Preparation, physico-biochemical characterization, and proteomic analysis of highly transparent corneal extracellular matrices for lamellar keratoplasty and tissue-engineered cornea construction.

Corneal opacity and deformation, which often require corneal transplantation for treatment, are among the leading causes of monocular blindness. To restore corneal clarity and integrity, there is a need for an artificial stroma that not only matches the transparency of donated human cornea but also effectively integrates to the corneal tissue. In this study, a transparent decellularized cornea was successfully developed using the high hydrostatic pressure method with processing conditions optimized for corneal decellularization. Biochemical analyses demonstrated the effective removal of cellular components from the transparent decellularized corneas, while preserving collagen and glycosaminoglycans. Proteome analysis also revealed that core matrisome and matrisome-associated proteins remained following decellularization, similar to the composition observed in untreated corneas. The light transmittance of the transparent decellularized corneas was 86.4 ± 1.5 % in the visible region, comparable to that of donated human corneas. No complications, such as angiogenesis, were observed following interlamellar corneal transplantation in rabbits. The grafts were almost imperceptible immediately following surgery and achieved complete transparency within a few days, becoming indistinguishable even under a microscope. The transparent decellularized cornea presented here has promising potential as a material for application in lamellar keratoplasty.

Fibrin Hydrogel Layer-Anchored Pericardial Matrix Prevents Epicardial Adhesion in the Severe Heart Adhesion-Induced Miniature Pig Model.

Postoperative adhesion is a very common and serious complication that occurs frequently in cardiac surgery. The purpose of this study was to evaluate the efficacy of a fibrin hydrogel layer-anchored decellularized pericardial matrix in preventing pericardial adhesions in a miniature pig model with a myocardial injury. Fibrin hydrogel layer-anchored decellularized pericardial matrix was prepared by spraying a mixture of fibrinogen and thrombin on a fibrinogen-doped decellularized pericardium. Cardiac injury was generated by abrading and desiccating the epicardial surface of a miniature pig to induce severe postoperative adhesions. The adhesion between the epicardial surface and fibrin hydrogel layer-anchored decellularized pericardial matrix in three different regions (left outer, front, and right outer) was evaluated macroscopically one month after surgery. The fibrin hydrogel layer-anchored decellularized pericardial matrix showed significantly less adhesion than an autologous pericardium (0.2 ± 0.7 in DPM-FHG0.5 and 0.4 ± 0.8 in DPM-FHG1, p < 0.01) and expanded polytetrafluoroethylene (ePTFE) (1.6 ± 0.5, p < 0.05). The fibrin hydrogel concentration had no effect on preventing postoperative adhesion. A thinner fibrin hydrogel layer was observed on the decellularized pericardial matrix one month after surgery; however, the inside of the matrix was filled with fibrin hydrogel. Fibrin hydrogel layer-anchored decellularized pericardial matrix prevented postoperative epicardial adhesions in a miniature pig model. Our findings suggest that pericardial closure using a fibrin hydrogel layer-anchored decellularized pericardial matrix is a promising method for preventing adverse outcomes in reoperative surgeries.

4-Arm PEG-Functionalized Decellularized Pericardium for Effective Prevention of Postoperative Adhesion in Cardiac Surgery.

Postoperative adhesions are a very common and serious complication in cardiac surgery, and the development of an effective anti-adhesion membrane showing resistance to the physical stimulus generated by the pulsation of the heart is desirable. In this study, an anti-adhesion material was developed through amine coupling between decellularized bovine pericardia (dBPCs) and 4-arm poly(ethylene glycol) succinimidyl glutarate (4-arm PEG-NHS) for the postoperative care of cardiac surgical patients. The efficacy of the 4-arm PEG-functionalized dBPCs in the prevention of adhesions after cardiac surgery was investigated in a rabbit heart adhesion model. The dBPCs meet the requirements for biocompatibility, flexibility, and sufficient suturable strength, and the 4-arm PEG moieties provide an anti-adhesion effect by the high excluded volume interactions of the PEG chains with proteins. The 4-arm PEG-functionalized dBPCs had a significantly greater anti-adhesion effect than the other materials tested and showed re-establishment of the mesothelial monolayer. These results suggested that the 4-arm PEG-functionalized dBPCs are a favorable material for an anti-adhesion membrane.

