Effect of luminal surface structure of decellularized aorta on thrombus formation and cell behavior.

Due to an increasing number of cardiovascular diseases, artificial heart valves and blood vessels have been developed. Although cardiovascular applications using decellularized tissue have been studied, the mechanisms of their functionality remain unknown. To determine the important factors for preparing decellularized cardiovascular prostheses that show good in vivo performance, the effects of the luminal surface structure of the decellularized aorta on thrombus formation and cell behavior were investigated. Various luminal surface structures of a decellularized aorta were prepared by heating, drying, and peeling. The luminal surface structure and collagen denaturation were evaluated by immunohistological staining, collagen hybridizing peptide (CHP) staining, and scanning electron microscopy (SEM) analysis. To evaluate the effects of luminal surface structure of decellularized aorta on thrombus formation and cell behavior, blood clotting tests and recellularization of endothelial cells and smooth muscle cells were performed. The results of the blood clotting test showed that the closer the luminal surface structure is to the native aorta, the higher the anti-coagulant property. The results of the cell seeding test suggest that vascular cells recognize the luminal surface structure and regulate adhesion, proliferation, and functional expression accordingly. These results provide important factors for preparing decellularized cardiovascular prostheses and will lead to future developments in decellularized cardiovascular applications.

In vitro evaluation of surface biological properties of decellularized aorta for cardiovascular use.

The aim of this study was to determine an in vitro evaluation method that could directly predict in vivo performance of decellularized tissue for cardiovascular use. We hypothesized that key factors for in vitro evaluation would be found by in vitro assessment of decellularized aortas that previously showed good performance in vivo, such as high patency. We chose porcine aortas, decellularized using three different decellularization methods: sodium dodecyl-sulfate (SDS), freeze-thawing, and high-hydrostatic pressurization (HHP). Immunohistological staining, a blood clotting test, scanning electron microscopy (SEM) analysis, and recellularization of endothelial cells were used for the in vitro evaluation. There was a significant difference in the remaining extracellular matrix (ECM) components, ECM structure, and the luminal surface structure between the three decellularized aortas, respectively, resulting in differences in the recellularization of endothelial cells. On the other hand, there was no difference observed in the blood clotting test. These results suggested that the blood clotting test could be a key evaluation method for the prediction of in vivo performance. In addition, evaluation of the luminal surface structure and the recellularization experiment should be packaged as an in vitro evaluation because the long-term patency was probably affected. The evaluation approach in this study may be useful to establish regulations and a quality management system for a cardiovascular prosthesis.

Elastic Modulus of ECM Hydrogels Derived from Decellularized Tissue Affects Capillary Network Formation in Endothelial Cells.

Recent applications of decellularized tissue have included the use of hydrogels for injectable materials and three-dimensional (3D) bioprinting bioink for tissue regeneration. Microvascular formation is required for the delivery of oxygen and nutrients to support cell growth and regeneration in tissues and organs. The aim of the present study was to evaluate the formation of capillary networks in decellularized extracellular matrix (d-ECM) hydrogels. The d-ECM hydrogels were obtained from the small intestine submucosa (SIS) and the urinary bladder matrix (UBM) after decellularizing with sodium deoxycholate (SDC) and high hydrostatic pressure (HHP). The SDC d-ECM hydrogel gradually gelated, while the HHP d-ECM hydrogel immediately gelated. All d-ECM hydrogels had low matrix stiffness compared to that of the collagen hydrogel, according to a compression test. D-ECM hydrogels with various elastic moduli were obtained, irrespective of the decellularization method or tissue source. Microvascular-derived endothelial cells were seeded on d-ECM hydrogels. Few cells attached to the SDC d-ECM hydrogel with no network formation, while on the HHP d-ECM hydrogel, a capillary network structure formed between elongated cells. Long, branched networks formed on d-ECM hydrogels with lower matrix stiffness. This suggests that the capillary network structure that forms on d-ECM hydrogels is closely related to the matrix stiffness of the hydrogel.

A hybrid small-diameter tube fabricated from decellularized aortic intima-media and electrospun fiber for artificial small-diameter blood vessel.

