Stromal vascular fraction promotes fibroblast migration and cellular viability in a hyperglycemic microenvironment through up-regulation of wound healing cytokines

Diabetic wounds have impaired healing and a propensity for further morbidity, which may result in amputations. Stromal vascular fraction (SVF) is an autologous source of heterogeneous cell population obtained from adipose tissue, which is rich in stem cells and presents little immunogenicity to the host. In this study, we hypothesized that murine fibroblasts subjected to hyperglycemic conditions co-treated with SVF exhibit greater functional activity through the colorimetric MTT assay and a cell-monolayer in-vitro scratch assay. We sought to establish the underlying mechanism of action via the utility of an ELISA chemiluminescence array on the supernatant medium of the cells. Our results demonstrate that the mean percentage gap closure at 24?h in the hyperglycemia?+?SVF group was significantly greater at 41.1%?±?1.6% compared to the hyperglycemia alone group 16.6%?±?1.5% (post-hoc Bonferroni test p?<?0.001, n?=?3) although there was no difference between the SVF and normoglycemia group. Further, this SVF group exhibited a significantly greater 2.4 fold increase in fibroblastic cell viability as compared to the hyperglycemia alone group (p?=?0.001, n?=?3). The supernatant medium of the cells upon testing with ELISA indicated that early phase wound healing cytokines including platelet-derived growth factor (p?=?0.012, n?=?3), interleukin-1 (p?=?0.003, n?=?3), basic fibroblast growth factor (p?=?0.003, n?=?3) and interleukin-10 (p?=?0.009, n?=?3) were expressed in significantly greater relative luminescent units in SVF as compared to hyperglycemia alone groups (Student t-test). Taken together and for the first time, our study shows that SVF is a promising therapeutic agent for up-regulating fibroblastic activity in a hyperglycemic microenvironment, and this result can be explained in part by the stimulation of wound-healing cytokines.     

Nonlinear permanent migration response to climatic variations but minimal response to disasters

