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. Author manuscript; available in PMC: 2018 Jul 1.
Published in final edited form as: J Vasc Surg. 2016 Sep 26;66(1):243–250. doi: 10.1016/j.jvs.2016.05.096

Fast degrading bioresorbable arterial vascular graft with high cellular infiltration inhibits calcification of the graft

Tadahisa Sugiura 1, Shuhei Tara 1, Hidetaka Nakayama 3, Tai Yi 1, Yong-Ung Lee 1, Toshihiro Shoji 1, Christopher K Breuer 1, Toshiharu Shinoka 1,2
PMCID: PMC5366287  NIHMSID: NIHMS805522  PMID: 27687327

Abstract

Objective

Bioresorbable vascular grafts are biologically active grafts which are entirely reconstituted by host-derived cells through an inflammation-mediated degradation process. Calcification is a detrimental condition that can severely impact graft performance. Therefore, prevention of calcification is of great importance to the success of bioresorbable arterial vascular grafts. The objective of this study is to test whether fast degrading bioresorbable arterial grafts with high cellular infiltration will inhibit calcification of grafts.

Methods

We created two versions of bioresorbable arterial vascular grafts: 1) Slow Degrading (SD) grafts and 2) Fast Degrading (FD) grafts. Both grafts had the same inner layer composed of a 50:50 poly (l-lactic-co-ε-caprolactone) copolymer (PLCL) scaffold. However, the outer layer of SD grafts was composed of poly (l-lactic acid) (PLA) nano-fiber whereas the outer layer of FD grafts was composed of a combination of PLA and polyglycolic acid (PGA) nano-fiber. Both grafts were implanted in 8–10 week old female mice (n = 15 in the SD group, n = 10 in the FD group) as infra-renal aortic interposition conduits. Animals were followed for 8 weeks.

Results

Von Kossa staining showed calcification in 7 out of 12 grafts in the SD group, but zero in the FD group (P<.01, chi-square test). The cell number in the outer layer of FD grafts was significantly higher than SD grafts (SD: 0.87 ± 0.65 × 103/mm2 vs. FD: 2.65 ± 1.91 × 103/mm2, P=.02).

Conclusions

The fast degrading bioresorbable arterial vascular graft with high cellular infiltration into the scaffold inhibited calcification of grafts.

Introduction

For patients with cardiovascular diseases, creating clinically acceptable prosthetic small-diameter arterial grafts (< 6 mm) as alternatives to autologous arterial or venous substitutes is paramount due to insufficient availability of autografts in patients with widespread atherosclerotic vascular disease.1

Bioresorbable vascular grafts have emerged as a potentially suitable replacement to prosthetic grafts. Bioresorbable vascular grafts are biologically active grafts, which, over time, are entirely reconstituted by host-derived cells via an inflammation-mediated scaffold degradation process.2 The application of bioresorbable vascular grafts has several advantages to other implantable prosthetics, such as continuous growth potential, favorable biocompatibility, and low risk of infection or rejection. In addition, clinical evidence has now shown that bioresorbable vascular grafts are safe and effective to use in pediatric patients undergoing extracardiac total cavopulmonary connection procedures.3, 4 We reported feasible late-term results (mean follow-up, 5.8 years) of tissue-engineered vascular grafts for pediatric patients with congenital heart disease.4 However, even with these advances, calcification remains one of the most detrimental events for long term arterial vascular grafts success, which could lead to thrombosis or graft rupture. Preventing calcification is thus of the utmost importance in long term success of arterial vascular grafts.

The scaffold material and structure play a crucial role in preventing calcification of grafts. A fast degrading elastomer, prepared from an elastic polymer, was shown to develop well-organized neotissue in a rapid remodeling process (within 90 days) without any calcification up to 1 year later.5, 6 Based on this finding, we hypothesized that fast degrading bioresorbable arterial vascular grafts with high cellular infiltration will inhibit calcification of grafts. To test this hypothesis we created two versions of the grafts: 1) Slow Degrading (SD) grafts and 2) Fast Degrading (FD) grafts. Both grafts have the same inner layer composed of a 50:50 poly (l-lactic-co-ε-caprolactone) copolymer (PLCL) scaffold. However, the outer layer of SD grafts was composed of poly (l-lactic acid) (PLA) nano-fibers, whereas the outer layer of FD grafts was composed of a combination of PLA and polyglycolic acid (PGA) nano-fibers.

