Evaluation method for cell-free in situ tissue-engineered vasculature monitoring: Proof of growth and development in a canine IVC model.

We previously developed a non-cell-dependent biodegradable scaffold to create in situ tissue-engineered vasculature (iTEV) and tested it in a canine inferior vena cava (IVC) model. As iTEV features change dramatically during tissue generation, practical, simple, and accurate methods to evaluate iTEV are needed. The present study examined the usefulness of a novel method to evaluate iTEV growth and remodeling according to a simple formula using angiography: hepatic vein (HV) index = (IVC-HV junction angle) ÷ (π × [minimal internal iTEV diameter ÷ 2]2). HV index strongly correlated with the pressure gradient across iTEV, which tended to improve during the tissue generation period up to 12 months post-implantation. Time-course changes in HV index reflected iTEV tissue development and in-vivo characteristics, such as hemodynamic congestion. In conclusion, HV index is useful to assess iTEV graft function because it represents both the morphometrics and hemodynamics of iTEV with only diagnostic imaging data.

Introduction

We previously reported the usefulness of a tissue-engineered graft (TEG) created with a biodegradable scaffold to accomplish vascular continuity in animal models [1-5] and clinical applications [6-9]. Implanted biodegradable materials act as templates for invasive cells from surrounding tissues, seeded cells, and/or newly generated tissues. However, with simultaneous changes in material degradation and tissue development, TEG features dramatically change as vascular tissue matures into native tissue [2].

In terms of the features of a developing TEG, our research focuses on the development of optimal materials for appropriate cell proliferation. These materials possess suitable repair characteristics to address the decrease in the internal diameter of TEGs during tissue generation. However, concerns of narrowing and stenosis during early tissue development of TEGs are still prevalent, even with tissue-engineering strategies that use better biodegradable constructs.

In-vivo tissue regeneration of TEGs is dependent on each individual’s self-remodeling/regeneration ability. These individualized variations equate to a range of pressure gradients across the TEG, which influence TEG narrowing and may cause non-crucial, but congestive, changes in the liver and other organs during tissue regeneration. As previously reported, changes in immature TEGs improve as the tissue generates and remodels into native-like tissue [3, 5, 10]. If there are pressure gradients across the TEG with congestive changes during this tissue generation period, the distal side of the suture line (namely the inferior vena cava [IVC] and hepatic vein [HV]) distends or dilates due to pressure overload. These unwanted changes were observed in our previous animal experiments. This negative characteristic prompted us to establish a novel evaluation method for TEG assessment. The idea was based on the notion that if we can identify the relationship among pressure gradient, TEG diameter, and IVC–HV junction angle, we can speculate the pressure gradient and evaluate the degree of liver congestion using imaging observations alone. Furthermore, we can compare changes in TEG features over time and calculate the degree of vascular remodeling using a logically calculated value. The present study was thus performed to assess the relationship among pressure gradient, TEG diameter, and IVC–HV junction angle in an animal model of TEG. Our findings may be applicable in a clinical setting.

In this study, a canine IVC model with in situ tissue-engineered vasculature (iTEV) was used because it does not require any cells, medication, or materials other than a tubular biodegradable scaffold to create a self-regenerated vasculature. As mentioned above, iTEV increases in wall thickness due to intimal hyperplasia, followed by healing and remodeling of related tissues to within a normal range; thus, narrowing or stenotic changes in iTEV are unavoidable [2, 5, 10]. The pressure gradient across iTEV increases, while the internal diameter decreases; however, this improves with time, allowing iTEV to develop into a native-like vessel [5]. In terms of the features of iTEV with proper tissue regeneration, iTEV ultimately remodels into the shape required by the living organism, without an unacceptable pressure gradient. However, because iTEV relies on each individual’s self-renewal ability, and each organism has its own healing/remodeling process, evaluating iTEV in each individual sample over time is difficult.

In the present study, iTEV was followed by catheterization to obtain angiographic imaging data and hemodynamic values. iTEV images were then analyzed to explore the usefulness of this novel evaluation method. We used a formula consisting of the smallest iTEV diameter and the IVC–HV junction angle. We named the novel score calculated with this simple formula the “HV index” to evaluate iTEV remodeling. The HV index enables congestive changes in iTEV to be monitored over time. An improvement in congestion suggests improvements in pressure gradient, remodeling, adaptation, and/or growth of iTEV, irrespective of body weight or height.

One of the issues with tissue-engineered vasculature is that the features of the vasculature dramatically change with time; thus, it is difficult to compare graft function by assessing the shape of iTEV itself. Furthermore, it is difficult to chronologically evaluate changes in iTEV using neither pressure gradient nor chronologically changing features alone because the pressure gradient and congestive changes do not recover simultaneously, and tissue generation patterns differ between individuals. Accordingly, the present study aimed to assess the usefulness of HV index for time-course evaluation of iTEV.

We also evaluated iTEV maturation using chronological development of vascular smooth muscle cell (VSMC) phenotypes to show that HV index yields robust results for the evaluation of iTEV from the perspective of biochemical analyses because VSMC maturity reflects iTEV maturity [2, 5, 10]. Cells in developing iTEV include cells found in healthy tissues, inflammatory cells, and adhesive cells, as well as fibrous tissue, and each of these components increase or decrease in number with tissue development. Thus, we speculated that an internal control such as beta-actin, which involves these inflammatory cell proteins, is not appropriate to examine VSMCs in developing iTEV. Furthermore, the cell composition and number of cells differ between individuals. Accordingly, we evaluated chronological changes in VSMC differentiation by examining the ratio of desmin, vimentin, and myosin heavy chain 11 (MYH11) proteins to α-smooth muscle actin (SMA) to demonstrate time-dependent differentiation of iTEV and to show the development of iTEV from the perspective of biochemical assessment. Accordingly, iTEV development was also assessed by examining the increment of differentiated VSMCs within the iTEV.

Herein, we describe HV index as a novel evaluation method. This method considers both morphometric and hemodynamic metrics of iTEV for precise evaluation of iTEV growth and remodeling. With the help of biochemical and immunohistological analyses, iTEV growth and remodeling are illustrated.

Materials and methods

In-vivo experimentation

All animals received care according to the National Institutes of Health (NIH) guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). The ethics committee of Animal Research at Tokyo Women’s Medical University reviewed and approved the study protocols (Permit Numbers: AE20-008-3-C, AE19-001, AE18-102, AE17-1, and AE16-1). All experiments were conducted at the animal experimentation facility of The Heart Institute of Japan.

In total, 26 healthy adult female beagles (Rikaken, Inc., Tokyo, Japan) with a mean weight of 9.4 kg (8.5–10.5 kg) were used in this study. Six animals were euthanized at 1, 3, and 6 months post-implantation of iTEV, and 8 animals were euthanized at 12 months for angiography, pressure studies, and histological and biochemical analyses.

