Intensification of extraction process through IVDV pretreatment from Eryngium creticum leaves and stems: Maximizing yields and assessing biological activities.

"Intensification of Vaporization by Decompression to the Vacuum" (IVDV) has initially emerged as a technology primarily employed for expanding and enhancing the texture of biological products. However, its recent applications have showcased significant promise in the realm of extracting bioactive molecules from various plant materials. In this context, optimization using response surface methodology was conducted to investigate the impact of IVDV pretreatment on the extractability of phenolic compounds from Eryngium creticum leaves and stems, as well as their biological activities. Using IVDV preceding the extraction led to higher total phenolic content (TPC) and enhanced antiradical activities in treated materials compared to untreated ones. The optimal processing conditions in terms of water content, steam pressure and treatment time were determined in order to maximize TPC (89.07 and 20.06 mg GAE/g DM in leaves and stems, respectively) and antiradical (DPPH) inhibition percentage (93.51% and 27.54% in leaves and stems, respectively). IVDV-treated extracts showed superior antioxidant, antibacterial and antibiofilm capacities compared to raw extracts. Using RP-UHPLC-PDA-MS, caffeic acid and rosmarinic acid were identified in IVDV-treated leaves. IVDV can be implemented as an innovative treatment applied prior to extraction to boost the recovery of biomolecules from plant matrices.

Chemical Composition and Biological Activities of Centranthus longiflorus Stems Extracts Recovered Using Ired-Irrad®, an Innovative Infrared Technology, Compared to Water Bath and Ultrasound.

Extraction of polyphenols from Centranthus longiflorus stems was conducted using ultrasound and infrared Ired-Irrad® techniques, and compared to the conventional water bath method. Response surface methodology was used to analyse the effect of time, temperature, and ethanol percentage, as well as to optimize the three extraction methods. The highest phenolic content (81 mg GAE/g DM) and antioxidant activity (76% DPPH inhibition) were recorded with the Ired-Irrad® extract obtained under the optimal conditions: 55 °C, 127 min, 48% (v/v) ethanol. Biological activities (antioxidant, antibacterial and antibiofilm) of the three extracts were assessed. All C. longiflorus stems extracts showed limited antibacterial effects regardless of the extraction method (MIC = 50 mg/mL), whereas Ired-Irrad® extract exhibited the highest biofilm eradication and prevention capacities (93% against Escherichia coli and 97% against Staphylococcus epidermidis, respectively). This bioactivity is likely related to abundant caffeoylquinic acid and quercetin rutinoside, as identified by RP-UHPLC-PDA-MS analysis. The results obtained further promote the effectiveness of Ired-Irrad® as a highly flexible and cost-efficient extraction technique.

Intensification of Polyphenols Extraction from Eryngium creticum Leaves Using Ired-Irrad® and Evaluation of Antibiofilm and Antibacterial Activities.

(1) Background: Eryngium creticum is a plant medicinally valued, and used in pharmacopeia to treat various diseases. No previous studies have been reported on E. creticum leaf extracts using an IR-assisted technique; thus, this study aimed to intensify polyphenol extraction using Ired-Irrad®, comparing it to the conventional water bath (WB) method. (2) Methods: Optimization of polyphenol extraction from E. creticum leaves was conducted using Response Surface Methodology. Ired-Irrad® was used and compared to the WB method. The biological activities (antiradical, antioxidant, antibacterial, and antibiofilm) of both extracts were assessed. UHPLC analysis was performed to analyze the phytochemical profile of both extracts. (3) Results: Under optimal conditions, IR improved the polyphenol extraction yield by 1.7 times, while lowering ethanol consumption by 1.5 times. Regarding the antibacterial activity, both WB and IR E. creticum leaf extracts exhibited the highest antibacterial activity against Staphylococcus epidermidis. The maximum biofilm prevention capacity was also noticed against S. epidermidis. UHPLC-MS analysis quantified two major phenolic compounds in both extracts: rutin and sinapic acid. (4) Conclusions: Ired-Irrad® technology proved to be an effective technique in intensifying polyphenol recovery, while preserving their quantity and quality.

Hyphenated LC-ABTS·+ and LC-DAD-HRMS for simultaneous analysis and identification of antioxidant compounds in Astragalus emarginatus Labill. extracts.

