Effective BMP-2 Release and Mineralization on a Graphene Oxide/Polyvinylpyrrolidone Hydrogel Forming Poly (ε-Caprolactone) Nanofibrous Scaffolds.

PCL nanofibrous scaffolds are widely used as bone scaffolds, and they can increase the efficiency of bone regeneration by loading drugs and/or growth factors onto them. However, to obtain a more effective bone regeneration effect, it is necessary to increase drug loading and release efficiency. In this study, conductive hydrogel forming nanofibrous scaffolds were prepared to increase drug efficiency. GO has an excellent conductivity and biocompatibility, making it an efficient conductive polymer for bone differentiation. Electrospun PCL was immersed in a mixed solution of GO and PVP and then crosslinked using gamma-ray irradiation. It was confirmed that GO/PVP-PCL was successfully prepared through its characterization (morphology, thermal, chemical, electrical, and biological properties). In addition, drug-release efficiency was confirmed by electrical stimulation after loading the sample with BMP-2, a bone-regeneration growth factor. Compared to PCL, it was confirmed that GO/PVP-PCL has an approximately 20% improved drug-release efficiency and an excellent mineralization of the scaffolds using SBF. After culturing MG63 cells on GO/PVP-PCL, a high effect on osteodifferentiation was confirmed by ALP activity. Therefore, GO/PVP-PCL prepared by a gamma-ray-induced crosslinking reaction is expected to be used as biomaterial for bone-tissue engineering.

Light and immunostimulant mediated in situ re-education of tumor-associated macrophages using photosensitizer conjugated mannan nanoparticles for boosting immuno-photodynamic anti-metastasis therapy.

In an immunosuppressive tumor microenvironment, tumor-associated macrophages (TAMs) are the most abundant cells displaying pro-tumorigenic M2-like phenotypes, encouraging tumor growth and influencing the development of resistance against conventional therapies. TAMs are highly malleable. They can be repolarized into tumoricidal M1-like cells. In this study, we report the synthesis of novel co-operative immuno-photodynamic nanoparticles involving TAM self-targeting acrylic acid grafted mannan (a polysaccharide) conjugated with the chlorin e6 (Ce6) photosensitizer and then loaded with resiquimod (R848), a toll-like receptor (TLR7/8) agonist. The mannan conjugated Ce6 loaded with R848 (MCR) as bioconjugate nanoparticles demonstrated selective targeting of anti-inflammatory M2-like cells. Using photodynamic therapy they were repolarized to pro-inflammatory M1-like cells with combined effects of reactive oxygen species (ROS)-triggered intracellular signaling and a small-molecule immunostimulant. The MCR also demonstrated a TAM-directed adaptive immune response, inhibited tumor growth, and prevented metastasis. Our results indicate that these MCR nanoparticles can effectively target TAMs and modulate them for cancer immunotherapy.

In Vivo Evaluation of Gamma-Irradiated and Heparin-Immobilized Small-Diameter Polycaprolactone Vascular Grafts with VEGF in Aged Rats.

The effectiveness of small-diameter vascular grafts depends on their antithrombogenic properties and ability to undergo accelerated endothelialization. The extreme hydrophobic nature of poly(ε-caprolactone) (PCL) hinders vascular tissue integration, limiting its use in medical implants. To enhance the antithrombogenicity of PCL as a biomaterial, we grafted 2-aminoethyl methacrylate (AEMA) hydrochloride onto the PCL surface using gamma irradiation; developed a biodegradable heparin-immobilized PCL nanofibrous scaffold using gamma irradiation and N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride/N-hydroxysuccinimide reaction chemistry; and incorporated vascular endothelial growth factor (VEGF) into the scaffold to promote vascular endothelial cell proliferation and prevent thrombosis on the vascular grafts. We assessed the physicochemical properties of PCL, heparin-AEMA-PCL (H-PCL), and VEGF-loaded heparin-AEMA-PCL (VH-PCL) vascular grafts using scanning electron microscopy, attenuated total reflection-Fourier transform infrared spectroscopy, toluidine blue O staining, and fibrinogen adsorption and surface wettability measurement. In addition, we implanted the vascular grafts into 24-month-old Sprague Dawley rats and evaluated them for 3 months. The H-PCL and VH-PCL vascular grafts improved the recovery of blood vessel function by promoting the proliferation of endothelial cells and preventing thrombosis in clinical and histological evaluation, indicating their potential to serve as functional vascular grafts in vascular tissue engineering.

