Direct Fabrication of Glycoengineered Cells via Photoresponsive Thiol-ene Reaction.

Three-dimensional printing of cell constructs with high-cell density, shape fidelity, and heterogeneous cell populations is an important tool for investigating cell sociology in living tissues but remains challenging. Herein, we propose an artificial intercellular adhesion method using a photoresponsive chemical cue between a thiol-bearing polymer and a methacrylate-bearing cell membrane. This process provided cell fabrication containing 108 cells/mL, embedded multiple cell populations in one structure, and enabled millimeter-sized scaleup. Our approach allows for the artificial cell construction of complex structures and is a promising bioprinting strategy for engineering tissues that are structurally and physiologically relevant.

High pressure pasteurization: Simultaneous native tissue decellularization and sterilization.

Introduction: Terminal sterilization is important for the clinical applicability of decellularized xenografts. High hydrostatic pressurization (HHP) process is a potential strategy for decellularization and decontamination of xenografts; however, its disinfection efficiency remains poorly elucidated. This study investigated the disinfection efficacy of the HHP process at physiologically relevant 36 °C against difficult-to-kill spore-forming bacteria. Methods: Bacillus atrophaeus and Geobacillus stearothermophilus were suspended in a pressurization medium with or without antibiotic agents and pressurized under two different HHP procedures: repeated and sustained pressurization. Results: The sustained pressurizing conditions, exploited for the conventional tissue decellularization, did not effectively eliminate the bacteria; however, repeated pressurization greatly increased the disinfection effect. Moreover, the antibiotic-containing pressurization medium further increased the disinfection efficiency to the level required for sterilization. Conclusions: The optimized high hydrostatic pressurization can be used to sterilize biological tissues during the decellularization process and is a promising strategy for manufacturing tissue-derived healthcare products.

Bone-targeting polyphosphodiesters that promote osteoblastic differentiation.

Polymers for pharmaceutical use have been attractive in medical treatments because of the conjugation of multifunctional components and their long circulation time in the blood stream. Bone-targeted drug delivery systems are also no exceptional, and several polymers have been proposed for the treatment of bone diseases, such as cancer metastasis and osteoporosis. Herein, we report that polyphosphodiesters (PPDEs) have a potential to enhance osteoblastic differentiation, and they have a targeting ability to bone tissues in vivo. Two types of PPDEs, poly (ethylene sodium phosphate) (PEP•Na) and poly (propylene sodium phosphate) (PPP•Na), have been synthesized. Regardless of the alkylene structure in the main chain of PPDEs, the gene expression of osteoblast-specific transcription factors and differentiation markers of mouse osteoblastic-like cells (MC3T3-E1 cells) cultured in a differentiation medium was significantly upregulated by the addition of PPDEs. Moreover, it was also clarified that the signaling pathway related to cytoplasmic calcium ions was activated by PPDEs. The mineralization of MC3T3-E1 cells has a similar trend with its gene expression and is synergistically enhanced by PPDEs with ß-glycerophosphate. The biodistribution of fluorescence-labeled PPDEs was also determined after intravenous injection in mice. PPDEs accumulated well in the bone through the blood stream, whereas polyphosphotriesters (PPTEs) tended to be excreted from the kidneys. Hydrophilic PEP•Na showed a superior bone affinity as compared with PPP•Na. PPDEs could be candidate polymers for the restoration of bone remodeling and bone-targeting drug delivery platforms.

Impact of REDV peptide density and its linker structure on the capture, movement, and adhesion of flowing endothelial progenitor cells in microfluidic devices.

Ligand-immobilization to stents and vascular grafts is expected to promote endothelialization by capturing flowing endothelial progenitor cells (EPCs). However, the optimized ligand density and linker structure have not been fully elucidated. Here, we report that flowing EPCs were selectively captured by the REDV peptide conjugated with a short linker. The microchannel surface was modified with the REDV peptide via Gly-Gly-Gly (G3), (Gly-Gly-Gly)3 (G9), and diethylene glycol (diEG) linkers, and the moving velocity and captured ratio were evaluated. On the unmodified microchannels, the moving velocity of the cells exhibited a unimodal distribution similar to the liquid flow. The velocity of the endothelial cells and EPCs on the peptide-immobilized surface indicated a bimodal distribution, and approximately 20 to 30% of cells moved slower than the liquid flow, suggesting that the cells were captured and rolled on the surface. When the immobilized ligand density was lower than 1 molecule/nm2, selective cell capture was observed only in REDV with G3 and diEG linkers, but not in G9 linkers. An in silico study revealed that the G9 linker tends to form a bent structure, and the REDV peptide is oriented to the substrate side. These results indicated that REDV captured the flowing EPC in a sequence-specific manner, and that the short linker was more adequate.

Particle-stabilized oil-in-water emulsions as a platform for topical lipophilic drug delivery.