For surgery, animals were anesthetized with intravenous injection of midazolam (0.1–0.3 mg/kg body weight); continuous intravenous injection of propofol (0.1–0.3 mg/kg/min); and intramuscular injection of medetomidine hydrochloride (5.0 μg/kg body weight), butorphanol tartrate (0.2 mg/kg body weight), and atropine sulfate (0.05 mg/kg body weight). Heparin (800 U/kg body weight) was administered intravenously a few minutes before cross-clamping the IVC for anticoagulation during anastomosis. The mean age of the animals upon the implantation of iTEV was 20.83 ± 1.069 months (range = 13.67–25.10 months).

Under general anesthesia, the seventh intercostal space was opened with a right thoracotomy incision. After heparinization, the IVC adjacent to the diaphragm and the right atrium adjacent to the right atrial–IVC junction were cross-clamped, and the IVC was incised and resected to anastomose the biodegradable scaffold to the greatest possible length using a simple clamping technique. The proximal end of the scaffold was sutured to the right atrium, and the distal end was sutured to the remaining IVC stump. Six excised native tissues were used for biochemical analysis.

The composite tubular scaffold was 10.5 mm in diameter, with a mean length of 26.03 ± 1.062 mm (23.00–33.00 mm). The tubular scaffold comprised multiple layers of L-lactide and ε-caprolactone copolymer (PLCL) sponge, spiraled fibers of 2–0 PLCL, and two 2–0 PLCL fiber warps placed in a long-axis direction for reinforcement to avoid constriction of the material after implantation. The outer side of the spiral was covered with 7–0 meshed PLCL fibers to support the PLCL sponge (Fig 1A–1C). Theoretically, the scaffold completely loses its strength within 6 months, and the material itself degrades within 8–12 months due to non-enzymatic hydrolysis [2, 5, 10].

Fig 1

Biodegradable scaffold and iTEV at 1 year post-implantation.

(A) Macroscopic images of the biodegradable scaffold used in this study (bar: 1 cm). (B) Higher magnification of a scaffold cross-section (bar: 500 μm). The spiraled fiber, the longitudinal warp, a cross-section of mesh fibers for scaffold reinforcement, and the honey-comb-shaped sponge are shown. (C) A schematic of the biodegradable scaffold in multiple layers is shown. From the inner side is the sponge composed of L-lactide and ε-caprolactone (PLCL) copolymer, a spiraled 2–0 PLCL fiber, two 2–0 PLCL fiber warps placed in a long-axis direction for reinforcement, a 7–0 meshed PLCL fiber, and the sponge.

Angiography and pressure measurements

Ten animals underwent both pressure studies and angiography at 0.5, 1, 3, 6 (n = 10), and 12 (n = 8) months post-implantation. For the control studies, 10 untreated animals not related to the present study underwent pressure studies and angiography using the same protocol. Animals were anesthetized using the protocol described above and placed in sterilized conditions for femoral vein dissection. An 8-Fr long-catheter sheath (Terumo, Tokyo, Japan) was placed through the femoral vein to the IVC adjacent to the HV, as previously described [2, 5, 10]. Animals were then placed in a lateral position for angiography. VascuTape® Radiopaque Tape (LeMaitre Vascular GK, Tokyo, Japan) was used with centimeter-long reference markings on the chest. A mobile C-arm imaging system (WHA-200 OPESCOPE ACTIVO; Shimadzu, Tokyo, Japan) was used to identify the sheath and catheter locations and to perform digital angiography. Approximately 10–15 ml of angiographic agent was injected to evaluate the features of the iTEV, IVC, and HV with breath-holding to avoid respiratory fluctuation. No anticoagulants were administered until euthanasia was performed [1–5, 10]. All angiographic data were saved to digital devices for further analysis.

Simultaneously, a Millar Mikro-Tip catheter (Millar Instruments Inc., Houston, TX) was used to continuously measure the pressure of the right atrium, the mid-portion of the iTEV, and the HV. Pressure waves were recorded using LabChart 8.1.5 data analysis software (ADInstruments Japan, Nagoya, Japan). The peak pressure of the right atrium, mid-portion of iTEV, and distal portion of iTEV were measured to analyze pressure gradients between the right atrium and the IVC. Ten samples of native IVC and HV from untreated (control) dogs were measured in the same manner as described above.

Digital angiographic imaging data were recorded in a lateral position. Data were used to measure the smallest internal diameter of iTEV and the angle formed by the distal sides of the IVC and HV (IVC–HV junction angle) using the Ruler tool in Photoshop CS6 Extended (Adobe, San Jose, CA). HV index, as a newly defined simple indicator for evaluating iTEV in a time-course-dependent manner, reflected both morphometry and hemodynamic metrics of iTEV. HV index was calculated according to the following formula: HV index = (IVC–HV junction angle) ÷ (π × [minimal internal iTEV diameter ÷ 2]2). To show the superiority of the HV index, the overall correlation between the pressure gradient and IVC–HV junction angle, the least estimated area of iTEV, and the HV index of iTEV were examined simultaneously.

Macroscopic and histological examination

After careful dissection, macroscopic sample overview images were taken with a digital camera. The length of iTEV was measured directly between the two suture lines. iTEV was longitudinally dissected to perform histological and biochemical analyses.

For histological examination, samples were fixed in 4% paraformaldehyde at pH 7.0 in phosphate-buffered saline, embedded in paraffin, and sectioned to 4–5 μm. Some sections were subjected to hematoxylin and eosin, Masson’s trichrome, Victoria blue van Gieson, or modified von Kossa stain. Immunostaining of remaining sections was performed using antibodies against factor VIII (1:2000; Abcam, Tokyo, Japan), α-SMA (clone 1A4, 1:2000; Dako, Tokyo, Japan), myosin heavy chain (MHC, 1:500; Sigma, St. Louis, MO), smooth muscle myosin heavy chain 1 (1:500; Yamasa, Tokyo, Japan), smooth muscle myosin heavy chain 2 (1:2000; Abcam), desmin (RD301, 1:500, ab8976; Abcam), vimentin (V9, 1:500, ab8069; Abcam), and MYH11 (1:200, ab53219; Abcam). Inflammatory cells in the specimens of iTEV at 1 and 12 months after implantation were identified by immunostaining for macrophage marker CD68 (1:500; Proteintech, Rosemont, IL). Histological examinations were performed as previously reported [1–5, 10].

All histological examinations and measurements were performed using light microscopy (Biozero Bz 8000; Keyence, Osaka, Japan), stereomicroscopy (MVX10; Olympus, Tokyo, Japan), and accompanying analytical software.

