹ lymphoid, myeloid, and mesothelial cells,³⁰ among others.³¹ DHAA is transported by the glucose transporters Glut1 into the mitochondria, but not the reduced form (AA).³² Subsequently, DHHA is reduced back to AA by protein disulfide isomerases. Thus, we assume that even if AA was oxidized during our experiments, there was a protective effect that could have been initiated by DHAA, which is ultimately reduced back to ascorbate in the mitochondria.

Our preliminary in vitro titration studies suggested that any dosage equal or above 100 μmol/L AA have a cytotoxic effect on cardiomyoblasts. In vivo, it has been reported that AA induces apoptosis of myeloid and lymphoid cells at concentrations above serum level (50 μmol/L).³³ The AA dosages utilized in our in vitro studies have also been previously reported as safe physiological dosage in other cell types.^10,32 Hence, we chose the lowest possible physiological dose (5 μmol/L) for our in vivo studies according to this rationale and the observation that after a 10-fold increase of AA dose there were no differences in cell survival in vitro.

Besides cytoprotection, AA seems to have an effect on cell differentiation. It has been shown that 100 μmol/L AA enhances differentiation of embryonic stem cell into cardiomyocytes in vitro,³⁴ as well as ex vivo differentiation of adult bone marrow stem cells into cardiomyocyte-like cells.³⁵ In our study, immunohistochemical assessment of MAGs after 3 and 5 days in culture revealed that AA induced morphological myogenic characteristics including elongation and cell fusion.³⁶ Expression of α-sarcomeric actin was observed among all groups at day 3, yet more organized z-line-like patterns were observed in the AA-enriched grafts at both days 3 and 5 in culture. These results suggest that AA may have an effect on H9C2 cardiomyoblast differentiation. However, this interesting observation should be further addressed in future studies.

To evaluate the AA effect on a bioartificial graft in vivo, we developed a simple and reproducible renal pouch model. To assess donor H9C2-Fluc-GFP cell survival within the graft, we performed noninvasive imaging at days 1, 3, and 6 postimplantation with a bioluminescence charge-coupled device camera. We observed that 1 week after graft implantation in a renal pouch for in vivo prevascularization, H9C2 cardiomyoblast survival decreases substantially. The effect was reflected by reduction of cell photon emission by nearly 75% of baseline in group B (plain MAGs). This observation is consistent with previously reported data from studies where BLI was used to assess myocardial graft survival in vivo in a heterotopic model of myocardial restoration in immunosuppressed rats.¹⁶ The authors have shown that H9C2 cardiomyoblasts injected in thin (3 × 3 × 1 mm) gelfoam grafts undergo massive cell death within 5 days after implantation into the ischemic myocardium. Early cardiomyoblast survival within the implanted grafts could only be improved by addition of collagen and growth factors¹⁶ or by cell transduction with the antiapoptotic hBcl-2 gene.³⁷ In addition, premature H9C2 cell death has been also documented after injection in healthy myocardium.³⁸ Here, we observed that by day 6 postimplantation cell, survival was significantly enhanced within the implanted myocardial artificial tissues via AA enrichment (group A). This indicates donor H9C2-Fluc-GFP cell survival and proliferation in the host in the AA-enriched MAGs during the first days postgraft implantation. Yet, confirmation of a proliferative effect of AA on H9C2 cells needs to be further addressed in future studies. Our results are of interest for cell transplantation models, given the fact that we used an allogeneic model in immunocompetent rats.

A limiting factor on cell or bioartificial tissue engraftment and survival is immune or inflammatory reaction. In past works we had demonstrated that the density of equine collagen scaffolds decreases by 30% after 2 weeks of implantation in skeletal muscle and triggers a heavy host cell infiltration.²³ Our previous and current studies also demonstrate a bioluminescence signal drop over the first week, indicating decrease in cell survival, regardless the administration of immunosuppresants (i.e., cyclosporine) or not.¹² It seems that the most sensitive and specific time-window to capture in vivo photon emission in MAGs—and also the most accurate—is the first week postimplantation in the renal pouch. Despite this short time-frame, it was sensitive enough in our hands to display the difference in bioluminescence signals in the AA-enriched MAGs. Cell viability after implantation has been demonstrated to be compromised in a time-dependent manner, even in models where one or two immunosuppresive agents have been used.^12,21 Further, in our previous study, donor cells were detected using a cell tracker up to day 21 after graft implantation in immunosupressed rats.²³ This demonstrates that, in current models, the fate of grafted tissue is limited by immunologic barriers even when immunosupression is used.

It is likely that the restorative effect of implanted bioartificial grafts is achieved via neovascularization/paracrine effects,^6,19 which in turn attenuate infarct expansion, rather than by cardiomyocyte regeneration, cell differentiation, or functional graft integration into the host's myocardium. Evidence has shown that most bioengineered tissues for myocardial restoration have mainly focused on multicellular allogeneic approaches to achieve neovascularization within bioengineered grafts.^8,39–43 With our study, we introduce a strategy to promote a natural pattern of angiogenic sprouting and graft vascularization in the graft recipient itself.

A prominent novelty of this study is the introduction of the pouch model for in vivo graft prevascularization, which has been proven to be an easy and efficient way to supply bioengineered myocardial tissues with blood vessels of autologous origin. Using neonatal rat cardiomyocytes cast in an equine collagen type I mesh, we have recently shown that graft survival after implantation is determined by host immune responses and degree of angiogenesis.⁴⁴ In such a model, vascularization could be seen only in the superficial layer of the graft after 14–21 days of implantation. Interestingly, the vascular density was significantly lower when the rats received immunosuppressive therapy.²³ Here we present a model with superior and faster angiogenic potential. Moreover, we demonstrate that the neovascularization process is enhanced in AA-enriched grafts.

In conclusion, the current model is a powerful one to vascularize engineered implants in vivo. The possible explanation for the superior AA-enriched graft viability in our allogeneic model might be both the antiapoptotic and proangiogenic effects exerted by AA. These findings render AA a considerable supplement for cell- and tissue transplant-based therapies. Yet, further studies should be done to evaluate the effect of AA in 3D bioartificial myocardial grafts containing human stem cells to confirm the translational potential of our approach. We are currently carrying out experiments aiming at postischemic myocardial restoration using prevascularized AA-enriched MAGs. Our approach provides a realistic perspective for the clinical setting: enhancement of cell survival in 3D bioengineered tissues both in vitro and in vivo, and a method to mature grafts for later autologous implantation.

Footnotes

Acknowledgments

The authors thank Ms. Diane Tan Ai Lin for technical contributions toward cloning and preparation of the lentiviral vectors, and Ms. Shera Lilyanna for technical help in maintaining cell cultures.

Disclosure Statement

No competing financial interests exist.

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