Biomolecules, Vol. 12, Pages 1776: Evaluation of Biointegration and Inflammatory Response to Blood Vessels Produced by Tissue Engineering—Experimental Model in Rabbits

1. IntroductionThe treatment of peripheral arterial disease (PAD) [1] with limb-threatening ischemia is complex, and surgical revascularization is often needed [2,3]. In cases where it is not possible to use an autologous vein for a conventional bypass, synthetic arterial substitutes, such as Dacron® or polytetrafluoroethylene (PTFE) prostheses can be used [4]. The major limitations for the use of synthetic material derive mainly from infections of the prosthesis or at the incision site, as well as the high risk of contamination of the prosthesis from distant infections [3]. In addition, for below-knee prosthetic bypass there is the limitation of poor arterial outflow or incompatibility between caliber and flow [2,4]. Endovascular procedures can be conducted but sometimes have limitations too. When revascularization is indicated but not carried out, the risk of amputation greatly increases [2]. Therefore, the tissue engineering of blood vessels (TEBV) represents a promising perspective to produce personalized vascular substitutes to be used in revascularization surgeries [5]. A major challenge for TEBV to widely reach clinical practice is the production of a functional, resistant, and three-dimensional blood vessel [6,7]. For that, the basis of development requires that the scaffold (synthetic or biological) be sufficient to provide the necessary support for the implanted stem cells to differentiate, renew the extracellular matrix (ECM) and do not induce immunological reactions [8,9,10].The use of decellularized blood vessels as scaffolds presents some initial biological advantages for TEBV [6,9]. They harbor their own characteristics such as the composition and resistance of the ECM [5,9]. However, for the use of biological material as a scaffold, it is necessary to attain efficient decellularization that guarantees an adequate elimination of cell surface antigens to avoid an immune-mediated rejection of the transplanted tissue and, consequently, the risks of graft loss, including the risk of death or contamination [11,12,13,14].

This study intended to evaluate 3D structures produced by the decellularization of rabbits’ inferior vena cava (IVC) with 1% sodium dodecyl sulfate (SDS) for 2 h (scaffolds), repopulated or not with ASCs (autologous and allogeneic) compared to allogeneic IVCs, implanted in the back of rabbits, maintained for 60 days, to study the inflammatory patterns and the behavior and reactions induced by these structures in the living organism.

2. Materials and Methods 2.1. Animal Handling Conditions and Tissue Acquisition

A total of 18 adult New Zealand rabbits were used, all nonpregnant females weighing between 2.5 and 3.5 kg. The IVCs were obtained from 6 donor animals, and 2 g of adipose tissue (AT) was also obtained from two of these animals to generate the allogeneic cell bank; one animal was the donor of allogeneic IVC to be used in natura at the time of implantation.

The animals were housed under controlled conditions and fed a standard pellet diet with water ad libitum. Before the surgical procedures to obtain the AT and IVC, the animals were given an intramuscular combination of ketamine 10 mg/kg (Dopalen®, 100 mg/mL, Paulínia, SP, Brazil), xylazine hydrochloride 3 mg/mL kg (Anasedan® 2%, 20 mg/mL, Paulínia, SP, Brazil), and acepromazine 0.1 mg/kg (Apromazin® 0.2%, 200 mg/mL, Barueri, SP, Brazil). In addition, a local anesthetic injection, lidocaine 7 mg/kg (Xylocaine 1% 20 mg/mL, Cristália, Butantã, SP, Brazil) was administered at the site designed for the surgical incision. Harvesting was carried out under aseptic conditions and at the end of the procedure, the animals were euthanized with pentobarbital.

2.2. Scaffolds ProductionThe IVCs were sectioned into segments of approximately 2 cm in length, totaling 12 fragments. Decellularization was carried out using the protocol with 1% SDS for 2 h under shaking in a Shaker (News Brunswick Scientific® Edison, NJ, USA), at 37 °C. (Supplementary file S1) [9,13,15]. The fragments were stored in a refrigerator at 4 °C in a sterile solution containing antibiotics and fungicide. 2.3. Obtaining Adipose-Derived Stem Cells (ASCs)

In order to obtain autologous ASCs, the animals were anesthetized, then 2 g of AT was surgically removed from the interscapular region and stored in a conical tube containing a solution of N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) supplemented with penicillin, 100 mg/mL streptomycin, and 25 mg/mL amphotericin B (2 mmol/L 1-glutamine; Invitrogen™, Waltham, MA, USA). ASCs were obtained by enzymatic dissociation with collagenase type I (Invitrogen™, Waltham, MA, USA). Cell culture procedures were performed with an initial cell count of 6 × 104 cells/cm2, obtained from 12 fragments of adipose tissue. These cells were seeded and expanded in 25 cm2 culture flasks using Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin, 25 mg/mL amphotericin B (2 mmol/L of l-glutamine; Invitrogen™, Waltham, MA, USA), 1% (v/v) of minimal essential medium (MEM), an essential amino acid solution (Invitrogen™, Waltham, MA, USA), and 0.5% (v/v) of 10 mM MEM nonessential amino acid solution (Invitrogen™, Waltham, MA, USA) until the number of cells required for the entire study was reached. ASCs were analyzed phenotypically by flow cytometry (FC) using CD45, CD44, CD90, and CD11b, and by tri-lineage differentiation techniques (StemPro™ adipogenesis, chondrogenesis, and osteogenesis differentiation kits; Invitrogen, Waltham, MA, USA).