The compounds in leaf and stem extracts of Astragalus emarginatus Labill. (AEL), a plant species used in traditional Lebanese medicine, were investigated for antioxidant properties. First, the activity of various extracts was assessed using the Trolox equivalent antioxidant capacity, oxygen radical absorption capacity, and 2,2-diphenyl-1-picryl-hydrazyl-hydrate assays. The extract obtained using 30% ethanol showed the greatest activity. The antioxidant compounds in this extract were screened using a hyphenated high-performance liquid chromatography-2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) radical (ABTS·+) system before being separated by ultra-high-performance liquid chromatography and identified using high-resolution mass spectrometry and ultra-violet-visible diode array detection. Approximately 40 compounds were identified. Hydroxycinnamates (caffeic, ferulic, and p-coumaric acid derivatives) and flavonoids (quercetin, luteolin, apigenin, and isorhamnetin derivatives) were the two main categories of the identified compounds. The active compounds were identified as caffeic acid derivatives and quercetin glycosides. In addition, the catechol moiety was shown to be key to antioxidant activity. This study showed that AEL is a source of natural antioxidants, which may explain its medicinal use.

Antibacterial and Immunomodulatory Properties of Acellular Wharton's Jelly Matrix.

Of all biologic matrices, decellularized tissues have emerged as a promising tool in the field of regenerative medicine. Few empirical clinical studies have shown that Wharton's jelly (WJ) of the human umbilical cord promotes wound closure and reduces wound-related infections. In this scope, we herein investigated whether decellularized (DC)-WJ could be used as an engineered biomaterial. In comparison with devitalized (DV)-WJ, our results showed an inherent effect of DC-WJ on Gram positive (S. aureus and S. epidermidis) and Gram negative (E. coli and P. aeruginosa) growth and adhesion. Although DC-WJ activated the neutrophils and monocytes in a comparable magnitude to DV-WJ, macrophages modulated their phenotypes and polarization states from the resting M0 phenotype to the hybrid M1/M2 phenotype in the presence of DC-WJ. M1 phenotype was predominant in the presence of DV-WJ. Finally, the subcutaneous implantation of DC-WJ showed total resorption after three weeks of implantation without any sign of foreign body reaction. These significant data shed light on the potential regenerative application of DC-WJ in providing a suitable biomaterial for tissue regenerative medicine and an ideal strategy to prevent wound-associated infections.

Investigation of squalene-doxorubicin distribution and interactions within single cancer cell using Raman microspectroscopy.

Intracellular distribution of doxorubicin (DOX) and its squalenoylated (SQ-DOX) nanoparticles (NPs) form in murine lung carcinoma M109 and human breast carcinoma MDA-MB-231 cells was investigated by Raman microspectroscopy. Pharmacological data showed that DOX induced higher cytotoxic effect than SQ-DOX NPs. Raman data were obtained using single-point measurements and imaging on the whole cell areas. These data showed that after DOX treatment at 1 µM, the spectral features of DOX were not detected in the M109 cell cytoplasm and nucleus. However, the intracellular distribution of SQ-DOX NPs was higher than DOX in the same conditions. In addition, SQ-DOX NPs were localized into both cell cytoplasm and nucleus. After 5 µM treatment, Raman bands of DOX at 1211 and 1241 cm-1 were detected in the nucleus. Moreover, the intensity ratio of these bands decreased, indicating DOX intercalation into DNA. However, after treatment with SQ-DOX NPs, the intensity of these Raman bands increased. Interestingly, with SQ-DOX NPs, the intensity of 1210/1241 cm-1 ratio was higher suggesting a lower fraction of intercalated DOX in DNA and higher amount of non-hydrolyzed SQ-DOX. Raman imaging data confirm this subcellular localization of these drugs in both M109 and MDA-MB-231 cells. These finding brings new insights to the cellular characterization of anticancer drugs at the molecular level, particularly in the field of nanomedicine.

Interaction of implant infection-related commensal bacteria with mesenchymal stem cells: a comparison between Cutibacterium acnes and Staphylococcus aureus.

