Didier Dréau

The susceptibility of prosthetic biomaterials to infection

BackgroundDespite the use of a sterile technique and the administration of prophylactic antibiotics during surgical procedures, mesh infection continues to complicate the use of biomaterials. The purpose of this study was to compare the susceptibility to infection of prosthetic biomaterials in a live-animal model.MethodsThe following seven prosthetic mesh biomaterials were used in this study. Expanded polytetrafluoroethylene (ePTFE) with silver/chlorhexidine (DM+), ePTFE (DM), porcine intestinal submucosa (S), polypropylene (M), ePTFE/polypropylene (X), hyaluronate/carboxymethylcellulose/polypropylene (SM), and human acellular dermal matrix (A). Lewis rats (n = 108) underwent creation of a single ventral hernia; 105 of them were repaired with a different mesh (2-cm2 piece). Twelve pieces of each mesh were inoculated at the time of hernia repair with 108 Staphylococcus aureus (n = 84). Three pieces of each mesh were placed without bacterial inoculation (n = 21). In three animals, no mesh was placed; instead, the peritoneum of the hernia defect was inoculated (n = 3). After 5 days, the animals were killed and the mesh was explanted (peritoneum for the nonmesh control). The mesh was vortex-washed and incubated in tryptic soy broth. Bacterial counts were determined using serial dilutions and spot plates and quantified in colony-forming units (CFU) per square centimeter of mesh present in the vortex wash fluid (wash count) and the soy broth (broth count). Data are presented as the mean log10, with analysis of variance (ANOVA) and Tukey’s test used to determine significance (p < 0.05).ResultsThe DM+ material had no detectable live bacteria in the wash or broth counts in 10 of 12 tested samples (p = 0.05). Of the samples that showed bacterial growth, the peritoneum control group had a lower wash count than A (p = 0.05) and the lowest broth count of all the materials except for DM+ (p = 0.05). In addition, SM had a significantly lower wash count than A (p = 0.05), with no broth count difference. In regard to wash and broth counts, DM, M, X, SM, S, and A were no different (p = NS).ConclusionsThe DM+ material was the least susceptible to infection. Impregnation with silver/chlorhexidine killed the inoculated bacteria, preventing their proliferation on the mesh surface. Other than DM+, native peritoneal tissue appears to be the least susceptible to infection. Silver/chlorhexidine appears to be an effective bactericidal agent for use with mesh biomaterials.

Mesenchymal stem cell-derived CCL-9 and CCL-5 promote mammary tumor cell invasion and the activation of matrix metalloproteinases

Stromal chemokine gradients within the breast tissue microenvironment play a critical role in breast cancer cell invasion, a prerequisite to metastasis. To elucidate which chemokines and mechanisms are involved in mammary cell migration we determined whether mesenchymal D1 stem cells secreted specific chemokines that differentially promoted the invasion of mammary tumor cells in vitro. Results indicate that mesenchymal D1 cells produced concentrations of CCL5 and CCL9 4- to 5-fold higher than the concentrations secreted by 4T1 tumor cells (P < 0.01). Moreover, 4T1 tumor cell invasion toward D1 mesenchymal stem cell conditioned media (D1CM), CCL5 alone, CCL9 alone or a combination CCL5 and CCL9 was observed. The invasion of 4T1 cells toward D1 mesenchymal stem CM was dose-dependently suppressed by pre-incubation with the CCR1/CCR5 antagonist met-CCL5 (P < 0.01). Furthermore, the invasion of 4T1 cells toward these chemokines was prevented by incubation with the broad-spectrum MMP inhibitor GM6001. Additionally, the addition of specific MMP9/MMP13 and MMP14 inhibitors prevented the MMP activities of supernatants collected from 4T1 cells incubated with D1CM, CCL5 or CCL9. Taken together these data highlight the role of CCL5 and CCL9 produced by mesenchymal stem cells in mammary tumor cell invasion.

