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.

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. SIGNIFICANCE: 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, studies deciphering the mechanisms that govern osteoblast commitment and differentiation are summarized. This article highlights the limitations of current translational applications of stem cells and the opportunities to use the bone-like nodule model for bone regenerative therapies.

Strontium-Substituted Bioceramics Particles: A New Way to Modulate MCP-1 and Gro-α Production by Human Primary Osteoblastic Cells.

BACKGROUND: To avoid morbidity and limited availability associated with autografts, synthetic calcium phosphate (CaP) ceramics were extensively developed and used as bone filling materials. Controlling their induced-inflammatory response nevertheless remained a major concern. Strontium-containing CaP ceramics were recently demonstrated for impacting cytokines' secretion pattern of human primary monocytes. The present study focuses on the ability of strontium-containing CaP to control the human primary bone cell production of two major inflammatory and pro-osteoclastogenic mediators, namely MCP-1 and Gro-α, in response to ceramics particles. METHODS: This in vitro study was performed using human primary osteoblasts in which their response to ceramics was evaluated by PCR arrays, antibody arrays were used for screening and real-time PCR and ELISA for more focused analyses. RESULTS: Study of mRNA and protein expression highlights that human primary bone cells are able to produce these inflammatory mediators and reveal that the adjunction of CaP in the culture medium leads to their enhanced production. Importantly, the current work determines the down-regulating effect of strontium-substituted CaP on MCP-1 and Gro-α production. CONCLUSION: Our findings point out a new capability of strontium to modulate human primary bone cells' communication with the immune system.

Biocompatibility study of lithium disilicate and zirconium oxide ceramics for esthetic dental abutments.

PURPOSE: The increasing demand for esthetically pleasing results has contributed to the use of ceramics for dental implant abutments. The aim of this study was to compare the biological response of epithelial tissue cultivated on lithium disilicate (LS2) and zirconium oxide (ZrO2) ceramics. Understanding the relevant physicochemical and mechanical properties of these ceramics will help identify the optimal material for facilitating gingival wound closure. METHODS: Both biomaterials were prepared with 2 different surface treatments: raw and polished. Their physicochemical characteristics were analyzed by contact angle measurements, scanning white-light interferometry, and scanning electron microscopy. An organotypic culture was then performed using a chicken epithelium model to simulate peri-implant soft tissue. We measured the contact angle, hydrophobicity, and roughness of the materials as well as the tissue behavior at their surfaces (cell migration and cell adhesion). RESULTS: The best cell migration was observed on ZrO2 ceramic. Cell adhesion was also drastically lower on the polished ZrO2 ceramic than on both the raw and polished LS2. Evaluating various surface topographies of LS2 showed that increasing surface roughness improved cell adhesion, leading to an increase of up to 13%. CONCLUSIONS: Our results demonstrate that a biomaterial, here LS2, can be modified using simple surface changes in order to finely modulate soft tissue adhesion. Strong adhesion at the abutment associated with weak migration assists in gingival wound healing. On the same material, polishing can reduce cell adhesion without drastically modifying cell migration. A comparison of LS2 and ZrO2 ceramic showed that LS2 was more conducive to creating varying tissue reactions. Our results can help dental surgeons to choose, especially for esthetic implant abutments, the most appropriate biomaterial as well as the most appropriate surface treatment to use in accordance with specific clinical dental applications.

In vitro and in vivo evaluation of the inflammatory potential of various nanoporous hydroxyapatite biomaterials.

AIM: To discriminate the most important physicochemical parameters for bone reconstruction, the inflammatory potential of seven nanoporous hydroxyapatite powders synthesized by hard or soft templating was evaluated both in vitro and in vivo. MATERIALS & METHODS: After physical and chemical characterization of the powders, we studied the production of inflammatory mediators by human primary monocytes after 4 and 24 h in contact with powders, and the host response after 2 weeks implantation in a mouse critical size defect model. RESULTS: In vitro results highlighted increases in the secretion of TNF-α, IL-1, -8, -10 and proMMP-2 and -9 and decreases in the secretion of IL-6 only for powders prepared by hard templating. In vivo observations confirmed an extensive inflammatory tissue reaction and a strong resorption for the most inflammatory powder in vitro. CONCLUSION: These findings highlight that the most critical physicochemical parameters for these nanoporous hydroxyapatite are, the crystallinity that controls dissolution potential, the specific surface area and the size and shape of crystallites.