Quantitative Photothermal Characterization with Bioprinted 3D Complex Tissue Constructs for Early-Stage Breast Cancer Therapy Using Gold Nanorods.

Plasmonic photothermal therapy (PPTT) using gold nanoparticles (AuNPs) has shown great potential for use in selective tumor treatment, because the AuNPs can generate destructive heat preferentially upon irradiation. However, PPTT using AuNPs has not been added to practice, owing to insufficient heating methods and tissue temperature measurement techniques, leading to unreliable and inaccurate treatments. Because the photothermal properties of AuNPs vary with laser power, particle optical density, and tissue depth, the accurate prediction of heat generation is indispensable for clinical treatment. In this report, bioprinted 3D complex tissue constructs comprising processed gel obtained from porcine skin and human decellularized adipose tissue are presented for characterization of the photothermal properties of gold nanorods (AuNRs) having an aspect ratio of 3.7 irradiated by a near-infrared laser. Moreover, an analytical function is suggested for achieving PPTT that can cause thermal damage selectively on early-stage human breast cancer by regulating the heat generation of the AuNRs in the tissue.

Synthesis and characterization of hydrogels based on carboxymethyl chitosan and poly(vinylpyrrolidone) blends prepared by electron beam irradiation having anticancer efficacy, and applications as drug carrier for controlled release of drug.

Herein, carboxymethyl chitosan and poly(vinylpyrrolidone) based hydrogels were synthesized by electron beam irradiation with dose variations (15 kGy, 30 kGy, and 45 kGy) for drug delivery applications. Irradiation crosslinked hydrogels were characterized for swellings in different medias, chemical, thermal, cell cytotoxicity, and drug release aspects. Swelling analysis was evaluated in distilled water, buffer, and saline solutions. Fourier transform infrared analysis revealed the establishment of physical interactions and confirmed the presence of functional groups present in the drug carriers. Scanning electron microscopy depicted the porous structure, which is responsible for swelling, drug loading, and release. Cell cytotoxicity assays indicated good cell viability on RAW 264.7 cells and anticancer activity on cancerous AGS cell lines. Cumulative drug release (%) of kanamycin in PBS at pH 7.4 was more than 90 % at 168 h. These drug carriers show promise to be developed as a sustained drug delivery system.

Biomimetic nonbiofouling polypyrrole electrodes grafted with zwitterionic polymer using gamma rays.

Bioelectrodes, including metallic and conductive polymer (CP) bioelectrodes, often suffer from biofouling by contamination from microbacteria and/or biomolecules in biological systems, which can cause substantial impairment of biofunctionality and biocompatibility. Herein, we have employed an in situ polymerization of methacryloyloxyethyl phosphorylcholine (MPC) by gamma radiation to introduce fouling-resistant properties onto the surface of the conductive polymer, polypyrrole (PPy). The concentrations of an MPC monomer were varied during the grafting. PPy electrodes modified with MPC (PPy-g-MPC) revealed excellent anti-biofouling properties, as demonstrated by multiple analyses, such as serum protein adsorption, fibroblast adhesion, bacteria adhesion, and scar tissue formation in vivo. Importantly, PPy-g-MPC, which was modified with 0.2 M MPC using gamma radiation, exhibited electrical properties similar to unmodified PPy electrodes, indicating that our MPC grafting strategies did not cause impairment of electrical/electrochemical properties of the original PPy electrodes while successfully introducing anti-biofouling properties. Zwitterionic MPC polymer grafting on PPy electrodes by in situ polymerization with gamma radiation will benefit the development of highly biocompatible and functional bioelectrodes, such as neural electrodes, stimulators, and biosensors.