Materials and Methods

Bioresorbable arterial vascular grafts

We created two versions of bioresorbable arterial vascular grafts: 1) Slow Degrading (SD) graft and 2) Fast Degrading (FD) graft. Both grafts have the same inner layer composed of a 50:50 poly (l-lactic-co-ε-caprolactone) copolymer (PLCL) scaffold. The scaffolds were constructed by pouring a solution of PLCL into a glass tube, then freeze-drying under a vacuum as previously described.7 Next, these scaffolds were reinforced by electrospinning fibers in the outer layer. The outer layer of SD grafts was composed of a poly (l-lactic acid) (PLA) nano-fibers, whereas the outer layer of FD grafts was composed of a combination of PLA and polyglycolic acid (PGA) nano-fiber. The proportion of the PLA and PGA nano-fibers is 50:50. The thickness of the nano-fiber layer and the inner layer in either group is around 40 μm and 200 μm, respectively. Luminal diameter of each graft was around 600 μm (Fig. 1A, B).

Fig. 1.

Fig. 1

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Mouse bioresorbable arterial vascular graft. A, A schematic of the mouse bioresorbable arterial vascular graft. B, An SEM image of the mouse bioresorbable arterial vascular graft. C, The mouse abdominal aortic interposition graft.

Animal model and surgical implantation

All animals received humane care in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mean arterial pressure of C57BL/6 mice is 110 ± 2 mmHg.8 The grafts of 3mm in length were implanted in 8–10 week old female mice (n = 15 in the SD group, n = 10 in the FD group) as infra-renal aortic interposition conduits using standard microsurgical techniques (Fig. 1C).9 Low pressure vascular clamps (Fine Science Tools, Foster City, CA) were used for cross clamping and 400 U/kg of heparin was injected intramuscularly 5 minutes before unclamping. The graft was anastomosed with running suture using a 10-0 polypropylene suture. Animals were followed for 8 weeks to evaluate neotissue formation. An aspirin-mixed diet (0.1% of diet) was fed to mice of each group 3 days before and 3 days after surgery to prevent acute thrombosis.

Histology, immunohistochemistry, and immunofluorescence

Explanted grafts at 8 weeks after implantation were fixed in 4% para-formaldehyde, embedded in paraffin, sliced (5 μm thick sections), and stained with Hematoxylin and Eosin (H&E) and von Kossa staining. Outer and luminal perimeters of the grafts were manually measured from H&E staining with Image J software (NIH, Bethesda, MD) to obtain luminal diameter and wall thickness measurements. Polarized microscopic images of H&E staining indicate the remaining polymer as white. One representative section from each explant was stained and imaged, which was used to measure the area fraction of the remaining polymer with Image J software. H&E staining was used for cell counting. One representative section from each explant was stained and imaged. Low magnification (5x) images were divided into nine sections (3×3). Four of these regions (upper middle, center, lower right, and lower left) were selected to obtain high magnification (20x) images, the area of which was 0.12 mm2. All positively stained nuclei were counted from high magnification images. Averages from these four regions represented the number of cells in each section.

Identification of M1 macrophages, and M2 macrophages were done by immunofluorescent staining of paraffin-embedded explant sections with rat anti-MAC3 antibody (1:75, DAKO, Carpinteria, CA), mouse anti-iNOS antibody (1:200, Abcam, Cambridge, MA), and rabbit anti-Mannose Receptor (CD206) antibody (1:100, Abcam), followed by Alexa Fluor 488 anti-rat IgG secondary antibody (1:300, Invitrogen, Carlsbad, CA), Alexa Fluor 647 anti-mouse IgG secondary antibody (1:300, Invitrogen), and Alexa Fluor 488 anti-rabbit IgG secondary antibody (1:300, Invitrogen), respectively. Immunofluorescent staining for CD31 as a marker of endothelial cells and for α-SMA was performed using rabbit anti-CD31 primary antibody (1:50, Abcam) and mouse anti-α-SMA primary antibody (1:500, DAKO), followed by Alexa Fluor 488 anti-rabbit IgG secondary antibody (1:300, Invitrogen), and Alexa Fluor 647 anti-mouse IgG secondary antibody (1:300, Invitrogen), respectively. Fluorescence images were obtained with an Olympus IX51 microscope (Olympus, Tokyo, Japan).