Staphylococcus aureus and Cutibacterium acnes are involved in several tissue infections and can encounter mesenchymal stem cells (MSCs) during their role in tissue regenerative process. C. acnes and S. aureus internalization by three types of MSCs derived from bone marrow, dental pulp and Wharton's jelly; and bacterial biofilm production were compared. Internalization rates ranged between 1.7-6.3% and 0.8-2.7% for C. acnes and S. aureus, respectively. While C. acnes strains exhibited limited cytotoxic effect on MSCs, S. aureus were more virulent with marked effect starting after only 3 h of interaction. Both bacteria were able to produce biofilms with respectively aggregated and monolayered structures for C. acnes and S. aureus. The increase in C. acnes capacity to develop biofilm following MSCs' internalization was not linked to the significant increase in number of live bacteria, except for bone marrow-MSCs/C. acnes CIP 53.117 with 79% live bacteria compared to the 36% before internalization. On the other hand, internalization of S. aureus had no impact on its ability to form biofilms composed mainly of living bacteria. The present study underlined the complexity of MSCs-bacteria cross-interaction and brought insights into understanding the MSCs behavior in response to bacterial infection in tissue regeneration context.

Poly-L-Lysine and Human Plasmatic Fibronectin Films as Proactive Coatings to Improve Implant Biointegration.

The success of stable and long-term implant integration implies the promotion, control, and respect of the cell microenvironment at the site of implantation. The key is to enhance the implant-host tissue cross talk by developing interfacial strategies that guarantee an optimal and stable seal of soft tissue onto the implant, while preventing potential early and late infection. Indeed, implant rejection is often jeopardized by lack of stable tissue surrounding the biomaterial combined with infections which reduce the lifespan and increase the failure rate of implants and morbidity and account for high medical costs. Thin films formed by the layer-by-layer (LbL) assembly of oppositely charged polyelectrolytes are particularly versatile and attractive for applications involving cell-material contact. With the combination of the extracellular matrix protein fibronectin (Fn, purified from human plasma) and poly-L-lysine (PLL, exhibiting specific chain lengths), we proposed proactive and biomimetic coatings able to guarantee enhanced cell attachment and exhibiting antimicrobial properties. Fn, able to create a biomimetic interface that could enhance cell attachment and promote extracellular cell matrix remodeling, is incorporated as the anionic polymer during film construction by the LbL technic whereas PLL is used as the cationic polymer for its capacity to confer remarkable antibacterial properties.

Biopolymers-calcium phosphate antibacterial coating reduces the pathogenicity of internalized bacteria by mesenchymal stromal cells.

A multifunctional material system that kills bacteria and drives bone healing is urgently sought to improve bone prosthesis. Herein, the osteoinductive coating made of calcium phosphate/chitosan/hyaluronic acid, named Hybrid, was proposed as an antibacterial substrate for stromal cell adhesion. This Hybrid coating possesses a contact-killing effect reducing by 90% the viability of Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Pseudomonas aeruginosa (P. aeruginosa) strains after 48 h of contact. In addition to the production of immunomodulatory mediators, Wharton's jelly (WJ-SCs), dental pulp (DPSCs) and bone marrow (BM-MSCs) derived stromal cells were able to release antibacterial and antibiofilm agents effective against S. aureus and P. aeruginosa strains, respectively. Studying the effect of the Hybrid coating on the internalization of S. aureus by the stromal cells, in acute-mimicking bone infection, highlighted an increase in the bacteria internalization by DPSCs and BM-MSCs when cultured on the Hybrid coating versus uncoated glass. Despite the internalization, Hybrid coating showed a beneficial effect by reducing the pathogenicity of the internalized bacteria. The formation of biofilm was reduced by at least 50% in comparison to internalized bacteria by stromal cells on uncoated glass. This work opens the route for the development of innovative antibacterial coatings by taking into account the internalization of bacteria by stromal cells.

Mechanobiologically induced bone-like nodules: Matrix characterization from micro to nanoscale.