Enhanced tendon restoration effects of anti-inflammatory, lactoferrin-immobilized, heparin-polymeric nanoparticles in an Achilles tendinitis rat model.

Gradual wear and tear can cause a local inflammatory response in tendons. The trauma and inflammatory reaction eventually impair the biomechanical properties of the tendon. In this study, we prepared lactoferrin-immobilized, heparin-anchored, poly(lactic-co-glycolic acid) nanoparticles (LF/Hep-PLGA NPs) and evaluated their in vitro anti-inflammatory effects on interleukin-1ß (IL-1ß)-treated tenocytes and in vivo tendon healing effects in a rat model of Achilles tendinitis. Long-term LF-deliverable NPs (LF/Hep-PLGA NPs) remarkably decreased mRNA levels of pro-inflammatory factors [cyclooxygenase-2 (COX-2), IL-1ß, matrix metalloproteinase-3 (MMP-3), MMP-13, IL-6, and tumor necrosis factor-α (TNF-α)] and increased mRNA levels of anti-inflammatory cytokines (IL-4 and IL-10) in both IL-1ß-treated tenocytes and the Achilles tendons of a collagenase-induced Achilles tendinitis rat model. Interestingly, anti-inflammatory LF/Hep-PLGA NPs greatly enhanced collagen content, mRNA levels of tenogenic markers [collagen type I (COL1A1), decorin (DCN), tenascin-C (TNC)], and biomechanical properties such as tendon stiffness and tensile strength. These results suggest that anti-inflammatory LF/Hep-PLGA NPs are effective at restoring tendons in Achilles tendinitis.

Therapeutic Efficacy of Intratendinous Delivery of Dexamethasone Using Porous Microspheres for Amelioration of Inflammation and Tendon Degeneration on Achilles Tendinitis in Rats.

Achilles tendinitis caused by overuse, aging, or gradual wear induces pain, swelling, and stiffness of Achilles tendon and leads to tendon rupture. This study was performed to investigate the suppression of inflammation responses in interleukin-1ß- (IL-1ß-) stimulated tenocytes in vitro and the suppression of the progression of Achilles tendinitis-induced rat models in vivo using dexamethasone-containing porous microspheres (DEX/PMSs) for a sustained intratendinous DEX delivery. DEX from DEX/PMSs showed the sustained release of DEX. Treatment of IL-1ß-stimulated tenocytes with DEX/PMSs suppressed the mRNA levels for COX-2, IL-1ß, IL-6, and TNF-α. The intratendinous injection of DEX/PMSs into Achilles tendinitis rats both decreased the mRNA levels for these cytokines and increased mRNA levels for anti-inflammatory cytokines IL-4 and IL-10 in tendon tissues. Furthermore, DEX/PMSs effectively prevented tendon degeneration by enhancing the collagen content and biomechanical properties. Our findings suggest that DEX/PMSs show great potential as a sustained intratendinous delivery system for ameliorating inflammation responses as well as tendon degeneration in Achilles tendinitis.

Gamma Ray-Induced Polymerization and Cross-Linking for Optimization of PPy/PVP Hydrogel as Biomaterial.

Conducting polymer (CP)-based hydrogels exhibit the behaviors of bending or contraction/relaxation due to electrical stimulation. They are similar in some ways to biological organs and have advantages regarding manipulation and miniaturization. Thus, these hydrogels have attracted considerable interest for biomedical applications. In this study, we prepared PPy/PVP hydrogel with different concentrations and content through polymerization and cross-linking induced by gamma-ray irradiation at 25 kGy to optimize the mechanical properties of the resulting PPy/PVP hydrogel. Optimization of the PPy/PVP hydrogel was confirmed by characterization using scanning electron microscopy, gel fraction, swelling ratio, and Fourier transform infrared spectroscopy. In addition, we assessed live-cell viability using live/dead assay and CCK-8 assay, and found good cell viability regardless of the concentration and content of Py/pTS. The conductivity of PPy/PVP hydrogel was at least 13 mS/cm. The mechanical properties of PPy/PVP hydrogel are important factors in their application for biomaterials. It was found that 0.15PPy/PVP20 (51.96 ± 6.12 kPa) exhibited better compressive strength than the other samples for use in CP-based hydrogels. Therefore, it was concluded that gamma rays can be used to optimize PPy/PVP hydrogel and that biomedical applications of CP-based hydrogels will be possible.