Biochemical protein analysis

After thawing, iTEV and native IVC samples were weighed, and total protein was extracted using the Minute™ Total Protein Extraction Kit (Invent Biotechnologies Inc., Eden Prairie, MN). Total protein content was determined using the TaKaRa Bradford Protein Assay Kit (Takara, Kusatsu, Shiga, Japan). Equal amounts (10 μg) of denatured protein were added per lane for separation in 4%‒15% Mini-PROTEAN® TGX™ Precast Gels (Bio-Rad, Tokyo, Japan) using Tris-glycine buffer (Bio-Rad) and transferred to polyvinylidene difluoride (PVDF) membranes (Trans-Blot® Turbo™ Mini PVDF Transfer Packs; Bio-Rad) using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). Western BLoT Rapid Detect v2.0 (Takara) and antibodies were used to detect immobilized target proteins on PVDF membranes, according to the manufacturer’s protocol. Sample immunoblots were incubated with antibodies against the following targets: α-SMA (1:1000, clone 1A4; Dako), MYH11 (1:1000, ab53219; Abcam), and desmin (1:1000, RD-301; ThermoFisher Scientific, Rockford, IL) or vimentin (1:2000, ab8069; Abcam) to detect three blots simultaneously on the same lane. Membranes were treated with a chemiluminescent reagent (Western BLoT Sensitive HRP Substrate; Takara) for enhancement, followed by analysis of blot images using a cooled charge-coupled device camera (Las 3000 Mini; Fujifilm, Tokyo, Japan) and image analysis software (Multi Gauge version 3.0; Fujifilm). MagicMark™ XP Western Protein Standard (Invitrogen, Carlsbad, CA) was used to determine protein size. Data were quantified as the ratio between the specified protein’s luminescence and α-SMA expression.

To assess collagen content in iTEV, high-performance liquid chromatography was used to measure 4-hydroxyproline in samples, as previously described [2, 5, 10]. A commercially available Fastin™ Elastin Assay kit (F2000; Biocolor, Antrim, UK) was used to quantify elastin in iTEV and native veins. Representative iTEV and native samples were weighed to obtain wet weight. Samples were then minced and digested at least three times with 0.25 M oxalic acid at 100°C for 1 h to extract mature insoluble elastin into the supernatant. Elastin content in each extract was measured using the Fastin Elastin Assay kit according to the manufacturer’s instructions, and the results were normalized to sample wet weight to yield the mass of insoluble elastin per tissue wet weight (μg/mg). The results from 12-month iTEV samples and native veins (n = 8 each) were used in this study. Absorbance was determined at 513 nm using the SpectraMax ABS Plus microplate reader (Molecular Devices, Tokyo, Japan), and all samples were tested in duplicate in each assay.

Statistical analysis

All data were tested for normality using the Shapiro–Wilk test (P < 0.05 indicates a non-normal distribution) and coefficients of skewness and kurtosis. The Wilcoxon rank-sum test was used to compare results between controls and 4-hydroxyproline or elastin samples. The Kruskal–Wallis test was used to evaluate statistical significance between samples at each time point. The Steel–Dwass test was used as a posthoc test to calculate pairwise comparisons of mean rank sums of independent samples between group levels. Spearman’s rank correlation coefficient (denoted by ρ; Rho) was used to measure the strength and direction of the association between pressure gradient and the HV index, the least estimated area of iTEV, and the IVC–HV junction angle by time (months). Furthermore, the equivalency of independent correlation coefficients was determined to examine whether correlation coefficients between HV index and pressure gradients at each month differed.

The maximum information coefficient (MIC), the maximum asymmetry score (MAS), the minimum cell number, MIC-R2, and the total information coefficient (TIC) were measured to test the linearity, monotonicity, complexity, and strength of the relationship between the pressure gradient and least estimated area of iTEV, the IVC–HV junction angle, and the HV index [11].

All data are expressed as mean ± standard error of the mean and/or median and range, and significance was assumed for differences with P values of <0.05.

All data management and analyses were conducted using Microsoft Excel (Microsoft Japan Co., Ltd., Tokyo, Japan) and R 3.5.1 (R Core Team [2018]; R: A language and environment for statistical computing; R Foundation for Statistical Computing, Vienna, Austria).

Results

Macroscopic overview of iTEV

All animals examined in this study remained alive and healthy without any serious complications until euthanasia. No ascites due to narrowing or stenosis of iTEV was noted. All animals with implanted iTEV underwent angiography and pressure studies before dissection. Scaffolds, which were sewn between the right atrium and the IVC, remodeled into native-like vessels (Fig 4B). No thrombosis or aneurysmal changes in iTEV were observed at dissection. iTEV length at dissection, when compared with the implanted scaffold length, indicated that all iTEV grafts changed in length over a period of 12 months, with this change ranging from −1.3 mm to 2.0 mm (0.463 ± 0.018 mm; n = 8).

Fig 4

Representative histological sections of iTEV at 1 month and 1 year post-implantation.

(A and B) Macroscopic view of 1-month iTEV (A) and 1-year iTEV (B). View of resected iTEV is shown in the lower panel (bars: 1 cm, RA: right atrium). (C and D) Masson’s trichrome and (E and F) Victoria blue staining. (G and H) Inflammatory cells in the specimens of iTEV were identified by immunoexpression against CD68, a known macrophage marker, in developing iTEV (1 month) and mature iTEV (1 year) (bars: 100 μm). Histological studies revealed that CD68-positive cells were expressed mainly in the vicinity of the LCL fibers and sponge, and some were observed in the neo-intima that developed on the surface of the biodegradable scaffold (⁎) (1M; G). In 1-year iTEV, CD68-positive cells were observed on the outer side of the iTEV, which consisted of the remaining scaffold or adhesive tissues (1Y; H). (I and J) α-Smooth muscle actin (SMA), (K and L) myosin heavy chain 11 (MYH11), (M and N) desmin and (O and P) vimentin immunostaining demonstrate differences in expression in vascular smooth muscle cells (VSMCs) and intermediate filaments in developing iTEV (1 month) and mature iTEV (1 year) (bars: 100 μm). α-SMA expression is clearly visible at 1 month and 1 year, but MYH11, desmin, and vimentin are not clearly observed in 1-month iTEV. Mature VSMCs and intermediate filaments are more visible in 1-year iTEV.

Angiography and relationship between pressure gradient and HV index

All animals were placed in a lateral position for angiographic and hemodynamic studies. An example of chronological changes in iTEV over time is shown in Fig 2. As shown by the yellow triangles and short dashed lines in Fig 2, all digital data were examined to calculate the smallest iTEV diameter, and the angle of the two dashed slanted lines was used to calculate HV index. Spearman’s Rho of the least estimated area of iTEV, the IVC–HV junction angle, and HV index were −0.695 (95% confidence interval [CI]: −0.792, −0.540, P < 0.001) (S2A Fig), 0.603 (95% CI: 0.40, 0.757, P < 0.001) (S2E Fig), and 0.718 (95% CI: 0.560, 0.818, P < 0.001), respectively (Table 1 and Fig 3A). As shown in Table 2, HV index showed a strong correlation with pressure gradient, monotonicity, linearity, the lowest MIC-R2 value, and the highest TIC. Considering these results and the Rho value of the HV index, we adopted HV index in this study.

Fig 2

Representative data of chronological changes in iTEV angiography in the same sample.

Left row: Yellow arrowheads indicate the site with the smallest iTEV diameter in each study. IVC: inferior vena cava, HV: hepatic vein. Right row: The angle was composed of two slanted dashed lines at the IVC–HV junction, which was measured to calculate the HV index. iTEV features, as well as native IVC, HV, and iTEV measurements change over time.