Low-environmental-impact emulsion systems for transdermal drug delivery in topical treatment have gained increasing interest. However, low stability and adverse systemic side effects severely decrease their efficiency. This study proposed a stable oil-in-water (O/W) emulsion loaded with bifonazole (BFZ) as a lipophilic drug stabilized by poly(2-isopropoxy-2-oxo-1,3,2-dioxaphospholane)-modified cellulose nanocrystals (CNC-g-PIPP) as vehicles for topical delivery of lipophilic drugs. We fully characterized stability, BFZ-loaded particle-stabilized emulsions (PEs) for morphology, droplet size, and its distribution. In addition, we evaluated the in vitro drug-releasing capacity and in vitro skin permeation of BFZ in a porcine skin animal model using a side-bi-side® diffusion cell. An O/W BFZ-loaded emulsion stabilized with CNC-g-PIPP particles (BFZ-loaded CP-PE) with a small mean droplet size of 2.54 ± 1.39 µm was developed and was stable for > = 15 days without a significant change in droplet size. The BFZ-loading efficiency in PEs was 83.1 %. BFZ was slowly released over an extended period, and the releasing ratio from BFZ-loaded CP-PE was only 17 % after 48 h. The BFZ-loaded CP-PE showed a ∼4.4-fold increase in BFZ permeation and penetration compared to a conventional surfactant-stabilized emulsion and BFZ control solution. Fluorescence-labeling studies showed that BFZ-loaded CP-PE could well penetrate skin layers from the stratum corneum (SC) to the dermis. In addition, histopathology studies of porcine skin treated with the PE formulation showed an intact SC with unaltered adjacent structures and no observed signs of inflammation. Therefore, the proposed CP-PE shows great potential as a transdermal drug carrier for enhancing lipophilic drug permeation.

Bone-targeting phospholipid polymers to solubilize the lipophilic anticancer drug.

Current chemotherapy methods have limited effectiveness in eliminating bone metastasis, which leads to a poor prognosis associated with severe bone disorders. To provide regional chemotherapy for this metastatic tumor, a bone-targeting drug carrier was produced by introducing the osteotropic bisphosphonate alendronate (ALN) units into an amphiphilic phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate). The polymer can form nanoparticles with a diameter of less than 30 nm; ALN units were exposed to the outer layer of the particle. A simple mixing procedure was used to encapsulate a hydrophobic anticancer drug, known as docetaxel (DTX), in the polymer nanoparticle, providing a uniform solution of a polymer-DTX complex in the aqueous phase. The complex showed anticancer activities against several breast cancer cell lines, and the complex formation did not hamper the pharmacological effect of DTX. The fluorescence observations evaluated by an in vivo imaging system and fluorescence microscopy showed that the addition of ALN to the polymer-DTX complex enhanced bone accumulation. Bone-targeting phospholipid polymers are potential solubilizing excipients used to formulate DTX and deliver the hydrophobic drug to bone tissues by blood administration.

Bone-targeting poly(ethylene sodium phosphate).

Poly(ethylene sodium phosphate) (PEP·Na) showed excellent cytocompatibility and in vivo bone affinity. Moreover, PEP·Na did not interact with thrombin, which is a coagulation-related protein. Because immobilization of therapeutic agents and imaging probes on PEP·Na is easily performed, PEP·Na is a promising polymer for bone-targeted therapies.

Label-Free Separation of Induced Pluripotent Stem Cells with Anti-SSEA-1 Antibody Immobilized Microfluidic Channel.

When induced pluripotent stem cells (iPSCs) are routinely cultured, the obtained cells are a heterogeneous mixture, including feeder cells and partially differentiated cells. Therefore, a purification process is required to use them in a clinical stage. We described a label-free separation of iPSCs using a microfluidic channel. Antibodies against stage-specific embryonic antigen 1 (SSEA-1) was covalently immobilized on the channel coated with a phospholipid polymer. After injection of the heterogeneous cell suspension containing iPSCs, the velocity of cell movement under a liquid flow condition was measured. The mean velocity of the cell movement was 2.1 mm/sec in the unmodified channel, while that in the channel with the immobilized-antibody was 0.4 mm/sec. The eluted cells were fractionated by eluting time. As a result, the SSEA-1 positive iPSCs were mainly contained in later fractions, and the proportion of iPSCs was increased from 43% to 82% as a comparison with the initial cell suspension. These results indicated that iPSCs were selectively separated by the microfluidic channel. This channel is a promising device for label-free separation of iPSCs based on their pluripotent state.

Individual evaluation of cardiac marker expression and self-beating during cardiac differentiation of P19CL6 cells on different culture substrates.