In bone tissue engineering, stem cells are known to form inhomogeneous bone-like nodules on a micrometric scale. Herein, micro- and nano-infrared (IR) micro-spectroscopies were used to decipher the chemical composition of the bone-like nodule. Histological and immunohistochemical analyses revealed a cohesive tissue with bone-markers positive cells surrounded by dense mineralized type-I collagen. Micro-IR gathered complementary information indicating a non-mature collagen at the top and periphery and a mature collagen within the nodule. Atomic force microscopy combined to IR (AFM-IR) analyses showed distinct spectra of "cell" and "collagen" rich areas. In contrast to the "cell" area, spectra of "collagen" area revealed the presence of carbohydrate moieties of collagen and/or the presence of glycoproteins. However, it was not possible to determine the collagen maturity, due to strong bands overlapping and/or possible protein orientation effects. Such findings could help developing protocols to allow a reliable characterization of in vitro generated complex bone tissues.

Osteoinductive Material to Fine-Tune Paracrine Crosstalk of Mesenchymal Stem Cells With Endothelial Cells and Osteoblasts.

While stem cell/biomaterial studies provide solid evidences that biomaterial intrinsic cues deeply affect cell fate, current strategies tend to neglect their effects on mesenchymal stem cells (MSCs) secretory activities and resulting cell-crosstalks. The present study aims to investigate the impact of bone-mimetic material (B-MM), with intrinsic osteoinductive property, on MSCs mediator secretions; and to explore underlying effects on cells involved in bone regeneration. Human MSCs were cultured, on B-MM, made from inorganic calcium phosphate supplemented with chitosan and hyaluronic acid biopolymers. Collected MSCs culture media were assessed for mediators release quantification and used further to stimulate endothelial cells (ECs) and alveolar bone derived osteoblasts (OBs). Without osteogenic supplements, MSCs committed into bone lineage forming thus 3D bone-like nodules after 21 days. Despite a weak percentage of cell commitment, our data elucidate new aspects of osteoinductive material effect on MSCs functions through the regulation of the secretion of mediators involved in bone regeneration and subsequently the MSCs/ECs indirect crosstalk with osteogenesis-boosting effect. Using MSCs culture media, we demonstrate a large potential of osteoinductive materials and MSCs in bone regenerative medicine. Such strategies could help to address some insights in cell-free therapies using MSCs derived media.

Boosting mesenchymal stem cells regenerative activities on biopolymers-calcium phosphate functionalized collagen membrane.

The regeneration of bone-soft tissue interface, using functional membranes, remains challenging and can be promoted by improving mesenchymal stem cells (MSCs) paracrine function. Herein, a collagen membrane, used as guided bone regeneration membrane, was functionalized by calcium phosphate, chitosan and hyaluronic acid hybrid coating by simultaneous spray of interacting species process. Composed of brushite, octacalcium phosphate and hydroxyapatite, the hybrid coating increased the membrane stiffness by 50%. After 7 days of MSCs culture on the hybrid coated polymeric membrane, biological studies were marked by a lack of osteoblastic commitment. However, MSCs showed an enhanced proliferation along with the secretion of cytokines and growth factors that could block bone resorption and favour endothelial cell recruitment without exacerbating polynuclear neutrophils infiltration. These data shed light on the great potential of inorganic/organic coated collagen membranes as an alternative bioactive factor-like platform to improve MSCs regenerative capacity, in particular to support bone tissue vascularization and to modulate inflammatory infiltrates.

Combining Calcium Phosphates with Polysaccharides: A Bone-Inspired Material Modulating Monocyte/Macrophage Early Inflammatory Response.

The use of inorganic calcium/phosphate supplemented with biopolymers has drawn lots of attention in bone regenerative medicine. While inflammation is required for bone healing, its exacerbation alters tissue regeneration/implants integration. Inspired by bone composition, a friendly automated spray-assisted system was used to build bioactive and osteoinductive calcium phosphate/chitosan/hyaluronic acid substrate (CaP-CHI-HA). Exposing monocytes to CaP-CHI-HA resulted in a secretion of pro-healing VEGF and TGF-ß growth factors, TNF-α, MCP-1, IL-6 and IL-8 pro-inflammatory mediators but also IL-10 anti-inflammatory cytokine along with an inflammatory index below 1.5 (versus 2.5 and 7.5 following CaP and LPS stimulation, respectively). Although CD44 hyaluronic acid receptor seems not to be involved in the inflammatory regulation, results suggest a potential role of chemical composition and calcium release from build-up substrates, in affecting the intracellular expression of a calcium-sensing receptor. Herein, our findings indicate a great potential of CaP-CHI-HA in providing required inflammation-healing balance, favorable for bone healing/regeneration.

