Bone tissue engineering with human mesenchymal stem cell sheets constructed using magnetite nanoparticles and magnetic force

An in vitro reconstruction of three-dimensional (3D) tissues without the use of scaffolds may be an alternative strategy for tissue engineering. We have developed a novel tissue engineering strategy, termed magnetic force-based tissue engineering (Mag-TE), in which magnetite cationic liposomes (MCLs) with a positive charge at the liposomal surface, and magnetic force were used to construct 3D tissue without scaffolds. In this study, human mesenchymal stem cells (MSCs) magnetically labeled with MCLs were seeded onto an ultra-low attachment culture surface, and a magnet (4000 G) was placed on the reverse side. The MSCs formed multilayered sheet-like structures after a 24-h culture period. MSCs in the sheets constructed by Mag-TE maintained an in vitro ability to differentiate into osteoblasts, adipocytes, or chondrocytes after a 21-day culture period using each induction medium. Using an electromagnet, MSC sheets constructed by Mag-TE were harvested and transplanted into the bone defect in the crania of nude rats. Histological observation revealed that new bone surrounded by osteoblast-like cells was formed in the defect area 14 days after transplantation with MSC sheets, whereas no bone formation was observed in control rats without the transplant. These results indicated that Mag-TE could be used for the transplantation of MSC sheets using magnetite nanoparticles and magnetic force, providing novel methodology for bone tissue engineering.     

The effect of RGD peptide-conjugated magnetite cationic liposomes on cell growth and cell sheet harvesting

Tissue engineering requires novel technologies for establishing 3D constructs, and the layered method of culturing cell sheets (cell sheet engineering) is one potentially useful approach. In the present study, we investigated whether coating the culture surface with RGD (Arg-Gly-Asp) peptide-conjugated magnetite cationic liposomes (RGD-MCLs) was able to facilitate cell growth, cell sheet construction and cell sheet harvest using magnetic force without enzymatic treatment. To promote cell attachment, an RGD-motif-containing peptide was coupled to the phospholipid of our original magnetite cationic liposomes (MCLs). The RGD-MCLs were added to a commercially available 24-well ultra-low-attachment plate the surface of which comprised a covalently bound hydrogel layer that was hydrophilic and neutrally charged. A magnet was placed on the underside of the well in order to attract the RGD-MCLs to the surface of the well, and then NIH/3T3 cells were seeded into the well. Cells adhered to the bottom of the culture surface, which was coated with RGD-MCLs, and the cells spread and proliferated to confluency. After incubation, the magnet was removed and the cells were detached from the bottom of the plates, forming a contiguous cell sheet. Because the sheets contained magnetite nanoparticles, they could be harvested using a magnet inserted into the well. These results suggest that this novel methodology using RGD-MCLs and magnetic force, which we have termed 'magnetic force-based tissue engineering (Mag-TE)', is a promising approach for tissue engineering.     

Novel methodology for fabrication of tissue-engineered tubular constructs using magnetite nanoparticles and magnetic force

Novel technologies for creating three-dimensional constructs with complex shapes would be highly useful in tissue engineering. In the present study, tubular structures were constructed using magnetic force. Magnetite nanoparticles in cationic liposomes were taken up by target cells. The magnetically labeled cells were seeded onto ultralow-attachment plates, and a magnet was placed under the wells. After 24 h of culture, the magnetically labeled cells formed a cell sheet. Subsequently, when a cylindrical magnet was rolled onto the cell sheet, the cell sheet was attracted to the magnet and formed a tube around it. The magnet was then removed, leaving behind a tubular structure. Two types of tissue were used to create tubular structures: urinary tissue, consisting of a monotypic urothelial cell layer; and vascular tissue, consisting of heterotypic layers of endothelial cells, smooth muscle cells, and fibroblasts. The present results suggest that this novel methodology using magnetite nanoparticles and magnetic force, which we have termed "magnetic force-based tissue engineering" (Mag-TE), is a promising approach to constructing tissue-engineered tubular structures.     