In vivo recellularization of xenogeneic vascular grafts decellularized with high hydrostatic pressure method in a porcine carotid arterial interpose model.

Autologous vascular grafts are widely used in revascularization surgeries for small caliber targets. However, the availability of autologous conduits might be limited due to prior surgeries or the quality of vessels. Xenogeneic decellularized vascular grafts from animals can potentially be a substitute of autologous vascular grafts. Decellularization with high hydrostatic pressure (HHP) is reported to highly preserve extracellular matrix (ECM), creating feasible conditions for recellularization and vascular remodeling after implantation. In the present study, we conducted xenogeneic implantation of HHP-decellularized bovine vascular grafts from dorsalis pedis arteries to porcine carotid arteries and posteriorly evaluated graft patency, ECM preservation and recellularization. Avoiding damage of the luminal surface of the grafts from drying significantly during the surgical procedure increased the graft patency at 4 weeks after implantation (P = 0.0079). After the technical improvement, all grafts (N = 5) were patent with mild stenosis due to intimal hyperplasia at 4 weeks after implantation. Neither aneurysmal change nor massive thrombosis was observed, even without administration of anticoagulants nor anti-platelet agents. Elastica van Gieson and Sirius-red stainings revealed fair preservation of ECM proteins including elastin and collagen after implantation. The luminal surface of the grafts were thoroughly covered with von Willebrand factor-positive endothelium. Scanning electron microscopy of the luminal surface of implanted grafts exhibited a cobblestone-like endothelial cell layer which is similar to native vascular endothelium. Recellularization of the tunica media with alpha-smooth muscle actin-positive smooth muscle cells was partly observed. Thus, we confirmed that HHP-decellularized grafts are feasible for xenogeneic implantation accompanied by recellularization by recipient cells.

Introduction of Cells into Porous Poly-l-Lactic Acid Scaffolds Using Impregnation Techniques.

Porous materials containing cells-prepared via cell seeding on scaffolds or gelation of cell-containing solutions-have been widely studied to investigate tissue regeneration and three-dimensional cultures. However, these methods cannot introduce cells into porous materials that have low water absorption or scaffolds that require cytotoxic solvents or processes for their production. In this study, first, three different impregnation treatments conditions (vacuum, pressure, and vacuum pressure impregnation: VPI) were applied to cell suspensions to evaluate the effect of each treatment on cells. Following all three treatments, fibroblasts adhered to the cell culture dish and proliferated in the same manner as untreated cells, which confirmed that the three impregnation treatments did not affect cell function. Second, cells were introduced into a poly-l-lactic acid (PLA) scaffold, which has low water absorption, using the same impregnation treatments. The PLA scaffolds subjected to the three impregnation treatments that exhibited a significantly greater amount of DNA than those subjected to immersion treatments and showed increasing amounts of DNA in the order vacuum treatment > VPI treatment > pressure treatment. Furthermore, the amount of DNA in the vacuum-treated and VPI-treated PLA scaffolds increased on the first, third, and fifth days of culture, and it was confirmed that the cells introduced into the PLA scaffolds proliferated. These results suggest that vacuum and VPI treatments may be useful methods for introducing cells into porous materials.

Preparation of gradient-type biological tissue-polymer complex for interlinking device.

The aim of this study was to investigate the monomer absorption behavior of decellularized dermis and prepare a gradient-type decellularized dermis-polymer complex. Decellularized dermis was prepared using sodium dodecyl sulfate, and its monomer absorption behavior was investigated using three types of hydrophobic monomer with different surface free energies. The results show that monomer absorption depends strongly on the tissue structure, regardless of the surface free energy, and the amount of absorbed monomer can be increased by sonication. Based on these results, we prepared a gradient-type decellularized dermis-poly(methyl methacrylate) complex by controlling the permeation time of the methyl methacrylate monomer and polymerization initiator into the decellularized dermis. The mechanical strength of this complex gradually increased from the dermis side to the polymer side, and combined the physical characteristics of the dermis and the polymer.

