UV-irradiated parylene surfaces for proliferation and differentiation of PC-12 cells.

PC-12 cells originate from neuroblastic cells, which have an ability to differentiate into neuronlike cells. In this work, the purpose was to estimate the influence of microenvironments on cell attachment and neuritogenesis capacity of PC-12 cells on parylene-N and parylene-C films with and without ultraviolet (UV) light treatment. The estimate of total cell number after incubation for 72h, the ratio of adherent to suspended cells, counting of neurite outgrowths on parylene-N or parylene-C films after UV exposure suggested that these films were suitable for proliferation as well as differentiation of PC-12 cells. The differences in surface properties of parylene-N and parylene-C films with and without UV exposure were analyzed by contact angle measurement, Fourier-transform infrared spectrometry, and X-ray photoelectron spectroscopy. According to these analyses, introduction of oxygen-related chemical functional groups was presumed to result in increased hydrophilicity and efficiency of protein immobilization on parylene-N and parylene-C films after UV treatment. According to fluorescent staining, western blotting, and cell cycle analysis, UV-treated parylene-C and parylene-N films appear to effectively facilitate simultaneous proliferation and differentiation of PC-12 cells with neurite outgrowth.

Astroglial Activation by an Enriched Environment after Transplantation of Mesenchymal Stem Cells Enhances Angiogenesis after Hypoxic-Ischemic Brain Injury.

Transplantation of mesenchymal stem cells (MSCs) has paracrine effects; however, the effects are known to be largely limited. Here we investigated the combination effects of cell transplantation and enriched environment (EE) in a model of hypoxic-ischemic brain injury. Brain damage was induced in seven-day-old mice by unilateral carotid artery ligation and exposure to hypoxia (8% O2 for 90 min). At six weeks of age, the mice were randomly assigned to four groups: phosphate-buffered saline (PBS)-control (CON), PBS-EE, MSC-CON, and MSC-EE. Rotarod and grip strength tests were performed to evaluate neurobehavioral functions. Histologic evaluations were also performed to confirm the extent of astrocyte activation and endogenous angiogenesis. An array-based multiplex ELISA and Western blot were used to identify growth factors in vivo and in vitro. Two weeks after treatment, levels of astrocyte density and angiogenic factors were increased in MSC-EE mice, but glial scarring was not increased. Eight weeks after treatment, angiogenesis was increased, and behavioral outcomes were synergistically improved in the MSC-EE group. Astrocytes co-cultured with MSCs expressed higher levels of angiogenic factors than astrocytes cultured alone. The mechanisms of this synergistic effect included enhanced repair processes, such as increased endogenous angiogenesis and upregulation of angiogenic factors released from activated astrocytes.

Can FLAIR hyperintense vessel (FHV) signs be influenced by varying MR parameters and flow velocities? A flow phantom analysis.

BACKGROUND: Fluid-attenuated inversion recovery (FLAIR) hyperintense vessels (FHVs) have been used to assess leptomeningeal collateral flow in acute ischemic stroke. However, prior FHVs studies showed inconsistent results, which may be ascribable to different magnetic resonance (MR) parameters used. PURPOSE: To evaluate whether FHVs could be influenced by varying MR parameters and flow velocities, using a flow phantom. MATERIAL AND METHODS: A total of 512 sets of FLAIRs were performed with varying parameters and flow velocities, using a flow phantom. Flow phantom was manufactured with 3.5% agarose solution, an 8-mm inner diameter silicone tube and non-pulsatile pump. Varying MR parameters were repetition time (TR)/inversion time (TI), echo time (TE), flip angle (FA) of refocusing pulse, and periodically rotated overlapping parallel lines with enhanced reconstruction (PROPELLER). The signal intensity of flow were measured and regarded as the degree of FHVs. Simple and multiple linear regression analyses were applied to evaluate the association between the degree of FHVs and varying MR parameters as well as flow velocities. RESULTS: On univariate analysis, PROPELLER technique (R(2 )= 0.448) demonstrated strongest correlation with the degree of FHV, followed by flow velocities (R(2 )= 0.204), FA (R(2 )= 0.126), and TE (R(2 )= 0.031), whereas TR/TI showed no significant correlations. On multivariate analysis, TE, FA, PROPELLER technique, and flow velocities were independent factors influencing the degree of FHVs (<0.001). CONCLUSION: Flow velocities, FA of refocusing pulse, TE, and PROPELLER technique significantly affected the degree of FHVs. Optimized MR parameters should be used consistently in future studies, which may provide more reliable results.