Preparation of Radiation Cross-Linked Poly(Acrylic Acid) Hydrogel Containing Metronidazole with Enhanced Antibacterial Activity.

Metronidazole (MD) is known as a periodontitis medicine and has been widely used in antibiotics for resistance to anaerobic bacteria, periodontal disease, and other threats. To treat diseases, drug delivery carriers are needed with a high bioadhesive property and enhanced drug penetration. Poly (acrylic acid) (PAA) hydrogel films have a good bioadhesive property and are able to localize the absorption site and increase the drug residence time. In this study, we fabricated a MD loaded PAA hydrogel with different MD content (0.1, 0.25, 0.5, and 1 wt%) using varying doses (25, 50, and 75 kGy) and the radiation doses (25, 50, or 75 kGy) in a one-step gamma-ray irradiation process. The chemical and physical structure were determined through a Fourier transformed infrared spectroscopy, X-ray photoelectron spectroscopy, gel content, and compressive strength. In addition, MD loaded PAA hydrogels were performed to MD release behaviors and cytotoxicity. Finally, we conducted antibacterial activity to demonstrate the prevention of growth of bacteria as a therapeutic dressing. The basic chemical structure analysis of MD was changed greatly at radiation doses of 50 and 75 kGy due to degradation by gamma-ray irradiation. However, when the absorbed dose was 25 kGy, the chemical structure analysis of MD did not change significantly, and the gel content and compressive strength of MD/PAA hydrogel were approximately 80% and 130 kPa, respectively. The MD/PAA hydrogels exhibited no cytotoxicity and good antibacterial activity against Escherichia coli, Staphylococcus aureus, and Streptococcus mutans. These results provide good evidence that MD/PAA hydrogel prepared by gamma-ray irradiation has potential as a competitive candidate for the therapeutic dressing.

Effect of Titanium Implants Coated with Radiation-Crosslinked Collagen on Stability and Osseointegration in Rat Tibia.

This study aimed to evaluate the titanium (Ti) implants coated with collagen type Ⅰ crosslinked using gamma-irrigation or glutaraldehyde (GA). The in vitro surface observations, quantification assay, and cell studies using human mesenchymal stem cells (hMSCs) were conducted. For in vivo experiments, the implants were divided into three groups and inserted into the rat tibias: control group (non-treated Ti implant), GA group (Ti implants coated with GA-crosslinked collagen) and 25 kGy group (Ti implants coated with gamma-radiation-crosslinked collagen at dose of 25 kGy). The animals were sacrificed at 4 weeks after implantation and the tissue sections were obtained. New bone volume (mm³) and bone-to-implant contact (BIC, %) within the region of interest (ROI) was measured. The in vitro results showed the highest osteogenic differentiation and levels of osteogenesis-related gene expressions in the 25 kGy group without cytotoxicity. The new bone volume of GA group was significantly higher than the control (p < 0.05). In the result of the BIC, the 25 kGy group was significantly higher than the control (p < 0.05). However, there was no significant difference between the experimental groups. Within the limitations of this study, Ti implant coated with gamma-radiation-crosslinked collagen has potential utility without side effects from chemical agents.

Preparation and evaluation of ß-glucan hydrogel prepared by the radiation technique for drug carrier applications.

