In vitro study on anti-inflammatory effects of epigallocatechin-3-gallate-loaded nano- and microscale particles.

PURPOSE: This study aimed to develop an anti-inflammation system consisting of epigallo-catechin-3-gallate (EGCG) encapsulated in poly(lactide-co-glycolic acid) (PLGA) particles to promote wound healing. METHODS: Nano- and microscale PLGA particles were fabricated using a water/oil/water emulsion solvent evaporation method. The optimal particle size was determined based on drug delivery efficiency and biocompatibility. The particles were loaded with EGCG. The anti-inflammatory effects of the particles were evaluated in an in vitro cell-based inflammation model. RESULTS: Nano- and microscale PLGA particles were produced. The microscale particles showed better biocompatibility than the nanoscale particles. In addition, the microscale particles released ~60% of the loaded drug, while the nanoscale particles released ~50%, within 48 hours. Thus, microscale particles were selected as the carriers. The optimal EGCG working concentration was determined based on the effects on cell viability and inflammation. A high EGCG dose (100 µM) resulted in poor cell viability; therefore, a lower dose (≤50 µM) was used. Moreover, 50 µM EGCG had a greater anti-inflammatory effect than 10 µM concentration on lipopolysaccharide-induced inflammation. Therefore, 50 µM EGCG was selected as the working dose. EGCG-loaded microparticles inhibited inflammation in human dermal fibroblasts. Interestingly, the inhibitory effects persisted after replacement of the drug-loaded particle suspension solution with fresh medium. CONCLUSION: The EGCG-loaded microscale particles are biocompatible and exert a sustained anti-inflammatory effect.

The Effects of Epigallocatechin-3-Gallate and Mechanical Stimulation on Osteogenic Differentiation of Human Mesenchymal Stem Cells: Individual or Synergistic Effects.

This study aims to investigate the roles and effects of EGCG (epigallocatechin-3-gallate) during the osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro. Recent studies have shown that proper mechanical stimuli can induce osteogenic differentiation of hMSCs apart from biochemical factors. In this study, the hMSC cultures were subjected to: (1) 25 uM EGCG alone or (2) 3% mechanical stretching (0.2 Hz for 4 h/day for 4 days) or (3) in combination with 3% mechanical stretching (0.2 Hz for 4 h/day for 4 days). The two factors were applied to the cell cultures separately and in combination to investigate the individual and synergistic effect of both mechanical stimulation and ECGC in the osteogenic differentiation of hMSCs. Utilizing real time PCR, we measured various osteogenic markers and even those related to intracellular signalings. Further investigation of mitochondria was performed that mitochondria biogenesis, antioxidant capacity, and morphological related markers were measured. hMSCs were to be osteogenic or myogenic differentiated when they were under 3% stretching only. However, when EGCG was applied along with stretching they were to be osteogenic differentiated rather than to be myogenic differentiated. This was supported by evaluating intracellular signalings: BMP-2 and VEGF. Therefore, the synergistical effects of simultaneous employment of stretching and EGCG on osteogenic differentiation were confirmed. Moreover, simultaneous employment was found positive in mitochondria biogenesis, antioxidant capacity, and morphological changes. Through this study, we came into the conclusion that the combination of proper mechanical stretching, 3% in this study, and EGCG promote osteogenic differentiation. Reflecting that EGCG can be obtained from plants not from the chemical syntheses, it is worth to be studied further either by animal tests or long-term experiments for clinical applications.

MG-63 cells proliferation following various types of mechanical stimulation on cells by auxetic hybrid scaffolds.