We present a microlevel study to simultaneously investigate the effects of variations in temperature and precipitation along with sudden natural disasters to infer their relative influence on migration that is likely permanent. The study is made possible by the availability of household panel data from Indonesia with an exceptional tracking rate combined with frequent occurrence of natural disasters and significant climatic variations, thus providing a quasi-experiment to examine the influence of environment on migration. Using data on 7,185 households followed over 15 y, we analyze whole-household, province-to-province migration, which allows us to understand the effects of environmental factors on permanent moves that may differ from temporary migration. The results suggest that permanent migration is influenced by climatic variations, whereas episodic disasters tend to have much smaller or no impact on such migration. In particular, temperature has a nonlinear effect on migration such that above 25 °C, a rise in temperature is related to an increase in outmigration, potentially through its impact on economic conditions. We use these results to estimate the impact of projected temperature increases on future permanent migration. Though precipitation also has a similar nonlinear effect on migration, the effect is smaller than that of temperature, underscoring the importance of using an expanded set of climatic factors as predictors of migration. These findings on the minimal influence of natural disasters and precipitation on permanent moves supplement previous findings on the significant role of these variables in promoting temporary migration.Human migration has been identified as a potentially important response to climate change. Where climate change makes habitation in certain places less desirable or even impossible, people may respond by moving elsewhere. However, the idea that environmental change induces people to migrate remains a widely contested topic, especially given recent findings suggesting that environmental changes may also constrain movement (1–3). Historically, there has been a paucity of empirical demonstrations of environmental effects on population mobility, partly due to sparse data and partly because migration studies have tended to focus on further exploration of social and economic predictors of migration that have already been established as primary drivers. More recently however, new empirical approaches to exploring the relationship between migration and climate change have emerged.Based on a review of the existing literature (SI Text, Literature Review), there is conflicting evidence on the effects of climatic variations and natural disasters on migration, partly arising from the inability to distinguish permanent moves from temporary ones, especially in the case of macrolevel studies that analyze aggregate flows of people. Furthermore, the effects may vary significantly by distance of migration destination, which may also confound the overall effect of environmental factors on migration. Above all, most studies at the microlevel do not simultaneously examine the effects of both disasters and climatic changes on migration, and often use only one aspect of climate, generally variation in rainfall. However, precipitation and temperature are historically correlated and to infer an unbiased effect of either one on migration probability, both need to be included in the model (4).The current study therefore attempts to improve on the existing studies. This study is, to our knowledge, the first at a microlevel to simultaneously explore the effects of sudden natural disasters and climatic variations on permanent migration of the whole household. (Household migration can take several forms: migration of a single member or individual migration; migration of one or more members of the household or split household migration; and migration of whole household, which includes migration of the entire household along with the head of household.) In doing so, we test the effect of temperature along with precipitation on migration decision. We include a summary that allows a quick comparison of the methodology used in our study compared with previous studies in terms of the choice of environmental variables to predict migration (Table S1). Prior studies have primarily examined individual migration behavior, which may capture both temporary and permanent migration. In contrast, province-level migration of entire households, as we show (with the use of data that follows households over a period of 15 y), tends to be more permanent. (We hereafter use the term “permanent.”) To our knowledge, the only other studies that focus on household migration explore the mechanisms by which natural disasters can deter migration of the household or its relatives (5, 6). By studying migration behavior of the whole household, we are able to focus on permanent migration, and therefore directly test how sudden disasters along with variations in rainfall and temperature affect permanent and relatively longer distance (province-to-province) migration as opposed to temporary movement; this allows us to complement the findings in some existing studies that generally conclude that natural disasters and rainfall result in temporary and short-distance moves while providing new evidence on the temperature–migration link in the context of microlevel studies.To achieve these goals, we chose Indonesia as our study site because as the world’s largest archipelago situated in a tectonically active location, the country is highly exposed to both geologic and climatic hazards (SI Text, Background on Indonesia). In addition, as the world’s fourth most populous country with ∼40% of the labor force engaged in agriculture and more than 60% of the total population living in the coastal areas, the country is extremely vulnerable to the impacts of climate variations and extreme events (SI Text, Background on Indonesia). At the same time, the availability of household panel data with an exceptional tracking rate allows us to use a difference-in-differences approach to study the migration of households before and after disasters as well as establish a plausibly causal link between climatic variations and migration. We use the Indonesian Family Life Survey (IFLS), a household panel survey representative of ∼83% of the Indonesian population from 13 of the 27 provinces in 1993 (Fig. S1). The IFLS provides data on 7,185 original households, followed over a period of 15 y (Materials and Methods). The migration outcome we predict captures migration of households, which is likely permanent. We track whole-household migration for a 15-y period during which the original households are followed with a very high retention rate even after they migrate. Some 95% of the migrant households end up migrating only once and do not return to their original province during the entire 15-y period. Therefore, at least in the Indonesian case, the provincial level migration of households seems to be permanent (see SI Text, Internal Migration in Indonesia, for more on internal migration in Indonesia).The household panel data are supplemented with data on natural disasters taken from DesInventar database, which provides disaster-related data using information on disasters of different intensities based on preexisting official data, academic records, newspaper sources, and institutional reports. Different types of disasters may not all affect migration in the same way, thus cancelling out each other’s effects on migration. For our analysis, we therefore separately estimate the effects of each type of disaster. Furthermore, we use multiple measures to capture the different ways in which a disaster may affect the population, for three primary reasons. First, we believe that the intensity of disaster rather than simply the occurrence of disaster should more accurately predict its effect. Measures such as number of deaths, number injured, number of houses destroyed, and amount of financial loss (captured by monetary loss measured in Indonesian Rupiah) from each type of disaster aggregated at the province level are used to capture both the frequency as well as the intensity of disasters. Second, relying on a single measure of a disaster may not capture its overall effect because a certain event may result in large financial losses but inflict little physical harm. Third, using alternate measures of disaster allows one to compare and confirm results, and can be used as a robustness check (Materials and Methods).Finally, for measures of climatic variations, we construct estimates of average temperature and precipitation for each province during each observation interval (Materials and Methods), because temperature and rainfall variations together provide a more complete measure of the extent of climate variations that may affect migration (7).     

A Comparative Study of BioAggregate and ProRoot MTA on Adhesion,Migration, and Attachment of Human Dental Pulp Cells

Introduction

The aim of the present study was to evaluate the effects of a novel bioceramic nanoparticular cement, BioAggregate (Innovative Bioceramix, Vancouver, BC, Canada), on the adhesion, migration, and attachment of human dental pulp cells (HDPCs) and to compare its performance with that of ProRoot mineral trioxide aggregate (MTA) (Dentsply, Tulsa, OK).

Methods

Primary cultured HDPCs were treated with various dilutions of BioAggregate and MTA extracts to assess the cell viability using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Cell adhesion assay was performed using type I collagen-coated plates. An in vitro scratch wound healing model was used to determine cell migration. Focal adhesion formation and cytoskeleton organization were further examined by double immunofluorescence labeling for vinculin and fibrous actin. To assess cell attachment, HDPCs were directly seeded onto the material surfaces and observed by scanning electron microscopy.

