Halima Kerdjoudj

Polyelectrolyte Films Boost Progenitor Cell Differentiation into Endothelium‐like Monolayers

Rapid differentiation of endothelial progenitor cells (EPCs) into confluent mature endothelial cells is important in tissue engineering for the design of autologous, nonthrombotic, vascular grafts. A new method based on EPC culture on poly(sodium-4- styrene-sulfonate)/poly(allylamine hydrochloride), that is, polyelectrolyte-multilayer-coated substrates, reduces the time from two months to two weeks.

O2 Level Controls Hematopoietic Circulating Progenitor Cells Differentiation into Endothelial or Smooth Muscle Cells

Background Recent studies showed that progenitor cells could differentiate into mature vascular cells. The main physiological factors implicated in cell differentiation are specific growth factors. We hypothesized that simply by varying the oxygen content, progenitor cells can be differentiated either in mature endothelial cells (ECs) or contractile smooth muscle cells (SMCs) while keeping exactly the same culture medium. Methodology/Principal Findings Mononuclear cells were isolated by density gradient were cultivated under hypoxic (5% O2) or normoxic (21% O2) environment. Differentiated cells characterization was performed by confocal microscopy examination and flow cytometry analyses. The phenotype stability over a longer time period was also performed. The morphological examination of the confluent obtained cells after several weeks (between 2 and 4 weeks) showed two distinct morphologies: cobblestone shape in normoxia and a spindle like shape in hypoxia. The cell characterization showed that cobblestone cells were positive to ECs markers while spindle like shape cells were positive to contractile SMCs markers. Moreover, after several further amplification (until 3rd passage) in hypoxic or normoxic conditions of the previously differentiated SMC, immunofluorescence studies showed that more than 80% cells continued to express SMCs markers whatever the cell environmental culture conditions with a higher contractile markers expression compared to control (aorta SMCs) signature of phenotype stability. Conclusion/Significance We demonstrate in this paper that in vitro culture of peripheral blood mononuclear cells with specific angiogenic growth factors under hypoxic conditions leads to SMCs differentiation into a contractile phenotype, signature of their physiological state. Moreover after amplification, the differentiated SMC did not reverse and keep their contractile phenotype after the 3rd passage performed under hypoxic and normoxic conditions. These aspects are of the highest importance for tissue engineering strategies. These results highlight also the determinant role of the tissue environment in the differentiation process of vascular progenitor cells.

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.

Endothelialized and preconditioned natural umbilical arteries with long term patency open the route for future human uses

The major challenge of vascular tissue engineering is to develop a small calibre vascular graft with a high patency rate. In native vessels, the thrombosis is prevented by the endothelium located at the luminal site of the vessel. The aim of this study was to develop a resistant endothelial lining on the inner surface of vascular graft using a polyelectrolyte multilayers (PEM) film. Umbilical arteries were de-endothelialized, coated with 3.5 bilayers of poly(styrene sulfonate) (PSS)/poly(allylamine hydrochloride) (PAH) and then cellularized with endothelial cells. The grafts were cultured for a week in static condition and preconditioned by exposure to a shear stress of at 1 Pa for three hours before implantation on the rabbit carotid site. Histological and confocal microscopy in vitro investigations showed that PEMs films improve cell adhesion and retention on the luminal surface after shear stress preconditioning. In vivo Doppler data showed that graft preconditioning is a crucial factor for graft patency. Indeed, preconditioned grafts remained over the whole experimental period, whereas unpreconditioned grafts were obstructed after only one week of implantation. These results open the route toward the development of a new generation of vascular substitutes having a long term patency.

Concise Review: In Vitro Formation of Bone-Like Nodules Sheds Light on the Application of Stem Cells for Bone Regeneration

Harnessing the differentiation of stem cells into bone‐forming cells represents an intriguing avenue for the creation of functional skeletal tissues. Therefore, a profound understanding of bone development and morphogenesis sheds light on the regenerative application of stem cells in orthopedics and dentistry. In this concise review, we summarize the studies deciphering the mechanisms that govern osteoblast differentiation in the context of in vitro formation of bone‐like nodules, including morphologic and molecular events as well as cellular contributions to mineral nucleation, occurring during osteogenic differentiation of stem cells. This article also highlights the limitations of current translational applications of stem cells and opportunities to use the bone‐like nodule model for bone regenerative therapies.

Chitosan/hydroxyapatite hybrid scaffold for bone tissue engineering.

BACKGROUND To favor regeneration following critical bone defect, a combination of autologous bone graft and biomaterials is currently used. Major drawbacks of such techniques remain the availability of the autologous material and the second surgical site, inducing pain and morbidity. OBJECTIVE Our aim was to investigate the biocompatibility in vitro of three dimensions hybrid biodegradable scaffolds combining osteoconductive properties of hydroxyapatite and anti-inflammatory properties of chitosan. METHODS Hybrid scaffolds were characterized by microscopic observations, equilibrium swelling ratio and overtime weight loss measurements. In vitro studies were performed using primary human bone cells cultured for 7, 14 and 21 days. Cell viability, proliferation, morphology and differentiation through alkaline phosphatase (ALP) activity measurement were assessed. RESULTS Characterization of our scaffolds demonstrated porous, hydrophilic and biodegradable characteristics. In vitro studies showed that these scaffolds have induced slight decrease in cell death and proliferation comparing to the culture plastic substrate control condition, as well as increased short term osteoinductive properties. CONCLUSIONS In this study, we have provided evidence that our hybrid hydroxyapatite/chitosan scaffolds could be suitable for bone filling.