BACKGROUND: Mechanical properties and cyto-compatibility of a composite scaffold which possessed negative (-) Poisson's ratio (NPR) was investigated for effective load transfer from auxetic scaffold to cell. METHODS: Organic/inorganic composite scaffolds were prepared by mixing hydroxyapatite (HA) to poly(lactide-co-glycolide) (PLGA). To induce NPR in composite scaffold, 3-directional volumetric compression was applied during the scaffold fabrication at adequate temperature(60°C). The pore size of scaffold ranged between 355-400 µm. RESULTS: Poisson's ratios of NPR scaffolds and control scaffolds were -0.07 and 0.16 at 10 % strain. For stable physical stimulating to loaded cells, ceramic/polymer composite scaffold was prepared by incorporating HA in PLGA to increase mechanical strength. Compressive strength of the HA/PLGA composite scaffold (15 wt. % HA to PLGA) was about 21.7 % higher than that of PLGA-only scaffold. The recovery rates of the NPR composite scaffold after applying compression in the dry and wet states were 90 % and 60 %, respectively. Also the composite scaffold was shown to have better hydrophilicity (61.9°) compared to the PLGA-only scaffolds (65.3°). Cell proliferation of osteoblast-like cell line (MG-63) in the composite scaffold was 20 % higher than in PLGA-only scaffold at static compressive stimulation. For dynamic compressive stimulation (15 min cyclic interval), cell proliferation in the composite scaffold was 2 times higher than that of in PLGA-only scaffold. In conclusion, NPR composite (HA/PLGA) scaffold was effective in isotropic compressive load delivery for osteogenic cell proliferation. CONCLUSION: This composite scaffold with stimulation can be used as tissue engineered scaffold and dynamic cell culture system for bone tissue regeneration.

MG-63 osteoblast-like cell proliferation on auxetic PLGA scaffold with mechanical stimulation for bone tissue regeneration.

BACKGROUND: Auxetic scaffolds (experimental) was fabricated by using poly(D, L-lactic-co-glycolic acid), 50:50, (PLGA) for effective bone cell proliferation with mechanical stimulation. METHODS: Negative Poisson's ratio in scaffold, 3-directional volumetric compression was applied during the scaffold fabrication at adequate temperature (60 °C). The pore size of scaffold ranged between 355 and 400 µm. RESULTS: The porous morphology of the prepared auxetic scaffolds had shown partially concave and dent shapes in SEM image as expected. The lowest Poisson's ratios of experimental group was -0.07 at 60 °C/10 min. Compressive strength of experimental group was shown about 3.12 times higher than control group (conventional scaffold) in dry state at 25 °C. The compressive strengths of both groups were tended to be decreased dramatically in wet state compared to in dry state. However, compressive strengths of experimental group were higher 3.08 times and 1.88 times in EtOH/PBS (25 °C) and EtOH/PBS/DMEM (37 °C) than control group in wet state, respectively. Degradation rate of the scaffolds showed about 16 % weight loss in 5 weeks. In cell attachment test, experimental group showed 1.46 times higher cell proliferation than control group at 1-day with compressive stimulation. In 3-day culture, the experimental group showed 1.32 times higher than control group. However, there was no significant difference in cell proliferation in 5-day cultivation. CONCLUSION: Overall, negative Poisson's ratio scaffolds with static mechanical stimulation could affect the cell proliferation at initial cultivation time.

Cationic Nanocylinders Promote Angiogenic Activities of Endothelial Cells.

Polymers have been used extensively taking forms as scaffolds, patterned surface and nanoparticle for regenerative medicine applications. Angiogenesis is an essential process for successful tissue regeneration, and endothelial cell-cell interaction plays a pivotal role in regulating their tight junction formation, a hallmark of angiogenesis. Though continuous progress has been made, strategies to promote angiogenesis still rely on small molecule delivery or nuanced scaffold fabrication. As such, the recent paradigm shift from top-down to bottom-up approaches in tissue engineering necessitates development of polymer-based modular engineering tools to control angiogenesis. Here, we developed cationic nanocylinders (NCs) as inducers of cell-cell interaction and investigated their effect on angiogenic activities of human umbilical vein endothelial cells (HUVECs) in vitro. Electrospun poly (L-lactic acid) (PLLA) fibers were aminolyzed to generate positively charged NCs. The aninolyzation time was changed to produce two different aspect ratios of NCs. When HUVECs were treated with NCs, the electrostatic interaction of cationic NCs with negatively charged plasma membranes promoted migration, permeability and tubulogenesis of HUVECs compared to no treatment. This effect was more profound when the higher aspect ratio NC was used. The results indicate these NCs can be used as a new tool for the bottom-up approach to promote angiogenesis.