2.4. Experimental Groups

For all experiments, the vessels (with or without cells) were surgically fixed in the subcutaneous tissue over the dorsal muscular fascia. Four groups were formed (in triplicate)—group 1: implantation of a fragment of allogeneic inferior vena cava in natura (allogeneic IVC); group 2: implantation of a decellularized vein fragment with SDS without the addition of cells (decellularized IVC-SDS); group 3: implantation of a decellularized vein fragment with SDS + 1 × 105 allogeneic ASCs (decellularized IVC-SDS + allogeneic ASCs); group 4: implantation of a decellularized vein fragment with SDS + 1 × 105 autologous ASCs (decellularized IVC-SDS + autologous ASCs).

2.5. Biological Scaffold and ASCs Seeding

The ASCs (1.3 × 105) were submitted to the cytoplasmic labeling process with Qtracker 605 (CellLabeling Kits—Invitrogen™, Waltham, MA, USA). A solution containing 10 nM of Qdots (1 µL of solution A mixed with 1 µL of solution B, for each 1 × 105 of cells), added in 200 µL of DMEM was used. This solution was homogenized with the cell pellet previously obtained and kept in a 5% CO2 incubator at 37 °C for a period of 45 min, protected from light. After the incubation period, the ASCs were washed with D-PBS and diluted in PuraMatrix® peptide hydrogel (BD Biosciences, Franklin Lakes, NJ, USA). A total of 10 µL of this solution was pipetted into the lumens of each scaffold. The experiment was carried out in triplicate, maintained for 2 h in culture for cell preadherence in a DMEM culture medium in a CO2 incubator. With a small fragment of the scaffold with ASCs, an immunofluorescence (IF) analysis was performed with Fluoroshield™ with DAPI (Sigma, San Luis, MO, USA) to visualize the ASC nuclei to confirm the beginning of scaffold recellularization.

2.6. Surgical Experimentation

For the implantation of the grafts, the animals were anesthetized as previously described, and a surgical aseptic technique was used. A small longitudinal incision, 2 cm in length, was done in the dorsal thoracic region of the animal, dissecting up to the limit of the most superficial fascia of the latissimus dorsi, where the study fragments were fixed (only one fragment per animal) with a simple nonabsorbable stitch of NYLON 4-0 (Ethicon®, Somerville, NJ, USA) at each end of the implant, for identification at the time of explantation. The animals were kept in special housing as previously described and monitored daily, being euthanized after the 60-day observation period. The fragments were surgically collected for a histomorphological analysis.

2.7. Analysis of Interleukins and Oxidative Stress

During the monitoring period, 1.5 mL of peripheral blood was collected from the marginal ear vein of each animal before the procedure and 1, 7, 14, 30, and 60 days after the surgical procedure. Each sample was centrifuged at 2000× g for 5 min to obtain plasma, then kept frozen at −80 °C until inflammatory interleukins (IL6, TNF-α) and anti-inflammatory interleukins (IL-4, IL10) were measured by an enzyme-linked immunosorbent assay (ELISA) (all of them, Invitrogem™, Waltham, MA, USA). At the end of the study, one fragment of the implanted tissue, 0.5 cm in length, was subjected to maceration and the same interleukins were evaluated.

In addition, analyses were conducted for the detection of oxidative stress by the method of the malonaldehyde (MDA) reaction with thiobarbituric acid (TBARS) [16]. The MDA dosage of the samples was intended to assess oxidative stress variations caused by implants. 2.8. Histomorphology

The collected fragments (60 days after implantation) were sectioned into two equal parts, one being placed in 10% buffered formaldehyde to make paraffin-embedded blocks, and the other kept frozen for a fresh frozen section stained with Qtracker (red) under a fluorescence microscope, to identify the ASCs, in addition to being macerated later to measure tissue ILs.

