Fabrication of a cell culture scaffold that mimics the composition and structure of bone marrow extracellular matrix.

Cell culture models that mimic tissue environments are useful for cell and extracellular matrix (ECM) function analysis. Decellularized tissues with tissue-specific ECM are expected to be applied as cell culture scaffolds, however, it is often difficult for seeded cells to permeate their structures. In this study, we evaluated the adhesion and proliferation of mouse fibroblasts seeded onto decellularized bone marrow scaffolds that we fabricated from adult and fetal porcine. Decellularized fetal bone marrow displays more cell attachment and faster cell proliferation than decellularized adult bone marrow. Our findings suggest that decellularized fetal bone marrow is useful as a cell culture scaffold with bone marrow ECM and structure.

Investigation of anti-adhesion ability of 8-arm PEGNHS-modified porcine pericardium.

In post-adhesion surgery, there is a clinical need for anti-adhesion membranes specifically designed for the liver, given the limited efficacy of current commercial products. To address this demand, we present a membrane suitable for liver surgery applications, fabricated through the modification of decellularized porcine pericardium with 20 KDa hexaglycerol octa (succinimidyloxyglutaryl) polyoxyethylene (8-arm PEGNHS). We also developed an optimized modification procedure to produce a high-performance anti-adhesion barrier. The modified membrane significantly inhibited fibroblast cell adherence while maintaining minimal levels of inflammation. By optimizing the modification ratio, we successfully controlled post-adhesion formation. Notably, the 8-arm PEG-modified pericardium with a molar ratio of 5 exhibited the ability to effectively prevent post-adhesion formation on the liver compared to both the control and Seprafilm®, with a low adhesion score of 0.5 out of 3.0. Histological analysis further confirmed its potential for easy separation. Furthermore, the membrane demonstrated regenerative capabilities, as evidenced by the proliferation of mesothelial cells on its surface, endowing anti-adhesion properties between the abdominal wall and liver. These findings highlight the membrane's potential as a reliable barrier for repeated liver resection procedures that require the removal of the membrane multiple times.

Characteristics of macrophage aggregates prepared by rotation culture and their response to polymeric materials.

Understanding the interaction between macrophages and biomaterials is important for the creation of new biomaterials and the development of technologies to control macrophage function. Since macrophages are strongly adhesive, caution is required when performing in vitro evaluations. Similarly, when THP-1 cells, macrophage precursor cells, are differentiated into macrophages using phorbol-12-myristate-13-acetate (PMA), it becomes difficult to detach them from the adherent substrate, which has been a problem on investigation of immunological responses to biomaterials. In this study, the interaction of THP-1 cell-differentiated macrophages with biomaterials was analyzed based on a new method of seeding THP-1 cells. THP-1 cells were cultured in static and rotation culture without and with PMA. In undifferentiated THP-1 cells, there was no change in cellular function between static and rotation cultures. In rotation culture with PMA, THP-1 cells differentiated and formed macrophage aggregates. IL-1ß and MRC1 expression in macrophage aggregates was examined after differentiation and M1/M2 polarization. Macrophage aggregates in rotation culture tended to be polarized toward M2 macrophages compared with those in static culture. In the evaluation of the responses of macrophage aggregates to several kinds of polymeric materials, macrophage aggregates showed different changes in MRC1 expression over time at 30, 50, and 70 rpm. Rotation speed of 30 rpm was considered most appropriate condition in that it gave stable results with the same trend as obtained with static culture. The use of macrophage aggregates obtained by rotational culture is expected to provide new insights into the evaluation of inflammatory properties of biomaterials.

Effect of multi arm-PEG-NHS (polyethylene glycol n-hydroxysuccinimide) branching on cell adhesion to modified decellularized bovine and porcine pericardium.

Implanting physical barrier materials to separate wounds from their surroundings is a promising strategy for preventing postoperative adhesions. Herein, we develop a material that switches from an anti-adhesive surface to an adhesive surface, preventing adhesion in the early stage of transplantation and then promoting recellularization. In this study, 2-arm, 4-arm, and 8-arm poly(ethylene glycol) succinimidyl glutarate (2-, 4-, 8-arm PEG-NHS) were used to modify the surface of decellularized porcine and bovine pericardium. The number of free amines on the surface of each material significantly decreased following modification regardless of the reaction molar ratio of NH2 and NHS, the number of PEG molecule branches, and the animal species of the decellularized tissue. The structure and mechanical properties of the pericardium were maintained after modification with PEG molecules. The time taken for the PEG molecules to detach through hydrolysis of the ester bonds differed between the samples, which resulted in different cell repulsion periods. By adjusting the reaction molar ratio, the number of PEG molecule branches, and the animal species of the decellularized pericardium, the duration of cell repulsion can be controlled and is expected to provide an anti-adhesion material for a variety of surgical procedures.

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.

Preparation of Cholesterol-Modified Hyaluronic Acid Nanogel-Based Hydrogel and the Inflammatory Evaluation Using Macrophage-like Cells.