Construction and harvest of multilayered keratinocyte sheets using magnetite nanoparticles and magnetic force

Novel technologies to establish three-dimensional constructs are desired for tissue engineering. In the present study, magnetic force was used to construct multilayered keratinocyte sheets and harvest the sheets without enzymatic treatment. Our original magnetite cationic liposomes, which have a positive surface charge in order to improve adsorption, were taken up by human keratinocytes at a concentration of 33 pg of magnetite per cell. The magnetically labeled keratinocytes (2x10(6) cells, which corresponds to 5 times the confluent concentration against the culture area of 24-well plates, in order to produce 5-layered keratinocyte sheets) were seeded into a 24-well ultralow-attachment plate, the surface of which was composed of a covalently bound hydrogel layer that is hydrophilic and neutrally charged. A magnet (4000 G) was placed under the well, and the keratinocytes formed a five-layered construct in low-calcium medium (calcium concentration, 0.15 mM) after 24 h of culture. Subsequently, when the five-layered keratinocytes were cultured in high-calcium medium (calcium concentration, 1.0 mM), keratinocytes further stratified, resulting in the formation of 10-layered epidermal sheets. When the magnet was removed, the sheets were detached from the bottom of the plates, and the sheets could be harvested with a magnet. These results suggest that this novel methodology using magnetite nanoparticles and magnetic force, which we have termed "magnetic force-based tissue engineering" (Mag-TE), is a promising approach for tissue engineering.     

Fabrication of complex three-dimensional tissue architectures using a magnetic force-based cell patterning technique

We describe the fabrication of three-dimensional tissue constructs using a magnetic force-based tissue engineering technique, in which cellular organization is controlled by magnetic force. Target cells were labeled with magnetite cationic liposomes (MCLs) so that the MCL-labeled cells could be manipulated by applying a magnetic field. Line patterning of human umbilical vein endothelial cells (HUVECs) labeled with MCLs was successfully created on monolayer cells or skin tissues using a magnetic concentrator device. Multilayered cell sheets were also inducible on a culture surface by accumulating MCL-labeled cells under a uniform magnetic force. Based on these results, we attempted to construct a complex multilayered myoblast C2C12 cell sheet. Here, patterned HUVECs were embedded by alternating the processes of magnetic accumulation of C2C12 cells for cell layer formation and magnetic patterning of HUVECs on the cell layers. This technique may be applicable for the fabrication of complex tissue architectures required in tissue engineering.     

Accelerated cell sheet recovery by co-grafting of PEG with PIPAAm onto porous cell culture membranes

Fabrication of functional tissue constructs from designed three-dimensional structures of cells using the layered method of cultured cell sheets could prove to be an attractive approach to tissue engineering. Rapid recovery of cell sheets is considered to be important as a basic technology for practical assembly of tissue-mimicking structures. To accelerate required culture substrate hydrophilic/hydrophobic functional changes according to the hydrated/dehydrated structural changes in response to culture temperature alteration, poly(N-isopropylacrylamide) (PIPAAm) was grafted with poly(ethylene glycol) (PEG) onto porous culture membranes by electron beam irradiation. Analyses by attenuated total reflection-Fourier transform infrared and electron spectroscopy for chemical analysis revealed that PIPAAm and PEG were successfully grafted to surfaces of porous membranes. PIPAAm-grafted porous membranes (PIPAAm-PM) were compared with porous membranes co-grafted with various amounts of PEG and PIPAAm (PIPAAm(PEG)-PM) for cell sheet detachment experiments. Approximately 35min incubation at 20 degrees C was required to completely detach cell sheets from PIPAAm-PM in a static condition, while only 19min to detach cell sheets from PIPAAm(PEG0.5%)-PM, which is co-grafted with PIPAAm and 0.5wt% of PEG. With porous membranes, water molecules were accessed by the PIPAAm molecules grafted on the surfaces from both underneath and peripheral to the attached cell sheet, resulting in more rapid hydration of grafted PIPAAm molecules and detachment of cell sheet than that for nonporous tissue culture polystyrene (TCPS) dish. With PIPAAm(PEG)-PMs, grafted PEG chains should accelerate the diffusion of water molecules to PIPAAm grafts, showing more rapid detachment of cell sheet compare to PIPAAm-PMs.     