Cell-based therapies using self-beating cardiomyocytes have been attracting great attention for use in cardiac regeneration, although an effective procedure to improve cardiac differentiation and self-beating induction is required. The purpose of this study is to clarify the effect of the culture substrate on cardiac maturation by separately evaluating the cardiac differentiation step and the beating induction step in vitro. To this end, the well-studied cardiomyocyte-like progenitor cell line P19CL6 and neonatal cardiomyocytes (NCMs) were selected and cultured on substrates coated with collagen type I (Col-I), gelatin (Gel), fibronectin (FN), or poly-l-lysine (PLL). It was found that the cardiac differentiation step, which was assessed using cardiac marker gene expression (GATA-binding protein 4 (GATA4), myocyte-specific enhancer factor 2D (MEF2D), and hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4)) in the P19CL6 embryonal carcinoma cells, was greatly enhanced on Col-I, Gel, and PLL. In contrast, the spontaneous beating step, which was directly assessed by counting the beating colonies and measuring contractile protein gene expression (α-myosin heavy chain (α-MHC), troponin C type 1 (TnC1), and troponin T type 2 (TnT2)) in the rat NCMs, was enhanced on the FN and PLL surfaces. In the present study, for the first time, it was found that PLL enhances both the cardiac differentiation and the beating induction steps of cardiac maturation, which can aid in preparing beating cardiomyocytes for regenerative medicine. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1166-1174, 2017.

Early tissue formation on whole-area osteochondral defect of rabbit patella by covering with fibroin sponge.

Large osteochondral defects have been difficult to repair via tissue engineering treatments due to the lack of a sufficient number of source cells for repairing the defect and to the severe mechanical stresses affecting the replacement tissue. In the present study, whole-area osteochondral defects of rabbit patella were covered and wrapped with a fibroin sponge containing chondrocytes, with or without Green Fluorescent Protein (GFP) transgenic marking, on the surface facing the osteochondral defect. Five of eight osteochondral defects that were covered with the chondrocyte-seeded fibroin sponges showed hyaline cartilage-like repair containing no fibroin fragments at 6 weeks after surgery. The repaired tissue showed a layer formation, which showed intensive safranin-O and toluidine blue staining, and which showed positive type II collagen immunostaining. The average surface coverage of the repaired cartilage was 53%. On average, 48% of the cells in the repaired tissue were derived from GFP transgenic chondrocytes, which had been seeded in the fibroin sponge. The fibroin-sponge covering had the potential to allow the early repair of large osteochondral defects. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1474-1482, 2016.

Porous Alpha-Tricalcium Phosphate with Immobilized Basic Fibroblast Growth Factor Enhances Bone Regeneration in a Canine Mandibular Bone Defect Model.

The effect of porous alpha-tricalcium phosphate (α-TCP) with immobilized basic fibroblast growth factor (bFGF) on bone regeneration was evaluated in a canine mandibular bone defect model. Identical bone defects were made in the canine mandible; six defects in each animal were filled with porous α-TCP with bFGF bound via heparin (bFGF group), whereas the other was filled with unmodified porous α-TCP (control group). Micro-computed tomography and histological evaluation were performed two, four and eight weeks after implantation. The bone mineral density of the bFGF group was higher than that of the control group at each time point (p < 0.05), and the bone mineral content of the bFGF group was higher than that of the control group at four and eight weeks (p < 0.05). Histological evaluation two weeks after implantation revealed that the porous α-TCP had degraded and bone had formed on the surface of α-TCP particles in the bFGF group. At eight weeks, continuous cortical bone with a Haversian structure covered the top of bone defects in the bFGF group. These findings demonstrate that porous α-TCP with immobilized bFGF can promote bone regeneration.

Lateral diffusion in a discrete fluid membrane with immobile particles.

Due to the coupling between the plasma membrane and the actin cytoskeleton, membrane molecules such as receptor proteins can become immobilized by binding to cytoskeletal structures. We investigate the effect of immobile membrane molecules on the diffusion of mobile ones by modeling the membrane as a two-dimensional (2D) fluid composed of hard particles and performing event-driven molecular dynamics simulations at a particle density where the system is in an isotropic liquid state. We show that the diffusion coefficient sharply decreases with increasing immobile fraction, dropping by a factor of ∼ 3 as the fraction of immobile particles increases from 0 to 0.1, in a system-size dependent manner. By combining our results with earlier calculations, we estimate that a factor-of-∼ 20 reduction in diffusion coefficients in live cell membranes, a puzzling finding in cell biology, can be accounted for when less than ∼ 22% of the particles in our model system is immobilized. Furthermore, we investigate the effects of confinement induced by a correlated distribution of immobile particles by calculating the distribution of the time it takes for particles to escape from a corral. In the regime where the particles can always escape from the corral, it is found that the escape times follow an exponential distribution, and the mean escape time grows exponentially with the density of obstacles at the corral boundary, increasing by a factor of 3-5 when immobile particles cover 50% of the boundary, and is approximately proportional to the area of the corral. We believe that our findings will be useful in interpreting (1) single molecule observations of membrane molecules and (2) results of particle based simulations that explore the effect of fluid dynamics on molecular transport in a 2D fluid.