Biocompatibility of sol-gel hydroxyapatite-titania composite and bilayer coatings

Titania-Hydroxyapatite (TiO2/HAP) reinforced coatings are proposed to enhance the bioactivity and corrosion resistance of 316L stainless steel (316L SS). Herein, spin- and dip-coating sol-gel processes were investigated to construct two kinds of coatings: TiO2/HAP composite and TiO2/HAP bilayer. Physicochemical characterization highlighted the bioactivity response of the TiO2/HAP composite once incubated in physiological conditions for 7days whereas the TiO2/HAP bilayer showed instability and dissolution. Biological analysis revealed a failure in human stem cells adhesion on TiO2/HAP bilayer whereas on TiO2/HAP composite the presence of polygonal shaped cells, possessing good behaviour attested a good biocompatibility of the composite coating. Finally, TiO2/HAP composite with hardness up to 0.6GPa and elastic modulus up to 18GPa, showed an increased corrosion resistance of 316L SS. In conclusion, the user-friendly sol-gel processes led to bioactive TiO2/HAP composite buildup suitable for biomedical applications.

Influence of serum percentage on the behavior of Wharton's jelly mesenchymal stem cells in culture

BACKGROUND Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several lineages with valuable applications in regenerative medicine. MSCs differentiation is highly dependent on physicochemical properties of the culture substrate, cell density and on culture medium composition. OBJECTIVE In this study, we assessed the influence of fetal bovine serum (FBS) level on Whartons jelly (WJ)-MSCs behavior seeded on polyelectrolyte multilayer films (PEMF) made of four bilayers of poly-allylamine hydrochloride (PAH) as polycation and poly-styrene sulfonate (PSS) as polyanion. METHODS MSCs isolated from WJ by explants method were amplified until the third passage. Their phenotypic characterization was performed by flow cytometry analyses. MSCs were seeded on PEMF, in Endothelial growth medium-2 (EGM-2) supplemented by either 5% or 2% FBS. Cells behavior was monitored for 20 days by optical microscopy and immunofluorescence. RESULTS Until 2 weeks on glass slides, no difference was observed whatever the FBS percentage. Then with 5% FBS, MSCs formed three-dimensional spheroids on PSS/PAH after 20 days of culture with a nuclear aggregate. Whereas, with 2% FBS, these spheroids did not appear and cells grown in 2D conserved the fibroblast-like morphology. CONCLUSIONS The decrease of FBS percentage from 5% to 2% avoids 3D cell spheroids formation on PAH/PSS. Such results could guide bioengineering towards building 2D structures like cell layers or 3D structures by increasing the osteogenic or chondrogenic differentiation potential of MSCs.

Toward completely constructed and cellularized blood vessels.

Vascular tissue engineering aims to develop implantable blood-vessels, exhibiting biological and biomechanical characteristics close to those of the native vessels. The ultimate goal of our group is to engineer suitable blood vessel substitutes which could be stored for a long time in vascular bank conditions.First attempts tried to develop coating procedures allowing endothelial cells (EC) differentiation, adhesion and retention on current vascular substitutes but the weak in vivo patency of these grafts was related. Since 2003, our group have been evaluated a new surface modification of internal surface of blood vessels based on polyelectrolyte films coating. The layer-by-layer self-assembly and the resulting polyelectrolyte multiplayer films (PEM) is a simple and versatile way to engineer surfaces with highly specific properties. Previous studies indicated that the poly(sodium-4 styrene sulfonate)/poly (allylamine hydrochloride) PSS/PAH multilayered films when ended by PAH, induce strong adhesion and retention of mature EC which spread and keep their phenotype as well on glass, on expanded polytetrafluoroethylene ePTFE and on cryopreserved arteries. The mechanical properties (compliance), leading to early intimal hyperplasia and graft failure, were lost after artery cryopreservation. We have demonstrated that the compliance and elasticity restoration of PEM treated cryopreserved arteries close to native arteries.In other respect, the use of the circulating progenitor which could be differentiated into matures vascular cell offers new opportunities in vascular engineering. Currents protocols, expend at least 1 month to observe both smooth muscle (SMCs) and endothelium (ECs)-like morphology and about two months for confluent monolayer cells. The progenitor cells cultivated on PEM treated glass showed mature and functional vascular cells (SMCs and ECs) development after only 14 days of culture. The morphological appearance, mature and healthy phenotype markers expression and repartition of differentiated cells are close to mature cells.Challenge now is to build up in less a month, an autologous cellularized vascular graft using patient peripheral stem cells.

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 Whartons 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.