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.

A new insight into the dissociating effect of strontium on bone resorption and formation.

Calcium phosphates are widely used as biomaterials and strontium (Sr) is known to have the ability to modify the bone balance towards osteosynthesis. In the present study we investigated the capacity of Sr-substituted sol-gel calcium phosphate to modify the expression of genes and proteins involved in extracellular matrix synthesis by primary bone cells. We first determined the most effective concentration of strontium using human primary bone cells. Sol-gel biphasic calcium phosphate (BCP) powders were then synthesised to obtain release of the optimal concentration of strontium. Finally, human osteoblasts obtained from explant cultures were cultured in the presence of sol-gel BCP, Sr-substituted BCP (5% Sr-substituted BCP, corresponding to a release of 5×10(-5)M [Sr(2+)] under the culture conditions (BCP(5%))) and medium containing strontium chloride (SrCl(2)). Viability, proliferation, cell morphology, protein production and protein activity were studied. We demonstrated that 5×10(-5)M SrCl(2) and BCP(5%) increased the expression of type I collagen and SERPINH1 mRNA and reduced the production of matrix metalloproteinases (MMP-1 and MMP-2) without modifying the levels of the tissue inhibitors of MMPs (TIMPs). Thus strontium has a positive effect on bone formation.

Cystic fibrosis transmembrane conductance regulator (CFTR) regulates the production of osteoprotegerin (OPG) and prostaglandin (PG) E2 in human bone.

Bone loss is an important clinical issue in patients with cystic fibrosis (CF). Whether the cystic fibrosis transmembrane conductance regulator (CFTR) plays a direct role in bone cell function is yet unknown. In this study, we provide evidence that inhibition of CFTR-Cl(-) channel function results in a significant decrease of osteoprotegerin (OPG) secretion accompanied with a concomitant increase of prostaglandin (PG) E(2) secretion of primary human osteoblast cultures (n=5). Our data therefore suggest that in bone cells of CF patients, the loss of CFTR activity may result in an increased inflammation-driven bone resorption (through both the reduced OPG and increased PGE(2) production), and thus might contribute to the early bone loss reported in young children with CF.

The effect of zinc on hydroxyapatite-mediated activation of human polymorphonuclear neutrophils and bone implant-associated acute inflammation.

Hydroxyapatite (HA) is widely used as coating biomaterial for prosthesis metal parts and as bone substitute. The release of HA particles induces an inflammatory response and, if uncontrolled, could result in implant loss. At the inflamed site, the polymorphonuclear cells (PMNs) represent the earliest phagocytic cells that predominate the cellular infiltrate. We have recently proposed that HA wear debris activate polymorphonuclear cells (PMNs) initiating and/or amplifying thereby the acute inflammatory response. Previous studies have shown that activation of monocytes by HA could be modulated by supplementing this latter with the divalent cation, Zinc. The purpose of this work was to investigate the modulation of PMNs activation following exposure to zinc-substituted HA. Our study demonstrate that addition of zinc to HA particles resulted in decreased levels of the pro-inflammatory mediator interleukin-8 (IL-8) and the matrix metallo-proteinase-9. We also show that these changes involve IL-8 receptors (CXCR-1 and CXCR-2).

Polymorphonuclear neutrophil response to hydroxyapatite particles, implication in acute inflammatory reaction.

Hydroxyapatite (HA) is widely used as a bone substitute or coating biomaterial in bone diseases or prosthesis metal parts. The release of HA particles induces an inflammatory response and, if uncontrolled, could result in implant loss. Among the hallmarks of such inflammatory response is early recruitment of the polymorphonuclear cells (PMNs). The purpose of this work is to investigate the response of PMNs following exposure to HA in terms of secreted mediators. Our study shows that HA particles increase the release of pro-inflammatory mediators such as interleukin-1alpha, as well as chemotactic factors such as interleukin-8, macrophage inflammatory protein-1alpha and macrophage inflammatory protein-1beta. HA also induces an increase in matrix metalloproteinase 9 expression. Taken together, our data demonstrate for the first time that HA is capable of activating PMNs, a phenomenon that could potentially contribute to the onset of implant-associated inflammation.