Table 1

Difference in Spearman’s Rho values by measuring methods.

	Lower 95% CI	Upper 95% CI	Rho	P value
Angle	0.405	0.757	0.603	5.41×e-07
Area	-0.792	-0.540	-0.695	1.57×e-09
HV index	0.560	0.818	0.718	< 2.20×e-16

Spearman’s Rho values of IVC–HV junction angle (Angle), least estimated area of iTEV (Area), and HV index. Lower and upper 95% confidence intervals (CIs) and P values are shown.

Fig 3

(A) Correlation between hepatic vein (HV) index and pressure gradient. Spearman’s rank correlation Rho value of the HV index is shown. Rho = 0.718 (95% confidence interval: 0.564, 0.826; P < 0.001). (B) Correlation between HV index and pressure gradient by month. In native IVC, Rho = 0.370, and in iTEV at 0.5 months, Rho = 0.709; at 1 month, Rho = 0.540; at 3 months, Rho = 0.794; at 6 months, Rho = 0.600; and at 12 months, Rho = 0.786. There was no difference in the correlation between months (P = 0.784, NS: not significant). (C and D) Changes in pressure gradient (C) and HV index (D) by month expressed as box and whisker plots. Lines represent the lower, median, and upper quartile values. Whiskers represent the extent of the remaining data. Circles represent mean values. Pressure gradient, P = 0.007; HV index, P < 0.001 using the Kruskal–Wallis test. The Steel–Dwass test was used for posthoc analysis. (C) Native vs. 1 month (*, P = 0.024). (D) Native vs. 0.5 months (**, P = 0.003); native vs. 1 month (***, P = 0.0002); native vs. 3 months (***, P = 0.0002); native vs. 6 months (***, P = 0.0002); and native vs. 12 months (***, P = 0.0007). (E and F) Each dot-line graph shows time-dependent changes in pressure gradient (E) and HV index (F) by sample.

Table 2

The linearity, monotonicity, complexity, and strength of the relationship between the pressure gradient and three different measuring methods.

	MIC	MAS	MCN	MIC.R2	TIC
Angle	1	0.090	5.322	0.590	82.27
Area	1	0.198	5.000	0.711	91.58
HV index	1	0.097	4.585	0.425	95.86

The maximum information coefficient (MIC), the maximum asymmetry score (MAS), the minimum cell number (MCN), MIC-R2, and the total information coefficient (TIC) of IVC–HV junction angle (Angle), least estimated area of iTEV (Area), and HV index are shown.

Next, we tested the correlation between HV index and pressure gradient by month. Spearman’s Rho values of HV index of native IVC and iTEV at 0.5, 1, 3, 6, and 12 months were 0.370, 0.709, 0.539, 0.794, 0.600, and 0.786, respectively (Fig 3F). When the equivalency of independent correlation coefficients was calculated, there was no difference in Spearman’s Rho value by month (Chi-squared value = 2.452, degrees of freedom = 5, P = 0.784). The pressure gradient of iTEV between the right atrium and the IVC is shown in Fig 3C. A significant difference in pressure gradient was only observed between native IVC (1.269 ± 0.236, median: 1.353, range: 0.022–2.162) and 1-month iTEV (4.693 ± 0.876, median: 4.656, range: 0.273–8.794) (P = 0.024). During tissue remodeling, although a change in scaffold diameter (S1 Fig and S1 Table) was observed, the scaffold maintained its patency with concurrent material degradation. A significant difference in HV index was observed between native IVC (0.297 ± 0.040, median: 0.301, range: 0.143–0.540) and 0.5-month iTEV (2.277 ± 0.436, median: 2.337, range: 0.281–4.543) (P = 0.003); 1-month iTEV (3.532 ± 0.589, median: 3.667, range: 0.752–6.532) (P = 0.0002); 3-month iTEV (3.381 ± 0.644, median: 3.241, range: 0.810–6.479) (P = 0.0002); 6-month iTEV (2.608 ± 0.515, median: 2.323, range: 0.599–5.056) (P = 0.0002); and 12-month iTEV (1.914 ± 0.541, median: 1.401, range: 0.644–5.126) (P < 0.001).

We measured HV index to evaluate time-dependent changes in each sample. Time-course changes in pressure gradient and HV index by sample are shown in Fig 3E and 3F. Individual differences are clearly shown because the peak pressure gradient appears between 1 and 6 months post-implantation. Furthermore, the pressure gradient of most samples decreased to native IVC levels; however, HV index recovery was not complete by 12 months post-implantation (Fig 3C to 3F).

Simultaneously, we tested the correlation between pressure gradient and the least estimated area of iTEV as well as the correlation between pressure gradient and the IVC–HV junction by month. Spearman’s Rho values of the least estimated area of native IVC and iTEV at 0.5, 1, 3, 6, and 12 months were 0.188, –0.636, –0.261, –0.503, –0.770, and –0.905, respectively (S2B Fig). When the equivalency of independent correlation coefficients was calculated, there was no difference in Spearman’s Rho value by month (Chi-squared value = 10.59, degrees of freedom = 5, P = 0.060). Spearman’s Rho values of the IVC–HV junction angle of native IVC and iTEV at 0.5, 1, 3, 6, and 12 months were 0.285, 0.503, 0.903, 0.770, 0.455, and 0.571, respectively (S2F Fig). When the equivalency of independent correlation coefficients was calculated, there was no difference in Spearman’s Rho value by month (Chi-squared value = 6.580, degrees of freedom = 5, P = 0.253).

The changes in least estimated area of iTEV and native IVC are shown in S2C Fig, and time-course changes in least estimated area by sample are shown in S2D Fig. Significant differences in the least estimated area were observed between native IVC (69.15 ± 7.014, median: 63.48, range: 45.91–124.2) and 0.5-month iTEV (29.19 ± 3.695, median: 27.81, range: 15.77–55.60) (P < 0.001); 1-month iTEV (18.21 ± 2.667, median: 16.49, range: 9.700–37.59) (P < 0.001); 3-month iTEV (18.81 ± 2.149, median: 17.92, range: 11.97–28.89) (P < 0.001); 6-month iTEV (22.90 ± 3.158, median: 22.26, range: 11.69–40.67) (P < 0.001); and 12-month iTEV (28.30 ± 4.774, median: 24.89, range: 13.71–53.24) (P = 0.002) (S2C Fig). Changes in IVC–HV angle of iTEV and native IVC are shown in S2G Fig, and time-course changes in IVC–HV angle by sample are shown in S2H Fig. Significant differences in the IVC–HV angle were observed between native IVC (18.69 ± 2.861, median: 17.03, range: 9.130–41.54) and 0.5-month iTEV (54.09 ± 6.903, median: 58.74, range: 15.63–82.47) (P = 0.013); 1-month iTEV (54.75 ± 4.620, median: 53.45, range: 28.28–71.73) (P < 0.001); 3-month iTEV (52.70 ± 6.263, median: 53.06, range: 18.31–80.35) (P = 0.003); 6-month iTEV (47.04 ± 4.990, median: 51.80, range: 24.35–67.95) (P = 0.002); and 12-month iTEV (40.84 ± 5.005, median: 39.76, range: 26.86–70.26) (P = 0.010) (S2G Fig).