PLA-Collagen Composite Scaffold Fabrication by Vacuum Pressure Impregnation.

Composite scaffolds are made by various methods, such as copolymerization, freeze gelation, and thermally induced phase separation, which can compound materials with different properties using solvents and heat. However, it is difficult to compound solvents and heat-sensitive materials such as natural polymers. In this study, we investigated a vacuum pressure impregnation (VPI) method for creating a composite of natural polymers. A collagen solution could not be introduced into a poly (l-lactide) (PLA) porous material using an immersing treatment, but it is possible using the VPI method. The resulting PLA-collagen composite scaffold had greater water adsorption and degradation than a PLA scaffold. These results indicate that VPI may be a promising method for creating composites of natural materials. Impact Statement This is the development of a method for introducing cells into a completed porous material in a short time. This technology is expected to be applied to tissue regeneration and 3D culture.

Re-epithelialization and remodeling of decellularized corneal matrix in a rabbit corneal epithelial wound model.

This study investigated the in vivo correlation between re-epithelialization and remodeling of a decellularized corneal matrix prepared by a high-hydrostatic pressure (HHP) method in rabbits. Decellularized corneal matrices were transplanted in a 6-mm-diameter recipient corneal interlamellar pocket with a 2 mm epithelial defect. The time course of graft status in rabbits was examined daily for 6 months by biomicroscopy and scored for clarity and re-epithelialization, after which the rabbits were sacrificed for histological analysis. Fluorescein staining revealed that the corneal epithelial cells had migrated onto the decellularized corneal matrix. Histological analysis revealed that the implanted decellularized corneal matrix was completely integrated with the recipient rabbit cornea and the stratified corneal epithelia consisting of multiple layers were regenerated, similar to that in the normal cornea. The recipient keratocytes infiltrated into the decellularized corneal matrix at 6 months after the operation and the decellularized corneal matrix was gradually remodeled into the recipient tissue. Transmission electron microscopy revealed that the ultrastructure of the decellularized corneal matrix was rearranged, similar to the normal cornea. These findings suggest that the decellularized corneal matrix serves as a template for remodeling. The decellularized corneal matrix obtained through HHP is a useful graft for corneal tissue regeneration.

A fibrin-coated pericardial extracellular matrix prevented heart adhesion in a rat model.

As most surgical treatments pose a risk of tissue adhesion, methods to prevent adhesion are needed across various surgical fields. In this study, we investigated the use of a decellularized pericardium with fibrin glue to prevent rat heart adhesion. Porcine pericardia were decellularized by a high-hydrostatic pressure method. Cells adhered to the resulting pericardial extracellular matrix (ECM) during an in vitro cell-seeding test, but fibrin-coated pericardial ECM showed reduced cell adhesion. In a rat surgical model of heart adhesion, the fibrin-coated pericardial ECM did not adhere to the heart and mesothelial cell adhesion was observed on the ECM surface. Notably, the anti-adhesion effect of fibrin-coated pericardial ECM was observed 4 weeks after surgery. These results support the utility of fibrin-coated pericardial ECM as an adhesion prevention material for cardiovascular surgery. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1088-1094, 2019.

[Rabbit Model for Evaluation of Anti-adhesive Materials after Open Heart Surgery].

Surgical trauma to the pericardial mesothelium during open heart procedures has formation of fibrovascular adhesions. Surgeons are confronted with cardiac adhesions, leading to an increased surgical risk such as intractable bleeding and possible catastrophic hemorrhage. In order to solve the problem, the anti-adhesion membrane has been developed and used. However, their performances are far from perfect, so it has been expected to develop a novel anti-adhesive material. For preparing an anti-adhesive material, there is 1 serious problem, a lack of golden standard of animal model for evaluation of anti-adhesivity. In this study, we tried to establish a standard system for evaluation of the performance of anti-adhesive materials for the chest-area surgery using rabbit. Setting the condition of the damage to heart, the objective evaluation system was established. And we performed experimental study to evaluate prevention of adhesions with pericardial substitutes and our product under development based on this model.

Evaluation of small-diameter vascular grafts reconstructed from decellularized aorta sheets.