Nanogels are candidate biomaterials for tissue engineering and drug delivery. In the present study, a cholesterol-hyaluronic acid hydrogel was developed, and the pro-inflammatory response of macrophages to the hydrogel was investigated to determine its use in biomedical applications. Hyaluronic acid modified with cholesterol (modification rate: 0-15%) and maleimide (Chol-HA) was synthesized. The Chol-HA nanogel was formed through self-assembly via hydrophobic cholesterol interactions in aqueous solution. The Chol-HA hydrogel was formed through chemical crosslinking of the Chol-HA nanogel via a Michael addition reaction between the maleimide and thiol groups of 4arm-PEGSH. We found that the Chol-HA hydrogels with 5, 10, and 15% cholesterol inhibited the pro-inflammatory response of HiBiT-THP-1 cells, suggesting that the cholesterol contributed to the macrophage response. Furthermore, Interleukin 4 (IL-4) encapsulated in the hydrogel of the Chol-HA nanogel enhanced the inhibition of the inflammatory response in HiBiT-THP-1 cells. These results provide useful insights into the biomedical applications of hydrogels.

Preparation of mineralized pericardium by alternative soaking for soft-hard interregional tissue application.

Recent applications of decellularized tissues include the ectopic use of sheets and powders for three-dimensional (3D) tissue reconstruction. Decellularized tissues are modified (or fabricated) with the desired functions for application to the target (transplanted or used) tissue, including soft-hard interregional tissues, such as ligaments, tendons, and periodontal ligaments. This study aimed to prepare a mineralized decellularized pericardium to construct a soft-hard interregional tissue by 3D fabrication of decellularized pericardium, for example, rolling up to a cylindrical form. The decellularized pericardial tissue was prepared using the high hydrostatic pressurization (HHP) and surfactants method. The pericardium consisted of bundles of aligned fibers, and the bundles were slightly disordered when prepared with the surfactant decellularization method compared with that prepared using the HHP decellularization method. Mineralization of the decellularized pericardium was performed using an alternate soaking process with various cycles. The surface of the decellularized pericardium was covered with calcium phosphate precipitates, which accumulated on the surface with an increasing number of soaking cycles. The inside of the HHP decellularized pericardium was mineralized uniformly, whereas the mineralization of the decellularized pericardium decreased toward the interior. These findings suggest that the decellularization method strongly affects the structure and mineralized parts of the decellularized pericardium. The mineralized decellularized pericardium could be a candidate material for reconstructing alternative interregional tissues, such as ligaments and tendons.

A novel approach for the endothelialization of xenogeneic decellularized vascular tissues by human cells utilizing surface modification and dynamic culture.

Decellularized xenogeneic vascular grafts can be used in revascularization surgeries. We have developed decellularization methods using high hydrostatic pressure (HHP), which preserves the extracellular structure. Here, we attempted ex vivo endothelialization of HHP-decellularized xenogeneic tissues using human endothelial cells (ECs) to prevent clot formation against human blood. Slices of porcine aortic endothelium were decellularized using HHP and coated with gelatin. Human umbilical vein ECs were directly seeded and cultured under dynamic flow or static conditions for 14 days. Dynamic flow cultures tend to demonstrate higher cell coverage. We then coated the tissues with the E8 fragment of human laminin-411 (hL411), which has high affinity for ECs, and found that Dynamic/hL411showed high area coverage, almost reaching 100% (Dynamic/Gelatin vs Dynamic/hL411; 58.7 ± 11.4 vs 97.5 ± 1.9%, P = 0.0017). Immunostaining revealed sufficient endothelial cell coverage as a single cell layer in Dynamic/hL411. A clot formation assay using human whole blood showed low clot formation in Dynamic/hL411, almost similar to that in the negative control, polytetrafluoroethylene. Surface modification of HHP-decellularized xenogeneic endothelial tissues combined with dynamic culture achieved sufficient ex vivo endothelialization along with prevention of clot formation, indicating their potential for clinical use as vascular grafts in the future.

Extraction and Biological Evaluation of Matrix-Bound Nanovesicles (MBVs) from High-Hydrostatic Pressure-Decellularized Tissues.

Decellularized tissues are widely used as promising materials in tissue engineering and regenerative medicine. Research on the microstructure and components of the extracellular matrix (ECM) was conducted to improve the current understanding of decellularized tissue functionality. The presence of matrix-bound nanovesicles (MBVs) embedded within the ECM was recently reported. Results of a previous experimental investigation revealed that decellularized tissues prepared using high hydrostatic pressure (HHP) exhibited good in vivo performance. In the current study, according to the hypothesis that MBVs are one of the functional components in HHP-decellularized tissue, we investigated the extraction of MBVs and the associated effects on vascular endothelial cells. Using nanoparticle tracking assay (NTA), transmission electron microscopy (TEM), and RNA analysis, nanosized (100-300 nm) and membranous particles containing small RNA were detected in MBVs derived from HHP-decellularized small intestinal submucosa (SIS), urinary bladder matrix (UBM), and liver. To evaluate the effect on the growth of vascular endothelial cells, which are important in the tissue regeneration process, isolated SIS-derived MBVs were exposed to vascular endothelial cells to induce cell proliferation. These results indicate that MBVs can be extracted from HHP-decellularized tissues and may play a significant role in tissue remodeling.