Results

HDPCs exposed to BioAggregate extracts showed the highest viabilities at all extract concentrations at 24 and 48 hours, whereas cells exposed to original MTA extracts displayed suppressed viabilities at 72 hours compared with the control. Treatment with BioAggregate extracts enhanced cellular adhesion and migration of HDPCs in a concentration-dependent manner, which was superior to the effects induced by MTA extracts. Immunofluorescence staining indicated that both BioAggregate and MTA optimized focal adhesion formation and stress fiber assembly. Furthermore, scanning electron microscopic analysis revealed that HDPCs attached onto BioAggregate were more flattened and exhibited better spreading than cells on MTA.

Conclusions

BioAggregate is able to promote cellular adhesion, migration, and attachment of HDPCs, indicating its excellent cytocompatibility. Therefore, BioAggregate appears to be a possible alternative to MTA for pulp capping.     

α2β1 integrin and RhoA mediates electric field‐induced ligament fibroblast migration directionality

Guided cell migration is important in tissue development, repair, and engineering. We have previously demonstrated that applied electric fields (EFs) enhanced and directed ligament fibroblast migration and collagen production, depending on EF parameters. Electrical stimulation is widely used for the treatment of pain and to promote wound healing. In orthopaedic practices, applied EFs promote bone healing and ligament repair in vivo. In the current study, stimulation waveforms used in physical therapy for promoting tissue repair were adapted to examine their effects on ACL fibroblast migration. Using different waveform and field strengths, we discovered a decoupling of cell motility and directionality, which suggests disparate mechanisms. Integrin, a major extracellular matrix receptor, polarized in response to applied EFs and controlled cell directionality and signaling. Furthermore, we demonstrated that RhoA is a mediator between integrin aggregation and directed cell migration. Polarization is essential in directed cell migration and our study establishes an outside‐in signaling mechanism for EF‐induced cell directionality. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 322–327, 2013     

A skin-selective homing mechanism for human immune surveillance T cells

Effective immune surveillance is essential for maintaining protection and homeostasis of peripheral tissues. However, mechanisms controlling memory T cell migration to peripheral tissues such as the skin are poorly understood. Here, we show that the majority of human T cells in healthy skin express the chemokine receptor CCR8 and respond to its selective ligand I-309/CCL1. These CCR8(+) T cells are absent in small intestine and colon tissue, and are extremely rare in peripheral blood, suggesting healthy skin as their physiological target site. Cutaneous CCR8(+) T cells are preactivated and secrete proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma, but lack markers of cytolytic T cells. Secretion of interleukin (IL)-4, IL-10, and transforming growth factor-beta was low to undetectable, arguing against a strict association of CCR8 expression with either T helper cell 2 or regulatory T cell subsets. Potential precursors of skin surveillance T cells in peripheral blood may correspond to the minor subset of CCR8(+)CD25(-) T cells. Importantly, CCL1 is constitutively expressed at strategic cutaneous locations, including dermal microvessels and epidermal antigen-presenting cells. For the first time, these findings define a chemokine system for homeostatic T cell traffic in normal human skin.     

Bevacizumab对人Tenon囊成纤维细胞迁移抑制作用的研究

目的：观察bevacizumab对人Tenon囊成纤维细胞迁移能力的影响,探讨青光眼术后滤过泡瘢痕化的对策。方法：采用从陕西省人民医院中心实验室细胞库中取出冻存的人Tenon囊成纤维细胞,采用细胞复苏法,遵循无菌原则,进行常规培养。创伤划痕试验：待细胞融合度达到80%时在单层细胞表面划出一条无细胞的刮除带,空白对照组加入不含血清的DMEM培养液,bevacizumab处理组加入 bevacizumab 浓度为1 mg/mL 不含血清的DMEM培养液,分时段(0,24,48,72h)观察并测量划痕宽度。结果：人Tenon囊成纤维传代细胞显微镜下观察呈长梭形,细胞核位于细胞的中部,核较大,胞浆丰富,生长时呈漩涡状排列走形,增殖能力强,符合成纤维细胞的一般形态。冻存和复苏后细胞的形态结构和生物学特点维持不变。细胞划痕试验显示：0h时,两组细胞划痕初始宽度相等;24 h 时两组细胞迁移距离基本一致;48 h 时bevacizumab处理组细胞迁移距离明显小于空白对照组;72 h时空白对照组划痕基本愈合, bevacizumab处理组细胞迁移距离较48 h无明显变化,且细胞死亡数目较多。结论：利用细胞复苏法可成功培养形态结构和生物学特性稳定的传代人Tenon囊成纤维细胞,为实验研究奠定细胞学基础。成纤维细胞本身具有较强的迁移能力,外源性bevacizumab作用可以明显抑制细胞的迁移,作用时间过长时,会造成细胞的过多死亡。抗新生血管药物对成纤维细胞的迁徙具有一定的抑制作用,未来很有可能成为眼科临床抗击青光眼术后滤过泡瘢痕化的重要手段。     