Histological evaluation of iTEV

Representative macroscopic sections of iTEV at 1 month and 1 year post-implantation are shown in Fig 4A and 4B to compare SMC marker expression. Masson’s trichrome staining (Fig 4C and 4D) shows SMCs in both 1-month and 1-year iTEV. Victoria blue staining (Fig 4E and 4F) shows elastin production in 1-year iTEV. In terms of inflammatory responses in iTEV, histological studies revealed that CD68-positive cells were expressed mainly in the vicinity of the LCL fibers and sponge one month after implantation. Some CD68-positive cells were observed at the developing intima that grew on the surface of the biodegradable scaffold (Fig 4G). As the fibers degrade with time, the number of CD68-positive cells also decreased significantly with time in the vessel wall of the iTEV; however, the CD68-positive cells were located on the outer side of the iTEV, which is composed of adhesive tissues and remaining fragments of biodegradable scaffold (Fig 4H).

α-SMA-positive cells are clearly shown in both 1-month and 1-year iTEV samples (Fig 4I and 4J). In contrast, MYH11 (Fig 4K), desmin (Fig 4M), and vimentin (Fig 4O) were expressed at low levels during the differentiation phase in 1-month iTEV samples. MYH11 (Fig 4L) and desmin (Fig 4N) exhibited higher expression levels in 1-year iTEV samples compared with 1-month iTEV samples.

Increments of differentiated SMC markers to α-SMA protein

Representative immunoreactive band densitometry for MYH11 (227 kDa), desmin (54 kDa), vimentin (57 kDa), and α-SMA (40 kDa) in 1–12-month iTEV samples is shown in Fig 5A. Densitometry ratios of MYH11, desmin, and vimentin to α-SMA were measured at 1, 3, 6, and 12 months post-implantation. The Kruskal–Wallis test revealed changes in MYH11/α-SMA over time (P = 0.025), but changes in desmin/α-SMA (P = 0.121) and vimentin/α-SMA (P = 0.228) were not observed. The Steel–Dwass test revealed a difference in the densitometry ratio of MYH11/α-SMA between 1-month iTEV and native IVC (P = 0.032). A slight difference was also observed between 1-month and 12-month samples (P = 0.052).

Fig 5

Biochemical protein analysis of iTEV.

(A) Western blot immunoreactive protein bands for myosin heavy chain 11 (MYH11), vimentin, desmin, and α-smooth muscle actin (α-SMA) were measured by densitometry. Immunoreactive protein bands of MYH11 are visible in a 1-month iTEV sample. (B) Box and whisker plots of MYH11, vimentin, and desmin normalized to α-SMA in native IVC and iTEV samples by month. Lines represent lower, median, and upper quartile values. Whiskers represent the extent of the remaining data. Circles represent mean values. Desmin, P = 0.121; MYH11, P = 0.026; and vimentin, P = 0.228 using the Kruskal–Wallis test. The Steel–Dwass test was used for posthoc analysis. *, P < 0.05 vs. native IVC. Box and whisker plots of 4-hydroxyproline (C) and elastin (D) in 12-month iTEV and native IVC samples. Lines represent lower, median, and upper quartile values. Whiskers represent the extent of the remaining data. Circles represent mean values. 4-Hydroxyproline, P < 0.001; elastin, P = 0.161 using the Wilcoxon rank-sum test.

Biochemical findings

A significant difference in hydroxyproline content was found between 12-month iTEV and native IVC, with hydroxyproline content measuring 44.53 ± 8.310 μmol/g (median: 36.57 μmol/g) and 141.6 ± 15.38 μmol/g (median: 36.57 μmol/g) of wet tissue weight, respectively (Fig 5D; P < 0.001).

No difference in elastin content was observed between 12-month iTEV and native IVC, with elastin content measuring 59.55 ± 6.300 μg/g and 81.39 ± 10.78 μg/g of wet tissue weight, respectively (Fig 5E; P = 0.161).

Discussion

After several modifications to the strategy of TEG procedures, we came to use a combination of a biodegradable scaffold and mononuclear bone marrow cells as a cell source to create TEGs in animal models and in the clinical setting of congenital heart surgery [3, 4, 7]. Acceptable results were obtained late after surgery [6-9], although material modifications were still required because not all patients achieved complete repair in terms of hemodynamics and graft features.

Postoperative evaluations depend on hemodynamic tests with invasive risks and morphometrics [12]. These evaluations are difficult because TEG features change dramatically with time as tissue and extracellular matrices develop to create mature vascular tissue. The diameter, size, and features of the original biodegradable scaffold are not maintained after implantation as the tissue develops, due to the unavoidable characteristics of tissue engineering, even though most TEGs spontaneously remodel into the size required by the living organism [12].

This time-dependent variation in TEG characteristics is due to differences in inflammatory responses [3] and individual growth potential because tissue regeneration mostly depends on the self-renewal ability of an organism. Moreover, grafts mostly decrease in diameter, and tissue development is strongly related to inflammatory reactions [3–5, 13]. Increased fibrous tissue and neotissue formation, followed by wall thickening, make it difficult to decide whether graft stenosis or narrowing has occurred.

We previously reported a scaffold that has less chance of exhibiting a decrease in graft diameter without any cell preparation to create TEGs with simplicity [2, 5, 10], which we named iTEV. iTEV demonstrated histological, biomechanical, and biochemical similarities to native vessels in a canine model with acceptable results. Chronological changes in iTEV observed through morphometric and pressure studies also differ between individuals. Even though iTEV eventually remodels into native-like tissue, inflammatory reactions followed by iTEV narrowing during tissue development are unavoidable, which might be a providence of nature.

An optimal evaluation method is thus needed to prove that TEGs grow and remodel with time, especially during the iTEV tissue generation period. Furthermore, it is difficult to evaluate TEG growth in clinical situations because the natural growth of children and the morphological/hemodynamic changes of TEGs do not always occur in parallel [6-9]. Moreover, because we must determine the need for further intervention, a simple but robust evaluation method during the tissue generation period is necessary, especially in a clinical setting. Cardiac echocardiography, though a simple and repeatable method, cannot accurately estimate the pressure gradient across TEGs used in extracardiac total cavo-pulmonary connection because the method is influenced by cardiac output and respiration, as well as several patient factors and technique-related factors. Considering these points, an appropriate evaluation method for graft function, which incorporates both hemodynamic and morphometric factors, is necessary for tissue-engineering vasculature in a venous position.