Dose-Volume Analysis of Lung and Heart according to Respiration in Breast Cancer Patients Treated with Breast Conserving Surgery.

PURPOSE: Adjuvant radiotherapy of breast cancer using a photon tangential field incurs a risk of late heart and lung toxicity. The use of free breathing (FB), expiration breath hold (EBH), and deep inspiration breath hold (DIBH) during tangential breast radiotherapy as a means of reducing irradiated lung and heart volume was evaluated. METHODS: In 10 women with left-sided breast cancer (mean age, 44 years) post-operative computed tomography (CT) scanning was done under different respiratory conditions using FB, EBH, and DIBH in 3 CT scans. For each scan, an optimized radiotherapy plan was designed with 6 MV photon tangential fields encompassing the clinical target volume after breast-conserving surgery. RESULTS: The results of dose-volume histograms were compared using three breathing pattern techniques for the irradiated volume and dose to the heart. A significant reduction dose to the irradiated heart volume for the DIBH breathing technique was compared to FB and EBH breathing techniques (p<0.05). CONCLUSION: This study demonstrated that the irradiated heart volume can be significantly reduced in patients with left-sided breast cancer using the DIBH breathing technique for tangential radiotherapy.

Biological responses of ligament fibroblasts and gene expression profiling on micropatterned silicone substrates subjected to mechanical stimuli.

In this study, ligament fibroblasts were cultivated on micropatterned silicone substrates and subjected to cyclic stretching to simulate the in vivo biomechanical environment during ligament healing. Without stretching, ligament fibroblasts were aligned parallel to the microgrooves on the silicone substrate surface. However, we previously reported that uniaxial cyclic stretching induces alignment perpendicular to the stretching axis. With stretching on a microgrooved surface, cell proliferation and collagen production were greatly enhanced. The exact functions of the micropatterned surface and mechanical stimuli are unknown. Therefore, in gene expression microarray experiments, genes whose expression is inhibited by subculture from passage 0 (P0) to passage 8 (P8) and enhanced by micropatterning and stretching were sought out. The following six genes were selected: MGP, GADD45A, UNC5B, TGFB1, COL4A1, and COL4A2. The selected genes play fundamental roles in cell proliferation, differentiation, apoptosis, and structural maintenance. On the basis of the obtained gene expression profiles, we identified candidate genes that might be involved in responses to a micropatterned surface and mechanical stretching.

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.

Chemotactic migration of human mesenchymal stem cells and MC3T3-E1 osteoblast-like cells induced by COS-7 cell line expressing rhBMP-7.

During bone development, remodeling, and repair, bone morphogenetic proteins (BMPs) induce the differentiation of mesenchymal progenitor cells (MPCs) that enter into the osteoblastic lineage, and enhance the recruitment of MPCs and osteogenic cells. The process of migration is believed to be regulated, in part, by growth factors stored within the bone matrix, which are released by bone resorption. In this study, primary human mesenchymal stem cells (hMSCs) and MC3T3-E1 osteoblasts were examined for chemotaxis in response to recombinant human BMP-7 (rhBMP-7) produced in COS-7 cells (co-culture system). In order to produce BMP-7 transfected cells (BTCs), which serve as suppliers of rhBMP-7 under in vitro culture conditions, the encoding DNA was transferred into the pTARGET expression vector and introduced into COS-7 cells by conventional genetic engineering techniques. In cell culture studies, the rhBMP-7 produced in BTCs stimulated the specific activity of ALP, the production of cAMP in response to PTH, and the synthesis of osteocalcin. Migration assays were conducted with a computer-aided time-lapse video-microscopy system, to allow the rapid and precise analysis of cell migration and for the dynamic measurement of cell position and morphology. The migration distance and speed of the MC3T3-E1 cells, or hMSCs, co-cultured with BTCs, using a band-type seeding method, were significantly increased (p < 0.001), compared to those of the MC3T3-E1 cells (or hMSCs) only. In conclusion, these studies revealed that rhBMP-7 plays a role in the migration of bone-forming cells, and that the co-culture model (co-culture of bone-forming cells with BMP-7-producing cells) using a computer-aided, time-lapse video-microscopy system, is useful for the chemotactic migration assay of other chemotactic growth factors.