Following small-diameter vascular grafting, blood vessels fail to retain excellent antithrombotic function and therefore require application of antithrombogenic drugs. We have previously reported early attachment of endothelial cells to the luminal surface of high hydrostatic pressure (HHP)-decellularized arteries after transplantation. In addition, the graft retained antithrombotic function by endothelialization and remained open for several weeks. To fabricate tube grafts of optimal size and shape for small-diameter vascular grafting, we evaluated decellularized porcine aorta sheets as a suitable antithrombogenic material. Porcine aortic sheets were decellularized using detergent-based or HHP methods. The HHP-, but not detergent-based-, decellularized aortic sheets were verified to be acellular, and the mechanical properties of the native aortic sheet remained intact. To fabricate vascular grafts, the decellularized aortic sheets were rolled into tubes and secured using fibrin glue bonding. After implantation into a rat carotid artery model, the tubular grafts withstood normal blood pressure, mechanical beating, and pulsatile flow. After 3 weeks, the tubular grafts remained patent and recipient cell infiltration and cell attachment were observed on the luminal surface. These results indicate that HHP-decellularized aortic sheets may be useful as an antithrombogenic material for tubular vascular grafts. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1293-1298, 2017.

Histological structure affects recellularization of decellularized arteries.

Decellularized arteries were prepared to evaluate the in vivo recellularization of biological material after implantation. Porcine aortas and radial arteries were decellularized using high-hydrostatic pressure to form materials with histologically-different structures. Successful removal of cells from decellularized arteries was evaluated by hematoxylin-eosin staining and measurement of residual DNA. Cell remnants were eliminated completely from the decellularized arteries, and histological structures were preserved. Cells adhered to all decellularized artery samples, but infiltration of cells was observed only from the adventitial side of the decellularized radial artery. Rats were implanted subcutaneously with a decellularized aorta or radial artery to evaluate in vivo performance. Decellularized aortic tissue prevented cell infiltration better than that of the decellularized radial artery, suggesting that the elastin lamina in decellularized tissues prevents cell infiltration and suppresses recellularization.

[New Intracoronary Shunt Cannula in Off-pump Coronary Artery Bypass Grafting].

In off-pump coronary artery bypass grafting(OPCAB), a bloodless operative field has great influence on the quality of anastomosis. In addition, maintenance of distal coronary flow during anastomosis stabilizes hemodynamics. We introduced a new intracoronary shunt cannula, Mini Shunt Pro (MSP). MSP adopts the step-tip, which allows easy insertion and reduces the risk of intimal injury. The distal and proximal sides of the tips of MSP are 0.25 mm different in diameter, which improves its fitness to the coronary artery and enhances a bloodless operative field. This new shunt cannula is considered to be safe and useful for high-quality anastomosis in OPCAB.

Ultrastructural analysis of the decellularized cornea after interlamellar keratoplasty and microkeratome-assisted anterior lamellar keratoplasty in a rabbit model.

The decellularized cornea has received considerable attention for use as an artificial cornea. The decellularized cornea is free from cellular components and other immunogens, but maintains the integrity of the extracellular matrix. However, the ultrastructure of the decellularized cornea has yet to be demonstrated in detail. We investigated the influence of high hydrostatic pressure (HHP) on the decellularization of the corneal ultrastructure and its involvement in transparency, and assessed the in vivo behaviour of the decellularized cornea using two animal transplantation models, in relation to remodelling of collagen fibrils. Decellularized corneas were prepared by the HHP method. The decellularized corneas were executed by haematoxylin and eosin and Masson's trichrome staining to demonstrate the complete removal of corneal cells. Transmission electron microscopy revealed that the ultrastructure of the decellularized cornea prepared by the HHP method was better maintained than that of the decellularized cornea prepared by the detergent method. The decellularized cornea after interlamellar keratoplasty and microkeratome-assisted anterior lamellar keratoplasty using a rabbit model was stable and remained transparent without ultrastructural alterations. We conclude that the superior properties of the decellularized cornea prepared by the HHP method were attributed to the preservation of the corneal ultrastructure.

Effect of decellularized tissue powders on a rat model of acute myocardial infarction.