Increased extracellular matrix density decreases MCF10A breast cell acinus formation in 3D culture conditions

The extracellular matrix (ECM) contributes to the generation and dynamic of normal breast tissue, in particular to the generation of polarized acinar and ductal structures. In vitro 3D culture conditions, including variations in the composition of the ECM, have been shown to directly influence the formation and organization of acinus‐like and duct‐like structures. Furthermore, the density of the ECM appears to also play a role in the normal mammary tissue and tumour formation. Here we show that the density of the ECM directly influences the number, organization and function of breast acini. Briefly, non‐malignant human breast MCF10A cells were incubated in increasing densities of a Matrigel®–collagen I matrix. Elastic moduli near and distant to the acinus structures were measured by atomic force microscopy, and the number of acinus structures was determined. Immunochemistry was used to investigate the expression levels of E‐cadherin, laminin, matrix metalloproteinase‐14 and ß‐casein in MCF10A cells. The modulus of the ECM was significantly increased near the acinus structures and the number of acinus structures decreased with the increase in Matrigel–collagen I density. As evaluated by the expression of laminin, the organization of the acinus structures present was altered as the density of the ECM increased. Increases in both E‐cadherin and MMP14 expression by MCF10A cells as ECM density increased were also observed. In contrast, MCF10A cells expressed lower ß‐casein levels as the ECM density increased. Taken together, these observations highlight the key role of ECM density in modulating the number, organization and function of breast acini. Copyright

The heterodimerization of platelet-derived chemokines

Chemokines encompass a large family of proteins that act as chemoattractants and are involved in many biological processes. In particular, chemokines guide the migration of leukocytes during normal and inflammatory conditions. Recent studies reveal that the heterophilic interactions between chemokines significantly affect their biological activity, possibly representing a novel regulatory mechanism of the chemokine activities. The co-localization of platelet-derived chemokines in vivo allows them to interact. Here, we used nano-spray ionization mass spectrometry to screen eleven different CXC and CC platelet-derived chemokines for possible interactions with the two most abundant chemokines present in platelets, CXCL4 and CXCL7. Results indicate that many screened chemokines, although not all of them, form heterodimers with CXCL4 and/or CXCL7. In particular, a strong heterodimerization was observed between CXCL12 and CXCL4 or CXCL7. Compared to other chemokines, the main structural difference of CXCL12 is in the orientation and packing of the C-terminal alpha-helix in relation to the beta-sheet. The analysis of one possible structure of the CXCL4/CXCL12 heterodimer, CXC-type structure, using molecular dynamics (MD) trajectory reveals that CXCL4 may undergo a conformational transition to alter the alpha helix orientation. In this new orientation, the alpha-helix of CXCL4 aligns in parallel with the CXCL12 alpha-helix, an energetically more favorable conformation. Further, we determined that CXCL4 and CXCL12 physically interact to form heterodimers by co-immunoprecipitations from human platelets. Overall, our results highlight that many platelet-derived chemokines are capable of heterophilic interactions and strongly support future studies of the biological impact of these interactions.

Progranulin Stimulated by LPA Promotes the Migration of Aggressive Breast Cancer Cells

Abstract Activator and inhibitor roles for the 88-kDa-secreted glycoprotein progranulin (PGRN) have been demonstrated in ovarian cancer cells. Here, we investigated the effects of PGRN in breast cancer migration. Testing MCF7, MDA-MB-453, and MDA-MB-231 human breast cancer cells and the MCF10A breast epithelial cell line, we demonstrate that LPA-induced PGRN stimulation led to a significant increase in cell invasion of MDA-MB-453 and MDA-MB-231 cells only (p < 0.05). Moreover, incubation with an anti-PGRN antibody, an inhibitor of the ERK pathway (PD98059) or both in combination inhibited the ability of MDA-MB-231 cells to invade. Furthermore, the expression of focal adhesion kinases promoted by LPA-induced PGRN was also inhibited by PD98059 alone or in combination with an anti-PGRN antibody (p < 0.05). Taken together, these results suggest that the LPA activation of PGRN involving the ERK pathway is critical to promote MDA-MB-231 breast cancer cell invasion.