Influence of in vitro biomimicked stem cell 'niche' for regulation of proliferation and differentiation of human bone marrow-derived mesenchymal stem cells to myocardial phenotypes: serum starvation without aid of chemical agents and prevention of spontaneous stem cell transformation enhanced by the matrix environment.

Niche appears important for preventing the spontaneous differentiation or senescence that cells undergo during in vitro expansion. In the present study, it was revealed that human bone marrow-derived mesenchymal stem cells (hBM-MSCs) undergo senescence-related differentiation into the myocardial lineage in vitro without any induction treatment. This phenomenon occurred over the whole population of MCSs, much different from conventional differentiation with limited frequency of occurrence, and was accompanied by a change of morphology into large, flat cells with impeded proliferation, which are the representative indications of MSC senescence. By culturing MSCs under several culture conditions, it was determined that induction treatment with 5-azacytidine was not associated with the phenomenon, but the serum-starvation condition, under which proliferation is severely hampered, caused senescence progression and upregulation of cardiac markers. Nevertheless, MSCs gradually developed a myocardial phenotype under normal culture conditions over a prolonged culture period and heterogeneous populations were formed. In perspectives of clinical applications, this must be prevented for fair and consistent outcomes. Hence, the biomimetic 'niche' was constituted for hBM-MSCs by cultivating on a conventionally available extracellular matrix (ECM). Consequently, cells on ECM regained a spindle-shape morphology, increased in proliferation rate by two-fold and showed decreased expression of cardiac markers at both the mRNA and protein levels. In conclusion, the outcome indicates that progression of MSC senescence may occur via myocardial differentiation during in vitro polystyrene culture, and this can be overcome by employing appropriate ECM culture techniques.

Fibroblast growth factor-2 induced by enriched environment enhances angiogenesis and motor function in chronic hypoxic-ischemic brain injury.

This study aimed to investigate the effects of enriched environment (EE) on promoting angiogenesis and neurobehavioral function in an animal model of chronic hypoxic-ischemic (HI) brain injury. HI brain damage was induced in seven day-old CD-1® mice by unilateral carotid artery ligation and exposure to hypoxia (8% O2 for 90 min). At six weeks of age, the mice were randomly assigned to either EE or standard cages (SC) for two months. Rotarod, forelimb-use asymmetry, and grip strength tests were performed to evaluate neurobehavioral function. In order to identify angiogenic growth factors regulated by EE, an array-based multiplex ELISA assay was used to measure the expression in frontal cortex, striatum, and cerebellum. Among the growth factors, the expression of fibroblast growth factor-2 (FGF-2) was confirmed using western blotting. Platelet endothelial cell adhesion molecule-1 (PECAM-1) and α-smooth muscle actin (α-SMA) were also evaluated using immunohistochemistry. As a result, mice exposed to EE showed significant improvements in rotarod and ladder walking performances compared to SC controls. The level of FGF-2 was significantly higher in the frontal cortex of EE mice at 8 weeks after treatment in multiplex ELISA and western blot. On the other hand, FGF-2 in the striatum significantly increased at 2 weeks after exposure to EE earlier than in the frontal cortex. Expression of activin A was similarly upregulated as FGF-2 expression pattern. Particularly, all animals treated with FGF-2 neutralizing antibody abolished the beneficial effect of EE on motor performance relative to mice not given anti-FGF-2. Immunohistochemistry showed that densities of α-SMA(+) and PECAM-1(+) cells in frontal cortex, striatum, and hippocampus were significantly increased following EE, suggesting the histological findings exhibit a similar pattern to the upregulation of FGF-2 in the brain. In conclusion, EE enhances endogenous angiogenesis and neurobehavioral functions mediated by upregulation of FGF-2 in chronic hypoxic-ischemic brain injury.

Tissue engineering of the intervertebral disc with cultured nucleus pulposus cells using atelocollagen scaffold and growth factors.