Beneficial effects of freezing rate determined by indirect thermophysical calculation on cell viability in cryopreserved tissues.

Many types of mammalian cells, such as sperm, blood, embryos, etc., have been successfully cryopreserved for the last few decades, while no optimal method for the cryopreservation of mammalian tissues or organs has been established, showing a poor survival after thawing with a low recovery of function. In this study, the freezing rate was determined by indirect thermodynamic calculation, and its potential effect on the cryoprotection of human saphenous veins and tissue-engineered bones was investigated. The vein segments were frozen according to the calculated freezing rate, using rate-controlled freezing devices, with a freezing solution composed of 10% dimethylsulphoxide and 20% fetal bovine serum in RPMI 1640 media. The efficacy of indirect calculation was assessed by the cell viability measured using fluorescence double-staining methods. The results indicated that the freezing rate determined by indirect calculation significantly (P < 0.05) maintained the post-thaw cellular viability of the blood vessel, particularly in terms of the endothelial cells. However, it exerted relatively less protective effect on the osteoblastic cell-cultured scaffolds. These results suggest that freezing-induced injuries may occur in tissues, and the freezing rate determined by indirect thermophysical calculation can be used for the optimization of tissue cryopreservation by minimizing the injuries.

Long-term preservation of human saphenous vein by green tea polyphenol under physiological conditions.

Polyphenolic compounds are well known as a functional food with various bioactivities. However, less attention has been paid to the effect of phenolic antioxidants on the preservation of blood vessels. In this study, the possible effects of green tea polyphenolic compounds (GTPCs) on the longterm preservation of the human saphenous vein (HSV) were investigated under physiological conditions. HSV segments were pretreated with GTPCs (0.5 or 1.0 mg/mL) for 1 day and then incubated for 1, 2, 4, or 8 weeks. After incubation, cellular viability, endothelial nitric oxide synthase (eNOS) expression level, biomechanical properties, and vein histology were evaluated. When HSV segments were incubated without GTPC treatment, endothelial cell viability was significantly (p < 0.05) reduced with incubation time, and none of the endothelial cells expressed eNOS after 2 weeks. Furthermore, nontreated veins demonstrated appreciable inferiority in such mechanical properties as failure strength, elastic modulus, and compliance, compared with fresh veins. These results were confirmed by histological observations, which showed severe structural changes in nontreated veins. On the other hand, these phenomena were markedly prevented by preincubating veins with GTPCs (1.0 mg/mL) at 37 degrees C in a CO(2) incubator for 1 day. GTPC-pretreated veins could be preserved for at least 2 weeks under physiological conditions, retaining cellular viability and eNOS expression level and maintaining both biomechanical properties and vascular structures without any morphological alterations. These results demonstrate that GTPC treatment may be a useful method for preserving the HSV and could be exploited to craft strategies for the long-term preservation of other tissues under physiological conditions.

Tissue-engineered semi-microporous segmented polyetherurethane vascular prostheses.