Age-related modifications of type I collagen impair DDR1-induced apoptosis in non-invasive breast carcinoma cells.

Type I collagen and DDR1 axis has been described to decrease cell proliferation and to initiate apoptosis in non-invasive breast carcinoma in three-dimensional cell culture matrices. Moreover, MT1-MMP down-regulates these effects. Here, we address the effect of type I collagen aging and MT1-MMP expression on cell proliferation suppression and induced-apoptosis in non-invasive MCF-7 and ZR-75-1 breast carcinoma. We provide evidence for a decrease in cell growth and an increase in apoptosis in the presence of adult collagen when compared to old collagen. This effect involves a differential activation of DDR1, as evidenced by a higher DDR1 phosphorylation level in adult collagen. In adult collagen, inhibition of DDR1 expression and kinase function induced an increase in cell growth to a level similar to that observed in old collagen. The impact of aging on the sensitivity of collagen to MT1-MMP has been reported recently. We used the MT1-MMP expression strategy to verify whether, by degrading adult type I collagen, it could lead to the same phenotype observed in old collagen 3D matrix. MT1-MMP overexpression abrogated the proliferation suppression and induced-apoptosis effects only in the presence of adult collagen. This suggests that differential collagen degradation by MT1-MMP induced a structural disorganization of adult collagen and inhibits DDR1 activation. This could in turn impair DDR1-induced cell growth suppression and apoptosis. Taken together, our data suggest that modifications of collagen structural organization, due to aging, contribute to the loss of the growth suppression and induced apoptosis effect of collagen in luminal breast carcinoma. MT1-MMP-dependent degradation and aging of collagen have no additive effects on these processes.

Chitosan/hyaluronic acid multilayer films are biocompatible substrate for Wharton's jelly derived stem cells.

BACKGROUND: Discovery of mesenchymal stem cells (MSCs) in various adult human tissues opened the way to new therapeutic strategies involving tissue engineering from these cells. More recently, vascular substitutes have opened the era of vascular engineering by making replacement vessels from purely biological material. The objective of our study was to create a vascular substitute from MSCs using a multilayer polyelectrolyte film based on natural polymers (Chitosan and Hyaluronic Acid). METHODS: Biocompatibility and cellular behavior were evaluated in this study using MSCs from the Wharton's jelly (WJ) of human umbilical cords. WJ-MSCs adherence was assessed and cells morphology was investigated by Scanning Electron Microscopy (SEM) and actin visualization (Phalloidin). RESULTS: The number of WJ-MSCs seeded on the (CHI/HA)10 films was greater than the number of cells seeded on the collagen, as the spectrophotometric measurement showed a large cell proliferation on (CHI/HA)10 in comparison with collagen. After adhesion, WJ-MSCs showed a fibroblastic morphology on CHI/HA as for control (collagen I). These results were confirmed by cytoskeleton staining. CONCLUSIONS: The biocompatibility of WJ-MSCs and (CHI/HA)10 showed the possibility to combine the use of WJ-MSCs and (CHI/HA)10 films in vascular tissue engineering.

Collagen-Based Medical Device as a Stem Cell Carrier for Regenerative Medicine.

Maintenance of mesenchymal stem cells (MSCs) requires a tissue-specific microenvironment (i.e., niche), which is poorly represented by the typical plastic substrate used for two-dimensional growth of MSCs in a tissue culture flask. The objective of this study was to address the potential use of collagen-based medical devices (HEMOCOLLAGENE®, Saint-Maur-des-Fossés, France) as mimetic niche for MSCs with the ability to preserve human MSC stemness in vitro. With a chemical composition similar to type I collagen, HEMOCOLLAGENE® foam presented a porous and interconnected structure (>90%) and a relative low elastic modulus of around 60 kPa. Biological studies revealed an apparently inert microenvironment of HEMOCOLLAGENE® foam, where 80% of cultured human MSCs remained viable, adopted a flattened morphology, and maintained their undifferentiated state with basal secretory activity. Thus, three-dimensional HEMOCOLLAGENE® foams present an in vitro model that mimics the MSC niche with the capacity to support viable and quiescent MSCs within a low stiffness collagen I scaffold simulating Wharton's jelly. These results suggest that haemostatic foam may be a useful and versatile carrier for MSC transplantation for regenerative medicine applications.