We focused on the specific characteristics of the iTEV tissue-generating process to develop an evaluation method. We also learned from our previous studies that changes in graft diameter and organismal growth do not always correlate [6-9]. Additionally, to develop a novel evaluation method for the evaluation of graft function, we had to consider the characteristics of iTEV development that differ between individuals. Consequently, we constructed a formula to calculate comparable data in each individual that are not influenced by the morphometrics and hemodynamics of iTEV, or by changes in body size. As shown in this study, HV index and pressure gradient are strongly associated; thus, we are convinced that HV index should be assessed as one of the evaluation methods during patient follow up after iTEV implantation.

We here demonstrate the usefulness of HV index in an animal model. We observed hemodynamic improvement, iTEV remodeling, and an increase in graft diameter simultaneously in this study. Tissue development differs between individuals, and HV index identified time-dependent changes in iTEV in individual samples. Moreover, HV index does not depend on the features of iTEV itself because it requires only the smallest graft diameter according to imaging data. Furthermore, the IVC–HV junction angle is not influenced by body size. Thus, no matter how iTEV or the living body changes, previous and upcoming data of the HV index are comparable to evaluate changes in iTEV. With the smallest internal diameter and the IVC–HV junction angle, the HV index can be calculated using imaging data. This means that data obtained from different modalities, such as computed tomography and magnetic resonance imaging, could be comparable.

Novel TEG evaluation methods must be developed to achieve robust graft assessment and to demonstrate tissue generation and growth. A combination of routine pressure studies and measurement of objective data using non-invasive examinations to demonstrate TEG growth are desired, since it is difficult to explain TEG features by examining morphometry or hemodynamic metrics alone, especially in humans [12]. Changes in TEG size and patient growth, which influence hemodynamics, do not always occur in parallel [6, 8, 9]; thus, data do not always show TEG growth with good hemodynamic conditions. More specifically, size changes in the TEG do not always involve a reduction in the pressure gradient across the vasculature, and TEG function cannot be evaluated without hemodynamic metrics. However, systemic blood volume, pulmonary vascular resistance, cardiac function, sedation, and anesthetic agents also influence the pressure gradient during catheterization. Invasive hemodynamic monitoring is not a repeatable test because it poses several risks in children. Furthermore, repeat catheterization, which is invasive and sometimes has catastrophic adverse outcomes in children, should be avoided.

We set the standard limit of pressure gradient at 2 mmHg in a previous clinical trial of TEGs. However, this is not always appropriate because some patients require repeat intervention to minimize the pressure gradient across the vasculature during growth [6-9]. Furthermore, some patients developed unwanted veno-venous shunts, even with a low-pressure gradient across the TEG. Thus, we must decide whether changes in TEG diameter are due to growth and whether patients require further invasive postoperative intervention to recover graft stenotic changes, especially in a clinical setting. It is difficult to decide whether patients require intervention using classic pressure studies and the smallest TEG diameter value, due to aforementioned changes in TEG features and patient growth over time.

HV index demonstrated a good correlation with pressure gradient across the iTEV, and therefore changes in HV index may also be associated with an improvement/worsening in congestion in organs of the lower body, such as the liver, caused by iTEV narrowing or stenosis during tissue development. Furthermore, we show that HV index accurately reflects the in-vivo situation; specifically, HV index recovered more slowly compared with the pressure gradient.

Given the above, we present HV index as a novel measurement to evaluate iTEV (and TEGs) in terms of tissue development, growth, and remodeling. Additionally, the success of new vascular tissue formation will ultimately depend on the success of tissue regeneration and on the ability of the scaffold to meet the essential requirements of hierarchical composites of vascular tissue in each individual living organism. The increase in mature VSMC proliferation observed in this study confirmed that the results of the HV index are robust and objective, thereby demonstrating its usefulness for decision making in graft evaluation. We speculate that HV index would suggest not only graft growth and hemodynamic improvement, but also the efficacy of catheter intervention when introduced in a clinical setting. Moreover, we speculate that HV index could be applied to evaluate TEGs and for careful patient monitoring, such as detecting warning signs of graft narrowing or stenosis, evaluating pulmonary vascular resistance, and monitoring cardiac function, especially after the Fontan procedure in patients with congenital heart disease who undergo routine artificial conduit grafting.

Study limitations

The present study is limited due to the small number of HV index measurements. Furthermore, angiographic data were measured only in a lateral position in this study. Therefore, measurements of IVC–HV junction angle in biplane studies might be preferable to calculate HV index for better evaluation of iTEV development.

Conclusions

Tissue-engineered vasculature has specific dynamics after implantation, and therefore a novel evaluation method is desired for safe medical monitoring, especially in a clinical setting. HV index is a less invasive measurement modality compared with cardiac catheterization; it is not influenced by organismal growth and can easily identify improvements in TEGs by comparison with previous data. We believe that the HV index may yield great benefit postoperatively in children who require TEG implantation therapy.

Minimal diameter of iTEV by each sample.

Time-course changes in minimal diameter of iTEV by sample are shown.

(TIF)

Click here for additional data file.

(A) Correlation between the least estimated area of iTEV and pressure gradient. Spearman’s rank correlation Rho value of the HV index is shown. Rho = -0.695 (95% confidence interval: -0.792, -0.540; P < 0.001). (B) Correlation between the least estimated area and pressure gradient by month (Estimated Rho = -0.542). In native IVC, Rho = 0.188, and in iTEV at 0.5 months, Rho = -0.636; at 1 month, Rho = -0.261; at 3 months, Rho = -0.503; at 6 months, Rho = -0.770; and at 12 months, Rho = -0.904. There was no difference in the correlation between months (P = 0.060, NS: not significant). (C) Changes in the least estimated area of iTEV by month expressed as box and whisker plots. Lines represent the lower, median, and upper quartile values. Whiskers represent the extent of the remaining data. Circles represent mean values. The least estimated area, P < 0.001 using the Kruskal–Wallis test. The Steel–Dwass test was used for posthoc analysis. Native vs. 0.5 month (*, P < 0.001); native vs. 1 month (***, P < 0.001); native vs. 3 months (***, P < 0.001); native vs. 6 months (***, P < 0.001); and native vs. 12 months (***, P = 0.0016). (D) Each dot-line graph shows time-dependent changes in the least estimate area by sample. (E) Correlation between the IVC-HV junction angle and pressure gradient. Spearman’s rank correlation Rho value of the HV index is shown. Rho = 0.603 (95% confidence interval: 0.405, 0.757; P < 0.001). (F) Correlation between the IVC-HV junction angle and pressure gradient by month (Estimated Rho = 0.638). In native IVC, Rho = 0.285, and in iTEV at 0.5 months, Rho = 0.503; at 1 month, Rho = 0.903; at 3 months, Rho = 0.770; at 6 months, Rho = 0.455; and at 12 months, Rho = 0.571. There was no difference in the correlation between months (P = 0.254, NS: not significant). (G) Changes in the IVC-HV junction angle of iTEV by month expressed as box and whisker plots. Lines represent the lower, median, and upper quartile values. Whiskers represent the extent of the remaining data. Circles represent mean values. The IVC-HV junction angle, P < 0.001 using the Kruskal–Wallis test. The Steel–Dwass test was used for posthoc analysis. Native vs. 0.5 month (*, P = 0.013); native vs. 1 month (***, P < 0.001); native vs. 3 months (**, P = 0.003); native vs. 6 months (**, P 0.002); and native vs. 12 months (*, P = 0.010). (H) Each dot-line graph shows time-dependent changes in the IVC-HV junction angle by sample.