STUDY DESIGN: In vitro experiment using rabbit nucleus pulposus (NP) cells seeded in atelocollagen scaffolds under the stimulation of growth factors. OBJECTIVE: To demonstrate the effect of anabolic growth factors in rabbit NP cells cultured in atelocollagen type I and type II. SUMMARY OF BACKGROUND DATA: Atelocollagen provides intervertebral disc (IVD) cells for a biocompatible environment to produce extracellular matrix. IVD cells with exogenous transforming growth factor-beta 1 (TGF-ß1) and bone morphogenetic protein-2 (BMP-2) also render an increase in matrix synthesis. However, the effect of anabolic growth factors in NP cells cultured in atelocollagens was not elucidated before. METHODS: Rabbit NP cell was harvested, enzymatically digested, and cultured. The NP cells were seeded to atelocollagen type I and type II scaffolds, and then cultures were exposed to TGF-ß1 (10 ng/mL) and/or BMP-2 (100 ng/mL). DNA synthesis was measured using [4H]-thymidine incorporation. Newly synthesized proteoglycan was measured using [35S]-sulfate incorporation. Reverse transcription-polymerase chain reactions (RT-PCRs) for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin were performed. RESULTS: Rabbit NP cells cultured in atelocollagen type I scaffold showed an increase (1.7 to 2.4-fold) in DNA synthesis in response to TGF-ß1 and/or BMP-2 (P < 0.05), whereas NP cultures in atelocollagen type II demonstrated a 30% increase in DNA synthesis only with combination of both growth factors compared with control (P < 0.05). Rabbit NP cells in atelocollagen type II scaffold with TGF-ß1 and combination of both growth factors exhibited robust 5.3- and 5.4-fold increases in proteoglycan synthesis (P < 0.05), whereas any cultures in atelocollagen type I failed to show any significant increase compared with control. Rabbit NP cells in atelocollagen type I and type II scaffolds with TGF-ß1 and/or BMP-2 demonstrated the upregulation of aggrecan, collagen type I, and collagen type II mRNA expression compared with saline control (P < 0.05). The response in transcriptional level was more robust in atelocollagen type II than in type I. In any event, there is no recognizable expression of osteocalcin (P < 0.05). CONCLUSION: NP cells in atelocollagens under the stimulation of TGF-ß1 and BMP-2 exhibited anabolic responses in transcriptional and translational levels. Hence, such an approach can provide a suitable engineered tissue for IVD regeneration with potential for robust refurbishment of matrix.

Potential of nucleofected human MSCs for insulin secretion.

The goal of this experiment was to generate insulin-producing human mesenchymal stem cells (hMSCs) as a therapeutic source for type I diabetes mellitus, which is caused by insulin deficiency due to the destruction of islet ß cells. In various trials for the treatment of type I diabetes, cell-based therapy using adult stem cells is considered to be one of the most useful candidates for the treatment. In this experiment, a non-viral method called nucleofection was used to transfect hMSCs with pEGFP-C2 and furin-cleavable human preproinsulin gene (hPPI) to produce insulin-secreting cells as surrogate ß cells. Transfection efficiency was determined using flow cytometry analysis. Expression and production of insulin were tested using RT-PCR and ELISA. The expression, production and maturation of insulin from the genetically engineered hMSCs showed an increase when compared with a non-transfected control group. Insulin expression from hMSCs using nucleofection in this study has shown the potential for type I diabetes therapy. For further study, an evaluation for in vivo experiments and clinical applications must be supplemented.

The effects of AEB071 (sotrastaurin) with tacrolimus on rat heterotopic cardiac allograft rejection and survival.