In Vitro Tissue Reconstruction Using Decellularized Pericardium Cultured with Cells for Ligament Regeneration.

Recent applications of decellularized tissues have included the ectopic use of their sheets and powders for three-dimensional (3D) tissue reconstruction. Decellularized tissues are fabricated with the desired functions to employ them to a target tissue. The aim of this study was to develop a 3D reconstruction method using a recellularized pericardium to overcome the difficulties in cell infiltration into tight and dense tissues, such as ligament and tendon tissues. Decellularized pericardial tissues were prepared using the high hydrostatic pressurization (HHP) and surfactant methods. The pericardium consisted of bundles of aligned fibers. The bundles were slightly disordered in the surfactant decellularization method compared to the HHP decellularization method. The mechanical properties of the pericardium were maintained after the HHP and surfactant decellularizations. The HHP-decellularized pericardium was rolled up into a cylindrical formation. Its mechanical behavior was similar to that of a porcine anterior cruciate ligament in tensile testing. NIH3T3, C2C12, and mesenchymal stem cells were adhered with elongation and alignment on the HHP- and surfactant-decellularized pericardia, with dependences on the cell type and decellularization method. When the recellularized pericardium was rolled up into a cylinder formation and cultured by hanging circulation for 2 days, the cylinder formation and cellular elongation and alignment were maintained on the decellularized pericardium, resulting in a layer structure of cells in a cross-section. According to these results, the 3D-reconstructed decellularized pericardium with cells has the potential to be an attractive alternative to living tissues, such as ligament and tendon tissues.

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.

Tumor growth suppression by implantation of an anti-CD25 antibody-immobilized material near the tumor via regulatory T cell capture.

In this study, we designed and synthesized an implantable anti-CD25 antibody-immobilized polyethylene (CD25-PE) mesh to suppress tumor growth by removing regulatory T cells (Tregs). The PE mesh was graft-polymerized with poly(acrylic acid), and the anti-mouse CD25 antibody was then immobilized using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide reaction. Immobilization of the antibody on the PE mesh was confirmed by immunostaining. The CD25-PE mesh could effectively and selectively capture CD25-positive cells through antigen-antibody interactions when the CD25-PE mesh was incubated with a suspension of mouse spleen cells, including CD25-positive cells. In addition, implantation of the CD25-PE mesh into mice subcutaneously demonstrated the Treg-capturing ability of the CD25-PE mesh with only a weak inflammatory reaction. In tumor-bearing mice, tumor growth was suppressed by subcutaneous implantation of the CD25-PE mesh near the tumor for 1 week. These results suggested that the anti-CD25 antibody-immobilized material could capture Tregs in vivo and inhibit tumor proliferation in a limited tumor-bearing mouse model. Further research is needed to facilitate cancer immunotherapy using implantable anti-CD25 antibody-immobilized material as a Treg-capturing device.

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.

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.

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.

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.

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.

Protein adsorption and cell adhesion behavior of engineering plastics plasticized by supercritical carbon dioxide.

We aimed to evaluate the biological properties of engineering plastics (PC, PSU, PAR) processed using supercritical carbon dioxide (scCO2). Conventional mold process was used to prepare disk-shaped samples that were then plasticized by scCO2 at temperatures lower than the glass transition temperature (Tg) of the polymers. Surface roughness, contact angle, and amount of adsorbed protein on the surface were increased after treatment. The surface roughness of PC was significantly changed by scCO2 treatment. Cell adhesion and proliferation changed according to the differences in surface roughness. Initially, the cell adhesion decreased in all scCO2-treated polymers. At 3 day, the cell proliferation on scCO2-treated PC was lower than that on non-treated PC, while that on treated and non-treated PSU and PAR samples remained unaltered. These results suggest that when supercritical treatment is performed under conditions that affect the surface properties of the material, we should consider that cell adhesion and proliferation may change.

Characterization of Engineering Plastics Plasticized Using Supercritical CO2.

The purpose of this study was to evaluate the physical and chemical properties of engineering plastics processed using supercritical CO2. First, we prepared disk-shaped test pieces via a general molding process, which were plasticized using supercritical CO2 at temperatures lower than the glass-transition points of engineering plastics. Amorphous polymers were plasticized, and their molecular weight remained nearly unchanged after treatment with supercritical CO2. The mechanical strength significantly decreased despite the unchanged molecular weight. The surface roughness and contact angle increased slightly, and electrical properties such as the rate of charging decreased significantly. These results suggest that supercritical CO2 could be used for a new molding process performed at lower temperatures than those used in general molding processes, according to the required properties.