Hybrid small-diameter tubes were fabricated by wrapping decellularized aortic intima-media sheets around a tubular stainless steel mandrel with diameter 4 mm, and then by coating with electrospun segmented polyurethane. The synthetic coat was deposited uniformly to a thickness of about 0.5-3.5 µm depending on the duration of electrospinning. Resistance to luminal pressure, burst strength, and stiffness increased with the thickness of the electrospun coat, suggesting that the synthetic fabric reinforces the reconstructed acellular aortic intima-media. Human umbilical vein endothelial cells seeded on the inner surface acquired flagstone morphology, while normal human dermal fibroblasts seeded on the outer surface proliferated well and partly migrated into deeper layers. Collectively, the data suggest that reinforcing decellularized aortic intima-media with electrospun fibers generates a small-diameter hybrid blood vessel with good biocompatibility and suitable mechanical properties. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1064-1070, 2019.

Induction of in Vivo Ectopic Hematopoiesis by a Three-Dimensional Structured Extracellular Matrix Derived from Decellularized Cancellous Bone.

An in vitro blood production system could be an alternative to blood donation. We constructed a hematopoietic microenvironment using decellularized cancellous bones (DCBs) as scaffolds to sustain hematopoietic stem cells and supporting cells. The subcutaneous implantation of DCBs into mice with or without human mesenchymal stem cells (hMSCs) revealed that regardless of the presence of hMSCs DCBs were recellularized by some host cells and induced hematopoiesis. The ability of DCB to promote hematopoiesis was investigated by focusing on the components and the structure of cancellous bone, specifically reticular and adipose tissues and trabecular bone. Two decellularization methods were used to prepare DCBs. The DCBs differed concerning reticular tissue and adipose tissue. DCBs with these tissues could be recellularized at the original cellular location. An implantation experiment with DCBs revealed that they were very favorable for the persistent homing of hematopoietic stem cells. In addition, DCBs promoted ectopic hematopoiesis. The findings indicate that reticular tissues are important in directing hematopoiesis of DCBs.

Surface Topography of PDMS Replica Transferred from Various Decellularized Aortic Lumens Affects Cellular Orientation.

Cells sense and respond to various surface topographies of substrates. Many types of topographical architectures have been developed for understanding cell-extracellular matrix (ECM) interactions and for their application in biomaterials. In the present study, as a topographical surface similar to native tissue, we developed a PDMS replica prepared using the transferring method of the decellularized aorta, which is an ECM assembly, and its cellular behaviors, such as orientation and elongation on it. Decellularized aortas were prepared by high hydrostatic pressure (HHP) and sodium dodecyl sulfate (SDS) methods for use as templates. Scanning electron microscopic observation of the SDS replica showed a randomly rough surface. Further, microscaled linear structures along the direction of the aortic longitudinal axis were observed on the HHP replica. These results indicated that the topographical surface of the HHP and SDS decellularized aorta could be replicated to their replicas at a microscale. Fibroblasts (NIH3T3) and endothelial cells (HUVECs) were cultured on their surfaces. Although they were randomly aligned on the SDS replica and flat surface, the high cellular alignment along with the direction of the aortic longitudinal axis was shown in the HHP replica and HHP decellularized aorta. These results suggest that the topographical structure similar to a native aorta could effectively induce the cell alignment, which is important to regulate cellular functions, and could provide important methodologies and knowledge for vascular biomaterials or culture substrates.

Decellularized porcine aortic intima-media as a potential cardiovascular biomaterial.

OBJECTIVES: The aim of this research is to investigate the histological and mechanical properties of decellularized aortic intima-media, a promising cardiovascular biomaterial. METHODS: Porcine aortic intima-media was decellularized using two methods: high hydrostatic pressurization (HHP) and sodium dodecyl sulphate (SDS). The histological properties were characterized using haematoxylin and eosin staining and Elastica van Gieson staining. The mechanical properties were evaluated using a tensile strength test. RESULTS: The structure of the HHP-treated samples was unchanged histologically, whereas that of the SDS-treated samples appeared structurally loose. Consequently, with regard to the mechanical properties of SDS-decellularized intima-media, elastic modulus and tensile strength were significantly decreased. CONCLUSIONS: The decellularization method affected the structure and the mechanical properties of the biomaterial. The HHP-treated sample was structurally and mechanically similar to the untreated control. Its mechanical properties were similar to those of human heart valves and the iliac artery and vein. Our results imply that porcine aortic intima-media that is decellularized with HHP is a potential cardiovascular biomaterial.

Tissue-engineered acellular small diameter long-bypass grafts with neointima-inducing activity.