Retinal pigment epithelium cell alignment on nanostructured collagen matrices

We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α(2) were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α(2) was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α(2) expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α(2)-mediated matrix binding was verified by preincubation with an α(2)-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriers.     

The cultivation of human retinal pigment epithelial cells on Bombyx mori silk fibroin

We have presently evaluated membranes prepared from Bombyx mori silk fibroin (BMSF), for their potential use as a prosthetic Bruch's membrane and carrier substrate for human retinal pigment epithelial (RPE) cell transplantation. Porous BMSF membranes measuring 3 μm in thickness were prepared from aqueous solutions (3% w/v) containing poly(ethylene oxide) (0.09%). The permeability coefficient for membranes was between 3 and 9 × 10(-5) cm/s by using Allura red or 70 kDa FITC-dextran respectively. Average pore size (±sd) was 4.9 ± 2.3 μm and 2.9 ± 1.5 μm for upper and lower membrane surfaces respectively. Optimal attachment of ARPE-19 cells to BMSF membrane was achieved by pre-coating with vitronectin (1 μg/mL). ARPE-19 cultures maintained in low serum on BMSF membranes for approximately 8 weeks, developed a cobble-stoned morphology accompanied by a cortical distribution of F-actin and ZO-1. Similar results were obtained using primary cultures of human RPE cells, but cultures took noticeably longer to establish on BMSF compared with tissue culture plastic. These findings encourage further studies of BMSF as a substrate for RPE cell transplantation.     

S100a7蛋白在体外培养人视网膜色素上皮细胞中的表达及功能

背景：S100a7蛋白在细胞增殖、血管形成等病理生理过程中发挥重要作用,但在视网膜色素上皮细胞中的作用少有报道。 目的：探讨S100a7蛋白在体外培养人视网膜色素上皮细胞中的表达及作用。 方法：体外培养ARPE-19细胞株,通过免疫荧光及Western blot技术,检测S100a7在ARPE-19细胞中的表达;不同稀释度(1∶1 000,1∶10 000,1∶100 000)S100a7蛋白抗体与ARPE-19细胞共培养,通过MTT技术检测S100a7蛋白在人视网膜色素上皮细胞增殖过程中的作用。 结果与结论：免疫荧光及Western blot技术证实该蛋白在ARPE-19细胞中表达;MTT实验结果显示,人视网膜色素上皮细胞加入S100a7抗体72 h后,各浓度处理组的吸光度值较对照组显著均降低(P < 0.05)。结果证实S100a7蛋白在人视网膜色素上皮细胞中的表达,并明显促进人视网膜色素上皮细胞增殖。     

Fabrication of transferable micropatterned-co-cultured cell sheets with microcontact printing

The purpose of the present study is to develop a novel method for the fabrication of transferable micropatterned cell sheets for tissue engineering. To achieve this development, microcontact printing of fibronectin on commercially available temperature-responsive dishes was employed. Primary rat hepatocytes were seeded on the dish surfaces printed with fibronectin. Under serum-free conditions, hepatocytes were attached onto fibronectin domains selectively. Then, a second cell type of endothelial cells was seeded in the presence of serum. Double fluorescent staining revealed that endothelial cells successfully adhered to the intervals of hepatocyte domains. Finally, all the cells were harvested as a single contiguous micropatterned cell sheet upon temperature-reduction. With a cell sheet manipulator having a gelatin layer for the support of harvested cell sheets, harvested micropatterned cell sheets were transferred to new dish surfaces. This technique would be useful for the fabrication of thick tissue constructs having a complex microarchitecture.     