RNA extraction and reverse transcription quantitative polymerase chain reaction

Explanted grafts at 8 weeks after implantation were frozen in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA), and sectioned into twenty 30 μm sections. Total RNA was extracted and purified using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Reverse transcription was performed using a High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA). All reagents and instrumentation for gene expression analysis were obtained from Applied Biosystems. Quantitative polymerase chain reaction (qPCR) was performed with a Step One Plus Real-Time PCR System using the TaqMan Universal PCR Master Mix Kit. Reference numbers for primers are: Pecam1 (Mm01242584_m1), Nos3 (Mm00435217_m1), Acta2 (Mm00725412_s1), Bmp2 (Mm01340178_m1), Tnfsf11 (Mm00441906_m1), Tnf (Mm00443258_m1), Nos2 (Mm00440502_m1), Mrc1 (Mm00485148_m1), Arg1 (Mm00475988_m1), and Hprt (Mm00446968_m1). The results were analyzed using the comparative threshold cycle method and normalized with Hprt as an endogenous reference, and reported as relative values (ΔΔ CT) to those of the SD group.

Statistical analysis

Numeric values are listed as mean ± standard deviation. The number of experiments is shown in each case. Data of continuous variables with normal distribution were evaluated by student’s t test. P values less than 0.05 indicated statistical significance.

Results

Animal Survival

All mice in the FD group survived the 8 week implantation period with patent grafts. There were no complications, such as bleeding, acute thrombosis, or graft rupture in the FD group. However, in the SD group, 3 mice were sacrificed due to lower limb paralysis from acute thrombosis. The grafts in the survived mice were all patent. These findings were confirmed by autopsy within 24 hours after death or severe paralysis of lower limbs.

No significant difference in the quantitative morphometric analysis

H&E staining showed cell infiltration within the scaffold in both groups (Fig. 2A). There was no statistical difference in the luminal diameter (SD: 699 ± 128 μm vs. FD: 776 ± 24 μm, P=.09) (Fig. 2B) or wall thickness (SD: 298 ± 53 μm vs. FD: 293 ± 15 μm, P=.79) (Fig. 2C) between the groups.

Fig. 2.

Fig. 2

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Histology and morphometric data of slow degrading (SD) and fast degrading (FD) bioresorbable arterial vascular grafts. A, H&E staining showed cell infiltration within the scaffold in both groups. B and C, There was no statistical difference in the luminal diameter or wall thickness between the groups (P=.09 and .79, respectively). SD: Slow degrading graft, FD: Fast degrading graft.

Less residual scaffold and greater number of cells infiltrated in the outer layer of FD grafts

H&E staining with polarized light microscopy was used for quantitative measurement of residual scaffold in each group (Fig. 3A). There was significantly less residual scaffold in the outer layer in FD grafts than SD grafts (SD: 16.8 ± 1.7 % vs. FD: 14.1 ± 1.7 %, P=.005) (Fig. 3C). Significantly greater number of cells infiltrated in the outer layer of FD grafts than SD grafts (SD: 0.87 ± 0.65 × 103/mm2 vs. FD: 2.65 ± 1.91 × 103/mm2, P=.02), though no significant difference was observed in the inner layer (SD: 1.75 ± 0.60 × 103/mm2 vs. FD: 1.80 ± 0.53 × 103/mm2, P=.85) (Fig. 3B, D).

Fig. 3.

Fig. 3

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Cell infiltration into the outer and inner layer of the bioresorbable arterial vascular graft. A, Representative images of H&E staining with polarized light microscopy. B, Representative high magnification H&E staining images of the inner and outer layer of the graft. C, There was significantly less residual scaffold in the outer layer in FD grafts than SD grafts. **: P < .01. D, The cell number in the outer layer of FD grafts was significantly higher than SD grafts *: P < .05, although no significant difference was observed in the inner layer of the grafts (P=.85). SD: Slow degrading graft, FD: Fast degrading graft.

Calcification occurred only in SD grafts, not in FD grafts

Von Kossa staining revealed that calcification occurred only in SD grafts (7 out of 12), not in FD grafts (P<.01, chi-square test) (Fig. 4 A, B).

Fig. 4.

Fig. 4

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Calcification data of bioresorbable arterial vascular graft. A, Representative images of von Kossa staining in SD and FD grafts after 8 weeks. B, Quantification of von Kossa staining. Von Kossa staining revealed that calcification occurred only in SD grafts (7 out of 12), not in FD grafts. **: P < .01 (chi-square test). SD: Slow degrading graft, FD: Fast degrading graft.