Immune and inflammation responses to a 3-day period of intensified running versus cycling

Functional overreaching has been linked to alterations in immunity and host pathogen defense, but little is known as to whether or not running and cycling evoke different responses. This study compared inflammation, muscle damage and soreness, and innate immune function responses to a 3-day period of intensified exercise in trained long distance runners (N=13, age 34.4±2.4year) and cyclists (N=22, age 36.6±1.7year, P=0.452). Upper respiratory tract infection (URTI) symptomatology was monitored for 12weeks using the Wisconsin Upper Respiratory Symptom Survey (WURSS), and subjects from both athletic groups came to the lab during week five and exercised 2.5h/day for 3days in a row at 70% VO2max. Blood samples were collected before and after the 3-day period of exercise, with recovery samples collected 1-, 14-, and 38h-post-exercise. Samples were analyzed for muscle damage [creatine kinase (CK), myoglobin (MYO)], inflammation (CRP, IL-6, IL-8, IL-10, MCP), and innate immunity [granulocyte and monocyte phagocytosis (GR-PHAG and MO-PHAG) and oxidative burst activity (GR-OBA and MO-OBA)]. Runners compared to cyclists experienced significantly more muscle damage (CK 133% and MYO 404% higher post-3days exercise), inflammation (CRP 87%, IL-6 256%, IL 8 61%, IL-10 32%, MCP 29%), and delayed onset of muscle soreness (DOMS, 87%). The 3-day period of exercise caused significant downturns in GR-PHAG, MO-PHAG, GR-OBA, MO-OBA by 14- and 38h-recovery, but the pattern of change did not differ between groups. No group differences were measured for 12-week URTI severity (18.3±5.6 and 16.6±4.0, P=0.803) and symptom scores (33.4±12.6 and 24.7±5.8, P=0.477). These data indicate that a 3-day period of functional overreaching results in substantially more muscle damage and soreness, and systemic inflammation in runners compared to cyclists, but without group differences for 12-week URTI symptomatology and post-exercise decrements in innate immune function.

Overcoming hypoxia to improve tissue‐engineering approaches to regenerative medicine

The current clinical successes of tissue engineering are limited primarily to low‐metabolism, acellular, pre‐vascularized or thin tissues. Mass transport has been identified as the primary culprit, limiting the delivery of nutrients (such as oxygen and glucose) and removal of wastes, from tissues deep within a cellular scaffold. While strategies to develop sufficient vasculature to overcome hypoxia in vitro are promising, inconsistencies between the in vitro and the in vivo environments may still negate the effectiveness of large‐volume tissue‐engineered scaffolds. While a common theme in tissue engineering is to maximize oxygen supply, studies suggest that moderate oxygenation of cellular scaffolds during in vitro conditioning is preferable to high oxygen levels. Aiming for moderate oxygen values to prevent hypoxia while still promoting angiogenesis may be obtained by tailoring in vitro culture conditions to the oxygen environment the scaffold will experience upon implantation. This review discusses the causes and effects of tissue‐engineering hypoxia and the optimization of oxygenation for the minimization of in vivo hypoxia. Copyright

Influence of Pistachios on Performance and Exercise-Induced Inflammation, Oxidative Stress, Immune Dysfunction, and Metabolite Shifts in Cyclists: A Randomized, Crossover Trial