BACKGROUND: AEB071 (sotrastaurin) is a specific inhibitor of protein kinase C that prevents T-cell activation. Our previous study demonstrated that AEB071 monotherapy could prevent acute cardiac allograft rejection in rats. Herein, we investigated the effects of AEB071 combined with various doses of tacrolimus (Tac) on the allograft rejection and survival in a rat heart transplantation model. MATERIALS AND METHODS: Heterotopic cardiac transplantation from Brown-Norway to Lewis rats was performed. Cardiac allograft survival was assessed by monitoring heartbeats in six recipients of each experimental group. Another four recipient rats were selectively sacrificed in each group at d 7 post-transplantation for histologic examination. Serum transaminases, blood urea nitrogen, and creatinine concentrations were measured. RESULTS: AEB071 monotherapy prolonged allograft mean survival time (MST) compared with the untreated control group. Also a combination of AEB071 and Tac prolonged MST compared with monotherapy groups with higher dose of Tac. In the cardiac graft histology, AEB071 combined with Tac 0.6 mg/kg/d significantly decreased the rejection grade as indicative of decreased inflammatory cell infiltration into the graft. No experimental group was found with any abnormal histologic or serologic evidence of liver and kidney toxicity. CONCLUSION: AEB071 combined with a smaller dosage of Tac may be clinically possible to establish calcineurin inhibitor (CNI) minimization protocol in solid organ transplantation.

The effect of succinylated atelocollagen and ablative fractional resurfacing laser on striae distensae.

Striae distensae are dermal atrophic scars with epidermal thinning and decreased collagen and elastic fiber. There is no 'gold standard' treatment modality in the treatment of striae distensae. Collagen is a major extracellular matrix component and is important in wound healing. The ablative CO(2) fractional laser is effective in various cutaneous scars and this study was attempted to evaluate the effect of succinylated atelocollagen and ablative CO(2) fractional laser in the treatment of striae distensae. Participants were divided into two groups and received three laser treatments at a 4-week interval. Clinical improvement was evaluated by participants and two blinded physicians by observing the comparative photographs. Skin biopsies were randomly taken from six participants. The ablative fractional resurfacing laser was effective in the clinical improvement of striae distensae. Statistically significant differences were partly observed between the collagen and placebo groups. Clinical improvement scored by doctors showed more improvement in the collagen group. However, scoring by participants did not show significant differences between the collagen and placebo groups. In conclusion, the ablative fractional resurfacing laser is effective in the treatment of striae distensae and succinylated atelocollagen may also be effective for striae distensae treatment. However, to prove the effect of succinylated atelocollagen, further research with a larger group of participants is needed.

Development and analysis of a collagen-based hemostatic adhesive.

BACKGROUND: Commercially available hemostatic-adhesive has the risk of disease transmission because it is derived from a plasma component. The purpose of this study is to manufacture a new hemostatic adhesive and evaluate its performances. METHODS: Two atelocollagen-based hemostatic adhesives were produced and named as Bleestop A (2% esterified atelocollagen in 75% ethanol + 1% CaCl(2) + 0.71% DOPA + 0.1% tranexamic acid in DW) and Bleestop B (2% esterified atelocollagen in DW + 1% CaCl(2) + 0.71% DOPA + 0.1% aminocaproic acid in DW). These are compared with the negative control group (no adhesives used group) and the positive control group (Tissucol Duo Quick group). The adult male Sprague-Dawley rat model was adopted and the hemostatic-adhesion activities of each group were assessed by the adhesion strength test and the morphologic features of adhesion. The liver tissues were used. Histologic assessment of adhesion was accessed using light microscopy. RESULTS: Bleeding was controlled immediately after application of Bleestop A and partial adhesion was observed after 30 s. More than 95% area of the resected surface attached after 1 min. In Bleestop B, partial adhesion was observed after 30 s. More than 95% area of the resected surface was attached after 45 s. Histologic evaluation showed that Bleestop A and B mediated organized adhesion rapidly between both resected tissue surfaces. The adhesion strength of Bleestop A and B was better than the negative control group and showed same adhesion strength as the positive control group after 3 min. CONCLUSION: Bleestop A and B showed significant hemostasis and adhesion capability within 1 min. They are comparable to the positive control group.

Therapeutic potential of bone-marrow-derived mesenchymal stem cells differentiated with growth-factor-free coculture method in liver-injured rats.