We investigated the biocompatibility of semi-microporous segmented polyetherurethane (SPEU) that was coated with human microvascular endothelial cells. Three types of prosthesis were examined: (1) non-porous (control), (2) SPEU without a collagen coating (PNC) and (3) collagen-coated SPEU (PC). The attachment and proliferation of endothelial cells on SPEU (PNC and PC prostheses) was twice as great as in the control prostheses and endothelial cell adhesion in the PC prostheses was 1.4-times greater than in the PNC prostheses. The degree of endothelial cell coverage in the control prostheses was significantly lower than in the PNC and PC prostheses (P < 0.05), and there was no significant difference between the PNC and PC prostheses in the degree of endothelial cell coverage. As regards the retention of endothelial cells under simulated blood-flow conditions, the PNC prostheses exhibited a greater decrease in endothelial cell coverage, as compared to the PC prostheses (P < 0.05). Similarly, the PNC prostheses exhibited less platelet adhesion than the PC prostheses (P < 0.05). In summary, the semi-microporous SPEU offered better surfaces to anchor and culture for endothelial cells, and had superior anti-coagulant properties, as compared to nonporous SPEU. The coating of SPEU with collagen influenced both the attachment of endothelial cells and blood coagulation on the SPEU.

Enhanced neurite outgrowth of rat neural cortical cells on surface-modified films of poly(lactic-co-glycolic acid).

Neural cortical cells, isolated from prenatal rat cerebra, were grown on surface-modified poly(lactic-co-glycolic acid, 65:35) (PLGA) films coated with poly-D: -lysine (PDL) with either laminin (LN), fibronectin (FN) or collagen (CN). Immunocytochemistry showed that the isolated cells were highly immunopositive for both neurofilament and MAP-2 with well-organized neurites and somatodendritic localization. The presence of PDL with LN or FN on the PLGA films was essential for increased neural cell growth. Also, PLGA films coated with either PDL/LN or PDL/FN mixtures had higher neurite outgrowth and regular differentiation.

Effects of green tea polyphenol on preservation of human saphenous vein.

The potential role of green tea polyphenol (GtPP) in preserving the human saphenous vein was investigated under physiological conditions. The vein segments were incubated for 1, 3, 5, 7 and 14 days, either after 4h of treatment with 1.0mg/ml GtPP or in the presence of GtPP at the same concentration. After incubation, the endothelial cell viability, endothelial nitric oxide synthase (eNOS) expression and the vein histology were evaluated. When the veins were not treated with GtPP, the viability of the endothelial cells was significantly reduced with the progress in the culture time, and none of the cells expressed eNOS after 5 days. Furthermore, severe histological changes and structural damage were observed in the non-treated veins. In contrast, incubating the veins after 4h of GtPP treatment significantly prevented these phenomena. The cellular viability of the GtPP-treated vein was approximately 64% after 7 days, and eNOS expression was maintained up to 40%, compared to that of the fresh vein. The histological observations showed that the vasculature was quite similar to that of the fresh vein. When incubated with GtPP, the vein could also be preserved for 1 week under physiological conditions retaining both its cellular viability (61%) and eNOS expression level (45%) and maintaining its venous structure without any morphological changes. These results demonstrate that GtPP treatment may be a useful method for preserving the HSV.

A study of compatibility between cells and biopolymeric surfaces through quantitative measurements of adhesive forces.

The mechanism of cell adhesion to biomaterials or components of the extracellular matrix is an important topic in the field of tissue engineering and related biotechnological processes. Many factors affect cell adhesion, and many biochemical and biological studies have attempted to identify their roles in the adhesion mechanism. Systematic studies of this nature require quantification of the adhesive force of a cell to identify the effect of a specific factor. However, most studies of cell adhesive force have used qualitative approaches. We propose a new technique for quantifying the force by which cells adhere to various biomaterial surfaces, which utilizes the relationship between the deflection of a cantilever beam and the required force. A micropipette was used as the cantilever beam. This technique was used to measure the attachment forces of chondrocytes seeded on three different biodegradable polymers commonly used in tissue engineering and medicine: poly epsilon-carprolactone (PCL), poly(L-lactide) (PLLA) and poly(lactic-co-glycolic acid) (PGLA, L/G = 75:25). The bond between the cells and the three polymers was evaluated using the quantified adhesive forces. The adhesive forces were also measured 8, 12, 24 h and 5 days after seeding the chondrocytes on the polymer surfaces. Results of statistical analysis showed that the cells attached to the PLLA had the strongest average attachment force for up to 24 h after seeding (P < 0.05).