Comparable endothelialization and smooth muscle cell proliferation in both graft groups

Vascular endothelial cells serve as the cellular barrier between the blood and underlying tissue constituents. Endothelial cell dysfunction leads to the recruitment and infiltration of leukocytes which could contribute to calcification. To evaluate endothelialization and smooth muscle cell (SMC) proliferation in the grafts, CD31 and α-SMA staining was employed, respectively. A layer of endothelial cell coverage on the luminal surface of the graft followed by a SMC layer was observed in both groups (Fig. 5A). For quantitative comparison of endothelialization between the groups, gene expression of Pecam1 and Nos3 in the grafts was measured and demonstrated no statistical difference between the groups (Pecam1, SD: 1.00 ± 0.45. vs. FD: 0.74 ± 0.20, P=.41; Nos3, SD: 1.00 ± 0.14 vs. FD: 1.16 ± 0.08, P=.07) (Fig. 5B). For the quantitative comparison of SMC proliferation between the groups, gene expression of Acta2 in the grafts was measured and demonstrated no statistical difference between the groups (SD: 1.00 ± 0.40 vs. FD: 0.92 ± 0.55, P=.94) (Fig. 5C).

Fig. 5.

Fig. 5

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Endothelialization and smooth muscle cell proliferation of the grafts at 8 weeks after implantation. A, Representative immunofluorescent images of endothelial cells (CD31) and smooth muscle cells (α-SMA). B, Gene expression was analyzed by RT-qPCR using the ΔΔ CT method. There was no statistical difference of relative gene expression of PECAM 1 or NOS3 between the groups (P=.41 and .07, respectively). C, There was no statistical difference of relative gene expression of ACTA2 between the groups (P=.94). SD: Slow degrading graft, FD: Fast degrading graft.

Comparable gene expression of transcription factors for osteogenesis and osteoclastgenesis in both graft groups

BMP2 and RANKL is one of the key transcription factors associated with osteogenesis and osteoclastgenesis, respectively.10, 11 For the quantitative comparison between the groups, gene expression of BMP2 and RANKL in the grafts were measured, and there were no statistical difference between the groups (BMP2, SD: 1.00 ± 0.65 vs. FD: 1.17 ± 0.46, P=.62; RANKL, SD: 1.00 ± 0.74 vs. FD: 0.93 ± 0.71, P=.88) (Data not shown).

No significant difference in the macrophage infiltration into the grafts

Immunofluorescent staining with anti-MAC3, anti-iNOS, and anti-CD206 antibody demonstrated that both M1 and M2 macrophages infiltrated into the grafts in both groups (Fig. 6A). For the quantitative comparison of M1 macrophage between the groups, gene expression of TNFα and iNOS in the grafts was measured, and there was no statistical difference between the groups (TNFα, SD: 1.00 ± 0.31 vs. FD: 0.78 ± 0.28, P=.23; iNOS, SD: 1.00 ± 0.59 vs. FD: 0.92 ± 1.00, P=.88) (Fig. 6B). For the quantitative comparison of M2 macrophage between the groups, gene expression of Mrc1 and Arg1 in the grafts was measured, and there was no statistical difference between the groups (Mrc1, SD: 1.00 ± 0.34 vs. FD: 1.13 ± 0.15, P=.69; Arg1, SD: 1.00 ± 0.84 vs. FD: 0.91 ± 1.06, P=.89) (Fig. 6C).

Fig. 6.

Fig. 6

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Macrophage infiltration into the bioresorbable arterial vascular grafts. A, Representative immunofluorescent images of MAC3 (macrophage) and iNOS (M1 macrophage), and MAC3 and CD206 (M2 macrophage). B, Gene expression was analyzed by RT-qPCR using the ΔΔ CT method. There was no statistical difference in gene expression of TNFα or iNOS between the groups (P=.23 and .88, respectively). C, There was no statistical difference in gene expression of Mrc1 or Arg1 between the groups (P=.69 and .89, respectively). SD: Slow degrading graft, FD: Fast degrading graft.

Discussion

In the previous report we demonstrated well-organized neotissue formation in bioresorbable arterial grafts, including endothelial cell coverage on the luminal surface of the graft surrounded by smooth muscle cells 12 month after implantation in a mouse model. 12 However calcification is one of the most detrimental events for bioresorbable arterial vascular grafts.13 The literature has revealed that inflammation-dependent calcification in vessels is due to the accumulation of macrophages and that end-stage calcification is concomitant with a reduction in inflammation.14–16 These observations suggest that inflammation is involved in the initiation and propagation of calcific mineral deposition.17 The literature also showed that a fast degrading elastomer developed well-organized neotissue in a rapid remodeling process, without any calcification in a rat aortic implantation model.5 In addition, our previous work demonstarted that a bioresorbable arterial vascular graft, constructed of a PLA nano-fiber, had abundant scaffold fibers remaining and increased inflammation in the neointima, which caused severe calcification.18 Based on these findings, we hypothsized that fast degrading (FD) grafts will leave less residual scaffold fragments, thereby producing less inflamation, and subsequently inhibit calcification of the grafts. In the present study, we demonstrated that calcification occurred only in slow degrading (SD) grafts; there was no calcification in FD grafts, which had significantly less residual scaffold at 8 weeks.

