Cycloamylose-nanogel drug delivery system-mediated intratumor silencing of the vascular endothelial growth factor regulates neovascularization in tumor microenvironment.

RNAi enables potent and specific gene silencing, potentially offering useful means for treatment of cancers. However, safe and efficient drug delivery systems (DDS) that are appropriate for intra-tumor delivery of siRNA or shRNA have rarely been established, hindering clinical application of RNAi technology to cancer therapy. We have devised hydrogel polymer nanoparticles, or nanogel, and shown its validity as a novel DDS for various molecules. Here we examined the potential of self-assembled nanogel of cholesterol-bearing cycloamylose with spermine group (CH-CA-Spe) to deliver vascular endothelial growth factor (VEGF)-specific short interfering RNA (siVEGF) into tumor cells. The siVEGF/nanogel complex was engulfed by renal cell carcinoma (RCC) cells through the endocytotic pathway, resulting in efficient knockdown of VEGF. Intra-tumor injections of the complex significantly suppressed neovascularization and growth of RCC in mice. The treatment also inhibited induction of myeloid-derived suppressor cells, while it decreased interleukin-17A production. Therefore, the CH-CA-Spe nanogel may be a feasible DDS for intra-tumor delivery of therapeutic siRNA. The results also suggest that local suppression of VEGF may have a positive impact on systemic immune responses against malignancies.

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.

Induction of osteogenic differentiation by the extracellular matrix of fetal bone tissues and adult cartilage.

Decellularized cortical bone powder derived from adult animals has been shown to induce bone remodeling. Furthermore, it is increasingly evident that the extracellular matrix (ECM) within decellularized tissues differs depending on the source tissue and the age of the animal, leading to distinct effects on cells. In this study, we prepared powders from decellularized fetal and adult porcine bone tissues and conducted biological analyses to determine if the decellularized tissue could induce adipose-derived stem cell differentiation. Decellularized fetal tissues and adult cortical bone were converted into powder by cryomilling, but decellularized adult bone marrow and cartilage were not powdered through this process. In vitro assessments revealed that decellularized fetal tissues, decellularized adult cartilage extract, and decellularized fetal cartilage powder can induce osteoblast differentiation. This study suggests that decellularized fetal bone tissues and adult cartilage contain ECM components that can induce osteoblast differentiation. Additionally, it highlights the utility of decellularized fetal cartilage powder for bone reconstruction.

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.

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.

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.

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.

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.

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.

Nanogel Tectonics for Tissue Engineering: Protein Delivery Systems with Nanogel Chaperones.

Amphiphilic polysaccharide self-assembled (SA) nanogels are promising protein carriers owing to their chaperone-like activity that allows them to nanoencapsulate proteins within their polymer networks. The chaperoning function is an important concept that has led to breakthroughs in the development of effective protein drug delivery systems by stabilizing formulations and controlling the quality of unstable proteins. Recently, nanogel-tectonic materials that integrate SA nanogels as building blocks have been designed as new hydrogel biomaterials. This article describes recent progress and applications of SA nanogel tectonic materials as protein delivery systems for tissue engineering.

Effects of a polysaccharide nanogel-crosslinked membrane on wound healing.

INTRODUCTION: Wound-dressing materials that promote wound healing while protecting wounds from infections are advantageous for clinical applications. Hence, we developed a cholesterol-bearing pullulan (CHP) nanogel that stimulated wound healing; however, it was mechanically weak and difficult to handle. Thus, the purpose of this study was to examine precisely the effects of a mechanically reinforced nanogel-crosslinked (NanoClik) membrane on wound healing. MATERIALS AND METHODS: NanoClik was prepared by mixing a thiol-terminated polyethylene glycol solution and an acryloyl group-modified CHP nanogel solution. A thin silicone sheet membrane, which was combined with NanoClik, was prepared. The NanoClick membranes and both positive and negative control membranes (collagen combined with silicone membrane and silicone membrane alone, respectively) were tested in vivo using a dorsal skin defect rat model. The rate and extent of wound healing was compared between groups after 7 and 14 days of implantation. RESULTS: In the NanoClik membrane group, the wound area was significantly reduced and neoepithelialization was promoted, compared with that observed in the other groups. In addition, extension and accumulation of collagen fibers were evident in the NanoClik membrane group. CONCLUSION: The NanoClik membrane is a strong candidate for use as an effective and safe wound-dressing material. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 544-550, 2017.