Slides were prepared from the paraffin blocks for standard hematoxylin–eosin (HE) histology and for immunohistochemistry (IHQ) with the analysis of anti-CD31 primary marker diluted 1 in 10 (Novus Biologicals™, Englewood, CO, USA) and marked with secondary antibody Dako EnVision™ + Dual Link System-HRP for the identification of endothelial cells. The slides made with fresh material were destined for a blinded analysis of cell nuclei by immunofluorescence with the 4′,6′-diamino-2-phenyl-indole (DAPI) fluoroshield sealant (Sigma™, Cotia, SP, Brazil). In addition, separate slides with anti-CD31 stained with Fluorescein-5-isothiocyanat (FITC) were made and taken for photomicrographs under a confocal microscope (LEICA™ TSC SP8, Wetzlar, Germany).

2.9. Statistical Analysis

The comparison between means at time points was conducted using a repeated measures design to assess the interaction between groups versus time points. In cases where the data presented a symmetrical distribution, an ANOVA for repeated measures was used followed by Tukey’s multiple comparison test adjusted to the design. When the distribution was skewed, a gamma distribution adjustment was conducted for the same design, followed by Wald’s multiple comparison tests. In all tests, the significance level was set at 5%. All analyses were conducted using SAS for Windows, v.9.4 (SAS software for Windows version 9.4, SAS Institute Inc, Cary, NC, USA).

4. DiscussionThe conventional treatment for PAD is clinical, aiming at the control of risk factors. In a situation of critical ischemia, however, the alternatives are endovascular treatments with angioplasty or revascularization surgeries (bridges/bypass using an autologous vein or synthetic material) [2]. However, for a portion of patients, revascularization treatments are not applied, increasing the risk for limb amputations [4]. Consequently, biotechnology becomes a partner for the development of biomimetic products, and numerous studies have pointed to the possibility of the production of organs or tissues that may be promising for the regeneration or replacement of damaged vessels, considering that there is already enough technology for the differentiation of ASCs into endothelium, smooth muscle, and other tissues [17,18,19,20].In a review article, Afra et al. [21] compared the results of preclinical research with the use of autologous or allogeneic ASCs for the development of blood vessels and concluded that autologous cells are preferred. Our work goes in the same direction, pointing out the advantages of the use of autologous cells. In that same review, the authors studied the potential of ASCs in engineering functional blood vessels compared to other cell types, such as bone marrow mononuclear cells, endothelium precursor cells, adult autologous smooth muscle cells, autologous endothelial cells, embryonic stem cells, and pluripotent stems [21]. It was concluded that ASCs were still the preferred cells for tissue engineering of blood vessels, mainly because of their multipotent potential and ease of obtaining them in quantity. These reasons also guided our work. ASCs can also secrete a broad spectrum of bioactive macromolecules that help the regeneration of injured tissues and have immunomodulatory properties [21].The most suitable scaffolds for tissue engineering of blood vessels should be able to withstand vascular stress without degeneration, do not promote thrombotic events, present good interaction with adjacent tissues and implanted cells, and enable nutrient transport through the porosity of their wall until they are fully developed [20]. More than one of our previous studies [15] have determined that the method used in the production of a biological scaffold produced by the decellularization of the vena cava in the rabbit model meets these requirements, and the current study demonstrates that the interaction with autologous ASCs promotes a favorable environment, generating the ideal adaptive conditions when it is implanted on the animal’s back.The immune response triggered by the implantation of scaffolds in the in vivo animal model varied between groups and according to the form of analysis. For the evaluation of serum results, no significant differences were found between the groups at the times analyzed, which could be explained by the volatility of the production of these cytokines after the first hours of surgery, the small aggression caused by the surgery, and by the small amount of tissue implanted. For the analysis of the homogenate of part of the tissue collected 60 days after implantation, unlike the serum analysis, there was some difference between the groups even in the latest period assessed. A greater proinflammatory reaction, mediated by TNFα, was found in group 2, which contained only decellularized tissue compared to the other groups, but this was not seen in the assessment of IL6. Such finding is expected since the scaffold basically behaves like a foreign body [22]. However, a decrease in the IL10-mediated anti-inflammatory action was seen in the animals of group 3, which received allogeneic ASCs, which may be a sign of a delayed rejection reaction [23,24]. Interestingly, our experiments did not detect serum or tissue IL4 in any group. IL4 was previously selected to assess whether ASCs would have any anti-inflammatory activity in the experiment with biological scaffolds. However, we could not find significant levels of IL4 in any of our experiments; perhaps this can be explained by the volatility of the cytokine in the samples, the sensitivity level of the test, or the animal model used, or technical problems in the experiment; however, this question remains open [25]. Regardless, the immunomodulatory role of ASCs has already been established in experiments of this nature, and it is likely that they perform this function while not differentiating in other cell types [26,27,28,29].

Regarding the cellular differentiation of ASCs and the structural organization of these neovessels, the implants of groups 3 and 4, composed of decellularized vena cava + ASCs, were more promising. The presence of these red-marked cells, derived from viable cultures, was detected in the tissue even after 60 days of implantation.

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