(TIF)

Click here for additional data file.

Raw data of minimal diameter of iTEV.

Raw data of time-course changes in minimal diameter of iTEV by dample are shown.

(TIF)

Click here for additional data file.

2 Feb 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Atsushi Asakura, Ph.D

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf"

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

3. Thank you for stating the following financial disclosure:

“This work was supported in part by KAKENHI (Grant-in-aid for Scientific Research) of the Japan Society for the Promotion of Science (JSPS) (C: number 17K10739, C: number 23591878) from The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and in part by an Open Research Grant from the Japan Research Promotion Society for Cardiovascular Diseases (2018 and 2019). Any opinions, findings, conclusions, or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the author(s)’ organization(s), JSPS, or MEXT.”

Please state what role the funders took in the study.  If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.""

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

4. Thank you for stating the following in the Funding Section of your manuscript:

“This work was supported in part by KAKENHI (Grant-in-aid for Scientific Research) of the Japan Society for the Promotion of Science (JSPS) (C: number 17K10739, C: number 23591878) from The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and in part by an Open Research Grant from the Japan Research Promotion Society for Cardiovascular Diseases (2018 and 2019). Any opinions, findings, conclusions, or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the author(s)’ organization(s), JSPS, or MEXT.”

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“This work was supported in part by KAKENHI (Grant-in-aid for Scientific Research) of the Japan Society for the Promotion of Science (JSPS) (C: number 17K10739, C: number 23591878) from The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and in part by an Open Research Grant from the Japan Research Promotion Society for Cardiovascular Diseases (2018 and 2019). Any opinions, findings, conclusions, or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the author(s)’ organization(s), JSPS, or MEXT.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

6. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

7. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript reported the new evaluation method of tissue engineered blood vessels in the IVC position in dog model. Twenty-six animals underwent IVC replacement surgeries with in-site tissue-engineered vasculature (iTEV). These data are valuable and important for all the scientists working on vascular tissue engineering. However, there are several questions and concerns in this manuscript.

Their data is not supporting their conclusion because control data is missing. The title is also misleading and not appropriate from their data (it is complicated, not simple and nobody would use this index as this is only applicable in dog model). In addition, the reviewer do not think that this HV index is useful for evaluation of iTEV. The HV angle would be changed by the timing of respiration, table rotation angle at angiography. Even individual anatomical variety would be also related. The reviewer also could not understand the reason why they plan to utilize their own Index, because simplest evaluation method would be the measurement of vascular diameter and length over time. This manuscript also need additional information in control animal group without spiral reinforcement to prove their concept.

I have suggestions and questions to improve this manuscript.

1. Describe the narrowest diameter of tubular grafts after implantation over time., in all cases.

2. Describe the age of animals at the implantation of iTEV.

3. Describe the changes in body weight of animals at 12 months.

4. Describe the reason why the authors used many different size grafts from 23-33 mm in length, although animal body weight is very similar. They should have used the graft with same diameter and same length.

5. HV Index means the angle divided by narrowest area. Describe the reason why the authors did not check the co-relation between the angle and PG, or between narrowest area and PG.

6. Show the angiography and HV Index of control animal (Gore-Tex tube interposition) without any stenosis in Figure 2.

7. If the authors believe this HV Index is useful to evaluate the stenosis of the grafts, they can make a simulation banding model in control animals instead of graft implantation. Please comment on this issue.

8. When revising this manuscript, the reviewer would recommend that the authors should forget and remove this index part, just focus on the remodeling process of iTEV in histology and mechanical properties over time.

9. Describe the reason why the authors chose spiraled shape for reinforcement instead of ringed grafts.

10. Explain the reason why the fiber should be colored in blue.

11. In the abstract and Figure 4, the authors followed the animal at 0.5, 1, 3, 6, and 12 months, but in the Materials and Methods part (page 5), there are no mentioning about the group of 0.5 month. Did the authors survive animals after catheterization and angiography at 0.5 month after implantation? Or The authors made additional group?

12. In figure 4, histology should be shown with macroscopic pictures. 1month’s data looked like more stenotic from the data in Figure 3. Inflammatory cells like macrophage should be stained at 1 month and 1 year.

13. When the authors compare the histology and PG at 1 month and 3 months after implantation, is there any relations between the inflammation & thickening of tissue and PG?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

17 Mar 2022

Response to the Reviewer:

Reviewers' comments:

Reviewer #1: This manuscript reported the new evaluation method of tissue engineered blood vessels in the IVC position in dog model. Twenty-six animals underwent IVC replacement surgeries with in-site tissue-engineered vasculature (iTEV). These data are valuable and important for all the scientists working on vascular tissue engineering. However, there are several questions and concerns in this manuscript.

Their data is not supporting their conclusion because control data is missing. The title is also misleading and not appropriate from their data (it is complicated, not simple and nobody would use this index as this is only applicable in dog model). In addition, the reviewer do not think that this HV index is useful for evaluation of iTEV. The HV angle would be changed by the timing of respiration, table rotation angle at angiography. Even individual anatomical variety would be also related. The reviewer also could not understand the reason why they plan to utilize their own Index, because simplest evaluation method would be the measurement of vascular diameter and length over time. This manuscript also need additional information in control animal group without spiral reinforcement to prove their concept.

I have suggestions and questions to improve this manuscript.

1. Describe the narrowest diameter of tubular grafts after implantation over time, in all cases.

We appreciate your suggestion. To describe the narrowest diameter of iTEV after implantation over time, we have constructed a figure and table that include these data. Please see S1: Fig 1, Minimal diameter of iTEV and S2: Table 1, Numerical raw data of minimal diameter of iTEV.

2. Describe the age of animals at the implantation of iTEV.

In accordance with your suggestion, we have added information regarding the age of the animals at the implantation of iTEV. Please see page 7, line number 149–151 of the revised manuscript.

3. Describe the changes in body weight of animals at 12 months.

We apologize that we do not have accurate data regarding this point. However, we measured the body weight of each animal upon every catheter examination to determine the adequate dose of the anesthetic agents. Thus, we note that the body weight at 12 months did not change markedly when compared with the body weight upon the initial surgery.

4. Describe the reason why the authors used many different size grafts from 23-33 mm in length, although animal body weight is very similar. They should have used the graft with same diameter and same length.

We used the same diameter of the scaffold for each implantation; however, in terms of length, we placed the vascular clamp as distal as possible to implant a longer scaffold. Because of anatomical variations, the length of the implanted scaffold differed.

5. HV Index means the angle divided by the narrowest area. Describe the reason why the authors did not check the co-relation between the angle and PG, or between narrowest area and PG.