Two-dimensional manipulation of cardiac myocyte sheets utilizing temperature-responsive culture dishes augments the pulsatile amplitude

Although cardiac myocytes adherent to tissue culture polystyrene (TCPS) dishes retain the spontaneous beating, the pulsatile amplitude is highly limited compared to that in vivo. One of the main reasons for the limited pulsation may be the interface between the cells and the TCPS surfaces. Release of these cells from rigid TCPS surfaces may augment their pulsatile amplitude. With this perspective, we have developed a novel cell manipulation technique to detach cultured cardiac myocytes from rigid surfaces and to rescue higher pulsatile amplitude of the cells using temperature-responsive culture dishes and discuss the possibility of improving this heart tissue model. Primary cardiac myocytes were cultured on the slightly hydrophobic dish surfaces grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide). Cells adhered and proliferated, forming confluent cardiac myocyte sheets in a fashion similar to those on ungrafted TCPS dishes. Decrease in culture temperature resulted in surface change of the polymer from slight hydrophobic to highly hydrophilic due to extensive hydration of the grafted polymer on the dishes. This results in release of cardiac myocyte sheets from the dishes without enzymatic or EDTA treatment. When no support was used, the detached cardiac myocyte sheets shrank to one-tenth size, which ceased their pulsation. When chitin membranes were used to support the confluent sheets to prevent cell shrinkage, the detached cell sheets could be transferred and readily adhered onto another virgin TCPS dishes. These transferred cell sheets preserved the similar cell morphology and pulsation to those before the detachment. When polyethylene meshes were used to support cell sheet transfer, detached cardiac myocyte sheets partially attached to the mesh threads. Then, the constructs were inverted and placed in another culture dish to prevent direct association to dish surfaces. Moreover, the cardiac myocyte sheets were reorganized to heart tissue-like structures by the unisotropic contraction orientated by the mesh threads, and the pulsatile amplitude increased more than 10 times higher. This technique would bring about new insight in tissue engineering as well as cultured heart model.     

Novel approach for achieving double-layered cell sheets co-culture: overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature-responsive culture dishes

Confluent human aortic endothelial cells (HAECs) cultured on thermo-responsive culture dish grafted with poly (N-isopropylacrylamide) were recovered as a contiguous cell sheet. The double-layered co-culture was achieved by placing the recovered HAEC sheet onto the rat hepatocyte layer directly. The double-layered structure of HAEC and hepatocytes remained in tight contact during culture. Hepatocytes in the layered co-culture system with the HAEC sheet maintained the differentiated cell shape and the albumin expression for over 41 days of culture, whereas the functions disappeared within 10 days of culture in control hepatocytes without the HAEC sheet. The layered co-culture of hepatocytes and the HAEC sheets, which allows for the expression of differentiated functions of hepatocyte continuously, such as liver lobule, offers a major advancement in liver tissue engineering.     

Infection of retinal epithelial cells with L. amazonensis impacts in extracellular matrix proteins

One of the manifestations of leishmaniases is eye injuries which main characteristics are the injury of the anterior chamber of the eye and the resistance to specific treatments. The retinal pigment epithelial (RPE) cells participate in pathogen-induced intraocular inflammatory processes. We investigated Leishmania amazonensis–RPE cells relationship and its impact in laminin and fibronectin production. Using RPE cell (ARPE-19), we demonstrated that L. amazonensis adhere to these cells in the first hour of infection, whereas parasite internalization was only observed after 6 h. Seventy-two hours after infection, vacuoles with parasites debris were observed intracellularly, and no parasite were observed intra- or extracellularly at the 96 h, suggesting that Leishmania can infect ARPE-19 cells although this cells are able to clear the infection. Fibronectin and laminin were associated with L. amazonensis–ARPE-19 interaction. Confocal analysis showed no substantial alterations in fibronectin presence in ARPE-19-infected or ARPE-19-noninfected cells, whereas laminin levels increased three times 10 h after L. amazonensis infection. After this time, laminin levels decreased in infected cells. These results suggest that L. amazonensis–ARPE-19 infection induces increased production of laminin in the beginning of infection which may facilitate parasite–host cell interactions.     