Many research groups are currently investigating new treatment modalities for myocardial infarction. Numerous aspects need to be considered for the clinical application of these therapies, such as low cell integration and engraftment rates of cell injection techniques. Decellularized tissues are considered good materials for promoting regeneration of traumatic tissues. The properties of the decellularized tissues are sustained after processing to powder form. In this study, we examined the use of decellularized tissue powder in a rat model of acute myocardial infarction. The decellularized tissue powders, especially liver powder, promoted cell integration and neovascularization both in vitro and in vivo. Decellularized liver powder induced neovascularization in the infarct area, resulting in the suppression of myocardial necrosis. The results of this study suggest that decellularized liver powder has good potential for application as a blood supply material for the treatment of myocardial infarction.

Corneal Regeneration by Deep Anterior Lamellar Keratoplasty (DALK) Using Decellularized Corneal Matrix.

The purpose of this study is to demonstrate the feasibility of DALK using a decellularized corneal matrix obtained by HHP methodology. Porcine corneas were hydrostatically pressurized at 980 MPa at 10°C for 10 minutes to destroy the cells, followed by washing with EGM-2 medium to remove the cell debris. The HHP-treated corneas were stained with H-E to assess the efficacy of decellularization. The decellularized corneal matrix of 300 µm thickness and 6.0 mm diameter was transplanted onto a 6.0 mm diameter keratectomy wound. The time course of regeneration on the decellularized corneal matrix was evaluated by haze grading score, fluorescein staining, and immunohistochemistry. H-E staining revealed that no cell nuclei were observed in the decellularized corneal matrix. The decellularized corneal matrices were opaque immediately after transplantation, but became completely transparent after 4 months. Fluorescein staining revealed that initial migration of epithelial cells over the grafts was slow, taking 3 months to completely cover the implant. Histological sections revealed that the implanted decellularized corneal matrix was completely integrated with the receptive rabbit cornea, and keratocytes infiltrated into the decellularized corneal matrix 6 months after transplantation. No inflammatory cells such as macrophages, or neovascularization, were observed during the implantation period. The decellularized corneal matrix improved corneal transparency, and remodelled the graft after being transplanted, demonstrating that the matrix obtained by HHP was a useful graft for corneal tissue regeneration.

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.

Porcine radial artery decellularization by high hydrostatic pressure.

Many types of decellularized tissues have been studied and some have been commercially used in clinics. In this study, small-diameter vascular grafts were made using HHP to decellularize porcine radial arteries. One decellularization method, high hydrostatic pressure (HHP), has been used to prepare the decellularized porcine tissues. Low-temperature treatment was effective in preserving collagen and collagen structures in decellularized porcine carotid arteries. The collagen and elastin structures and mechanical properties of HHP-decellularized radial arteries were similar to those of untreated radial arteries. Xenogeneic transplantation (into rats) was performed using HHP-decellularized radial arteries and an untreated porcine radial artery. Two weeks after transplantation into rat carotid arteries, the HHP-decellularized radial arteries were patent and without thrombosis. In addition, the luminal surface of each decellularized artery was covered by recipient endothelial cells and the arterial medium was fully infiltrated with recipient cells.

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.

Application of a vacuum pressure impregnation technique for rehydrating decellularized tissues.

Most of the clinically available decellularized tissues are preserved in a freeze-dried state. Freeze-dried (FD) tissues can be preserved for long term, although a rehydration process is necessary before use. Currently, an immersion method is most commonly used in clinical procedures, but it is difficult for complicated and thick structure tissue rehydration. In this study, we tried to apply a vacuum pressure impregnation (VPI) technique for FD tissue rehydration. The water content of decellularized tissues can reach the water content of native tissues within 30 min using VPI, whereas it took 6 h to reach the same water content using the immersion method. Furthermore, heparin rehydrated aortas by VPI had more heparin release at each time point and therefore appeared more anticoagulant activity. We found that the VPI treatment promotes solution infiltration into materials, achieves complete rehydration of the decellularized tissues, and deep infiltration of heparin into the decellularized tissues, suggesting that VPI treatment could be applied as a rehydration method for biological materials.