To this point, we have added Tables 1 and 2 to demonstrate that the HV index yielded the best results to speculate the PG of iTEV. In accordance with your suggestion, we have also included data regarding the correlation between the angle and PG, and between the narrowest area and PG calculated with Spearman’s rank correlation. Please see S3 Fig.

6. Show the angiography and HV Index of control animal (Gore-Tex tube interposition) without any stenosis in Figure 2.

Thank you for your suggestion. We have revised Figure 2 to include the HV index of the control animal.

7. If the authors believe this HV Index is useful to evaluate the stenosis of the grafts, they can make a simulation banding model in control animals instead of graft implantation. Please comment on this issue.

It is valuable to know the relationship between the minimal diameter of iTEV and PG; however, in an acute experiment, the angle of HV will not change. We believe that the relationship between banded diameter and PG can be tested in a mock circuit, and therefore unnecessary animal experiments can be avoided. In terms of experiments that involve banding the IVC of the experimental animals, though the response is dependent on the tightness of the banding, we believe that banding the IVC results in rapid stress to the liver of the animal. If it is possible to control the banding after the operation through such means as adjustable banding, and if we are able to obtain adequate data with intermittently repeated catheter studies, then this approach may serve as a good control. However, considering animal welfare, and the principle goal of lessening the number of experiments performed on animals that involve the sacrifice of life, it might not be a useful experiment. Conversely, iTEV decreases its internal diameter, but the changes are gradual and followed by improvement. Accordingly, the experimental animals are able to endure the biological responses to the reduction of iTEV diameter over time.

8. When revising this manuscript, the reviewer would recommend that the authors should forget and remove this index part, just focus on the remodeling process of iTEV in histology and mechanical properties over time.

The concept of this study is to explore a method that is not influenced by the morphological/hemodynamical changes of iTEV and/or size of the living body to examine small changes in iTEV over time through imaging data and demonstrate iTEV improvement. Thus, we evaluated the correlation between the pressure gradient (PG) of iTEV and several measurements obtained by imaging data to speculate PG differences in the time course. As the reviewer has mentioned, the HV index seems to be applicable only in the dog model. However, we believe that the HV index can also be applied in patients who have undergone total cavo-pulmonary connection (TCPC: Fontan type operation). According to our previous data in the clinical trials, when images from catheter study, 3D-image computed tomography, and/or magnetic resonance imaging are acquired from the front side, we can calculate the HV index to compare the present and previous data to evaluate the improvement/worsening of hemodynamics. Additionally, there is a chance to avoid repeated catheter studies in the patient with this method. Furthermore, in terms of laboratory experiments, histological developments can be speculated without the sacrifice of the experimental animals. In terms of manufacturing the new scaffold, this method can be applied to assess the quality of improved scaffolds in this IVC model, with less sacrifice of experimental animals. Because the changes in vascular diameter are so small, as shown in the present study, it is difficult to determine the remodeling process without catheter pressure study; nonetheless, catheter pressure study and angiography are affected by factors including respiration, the direction of irradiation, and changes in physical status. Thus, we concluded that the HV index is useful, but because several pitfalls of catheter pressure study remain, we performed angiography upon breath-holding to avoid respiratory fluctuation.

Furthermore, when the biodegradable scaffold is applied in a patient who requires TCPC, there are no data to indicate the suitable size of the graft in growing patients with a size-changing tissue-engineered graft. Thus, a post-operative monitoring method to evaluate the condition of the tissue-engineered graft is desirable. There are multiple factors that influence the pressure gradient of the graft in Fontan patients, including pulmonary resistance, left side AV valve regurgitation, LV end-diastolic pressure, LV function, and collaterals. With a graft diameter and/or PG, total graft function is not fully evaluated. Thus, we performed this study to explore a new evaluation method. For these reasons, we would like to include the HV index in this manuscript.

9. Describe the reason why the authors chose spiraled shape for reinforcement instead of ringed grafts.

In our experimental design, we initially attempted to create a biodegradable scaffold that imitated a ringed Gore-Tex graft. It is possible to create ring-shaped biodegradable material; however, it is impossible to achieve similar mechanical strength with biodegrading ring-shaped materials. Furthermore, it was difficult to determine the optimal ring-to-ring distance. Ultimately, it was difficult to use the ring-shaped structure in our manufacturing process. Because we have studied vascular tissue engineering with spiraled biodegradable scaffolds since 2005 using the same implantation methods and protocol, we elected to use the fiber created with p(LA/CL) with marketing approval in this study.

10. Explain the reason why the fiber should be colored in blue.

As mentioned above, we used fiber from commercially available surgical sutures as a raw material. There is no reason for the specific color of the fiber. However, the use of colored fiber is advantageous because it facilitates easier recognition of the fiber’s location and helps prevent pricking of the rigid fiber when suturing the graft.

11. In the abstract and Figure 4, the authors followed the animal at 0.5, 1, 3, 6, and 12 months, but in the Materials and Methods part (page 5), there are no mentioning about the group of 0.5 month. Did the authors survive animals after catheterization and angiography at 0.5 month after implantation? Or The authors made additional group?

As mentioned in page 8 line 169-, 10 dogs underwent catheterization study at 0.5, 1, 3, 6, and 12 months. We did not include 0.5-month iTEV samples in histological and biochemical studies because our previous studies showed that two weeks post-implantation was an insufficient amount of time to observe histological/biochemical changes in the iTEV. Furthermore, excluding this time point allowed us to minimize the number of the experimental animals to be sacrificed.

12. In figure 4, histology should be shown with macroscopic pictures. 1month’s data looked like more stenotic from the data in Figure 3. Inflammatory cells like macrophage should be stained at 1 month and 1 year.

In accordance with your helpful advice, we have revised Figure 4 to include macrophage evaluation at 1 month and 1 year. Thank you for your suggestion.

13. When the authors compare the histology and PG at 1 month and 3 months after implantation, is there any relations between the inflammation & thickening of tissue and PG?

Thank you for this insightful question. As shown in Figure 3 and S1 Fig, the time-dependent changes in pressure gradient, minimal diameter of iTEV, HV index, etc., differ in each sample and have no regularity in terms of tissue regeneration, especially within 6 months after implantation. This might be related to the differences in inflammatory response in each individual sample. According to our previous study, the wall thickness of iTEV seems strongly related to the internal diameter; thus we used the minimal area of iTEV to calculate the HV index. Conversely, we might speculate the wall thickness of iTEV from the internal diameter. However, the relationship between PG and wall thickness of iTEV was not evaluated in this study because we did not sacrifice animals at 1 and 3 months for the HV index study; therefore, some of the opinions written here are only speculative.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

6 Apr 2022

Evaluation method for cell-free in-situ tissue-engineered vasculature monitoring: proof of growth and development in a canine IVC model

PONE-D-21-26967R1

Dear Dr. MATSUMURA,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Atsushi Asakura, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

8 Apr 2022

PONE-D-21-26967R1

Evaluation method for cell-free in situ tissue-engineered vasculature monitoring: proof of growth and development in a canine IVC model

Dear Dr. MATSUMURA:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Atsushi Asakura

Academic Editor

PLOS ONE