Researchers have attempted to develop efficient antithrombogenic surfaces, and yet small-caliber artificial vascular grafts are still unavailable. Here, we demonstrate the excellent patency of tissue-engineered small-caliber long-bypass grafts measuring 20-30 cm in length and having a 2-mm inner diameter. The inner surface of an acellular ostrich carotid artery was modified with a novel heterobifunctional peptide composed of a collagen-binding region and the integrin α4ß1 ligand, REDV. Six grafts were transplanted in the femoral-femoral artery crossover bypass method. Animals were observed for 20 days and received no anticoagulant medication. No thrombogenesis was observed on the luminal surface and five cases were patent. In contrast, all unmodified grafts became occluded, and severe thrombosis was observed. The vascular grafts reported here are the first successful demonstrations of short-term patency at clinically applicable sizes.

Micro-CT evaluation of high pressure-decellularized cardiovascular tissues transplanted in rat subcutaneous accelerated-calcification model.

We have succeeded in reducing the calcification of acellular aortas or valves in porcine allogeneic system by removing the DNA and phospholipids, but its further reduction is desirable. Here, the calcification of the acellular tissue was evaluated in rat subcutaneous transplantation model which is known as calcification model. Acellular samples prepared by high-hydrostatic pressure (HHP) protocols with different washing media were implanted and the calcification was monitored under micro-computed tomography for 1 and 3 months. The amount of the calcium deposition was quantitatively evaluated by atomic absorption spectroscopy. A cell culture medium showed very good cell removal ability but led to severe calcification at 1 month, and surprisingly the calcium deposition increased as the washing period increased. This calcification was suppressed by removing the DNA fraction with high DNase concentration. On the other hand, the calcification was greatly reduced when washed with saline even at low DNase concentration after 2 weeks washing. These results suggest that the ion species in the washing medium and the residual DNase cooperatively affect the tendency of in vivo calcification, which led us to the possibility of reduced calcification of acellular cardiac tissues.

Relation between the tissue structure and protein permeability of decellularized porcine aorta.

The aim of this study was to assess the suitability of decellularized porcine aorta as a vascular graft material by measuring its permeability to protein. Aorta samples were decellularized by treatment with either high hydrostatic pressurization (HHP) or sodium dodecyl sulfate (SDS). Histological evaluation showed that the structure of an HHP-treated sample was similar to that of an untreated sample, while the structure of an SDS-treated sample was surfactant-damaged. A two-chamber diffusion system was used to measure permeability to lysozyme and bovine serum albumin. The lysozyme and bovine serum albumin mass transfer coefficients calculated for an SDS-treated sample were significantly larger than those calculated for an untreated sample, while the mass transfer coefficients for an HHP-treated sample were similar to those for an untreated sample. The mass transfer coefficients showed very good agreement with the tissue structure characterization, which means that differences in permeability reflected differences in tissue structure.

In vivo characterization of a decellularized dermis-polymer complex for use in percutaneous devices.

To develop a method for making percutaneous devices that have high biocompatibility and do not induce downgrowth of epidermal cells, we prepared a partial decellularized dermis (DD)/poly(methyl methacrylate) (PMMA) complex (PDPC) with a PMMA rod firmly stabilized inside. The porcine decellularized tissue was chosen because of its high biocompatibility and mechanical properties, and MMA was used because it would adhere firmly to a polymer such as a catheter. The MMA filled the cavities in the dermis and polymerized, anchoring to the collagenous fibrils inside the porcine DD. The PDPC was cemented to the PMMA rod tightly and it was integrated with the surrounding tissue within 12 weeks of implantation. Furthermore, no downgrowth of the epidermis, which may cause clinical problems, was observed. We consider that the tissue-polymer complex may be a suitable candidate for use in percutaneous devices.

Complete cell killing by applying high hydrostatic pressure for acellular vascular graft preparation.

Pressure treatment has been developed in tissue engineering application. Although the tissue scaffold prepared by a ultrahydrostatic pressure treatment has been reported, an excessive pressure has a potential to disrupt a structure of extracellular matrix through protein denaturation. It is important to understand the suitable low-pressure condition and mechanisms for cell killing. In this study, cellular morphology, mitochondria activity, and membrane permeability of mammalian cells with various pressure treatments were investigated with in vitro models. When the cells were treated with a pressure of 100 MPa for 10 min, cell morphology and adherence were the same as an untreated cells. Dehydrogenase activity in mitochondria was almost the same as untreated cells. On the other hand, when the cells were treated with the pressure of more than 200 MPa, the cells did not adhere, and the dehydrogenase activity was completely suppressed. However, green fluorescence was observed in the live/dead staining images, and the cells were completely stained as red after above 500 MPa. That is, membrane permeability was disturbed with the pressure treatment of above 500 MPa. These results indicated that the pressure of 200 MPa for 10 min was enough to induce cell killing through inactivation of mitochondria activity.