Pro-migratory actions of the prostacyclin receptor in human breast cancer cells that over-express cyclooxygenase-2

Metastasis is the major cause of death in cancer patients. Elevated expression of cyclooxygenase-2 (COX-2) is observed in many human cancers and over-production of downstream prostaglandins (PGs) has been shown to stimulate metastasis. A role for increased PGE₂ production has been proposed, but whether other PGs contribute is currently unclear. In this study the pro-migratory actions of individual PGs were evaluated in MDA-MB-468 breast cancer cells that stably over-expressed COX-2 (MDA-COX-2 cells); cell migration was quantified using 3D-matrigel droplet assays. Inhibition of the prostacyclin and PGE synthases, but not alternate prostanoid synthases, prevented the increase in MDA-COX-2 cell migration produced by arachidonic acid (AA); direct treatment of cells with the stable prostacyclin analogue cicaprost also promoted migration. Pharmacological antagonism and knockdown of the IP receptor decreased cell migration, while antagonists of the alternate DP, EP2, FP, and TP prostanoid receptors were inactive. In support of these findings, activation of the IP receptor also enhanced migration in the MDA-MB-468, MDA-MB-231 and A549 cell lines, and IP receptor knock-down in MDA-COX-2 cells decreased the expression of a number of pro-migratory genes. In further studies, the prostacyclin/IP receptor and PGE₂/EP4 receptor pathways were found to be functionally independent and the inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK) selectively impaired the IP-receptor-dependent migration in MDA-COX-2 cells. Taken together, the prostacyclin/IP/PI3K-p38 MAPK axis has emerged as a novel pro-migratory pathway in breast cancer cells that over-express COX-2. This information could be utilized in novel treatment strategies to minimize tumor metastasis.     

黄药子醇提物抑制胃癌细胞功能的研究

目的研究黄药子醇提物对人胃癌细胞增殖、克隆形成和迁移能力的影响。方法制备黄药子醇提物,根据细胞培养液所含黄药子醇提物的不同浓度,将实验分为黄药子醇提物高浓度组(120 mg/L)、低浓度组(60 mg/L)和对照组(等量无菌水)。通过体外MTT、克隆形成实验、细胞迁移实验,分别检测黄药子醇提物对人胃癌细胞系MGC803增殖、克隆形成和迁移能力的影响。结果与对照组相比,黄药子醇提物高浓度组和低浓度组胃癌细胞体外增殖速度明显减慢(培养第2~6天时,F分别为29.130、21.864、67.826、36.015、43.656,均P<0.01)、平板克隆形成率明显减少(F=11.918,P<0.01),黄药子醇提物高浓度组的细胞迁移能力明显降低(F=4.258,P<0.05)。结论黄药子醇提物可以显著抑制胃癌细胞增殖、克隆形成和迁移能力,具有潜在的抑制胃癌细胞转移的能力。     

Safety assessment for octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)-propionate (CAS Reg. No. 2082-79-3) from use in food contact applications

Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate (CAS Reg. No. 2082-79-3), currently marketed as Irganox 1076 (I-76), is a sterically hindered phenolic antioxidant used in a variety of organic substrates, including those used in the manufacture of food contact articles. In 2012, the US Food and Drug Administration (USFDA), Office of Food Additive Safety (OFAS), initiated a post-market re-evaluation of the food contact applications of I-76. This project aimed to ensure that current dietary exposures from the use of I-76 in food contact articles are accurately captured and the safety assessment considered all relevant and available toxicological information. To accomplish these aims, the USFDA reviewed the available toxicological studies and chemistry information on food contact applications of I-76. Based on this in-depth analysis, a NOAEL of 64 mg/kg-bw/d (female rats) from a chronic rat study and a cumulative estimated dietary intake (CEDI) of 4.5 mg/p/d, was used to calculate a margin of exposure (MOE) of ∼850. We concluded that the previous and current exposure levels provide an adequate margin of safety (MOS) and remain protective of human health for the regulated uses.