Upregulation of endothelial gene markers in Wharton's jelly mesenchymal stem cells cultured on polyelectrolyte multilayers.

Designing convenient substrates is a pertinent parameter that can guide stem cell differentiation. Current research is directed toward differentiating mesenchymal stem cells (MSCs) into endothelial cells (ECs). It is generally accepted that MSCs cannot be easily differentiated into ECs without high concentrations of proangiogenic factors. To guide either bone marrow-derived mesenchymal stem cells (BM-MSCs) and Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into ECs-like phenotype, poly(allylamine-hydrochloride)/poly(styrene-sulfonate) multilayers film (PAH/PSS) was used as culture coating and compared to type I collagen (as control coating). After 2 weeks of culture and in absence of angiogenic growth factors, PAH/PSS upregulated KDR, PECAM-1, and CDH5 genes, whereas combining PAH/PSS with endothelial growth media (EGM-2® ) led to the production of respective proteins by WJ-MSCs. In contrast, not fully EC-like phenotype is obtained from the differentiation of BM- or WJ-MSCs cultured on type I collagen. Thus, using PAH/PSS coating in synergy with EGM-2® appears as an ideal condition promoting WJ-MSCs differentiation into ECs-like. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 292-300, 2017.

Corrigendum: Discoidin Domain Receptors: Potential Actors and Targets in Cancer.

[This corrects the article on p. 55 in vol. 7, PMID: 27014069.].

Discoidin Domain Receptors: Potential Actors and Targets in Cancer.

The extracellular matrix critically controls cancer cell behavior by inducing several signaling pathways through cell membrane receptors. Besides conferring structural properties to tissues around the tumor, the extracellular matrix is able to regulate cell proliferation, survival, migration, and invasion. Among these receptors, the integrins family constitutes a major class of receptors that mediate cell interactions with extracellular matrix components. Twenty years ago, a new class of extracellular matrix receptors has been discovered. These tyrosine kinase receptors are the two discoidin domain receptors DDR1 and DDR2. DDR1 was first identified in the Dictyostelium discoideum and was shown to mediate cell aggregation. DDR2 shares highly conserved sequences with DDR1. Both receptors are activated upon binding to collagen, one of the most abundant proteins in extracellular matrix. While DDR2 can only be activated by fibrillar collagen, particularly types I and III, DDR1 is mostly activated by type I and IV collagens. In contrast with classical growth factor tyrosine kinase receptors which display a rapid and transient activation, DDR1 and DDR2 are unique in that they exhibit delayed and sustained receptor phosphorylation upon binding to collagen. Recent studies have reported differential expression and mutations of DDR1 and DDR2 in several cancer types and indicate clearly that these receptors have to be taken into account as new players in the different aspects of tumor progression, from non-malignant to highly malignant and invasive stages. This review will discuss the current knowledge on the role of DDR1 and DDR2 in malignant transformation, cell proliferation, epithelial to mesenchymal transition, migratory, and invasive processes, and finally the modulation of the response to chemotherapy. These new insights suggest that DDR1 and DDR2 are new potential targets in cancer therapy.

Stem cells: a promising source for vascular regenerative medicine.

The rising and diversity of many human vascular diseases pose urgent needs for the development of novel therapeutics. Stem cell therapy represents a challenge in the medicine of the twenty-first century, an area where tissue engineering and regenerative medicine gather to provide promising treatments for a wide variety of diseases. Indeed, with their extensive regeneration potential and functional multilineage differentiation capacity, stem cells are now highlighted as promising cell sources for regenerative medicine. Their multilineage differentiation involves environmental factors such as biochemical, extracellular matrix coating, oxygen tension, and mechanical forces. In this review, we will focus on human stem cell sources and their applications in vascular regeneration. We will also discuss the different strategies used for their differentiation into both mature and functional smooth muscle and endothelial cells.