ß-Glucan can provide excellent environment to apply to drug carrier due to its immunological and anti-inflammatory effect. Minocycline hydrochloride (MH) has excellent oral bioavailability pharmacological properties. Specifically, MH is effectively absorbed into the gingiva for periodontal disease treatment. In this study, we attempt to develop MH loaded ß-glucan hydrogel for periodontal disease treatment through radiation-crosslinking technique. In addition, MH loaded ß-glucan hydrogels were tested for their cytotoxicity and antibacterial activity. Finally, we conducted an in vivo study to demonstrate the potential to prevent the invasion of bacteria to treat periodontal disease. The gel content and compressive strength of the ß-glucan hydrogels increased as the ß-glucan content and the absorbed dose (up to 7 kGy) increased. For a radiation dose of 7 kGy, the gelation and the compressive strength of a 6 wt% ß-glucan hydrogel were approximately 92% and 270 kPa, respectively. As a drug, MH was consistently released from ß-glucan hydrogels, reaching 80% at approximately 90 min. Furthermore, the MH loaded ß-glucan hydrogels showed no cytotoxicity. The MH loaded ß-glucan hydrogels exhibited good antibacterial activity against Porphyromonas gingivalis. In addition, MH loaded ß-glucan hydrogel demonstrated the potential of a good capability to prevent the invasion of bacteria and to treat wounds.

Effective gamma-ray sterilization and characterization of conductive polypyrrole biomaterials.

Conductive polymers, including polypyrrole (PPy), have been extensively explored to fabricate electrically conductive biomaterials for bioelectrodes and tissue engineering scaffolds. For their in vivo uses, a sterilization method without severe impairment of original material properties and performance is necessary. Gamma-ray radiation has been commonly applied for sterilization of medical products because of its simple and uniform sterilization without heat generation. Herein we describe the first study on gamma-ray sterilization of PPy bioelectrodes and its effects on their characteristics. We irradiated PPy bioelectrodes with different doses (0-75 kGy) of gamma-rays. Gamma-ray irradiation of the PPy (γ-PPy) increased the oxygenation and hydrophilicity of the surfaces. Interestingly, gamma-ray irradiation did not alter the electrical impedances and conductivities of the PPy substrates. Additionally, γ-PPy prepared with various dopants (e.g., para-toluene sulfonate, polystyrene sulfonate, and chlorine) showed the electrochemical properties similar to the non-irradiated control. Gamma-ray irradiation at doses of ≥15 kGy was required for effective sterilization as evidenced by complete eradication of gram positive and negative bacteria. γ-PPy substrates also showed cytocompatibility similar to untreated control PPy, indicating no substantial alteration of cytocompatibility. In conclusion, gamma ray sterilization is a viable method of sterilization of conducting polymer-based biomaterials for biomedical applications.

One-step synthesis of gene carrier via gamma irradiation and its application in tumor gene therapy.

INTRODUCTION: Although numerous studies have been conducted with the aim of developing drug-delivery systems, chemically synthesized gene carriers have shown limited applications in the biomedical fields due to several problems, such as low-grafting yields, undesirable reactions, difficulties in controlling the reactions, and high-cost production owing to multi-step manufacturing processes. MATERIALS AND METHODS: We developed a 1-step synthesis process to produce 2-aminoethyl methacrylate-grafted water-soluble chitosan (AEMA-g-WSC) as a gene carrier, using gamma irradiation for simultaneous synthesis and sterilization, but no catalysts or photoinitiators. We analyzed the AEMA graft site on WSC using 2-dimensional nuclear magnetic resonance spectroscopy (2D NMR; 1H and 13C NMR), and assayed gene transfection effects in vitro and in vivo. RESULTS: We revealed selective grafting of AEMA onto C6-OH groups of WSC. AEMA-g-WSC effectively condensed plasmid DNA to form polyplexes in the size range of 170 to 282 nm. AEMA-g-WSC polyplexes in combination with psi-hBCL2 (a vector expressing short hairpin RNA against BCL2 mRNA) inhibited tumor cell proliferation and tumor growth in vitro and in vivo, respectively, by inducing apoptosis. CONCLUSION: The simple grafting process mediated via gamma irradiation is a promising method for synthesizing gene carriers.