Serum albumin-alginate coated microspheres: role of the inner gel in binding and release of the KRFK peptide.

In continuation with our previous study using fluorescein-isothiocyanate (FITC)-Lys-Arg-Phe-Lys (KRFK) peptide, the aim of this work was to study the interaction of the unlabelled KRFK with calcium alginate gel microspheres coated with a serum albumin (HSA)-alginate membrane prepared using a transacylation method. Coated microspheres were prepared with two main sizes and two gel strengths. Control microspheres made of cross-linked alginate-HSA without calcium alginate gel were also prepared. A series of loading and release assays conducted with methylene blue showed the requirement of inner gel for binding the cationic molecule. Release experiments were performed in different media using unlabelled KRFK and coated microspheres. A plateau was reached within 1h, in contrast with the slow release of the FITC-peptide observed in our previous work. This discrepancy was attributed to modified properties of the labelled peptide. Adsorption assays of KRFK on coated microspheres were performed in the presence of growing concentrations of NaCl or imidazole. The ions were able to displace the peptide from the particles, which demonstrated ionic interactions, probably involving carboxylate groups of alginate. Adsorption isotherms showed that gel strength influenced affinity (4x10(5) L/mol or 8x10(5) L/mol for gelation with 5% or 20% CaCl(2), respectively). Binding site number doubled (from 2.6x10(-7) mol/mg to more than 5x10(-7) mol/mg) when microsphere size decreased from 450 microm to 100 microm. Binding sites were assumed to be located in the gel underneath the membrane.

Albumin-alginate-coated microspheres: resistance to steam sterilization and to lyophilization.

The paper describes the effect of different thermal treatments on the morphology and binding properties of particles prepared using a transacylation reaction between two biocompatible polymers, namely propylene glycol alginate and human serum albumin. Compared to control alginate gel microspheres, albumin-alginate covalent network offers a better resistance to the microspheres towards freezing, lyophilization and sterilization. The binding properties for methylene blue were not altered by the treatments. Moreover, stability in physiological environments opens interesting applications in biological and pharmaceutical fields.

Involvement of toll-like receptor 4 in the inflammatory reaction induced by hydroxyapatite particles.

Hydroxyapatite (HA) is widely used to coat metal parts in order to improve their biocompatibility. Analysis of retrieved tissues associated with failed implants, suggest that phagocytosis of HA wear debris by monocytes/macrophages might provide a potent stimulus for the release of a variety of cytokines. Phagocytosis involved a large variety of cellular receptors like toll-like receptors that results in activation of the transcriptional nuclear factor-kappaB (NF-kappaB) via a cell-signalling pathway. In the present paper, we aimed to evaluate the role of the toll-like receptor 4 (TLR4) in the production of inflammatory cytokines induced by HA particles using TLR4(+) and TLR4(-) peritoneal macrophages. We investigated the production of TNF-alpha and the activation of the nuclear transcription factor NF-kappaB. Our data clearly show for the first time that the production of TNF-alpha by macrophages exposed to HA particles was TLR4 dependent but not the activation of NF-kappaB. All these results open future therapies to reduce the inflammatory response induced by HA biomaterials.

Influence of the zinc concentration of sol-gel derived zinc substituted hydroxyapatite on cytokine production by human monocytes in vitro.

A possible complication associated with the implantation of hydroxyapatite (HA)-based prosthesis is the release of particles. These particles can be phagocyted by monocytes that are among the first cells to colonize the inflammatory site. The activated monocytes produce inflammatory mediators such as cytokines that cause osteoclasts activation. The present work, describes studies on the effect of sol-gel derived zinc-substituted HA particles with various zinc substitution percentages (0.5-2%) on cytokine production (TNF-alpha, IL-1beta, IL-6, IL-10, and IL-8) by both LPS-stimulated or unstimulated human monocytes. Our data demonstrates that the zinc has an effect on cytokines production. It decreases the production of TNF-alpha and increases the production of IL-8 by unstimulated cells. Using LPS-stimulated cells, it decreases the production of inflammatory cytokines and increases the production of anti-inflammatory cytokine (IL-10), indicating that zinc-substituted hydroxyapatite has favourable effects on the cytokines production by monocytes.