Macrophages have a critical role in vascular neotissue formation of grafts.19 There is a lot of literature supporting the critical role of monocytes and macrophages on various types of vascular repair or remodeling processes.20–22 In addition, macrophage related inflammation is understood to be a key contributor to osteogenesis in the early stages of atherosclerotic intimal calcification.15 Macrophages are involved in the early, pro-inflammatory phase of calcification by releasing extracellular vesicles containing a phosphatidylserine-annexin V-S100A9 complex that facilitates mineralnucleation.23 These procalcific macrophages exhibit a pro-inflammatory M1 macrophage phenotype. We assumed that the increased amounts of fragmented scaffold polymer in the SD group polarized the macrophages into the M1 macrophage phenotype which lead to calcification of the graft. However, the role of macrophage polarization in cardiovascular calcification remains largely unexplored.

Nano-fibers created by an electrospinning technique are a desirable material for fabricating bioresorbable arterial vascular grafts. Therefore, nano-fiber based scaffolds using biodegradable polymers have become a commonly used technique for constructing bioresorbable arterial vascular grafts; they have also demonstrated favorable surgical and mechanical properties with a high patency rate in arterial implantation models.24–26 However, in our previous study we demonstarted that the bioresorbable arterial vascular graft, which was constructed of slow degrading PLA nano-fibers, had severe calcification with abundant scaffold fragments remaining 12 months after implantation.18 This led us to create a fast degrading nano-fiber layer by combining PLA and PGA nano-fibers, which have a degradation period of 6–12 months and 2–3 weeks, respectively.1 After the PGA in the outer layer of FD graft degraded, inflammatory cells could infiltrate into the scaffold, thereby enabling the grafts to have both durability to the arterial pressure and inhibition of calcification because of fast scaffold degradation.

We assessed the grafts at 8 weeks after implantation. This time period was based on our previous report, which showed organized vascular neotissue in the internal lumen of the scaffold after 6 weeks in the mouse aortic implantation model.7 Therefore an 8-week time point was deemed the optimal end-point for evaluating neotissue formation of the graft.

There are several limitations in this study. First, in this study, we demonstrated that calcification occurred only in the SD group, not in the FD group. However, we could not show the mechanism underlying the prevention of graft calcification. Although we assessed the relationship between calcification and macrophage phenotype by measuring the M1 and M2 macrophage markers, we could not show the statistical difference. Second, calcification of SD grafts appeared to have occurred exclusively in the inner layer; however, the differences between SD and FD graft composition, residual scaffold, or cell infiltration were confined to the outer layer of the grafts. Third, some mice had acute thrombosis after graft implantation. In our prior experiences there was a propensity for acute thrombosis occurred at the percentage of 13 27 to 40. Thrombogenicity of the graft should be improved in the future experiments. Finally the lumen size of the grafts increased approximately 10% for the SD group and 30% for the FD group 8 weeks after implantation. Actually tensile strength of the SD grafts is greater than the FD grafts (Data not shown). The nano-fiber layer of the FD grafts may not be strong enough to endure arterial pressure and it could be possible that aneurysmal formation will occur at a later time point.

Conclusions

In this study, we demonstarted that calcification occurred only in the SD group, not in the FD group. Eight weeks after implantation FD grafts had significantly less residual scaffold and greater numbers of cells infiltrate into the outer layer than SD grafts. The fast degrading bioresorbable arterial vascular grafts with high cellular infiltration shows promise for the prevention of graft calcification.

Clinical Relevance.

Bioresorbable vascular grafts are biologically active grafts which are entirely reconstituted by host-derived cells throughout the lifespan of the patient. Calcification of the graft is a detrimental condition that can severely impact graft performance. Therefore, preventing graft calcification is of great importance to success of bioresorbable vascular grafts. We hypothesized that remaining scaffold polymer could affect graft calcification. Therefore we created fast degrading bioresorbable arterial vascular grafts. We report here that no calcification occurred in the fast degrading grafts although calcification occurred in the slow degrading grafts. These findings provide further strategies of prevention for calcification after implantation of bioresorbable vascular grafts in the clinical setting.

Footnotes

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