Preparation and Characterization of Resorbable Bacterial Cellulose Membranes Treated by Electron Beam Irradiation for Guided Bone Regeneration.

Bacterial cellulose (BC) is an excellent biomaterial with many medical applications. In this study, resorbable BC membranes were prepared for guided bone regeneration (GBR) using an irradiation technique for applications in the dental field. Electron beam irradiation (EI) increases biodegradation by severing the glucose bonds of BC. BC membranes irradiated at 100 kGy or 300 kGy were used to determine optimal electron beam doses. Electron beam irradiated BC membranes (EI-BCMs) were evaluated by scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, thermal gravimetric analysis (TGA), and using wet tensile strength measurements. In addition, in vitro cell studies were conducted in order to confirm the cytocompatibility of EI-BCMs. Cell viabilities of NIH3T3 cells on 100k and 300k EI-BCMs (100 kGy and 300 kGy irradiated BC membranes) were significantly greater than on NI-BCMs after 3 and 7 days (p < 0.05). Bone regeneration by EI-BCMs and their biodegradabilities were also evaluated using in vivo rat calvarial defect models for 4 and 8 weeks. Histometric results showed 100k EI-BCMs exhibited significantly larger new bone area (NBA; %) than 300k EI-BCMs at 8 weeks after implantation (p < 0.05). Mechanical, chemical, and biological analyses showed EI-BCMs effectively interacted with cells and promoted bone regeneration.

The Efficacy of Electron Beam Irradiated Bacterial Cellulose Membranes as Compared with Collagen Membranes on Guided Bone Regeneration in Peri-Implant Bone Defects.

Bacterial cellulose (BC) is a natural polysaccharide produced by some bacteria, and consists of a linear polymer linked by ß-(1,4) glycosidic bonds. BC has been developed as a material for tissue regeneration purposes. This study was conducted to evaluate the efficacy of resorbable electron beam irradiated BC membranes (EI-BCMs) for guided bone regeneration (GBR). The electron beam irradiation (EI) was introduced to control the biodegradability of BC for dental applications. EI-BCMs had higher porosity than collagen membranes (CMs), and had similar wet tensile strengths to CMs. NIH3T3 cell adhesion and proliferation on EI-BCMs were not significantly different from those on CMs (p > 0.05). Micro-computed tomography (µCT) and histometric analysis in peri-implant dehiscence defects of beagle dogs showed that EI-BCMs were non-significantly different from CMs in terms of new bone area (NBA; %), remaining bone substitute volume (RBA; %) and bone-to-implant contact (BIC; %) (p > 0.05). These results suggest resorbable EI-BCMs can be used as an alternative biomaterial for bone tissue regeneration.

The Effect of Thickness of Resorbable Bacterial Cellulose Membrane on Guided Bone Regeneration.

This study introduces the effect of the thickness of a bacterial cellulose membrane by comparing the bone regeneration effect on rat skulls when using a collagen membrane and different thicknesses of resorbable bacterial cellulose membranes for guided bone regeneration. Barrier membranes of 0.10 mm, 0.15 mm, and 0.20 mm in thickness were made using bacterial cellulose produced as microbial fermentation metabolites. Mechanical strength was investigated, and new bone formation was evaluated through animal experimental studies. Experimental animals were sacrificed after having 2 weeks and 8 weeks of recovery, and specimens were processed for histologic and histomorphometric analyses measuring the area of bone regeneration (%) using an image analysis program. In 2 weeks, bone-like materials and fibrous connective tissues were observed in histologic analysis. In 8 weeks, all experimental groups showed the arrangement of osteoblasts surrounding the supporting body on the margin and center of the bone defect region. However, the amount of new bone formation was significantly higher (p < 0.05) in bacterial cellulose membrane with 0.10 mm in thickness compared to the other experimental groups. Within the limitations of this study, a bacterial cellulose membrane with 0.10 mm thickness induced the most effective bone regeneration.