Collagen and elastin cross-linking: a mechanism of constrictive remodeling after arterial injury.

Constrictive remodeling after arterial injury is related to collagen accumulation. Cross-linking has been shown to induce a scar process in cutaneous wound healing and is increased after arterial injury. We therefore evaluated the effect of cross-linking inhibition on qualitative and quantitative changes in collagen, elastin, and arterial remodeling after balloon injury in the atherosclerotic rabbit model. Atherosclerotic-like lesions were induced in femoral arteries of 28 New Zealand White rabbits by a combination of air desiccation and a high-cholesterol diet. After 1 mo, balloon angioplasty was performed in both femoral arteries. Fourteen rabbits were fed beta-aminopropionitrile (beta-APN, 100 mg/kg) and compared with 14 untreated animals. The remodeling index, i.e., the ratio of external elastic lamina at the lesion site to external elastic lamina at the reference site, was determined 4 wk after angioplasty for both groups. Pyridinoline was significantly decreased in arteries from beta-APN-treated animals compared with controls, confirming inhibition of collagen cross-linking: 0.30 (SD 0.03) and 0.52 (SD 0.02) mmol/mol hydroxyproline, respectively (P = 0.002). Scanning and transmission electron microscopy showed a profound disorganization of collagen fibers in arteries from beta-APN-treated animals. The remodeling index was significantly higher in beta-APN-treated than in control animals [1.1 (SD 0.3) vs. 0.8 (SD 0.3), P = 0.03], indicating favorable remodeling. Restenosis decreased by 33% in beta-APN-treated animals: 32% (SD 16) vs. 48% (SD 24) (P = 0.02). Neointimal collagen density was significantly lower in beta-APN-treated animals than in controls: 23.0% (SD 3.8) vs. 29.4% (SD 4.0) (P = 0.004). These findings suggest that collagen and elastin cross-linking plays a role in the healing process via constrictive remodeling and restenosis after balloon injury in the atherosclerotic rabbit model.

Coating alginate microspheres with a serum albumin-alginate membrane: application to the encapsulation of a peptide.

Calcium alginate gel microspheres coated with a human serum albumin (HSA)-alginate membrane were prepared adapting a transacylation method previously applied to large beads. The procedure involved emulsification of an aqueous solution of sodium alginate and propylene glycol alginate (PGA) in an oily phase, followed by addition of CaCl(2). The resulting gel microspheres were transferred in an aqueous solution of HSA. The addition of 0.5 M NaOH started the reaction between PGA and HSA, producing amide bonds and forming a membrane around the particles. An optimization study was conducted, notably exploring the addition of HSA to the internal phase. The microcapsules were studied with respect to morphology (optical and scanning electron microscopy) and size (laser granulometry), in comparison with uncoated gel microspheres. Biocompatibility was checked in osteoblast cultures. Lysine-arginine-phenylalanine-lysine (KRFK) was encapsulated and the release kinetics was studied in vitro. The method provided stable microspheres (size around 60 microm), with a membrane surviving a treatment with citrate and resisting lyophilization. The microcapsules were shown biocompatible. The release of KRFK was slower (release time>8 days) than that of uncoated microspheres. These microcapsules might be useful as peptide containers to be combined with prosthetic materials for improving osteointegration.

Importance of the surface area ratio on cytokines production by human monocytes in vitro induced by various hydroxyapatite particles.

A possible complication associated with the implantation of hydroxyapatite (HA)-based prosthesis is the release of particles. Those particles can be phagocyted by monocytes that are among the first cells to colonize the inflammatory site. The activated monocytes produce inflammatory mediators, such as cytokines, which cause osteoclasts activation. It has previously been demonstrated using a surface area ratio (ratio of the total surface of the given particles to the surface area of cells) of 1 to 1 that there was a correlation between the expression and production of cytokines induced by HA. The present work studies the effect of physical characteristics of HA particles on the production of various inflammatory cytokines (tumour necrosis factor-alpha, interleukin (IL)-6, and IL-8) and anti-inflammatory cytokine (IL-10). However, the experiments were performed using a surface area ratio of 10 to 1. Our data demonstrate that all the particles, whatever their characteristics, induced a high expression of cytokines but the production was different, meaning that there was a post-transcriptional regulation. The size and sintering temperature seemed to be a characteristics that were less important compared to the shape; the needle particles appeared to induce the most important production of all the cytokines studied.