Mesenchymal stem cell (MSC) differentiation by growth factors may be improper due to possibility of clinical risk. We have previously developed a growth-factor-free coculture method and observed rat MSCs differentiated into hepatic progenitor cells. This study was aimed to validate hepatic differentiation potential in vivo. MSCs from bone marrow of green fluorescent protein-transgenic Sprague-Dawley rats were cocultured with hepatocytes from normal Sprague-Dawley rats, sharing growth-factor-free media. After 14 days, cells were implanted into the spleen of carbon tetrachloride (CCl4)-injured rats and kept for 4 weeks. Fibrosis remarkably decreased in CCl4/cocultured MSC at weeks 1, 3, and 4. Immunohistochemistry revealed that albumin, alpha-fetoprotein, and cytokeratin 19 (CK19) expression was high in CCl4/cocultured MSC only at week 1. Reverse transcription-polymerase chain reaction and Western blot revealed that CCl4/cocultured MSC had reduced alpha-fetoprotein expression at week 4, whereas CK18 and CK19 exhibited stronger expression. Albumin in CCl4/cocultured MSC increased at week 4 only in protein level. We assume that cocultured MSCs had stayed at hepatic progenitor stage until week 3, and differentiated into hepatocytes or bile-ductal epithelial cells afterward. Hepatic progenitor cells from MSC differentiation in the growth-factor-free coculture system may contribute to the therapeutic effect for liver disease in vivo.

Corneal dystrophy-associated R124H mutation disrupts TGFBI interaction with Periostin and causes mislocalization to the lysosome.

The 5q31-linked corneal dystrophies are heterogeneous autosomal-dominant eye disorders pathologically characterized by the progressive accumulation of aggregated proteinaceous deposits in the cornea, which manifests clinically as severe vision impairment. The 5q31-linked corneal dystrophies are commonly caused by mutations in the TGFBI (transforming growth factor-beta-induced) gene. However, despite the identification of the culprit gene, the cellular roles of TGFBI and the molecular mechanisms underlying the pathogenesis of corneal dystrophy remain poorly understood. Here we report the identification of periostin, a molecule that is highly related to TGFBI, as a specific TGFBI-binding partner. The association of TGFBI and periostin is mediated by the amino-terminal cysteine-rich EMI domains of TGFBI and periostin. Our results indicate that the endogenous TGFBI and periostin colocalize within the trans-Golgi network and associate prior to secretion. The corneal dystrophy-associated R124H mutation in TGFBI severely impairs interaction with periostin in vivo. In addition, the R124H mutation causes aberrant redistribution of the mutant TGFBI into lysosomes. We also find that the periostin-TGFBI interaction is disrupted in corneal fibroblasts cultured from granular corneal dystrophy type II patients and that periostin accumulates in TGFBI-positive corneal deposits in granular corneal dystrophy type II (also known as Avellino corneal dystrophy). Together, our findings suggest that TGFBI and periostin may play cooperative cellular roles and that periostin may be involved in the pathogenesis of 5q31-linked corneal dystrophies.

Dexamethasone increases angiopoietin-1 and quiescent hematopoietic stem cells: a novel mechanism of dexamethasone-induced hematoprotection.

Angiopoietin-1 (Ang-1) is known to have hematoprotective effects by increasing the quiescence of hematopoietic stem cells. However, it remains to be determined if the upregulation of Ang-1 and the subsequent increase in the quiescence of hematopoietic stem cells are also involved in the dexamethasone (Dex)-mediated bone marrow protection. Here Western blotting and flow cytometric analyses demonstrate that Dex increases the levels of Ang-1 in mouse bone marrow and the quiescence of hematopoietic stem cells. Our data for the first time suggest that the increased quiescence of hematopoietic stem cells provides a novel mechanism of Dex-induced hematoprotection.

A polyethylene glycol grafted bi-layered polyurethane scaffold: preliminary study of a new candidate prosthesis for repair of a partial tracheal defect.

The purpose of this study was to develop an artificial prosthesis for use in the reconstruction of a tracheal defect due to trauma, malignancy, stenosis, or other causes. Bi-layered porous-dense film polyurethane (PU) was manufactured for the main framework. Polyethylene glycol (PEG) was grafted onto the inner surface of the PU scaffold to act as a surfactant. The scaffold was transplanted into three beagles. An endoscopic examination was performed for the evaluation of the formation granulation tissue, to evaluate the status of the respiratory mucosa and the amount of crust at 1, 4, 8, and 12 weeks after surgery. A histological examination was also performed at 4, 8, and 12 weeks after surgery. All three beagles studied survived to the expected date. The endoscopic examination showed formation of granulation tissue; the crust was not very severe and the circular tracheal framework was well preserved. The histological examination showed that a large amount of fibrous tissue had grown through the pores of the porous scaffold. Pseudostratified columnar ciliated mucosa was also noted on the surface of scaffold, as visualized by scanning electron microscopy. The use of a bi-layered polyethylene grafted polyurethane scaffold is a good candidate prosthesis for tracheal reconstruction.