Polarized secretion of IL-6 and IL-8 by human retinal pigment epithelial cells

A number of cell types situated along interfaces of various tissues and organs such as the peritoneum and the intestine have been shown to secrete inflammatory cytokines in a polarized fashion. Retinal pigment epithelial (RPE) cells are positioned at the interface between the vascularized choroid and the avascular retina, forming part of the blood–retina barrier. These cells are potent producers of inflammatory cytokines and are therefore considered to play an important role in the pathogenesis of ocular inflammation. Whether cytokine secretion by these cells also follows a vectorial pattern is not yet known, and was therefore the subject of this study. Monolayers of human RPE cells (primary cultures and the ARPE-19 cell line) cultured on transwell filters were stimulated to produce IL-6 and IL-8 by adding IL-1β (100 U/ml) to either the upper or the lower compartment. After stimulation, the human RPE cell lines showed polarized secretion of IL-6 and IL-8 towards the basal side, irrespective of the side of stimulation. The ARPE-19 cell line also secreted IL-6 and IL-8 in a polarized fashion towards the basal side after basal stimulation; polarized secretion was, however, not apparent after apical stimulation. The observation that human RPE cells secrete IL-6 and IL-8 in a polarized fashion towards the choroid may represent a mechanism to prevent damage to the adjacent fragile retinal tissue.     

Electric impedance of human embryonic stem cell-derived retinal pigment epithelium

The barrier properties of epithelium are conventionally defined by transepithelial resistance (TER). TER provides information about the tightness of the epithelium. Electrical impedance spectroscopy (EIS) provides additional information regarding cell membrane properties, such as changes in electric capacitance and possible parallel or serial pathways that may correlate with the morphology of the cell layer. This study presents EIS of retinal pigment epithelial (RPE) cell model of the putative RPE differentiated from human embryonic stem cells (hESC-RPE). The generally utilized RPE cell model, ARPE-19, was used as immature control. The measured EIS was analyzed by fitting an equivalent electrical circuit model describing the resistive and capacitive properties of the RPE. Our results indicated that TER of hESC-RPE cells was close to the values of human RPE presented in the literature. This provides evidence that the stem cell-derived RPE in vitro can reach high-barrier function. Furthermore, hESC-RPE cells produced impedance spectra that can be modeled by the equivalent circuit of one time constant. ARPE-19 cells produced low-barrier properties, that is, an impedance spectra that suggested poor maturation of ARPE-19 cells. To conclude, EIS could give us means for non-invasively estimating the functionality and maturation of differentiated-RPE cells.     

Structural characterization of bioengineered human corneal endothelial cell sheets fabricated on temperature-responsive culture dishes

For the purpose of corneal regenerative medicine, we fabricated human corneal endothelial cell sheets on temperature-responsive dishes, which could be non-invasively harvested as intact, transplantable sheets by simply reducing the culture temperature. Cells demonstrated hexagonal cell shape with numerous microvilli and cilia, and also exhibited abundant cytoplasmic organelles similar to these cells in vivo. Immunofluorescence for type IV collagen and fibronectin revealed that abundant extracellular matrix (ECM) was deposited on the basal surface throughout culture, and the deposited ECM was harvested along with the cell sheets by reducing culture temperature to 20 degrees C. Faint ECM remnants were observed on the dish surfaces after cell sheet detachment. Immunofluorescence for ZO-1 showed that tight junctions were established between cells, and immunoblotting indicated that intact ZO-1 was maintained during cell sheet harvest, while conventional proteolytic cell harvest methods resulted in the degradation of ZO-1. These results suggest that these transplantable corneal endothelial cell sheets can be applied to treat patients with damaged corneas.     