Fabrication of a heparin-PVA complex hydrogel for application as a vascular access.

A high hydrostatic pressure method, which can apply over 600 MPa pressure was employed for preparing a hydrogel of poly(vinyl alcohol) (PVA) loaded with heparin. The aim of this study was to fabricate a heparin-PVA hydrogel conduit and evaluate its potential for vascular access. Heparin-PVA complex hydrogel showed suppressed heparin release and prevented clot formation, depending on the molecular weight of the PVA. Strength of the hydrogel conduit was increased by embedding a Dacron mesh between two PVA layers. The tubular heparin-PVA complex hydrogel displayed a burst pressure of 750 mmHg. The tubular heparin-PVA complex hydrogel did not show any occlusion or burst for 2 weeks after implantation, implying that this heparin-PVA complex hydrogel shows high potential for use as a vascular access. This is the first report on the preparation of a multilayered PVA hydrogel with heparin embedded on one side only. The proposed approach could be expanded to the fabrication of various biomaterials for specific purposes.

Decellularized dermis-polymer complex provides a platform for soft-to-hard tissue interfaces.

To develop a soft-to-hard tissue interface, we made a decellularized dermis/poly(methyl methacrylate) (PMMA) complex by soaking the decellularized dermis in methyl methacrylate (MMA) and an initiator, and then polymerizing the MMA. The decellularized tissue was chosen because of its good biocompatibility and the easiness of suturing it, and MMA because of its hard tissue compatibility and wide use in the biomedical field. The MMA filled the cavities in the dermis and polymerized within 10 min. No leaking or polymer aggregation was observed, implying that a homogenous tissue-polymer complex had formed. The cell infiltration and the integration between the tissue and the dermis occurred in vivo, whereas the cells could not infiltrate the tissue-polymer complex. This implies that the interface tissue should possess both complex and noncomplex parts, where the cells infiltrate the noncomplex part and stop when they encounter the complex part, integrating the soft and hard tissue or hard polymer.

Preparation of a Nanoscaled Poly(vinyl alcohol)/Hydroxyapatite/DNA Complex Using High Hydrostatic Pressure Technology for In Vitro and In Vivo Gene Delivery.

Our previous research showed that poly(vinyl alcohol) (PVA) nanoparticles incorporating DNA with hydrogen bonds obtained by high hydrostatic pressurization are able to deliver DNA without any significant cytotoxicity. To enhance transfection efficiency of PVA/DNA nanoparticles, we describe a novel method to prepare PVA/DNA nanoparticles encapsulating nanoscaled hydroxyapatites (HAps) prepared by high hydrostatic pressurization (980 MPa), which is designed to facilitate endosomal escape induced by dissolving HAps in an endosome. Scanning electron microscopic observation and dynamic light scattering measurement revealed that HAps were significantly encapsulated in PVA/HAp/DNA nanoparticles. The cytotoxicity, cellular uptake, and transgene expression of PVA/HAp/DNA nanoparticles were investigated using COS-7 cells. It was found that, in contrast to PVA/DNA nanoparticles, their internalization and transgene expression increased without cytotoxicity occurring. Furthermore, a similar level of transgene expression between plasmid DNA and PVA/HAp/DNA nanoparticles was achieved using in vivo hydrodynamic injection. Our results show a novel method of preparing PVA/DNA nanoparticles encapsulating HAp nano-crystals by using high hydrostatic pressure technology and the potential use of HAps as an enhancer of the transfection efficiency of PVA/DNA nanoparticles without significant cytotoxicity.

The effect of decellularized bone/bone marrow produced by high-hydrostatic pressurization on the osteogenic differentiation of mesenchymal stem cells.

Decellularized bone/bone marrow was prepared to provide a microenvironment mimicking that of the bone marrow for three-dimensional culture in vitro. Bone/bone marrows were hydrostatically pressed at 980 MPa at 30 °C for 10 min to dismantle the cells. Then, they were washed with EGM-2 and further treated in an 80% EtOH to remove the cell debris and lipid, respectively. After being rinsed and shaken with PBS again, treated bone/bone marrows were stained with hematoxylin and eosin (H-E) to assess the efficacy of decellularization. Cells were determined to have been completely removed through H-E staining of their sections and DNA quantification. Rat mesenchymal stem cells (rMSCs) were seeded on the decellularized bone/bone marrows and cultured for 21 days. The adhesion of rMSCs on or into decellularized bone/bone marrows was confirmed and proliferated over time in culture. The osteogenic differentiation effect of decellularized bone/bone marrows on rMSCs in the presence or absence of dexamethasone was investigated. Decellularized bone/bone marrows without dexamethasone significantly increased alkaline phosphatase (ALP) activity, indicating promoted osteogenic differentiation of rMSCs. In an animal study, when decellularized bone/bone marrows were implanted into the rat subcutaneous, no immune reaction occurred and clusters of the hematopoietic cells could be observed, suggesting the decellularized bone/bone marrows can provide a microenvironment in vivo.