The effect of PLGA sphere diameter on rabbit mesenchymal stem cells in adipose tissue engineering.

To investigate the effect of injectable PLGA sphere's diameter on adipose tissue engineering, rabbit mesenchymal stem cells were attached to various diameters of injectable PLGA spheres (<75; 75-100; 100-150; 150-200; and 200-250 microm). These five groups were cultured in adipogenic media for 2 weeks in vitro and injected into necks of nude mice. Prior to in vivo study, cell proliferation and adipogenic differentiation were determined by hexosaminidase assay and Oil red O staining after 2 weeks. Group C (100-150 microm) showed the highest adipogenic differentiation and the proliferation capacity of Group B (75-100 microm) was significantly higher than that of any other group. We harvested newly formed tissues from necks of nude mice after 1 and 4 weeks. Although PLGA spheres have not been degraded and there was no significant histological difference among various sizes of spheres after 1 week, well-organized fat pads (PLGA spheres were completely degraded) could be observed, and the histology of the 100-150 microm groups resembled that of native tissue after 4 weeks. Based on these experiments, we could conclude that the optimal size of PLGA spheres for adipogenesis was 100-150 microm.

Preparation and characterization of biodegradable anti-adhesive membrane for peritoneal wound healing.

Postoperative adhesions remain a significant complication of abdominal surgery although the wide variety of physical barriers has been developed to reduce the incidence of adhesion. In this study, the bilayered composite membrane formed by the association of a methoxy poly (ethylene glycol)-poly (L-lactide-co-glycolide) (mPEG-PLGA) film and a crosslinked collagen-hyaluronic acid (Col-HA) membrane with fibronectin (FN) coating was prepared for promoting wound healing and providing tissue adhesion resistance simultaneously. In vitro adhesion test revealed that fibroblasts attached better on Col-HA membrane compared to those on mPEG-PLGA film, PLGA film or Interceed (oxidized cellulose) while mPEG-PLGA film had the lowest cell adhesive property. In confocal microscopic observation, the actin filaments were significantly further polymerized when 50 or 100 microg/cm(3) fibronectin was incorporated on the COL-HA membranes. After 7-day culture, fibroblasts penetrated throughout the Col-HA-FN network and the cell density increased whereas very few cells were found attached on the surface of the mPEG-PLGA film. In vivo evaluation test showed that the composite membrane could remain during the critical period of peritoneal healing and did not provoke any inflammation or adverse tissue reaction.

Generation of insulin-producing human mesenchymal stem cells using recombinant adeno-associated virus.

The purpose of current experiment is the generation of insulin-producing human mesenchymal stem cells as therapeutic source for the cure of type 1 diabetes. Type 1 diabetes is generally caused by insulin deficiency accompanied by the destruction of islet beta-cells. In various trials for the treatment of type 1 diabetes, cell-based gene therapy using stem cells is considered as one of the most useful candidate for the treatment. In this experiment, human mesenchymal stem cells were transduced with AAV which is containing furin-cleavable human preproinsulin gene to generate insulin-producing cells as surrogate beta-cells for the type 1 diabetes therapy. In the rAAV production procedure, rAAV was generated by transfection of AD293 cells. Human mesenchymal stems cells were transduced using rAAV with a various multiplicity of infection. Transduction of recombinant AAV was also tested using beta-galactosidse expression. Cell viability was determined by using MTT assay to evaluate the toxicity of the transduction procedure. Expression and production of Insulin were tested using reverse transcriptase-polymerase chain reaction and immunocytochemistry. Secretion of human insulin and C-peptide from the cells was assayed using enzyme-linked immunosorbent assay. Production of insulin and C-peptide from the test group represented a higher increase compared to the control group. In this study, we examined generation of insulin-producing cells from mesenchymal stem cells by genetic engineering for diabetes therapy. This work might be valuable to the field of tissue engineering for diabetes treatment.