The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets

Primary canine oral mucosal epithelial cells were cultured on temperature-responsive dishes and cell culture inserts to fabricate transplantable epithelial cell sheets. When 3T3 feeder layers and fetal bovine serum were eliminated from dish culture, the harvested cell sheets became significantly more fragile. In contrast, when epithelial cells were cultured on inserts having submicron-scale pores, cell sheet fragility was eliminated. Keratin expression profiles showed no differences among the harvested cell sheets, but the expression of p63, a putative stem/progenitor marker, was strongly dependent on the presence of 3T3 feeder layers and serum. These results suggest that the maintenance of stem/progenitor cells is influenced by the apical/basal supply of nutrients as well as culture supplements.     

Down-regulation of microRNA-27b promotes retinal pigment epithelial cell proliferation and migration by targeting Nox2

Aberrant proliferation and migration of retinal pigment epithelium (RPE) cells contributes to the pathology of various ocular diseases. miR-27b has been reported to be crucial in the regulation of cell differentiation, proliferation, apoptosis, and migration. However, the role of miR-27b on RPE proliferation and migration remains largely unknown. Here the effect of miR-27b on ARPE-19 cells under platelet-derived growth factor (PDGF)-BB stimulation was explored. In this study, we found that the expression level of miR-27b was significantly reduced in ARPE-19 cells under PDGF-BB stimulation. Ectopic expression of miR-27b remarkably inhibited PDGF-BB-induced proliferation and migration in ARPE-19 cells. Furthermore, bioinformatic analysis and luciferase reporter assay showed that NADPH oxidase 2 (Nox2) was a direct target for miR-27b, and that knockdown of Nox2 expression mimicked the inhibitory effect of miR-27b on PDGF-BB ?induced proliferation and migration in ARPE-19 cells, whereas, restoration of Nox2 expression showed an opposite effect. In addition, the ROS production and the activation of P13K/AKT/mTOR signaling induced by PDGF-BB were also suppressed by miR-27b overexpression or Nox2 silencing. Thus, these findings indicated that miR-27b exerted its protective role in RPE cells under PDGF-BB stimulation was partially through regulation of Nox2 and its downstream P13K/AKT/mTOR signaling, which might be a potential therapeutic approach for treatment of diseases caused by RPE proliferation, and migration.     

Transplantation of tissue-engineered retinal pigment epithelial cell sheets in a rabbit model

The retinal pigment epithelium (RPE) plays an important role in maintaining a healthy neural retina. With changes due to age, morbidity or removal of choroidal neovascularis developed as a means ofation, damage or defects of the RPE occur. Accordingly, RPE transplantation techniques have been repairing the damaged RPE. We conducted a study to transplant tissue-engineered RPE cell sheets in a rabbit model. RPE cells were isolated from pigmented rabbit eyes and seeded on temperature-responsive culture surfaces. Cultured RPE cells were arranged as a monolayer with a cobblestone cell shape that is characteristic of native RPE. The pigmented RPE cell sheets were non-invasively harvested without enzymatic treatment simply by reducing the culture temperature. Using 3-port vitrectomy, RPE cell sheets were transplanted into the subretinal space of albino rabbits. Seven days after surgery, the rabbits were sacrificed, and the eyes were enucleated and examined under both light and electron microscopy. After transplantation, our results show that the RPE cell sheets attached to the host tissues in the subretinal space more effectively than with the injection of isolated cell suspensions. Although the cell sheets maintained a monolayer structure in most areas, they were slightly folded or wrinkled in some regions. We conclude that tissue-engineered RPE cell sheets harvested from temperature-responsive culture dishes can be effectively transplanted beneath the neural retina.