Capture and release of cells using a temperature-responsive surface that immobilizes an antibody through DNA duplex formation.

We synthesized a temperature-responsive surface that immobilized an antibody via DNA duplex formation for selective capture and release of target cells. Polyethylene films were modified by grafting poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAAm-co-AAc)), which were prepared at various ratios of NIPAAm/AAc. The increased hydrophilicity of P(NIPAAm-co-PAA) film with decreased temperature was confirmed by water contact angle measurement. Single strand DNA (20mer) was chemically immobilized on the surface and then antibody (anti-mouse CD45, mCD45) modified with the complementary single strand DNA was immobilized on the surface through DNA duplex formation. The mCD45 antibody immobilization was confirmed by immunostaining. HeLa cells (mCD45 negative) and mouse bone marrow (BM) cells (mCD45 positive) were adhered on the surfaces at 37 °C. Although HeLa cells were detached by 4 °C incubation, BM cells were still adhered on the surface and then the adhered cells were released by DNase treatment. From these results, it was suggested that cells could be selectively captured and collected by using a film having surface that immobilizes an antibody via DNA duplex formation.

Nanogel tectonic porous gel loading biologics, nanocarriers, and cells for advanced scaffold.

We developed a new self-assembled amphiphilic nanogel-crosslinked porous (NanoCliP) gel that can trap proteins, liposomes, and cells. The NanoCliP gel was prepared by Michael addition of a self-assembled nanogel of acryloyl group-modified cholesterol-bearing pullulan to pentaerythritol tetra (mercaptoethyl) polyoxyethylene, followed by freezing-induced phase separation. Dynamic rheological analysis revealed that the storage modulus (G') of the NanoCliP gel was approximately 10 times greater than that of a nonporous nanogel-crosslinked gel. Two-photon excitation deep imaging revealed that the NanoCliP gel comprises interconnected pores of several hundred micrometers in diameter. The NanoCliP gel trapped proteins and liposomes via hydrophobic interactions because its amphiphilic nanogels exhibit chaperone-like activity. Mouse embryonic fibroblasts penetrated the interconnected pores and adhered to the porous surface of fibronectin-complexed NanoCliP gel. In vivo, the NanoCliP gel enhanced cell infiltration, tissue ingrowth, and neovascularization without requiring exogenous growth factors, suggesting that the NanoCliP gel is a promising scaffold for tissue engineering.

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.

Therapeutic effect of nanogel-based delivery of soluble FGFR2 with S252W mutation on craniosynostosis.

Apert syndrome is an autosomal dominantly inherited disorder caused by missense mutations in fibroblast growth factor receptor 2 (FGFR2). Surgical procedures are frequently required to reduce morphological and functional defects in patients with Apert syndrome; therefore, the development of noninvasive procedures to treat Apert syndrome is critical. Here we aimed to clarify the etiological mechanisms of craniosynostosis in mouse models of Apert syndrome and verify the effects of purified soluble FGFR2 harboring the S252W mutation (sFGFR2IIIcS252W) on calvarial sutures in Apert syndrome mice in vitro. We observed increased expression of Fgf10, Esrp1, and Fgfr2IIIb, which are indispensable for epidermal development, in coronal sutures in Apert syndrome mice. Purified sFGFR2IIIcS252W exhibited binding affinity for fibroblast growth factor (Fgf) 2 but also formed heterodimers with FGFR2IIIc, FGFR2IIIcS252W, and FGFR2IIIbS252W. Administration of sFGFR2IIIcS252W also inhibited Fgf2-dependent proliferation, phosphorylation of intracellular signaling molecules, and mineralization of FGFR2S252W-overexpressing MC3T3-E1 osteoblasts. sFGFR2IIIcS252W complexed with nanogels maintained the patency of coronal sutures, whereas synostosis was observed where the nanogel without sFGFR2S252W was applied. Thus, based on our current data, we suggest that increased Fgf10 and Fgfr2IIIb expression may induce the onset of craniosynostosis in patients with Apert syndrome and that the appropriate delivery of purified sFGFR2IIIcS252W could be effective for treating this disorder.

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.