High-hydrostatic pressure technique is an effective method for the preparation of PVA-heparin hybrid gel.

To develop an antithrombotic material for preparation of small-diameter vascular graft, we describe a novel method to prepare a poly(vinyl alcohol) (PVA)-heparin hydrogels prepared by high-hydrostatic pressure (HHP, 980 MPa), which is designed for sustained release of heparin. Antithrombogenic test revealed that HHP method would not affect the antithrombin III (ATIII) activity of the released heparin. The distribution of heparin in the polymer matrix was homogeneous than freeze-thawing gel, due to the fast gelling affect of PVA which takes approximately 10 min for gel formation. The formation of intra- and intermolecular hydrogen bonds between PVA chains has trapped the heparin inside, suppressing the phase separation between PVA and heparin. Furthermore, evenly distribution of heparin induced the formation of heparin and PVA molecular complex, which brought the sustained release of heparin from the PVA despite the high swelling ratio. Our results show that it is possible to prepare a PVA-heparin hybrid gel which can be applied as an effective material for an antithrombotic system without using any chemical agent.

Preparation and characterization of decellularized cornea using high-hydrostatic pressurization for corneal tissue engineering.

To prepare acellular corneal scaffold, we used high-hydrostatic pressurization (HHP) to decellularize porcine cornea. The HHP method disrupts cells by hydrostatic pressurization, and then the disrupted cells' components are removed by washing with a cell culture medium. Porcine corneas were hydrostatically pressed at 980 MPa at 10 or 30 degrees C for 10 min to make them opaque. There was no change in the thickness of the corneas immediately after the pressurization, but they swelled during the washing process. The cornea swelling caused by HHP was suppressed when medium containing 3.5% w/v dextran was used. For H-E staining of the cornea decellularized with the HHP method, the complete removal of corneal cells was confirmed. Furthermore, when the corneas were immersed in glycerol for 1 hour, their optical properties were restored to those of native corneas. In an animal study, when acellular porcine corneas were implanted into rabbit cornea, no immune reaction occurred and the turbid corneas became clear. The decellularized corneas obtained through HHP could be useful as a corneal scaffold for tissue regeneration.

The use of high-hydrostatic pressure treatment to decellularize blood vessels.

A decellularization method using high-hydrostatic pressure (HHP) technology (>600MPa) is described. The HHP disrupts the cells inside the tissue. The cell debris can be eliminated with a simple washing process, producing clean, decellularized tissue. In this study, porcine aortic blood vessel was decellularized by HHP. The mechanical properties and in vivo performance of the decellularized tissue were evaluated. Mechanical properties of the decellularized tissue were not altered by the HHP treatment. Reduced inflammation of the decellularized tissue was confirmed by xenogenic transplant experimentation. An allogenic transplantation study showed that decellularized blood vessel endured the arterial blood pressure, and there was no clot formation on the luminal surface. In addition, cellular infiltration into the vessel wall was observed 4 weeks after implantation, suggesting that HHP treatments could be applied widely as a high-quality decellularization method.

Expression behavior of high-pressure-compacted plasmid DNA in mammalian cell.

We have been developing a novel compaction method of plasmid DNA using high pressure technology, and previously found that the size of the plasmid DNA was decreased with increasing the pressurizing-strength and time. In the present study, we investigated the tertiary structural change and the expression behavior of the pressure-compacted plasmid DNA in cell in vitro. When the pressure-compacted plasmid DNA was reacted with restriction enzyme (EcoRI), a large amount of the EcoRI was required to cleave the pressure-compacted plasmid DNA than the non-pressurized plasmid DNA, suggesting that the structural change of plasmid DNA was induced by the pressurization. The expression of the pressure-compacted plasmid DNA injected into cells using microinjection method was analyzed. It was clear that the pressure-compacted plasmid DNA could effectively delay gene expression, suggesting that the controlled compaction of plasmid DNA by high pressurization would be regulate the transgene expression.