Effects of sinusoidal electromagnetic field on structure and function of different kinds of cell lines.

This study investigated that whether a 2 mT, 60 Hz, sinusoidal electromagnetic field (EMF) alters the structure and function of cells. This research compared the effects of EMF on four kinds of cell lines: hFOB 1.19 (fetal osteoblast), T/G HA-VSMC (aortic vascular smooth muscle cell), RPMI 7666 (B lymphoblast), and HCN-2 (cortical neuronal cell). Over 14 days, cells were exposed to EMF for 1, 3, or 6 hours per day (hrs/d). The results pointed to a cell type-specific reaction to EMF exposure. In addition, the cellular responses were dependent on duration of EMF exposure. In the present study, cell proliferation was the trait most sensitive to EMF. EMF treatment promoted growth of hFOB 1.19 and HCN-2 compared with control cells at 7 and 14 days of incubation. When the exposure time was 3 hrs/d, EMF enhanced the proliferation of RPMI 7666 but inhibited that of T/G HA- VSMC. On the other hand, the effects of EMF on cell cycle distribution, cell differentiation, and actin distribution were unclear. Furthermore, we hardly found any correlation between EMF exposure and gap junctional intercellular communication in hFOB 1.19. This study revealed that EMF might serve as a potential tool for manipulating cell proliferation.

Construction of functional soft tissues from premodulated smooth muscle cells using a bioreactor system.

Artificial smooth muscle tissues should be constructed with well-differentiated and aligned smooth muscle cells (SMCs) for proper functioning. In a previous study, we produced cell/scaffold hybrids composed of consistently aligned SMCs in a contractile state using cyclic mechanical strain. In this study, the preconditioned hybrids were organized as functional smooth muscle constructs, which had a high cellular density, using a bioreactor system. We determined that the alignment and contractile phenotype of the initially generated SMCs would be retained after a 7-day culture period in a bioreactor. Mechanical properties of the smooth muscle constructs were measured and compared with those of native smooth muscle tissues and acellular scaffolds. The constructs had a denser cell concentration than the preconditioned hybrids, although they were not fully filled with cells. The premodulated cell alignment and contractile phenotype were retained after culture in a bioreactor. The 7-day-cultured constructs had similar allowed stress levels to native tissues while their stiffness was much lower, suggesting that they had malleable and durable characteristics. These results suggest that functional smooth muscle tissues with mechanical stability can be produced using premodulated SMCs and a bioreactor system.

Time-dependent modulation of alignment and differentiation of smooth muscle cells seeded on a porous substrate undergoing cyclic mechanical strain.

The orientation of cellular alignment in smooth muscle tissue engineering is directly related to optimal movement of engineered tissue when it is transplanted in vivo. Cyclic mechanical strain has been applied to modulate the alignment, proliferation, and differentiation of smooth muscle cells. This study was conducted to investigate the effects of cyclic mechanical strain on primary cultured myofibroblasts seeded onto three-dimensional polymeric scaffolds, and to determine the optimal mechanical treatment time required to produce artificial smooth muscle. The cells were primary cultured from rabbit esophageal smooth muscle layer, and a self-designed stretching chamber was used to modulate the cells on porous polyurethane (PU) scaffolds with 10% strain at a frequency of 1 Hz. The applied cyclic strain induced cellular alignment. In particular, cellular alignment perpendicular to the direction of strain was generated in the condition strained over 18 h. In terms of proliferation, the strained groups differed significantly from the statically cultured group, but no difference was observed between groups that were subjected to straining for different lengths of time. Quantitative analysis of alpha-smooth muscle actin (SMA) showed that differentiation was significantly promoted at 18 h of strain. Penetration of primary cultured cells into the pores of PU scaffolds was shown after cyclic strain application, especially in 18 and 24 h of strain. Consequently, it is expected that myofibroblast/scaffold hybrids, cyclically strained in the defined time course, could be practically applied to organize functional smooth muscle tissues having consistent cell alignment and up-regulated SMA.