Objectives Pistachio nut ingestion (3 oz./d, two weeks) was tested for effects on exercise performance and 21-h post-exercise recovery from inflammation, oxidative stress, immune dysfunction, and metabolite shifts. Methods Using a randomized, crossover approach, cyclists (N = 19) engaged in two 75-km time trials after 2-weeks pistachio or no pistachio supplementation, with a 2-week washout period. Subjects came to the lab in an overnight fasted state, and ingested water only or 3 oz. pistachios with water before and during exercise. Blood samples were collected 45 min pre-exercise, and immediately post-, 1.5-h post-, and 21-h post-exercise, and analyzed for plasma cytokines, C-reactive protein (CRP), F2-isoprostanes (F2-IsoP), granulocyte phagocytosis (GPHAG) and oxidative burst activity (GOBA), and shifts in metabolites. Results Performance time for the 75-km time trial was 4.8% slower under pistachio conditions (2.84±0.11 and 2.71±0.07 h, respectively, P = 0.034). Significant time effects were shown for plasma cytokines, CRP, F2-IsoP, GPHAG, and GOBA, with few group differences. Metabolomics analysis revealed 423 detectable compounds of known identity, with significant interaction effects for 19 metabolites, especially raffinose, (12Z)-9,10-Dihydroxyoctadec-12-enoate (9,10-DiHOME), and sucrose. Dietary intake of raffinose was 2.19±0.15 and 0.35±0.08 mg/d during the pistachio and no pistachio periods, and metabolomics revealed that colon raffinose and sucrose translocated to the circulation during exercise due to increased gut permeability. The post-exercise increase in plasma raffinose correlated significantly with 9,10-DiHOME and other oxidative stress metabolites. Conclusions In summary, 2-weeks pistachio nut ingestion was associated with reduced 75-km cycling time trial performance and increased post-exercise plasma levels of raffinose, sucrose, and metabolites related to leukotoxic effects and oxidative stress. Trial Registration ClinicalTrials.gov NCT01821820

Bioengineered silk scaffolds in 3D tissue modeling with focus on mammary tissues

In vitro generation of three-dimensional (3D) biological tissues and organ-like structures is a promising strategy to study and closely model complex aspects of the molecular, cellular, and physiological interactions of tissue. In particular, in vitro 3D tissue modeling holds promises to further our understanding of breast development. Indeed, biologically relevant 3D structures that combine mammary cells and engineered matrices have improved our knowledge of mammary tissue growth, organization, and differentiation. Several polymeric biomaterials have been used as scaffolds to engineer 3D mammary tissues. Among those, silk fibroin-based biomaterials have many biologically relevant properties and have been successfully used in multiple medical applications. Here, we review the recent advances in engineered scaffolds with an emphasis on breast-like tissue generation and the benefits of modified silk-based scaffolds.

Mammary epithelial cell adhesion, viability, and infiltration on blended or coated silk fibroin-collagen type I electrospun scaffolds.

Interactions between cells and the extracellular matrix (ECM) play a crucial role in regulating biological tissue function. Silk biomaterials from Bombyx mori (B. mori) silkworm silk are widely used in tissue engineering. As this silk fibroin (SF) contains no strong adhesion sites, we assessed whether the blending or coating of SF with collagen would further improve SF biocompatibility, in part through the addition of the specific integrin recognition sequences. In the present study, electrospun scaffolds were developed by blending 7% SF and 7% type I collagen solutions at ratios of 100:0 (pure SF), 95:5, 90:10, and 85:15 (SF:collagen, v/v) prior to electrospinning. Pure SF scaffolds were further coated with collagen type I. The physical and mechanical properties of these scaffolds and MCF10A mammary epithelial cell adhesion, viability, and infiltration into these blended or coated SF-collagen (SF-C) scaffolds were determined. The blending of SF with collagen decreased average pore sizes and fiber diameters of the electrospun scaffolds regardless of the ratio (p<0.01). The mechanical strength of these scaffolds did not change in their hydrated state (ns), but was decreased for 85:15 SF-C blended scaffolds in the dry state (p<0.05). The adhesion of MCF10A cells was significantly increased in SF-C blended or coated scaffolds compared to pure SF scaffolds (p<0.01). MCF10A cell viability and infiltration on SF-C coated scaffolds were significantly higher compared to all other conditions tested (p<0.01).