Metalloproteinase and cytokine production by THP-1 macrophages following exposure to chitosan-DNA nanoparticles.

The use of nanoparticles for gene therapy is gaining more and more interest for medical applications. Chitosan is among the candidate polymers that have a potential application as a gene delivery system. Before using chitosan-DNA nanoparticles in vivo, one must study their interaction and cell's behavior. Since macrophages play an important role in inflammatory processes, this study was performed to investigate the effects of chitosan-DNA nanoparticles on human THP-1 cell line. Cytokine (TNF-alpha, IL-1beta, IL-6 and IL-10) and metalloproteinase (MMP-2 and MMP-9) release as well as their inhibitors (TIMP-1 and TIMP-2) were assessed after time course incubation with different amount of nanoparticles. Their secretion was quantified by enzyme-linked immunosorbent assay. Gelatinolytic activity of MMP-2 and MMP-9 was determined by zymography in cell supernatants and lysates. Cytokine secretion was not detected even in the presence of high amount of nanoparticles. On the contrary, the secretion of MMP-9 in cell supernatants increased significantly after 24 and 48 h in comparison with non-treated cells. MMP-2 secretion was augmented only after 48 h for the highest concentrations of nanoparticles (10 and 20 microg/ml DNA content). However, zymography studies showed that the secreted MMPs were in the proactive forms, while the active form of MMP-9, but not MMP-2, was detected in cell lysates when 10 and 20 microg/ml DNA containing nanoparticles were used. In conclusion, exposure of THP-1 macrophages to Ch-DNA nanoparticles did not induce release of proinflammatory cytokines. The presence of active MMP-9 within the macrophages could possibly be related to nanoparticle phagocytosis and degradation rather than to inflammatory reactions.

The effect of the physical characteristics of hydroxyapatite particles on human monocytes IL-18 production in vitro.

Hydroxyapatite (HA) is widely used to coat the metal parts of prosthetic implants in order to improve their biocompatibility and as a bone defect filling material. HA has been demonstrated to produce particles at the prosthetic interface that lead to an activation of phagocytic cells that induce a cascade reaction leading to bone resorption and aseptic loosening. Monocytes/macrophages are commonly observed in the interface tissue, and are among the first cells to colonize the inflammatory site where they play a key role in the immune response. IL-18 is a pro-inflammatory cytokine. Monocytes/macrophages were described as IL-18 producing cells. IL-18 works antagonistically to IL-6, which activates osteoclastogenesis. In the present study, we investigated the ability of HA particles to induce the production of active IL-18 by human monocytes according to particle characteristics (size, sintering temperature and shape). Our study demonstrates, for the first time, that HA particles are capable of stimulating the production of the proinflammatory cytokine IL-18 in human monocytes according to their particle characteristics. The expression and the production of IL-18 was modified by the parameter studied. The difference observed between the expression and the production could be explain by the production of ICE. The needle shaped particles induced the larger production of IL-18.

MMP-2, MMP-9 and their inhibitors TIMP-2 and TIMP-1 production by human monocytes in vitro in the presence of different forms of hydroxyapatite particles.

After calcium-phosphates biomaterials based implantation like hydroxyapatite (HA) coating, particles are released in the periprosthetic tissues. Wear-debris induced fibrous membranes contain macrophage subsets that can produce metalloproteinases (MMPs), which are considered to be key enzymes in extra-cellular matrix turnover. Tissue inhibitors of metalloproteinases (TIMPs) are important regulator of MMPs activity. Interleukin-1 mainly produced by monocytes can also regulate MMPs production. In the present work, we have evaluated the effect of HA particles characteristics (size, shape and sintering temperature) on the MMP-2, -9 and their respective inhibitors TIMP-2, -1 production. Our results demonstrate that sintering temperature (that modify crystal size and surface area) have little effect on MMPs and TIMPs production. Non-phagocytable particles induced more MMP-9, although phagocytable particles induced more IL-1beta release. The shape of the particles was the most important factor since needle-shaped particles induced the most significant up-regulated expression of MMPs and IL-1beta.