Quantification of cell co-migration occurrences during cell aggregation on fibroin substrates.

A quantitative analytical method was proposed for measuring cell co-migration, which was defined as two or more cells migrating together. To accurately identify and quantify this behavior, cell migration on fibroin substrates was analyzed with respect to intercellular distance. Specifically, cell size was characterized by major diameter, and then, based on these measurements and cell center data, a specific threshold distance for defining co-migration was determined after analyzing cell motion using the Voronoi diagram method. The results confirmed that co-migration occurrences of rounded cells were significantly more stable on fibroin than on ProNectin substrates under the present experimental conditions. The cell co-migration analysis method in this article was shown to be successful in evaluating the stability of cell co-migration and also suggested the presence of "critical distance" where two cells interact on fibroin substrates. With further research, the cell co-migration analysis method and "critical distance" may prove to be capable of identifying the aggregation behavior of other cells on different materials, making it a valuable tool that can be used in tissue engineering design.

How do chondrocytes aggregate on fibroin substrate.

The effects of substrate material on the spatio-temporal behavior of cells is an important issue. Although cell aggregation has been observed on various fibroin substrates, the mechanisms of this aggregation have yet to be fully clarified. In this study, cell aggregation behavior on fibroin substrates were evaluated, focusing on the distance between each cell and the direction of individual cell migration. Our results showed that on fibroin substrates cells did not attract each other. However cells stayed close to adjacent cells over 24 hours of cultivation.

Quantitative evaluation of fibroblast migration on a silk fibroin surface and TGFBI gene expression.

Cell migration plays important roles in natural processes involving embryonic development, inflammation, wound healing, cancer metastasis and angiogenesis. Cell migration on various biomaterials is also believed to improve the rate of wound healing and implant therapies in the tissue-engineering field. This study measured the distance traversed, or mileage, of mouse fibroblasts on a silk fibroin surface. Fibroblasts on the fibroin surface moved with better progress during 24 h than cells on collagen or fibronectin surfaces. Results obtained by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) revealed that fibroblasts on the fibroin surface expressed transforming growth factor ß-induced protein (TGFBI), which is an extracellular matrix (ECM) protein, stronger than on other surfaces in the early cell-culture stages. These results demonstrate that the fibroin surface shows higher potential to enhance cell migration and the production of ECM than a collagen or fibronectin surface.

Observation and quantification of chondrocyte aggregation behavior on fibroin surfaces using Voronoi partition.

Cell migration is one of the fundamental processes in histogenesis, and it is necessary to investigate such multicellular behavior quantitatively in cell regeneration studies. In this study, Voronoi diagram analysis was first confirmed in simulation testing, and then used to evaluate the multicellular behavior of chondrocytes on three different substrates: (1) wild-type fibroin (FIB); (2) L-RGDSx2 transgenic fibroin; (3) and collagen. The indices for the round factor average, round factor homogeneity, and area disorder (AD), calculated from Voronoi diagram analysis, were used to characterize the difference in spatiotemporal changes for the different chondrocyte populations, and a regression analysis of the AD index was used to measure the speed of cell aggregation. The results suggested that the arginine-glycine-aspartic acid-serine sequence affects aggregate formation of chondrocytes cultured on FIB. The Voronoi diagram analysis represents one of the promising quantitative analyses for cell regeneration studies.

Observation of chondrocyte aggregate formation and internal structure on micropatterned fibroin-coated surface.

Condensation/aggregation process of rabbit-derived chondrocytes on a fibroin-coated patterned substrate was observed to estimate initial aggregation process in fibroin sponge. Chondrocytes were seeded on array of 160 microm diameter pits in three densities: 5 cells/pit (2.5 x 10(4) cells/cm(2), LOW), 15 cells/pit (7.5 x 10(4) cells/cm(2), MID) and 25 cells/pit (12.5 x 10(4) cells/cm(2), HIGH). In the MID and HIGH groups, cells tended to form aggregates after 24 h after cell seeding. In the LOW group, cell aggregate were not seen in a majority of the pits. Observation of aggregates using confocal laser scanning microscope showed that the chondrocytes at the interface of the fibroin surface tended to extend to the surface, developing an extensive network of stress fibers throughout the cytoplasm. On the other hand, chondrocytes in the other part of the aggregates maintained spherical shape, and most of the actin was localized in the cell cortex as opposed to in stress fibers. These results suggest two functional structures in the aggregates, which may explain the good balance between the maintenance of their differentiated phenotype and proliferation rate in the fibroin sponge.