Chestnut Honey Impregnated Carboxymethyl Cellulose Hydrogel for Diabetic Ulcer Healing.

Honey-based wound dressings have attracted a lot of attention from modern scientists owing to their anti-inflammatory and antibacterial effects without antibiotic resistance. Such dressings also promote moist wound healing, and have been considered natural, abundant, and cheap materials for folk marketing. This study investigated the various behaviors and characteristics of chestnut honey-impregnated carboxymethyl cellulose sodium hydrogel paste (CH⁻CMC) as a therapeutic dressing, such as its moist retention, antibacterial activity for inhibiting the growth of Staphylococcus aureus and Escherichia coli, and the rate of wound healing in db/db mice. The results provide good evidence, suggesting that CH⁻CMC has potential as a competitive candidate for diabetic ulcer wound healing.

Engineering an aligned endothelial monolayer on a topologically modified nanofibrous platform with a micropatterned structure produced by femtosecond laser ablation.

A monolayer of endothelial cells (ECs) aligned along the direction of blood flow plays crucial roles in the regulation of anti-thrombogenic and pro-inflammatory reactions in the blood vessel wall. Thus, many researchers have attempted to mimic the aligned structure of ECs in vascular grafts or tissue-engineered blood vessels. In the present study, we fabricated micro-groove patterned nanofibers using a femtosecond laser ablation technique to recapitulate the densely organized anisotropic architecture of the endothelial layer. Femtosecond laser ablation enabled us to generate high-resolution groove patterns (10 µm width) with 20 or 80 µm gaps on randomly oriented electrospun nanofibers. The patterned nanofibers exhibited anisotropic (transverse: 101.1 ± 4.0° and longitudinal: 123.5 ± 9.4°) water contact angles; however, the mechanical properties were consistent in both directions. The micropatterned nanofibers modulated the aligned structure or aspect ratio (20 µm: 0.23 ± 0.11 and 80 µm: 0.42 ± 0.18) of ECs along the pattern direction. In particular, the engineered aligned endothelial layer was effective in eliciting an anti-inflammatory response (approximately 50% greater than that of random or aligned nanofibers), thereby effectively preventing monocyte adhesion following activation by TNF-α treatment. Therefore, micropatterning by laser ablation can be utilized to generate high-resolution microgrooves on various substrates, thereby providing fundamental platforms for vascular tissue engineering.

Construction of 3-D Cellular Multi-Layers with Extracellular Matrix Assembly Using Magnetic Nanoparticles.

Construction of 3-dimensional (3-D) engineered tissue is increasingly being investigated for use in drug discovery and regenerative medicine. Here, we developed multi-layered 3-D cellular assembly by using magnetic nanoparticles (MNP) isolated from Magnetospirillum sp. AMB-1 magnetotactic bacteria. Magnetized human dermal fibroblasts (HDFBs) were prepared by treatment with the MNP, induced to form 3-D assembly under a magnetic field. Analyses including LIVE/DEAD assay, transmission electron microscopy revealed that the MNP were internalized via clathrin-mediated endocytosis without cytotoxicity. The magnetized HDFBs could build 3-D structure as a function of seeding density. When the highest seeding density (5 × 105 cells/mm2 was used, the thickness of assembly was 41.90 ± 1.69 µm, with approximately 9.3 ± 1.6 cell layers being formed. Immunofluorescence staining confirmed homogeneous distribution of ECM and junction proteins throughout the 3-D assembly. Real-time PCR analysis showed decrease in expression levels of collagen types I and IV but increase in that of connexin 43 in the 3-D assembly compared with the 2-D culture. Finally, we demonstrated that the discernible layers can be formed hierarchically by serial assembly. In conclusion, our study showed that a multi-layered structure can be easily prepared using magnetically-assisted cellular assembly with highlighting cell-cell and cell-ECM communication.