Macrophage-mediated angiogenic activation of outgrowth endothelial cells in co-culture with primary osteoblasts.

The successful vascularisation of complex tissue engineered constructs for bone regeneration is still a major challenge in the field of tissue engineering. In this context, co-culture systems of endothelial cells and osteoblasts represent a promising approach to advance the formation of a stable vasculature as well as an excellent in vitro model to identify factors that positively influence bone healing processes, including angiogenesis. Under physiological conditions, the activation phase of angiogenesis is mainly induced by hypoxia or inflammation. Inflammatory cells such as macrophages secrete proinflammatory cytokines and proangiogenic growth factors, finally leading to the formation of new blood vessels. The aim of this study was to investigate if macrophages might positively influence the formation of microvessel-like structures via inflammatory mechanisms in a co-culture system consisting of human outgrowth endothelial cells (OECs) and primary osteoblasts. Treatment of co-cultures with macrophages (induced from THP-1) resulted in a higher number of microvessel-like structures formed by OECs compared to the co-culture. This change correlated with a significantly higher concentration of the proangiogenic VEGF in cell culture supernatants of triple-cultures and was accompanied by an increase in the expression of different proinflammatory cytokines, such as IL-6, IL-8 and TNFα. In addition, the expression of E-selectin and ICAM-1, adhesion molecules which are strongly involved in the interaction between leukocytes and endothelial cells during the process of inflammation was also found to be higher in triple-cultures compared to the double co-cultures, documenting an ongoing proinflammatory stimulus. These results raise the possibility of actively using pro-inflammatory stimuli in a tissue engineering context to accelerate healing mechanisms.

Tissue reaction to sealing materials: different view at biocompatibility.

The biodegradability of root canal sealers in areas other than the root canal system is crucial to the overall success rate of endodontic treatment. The aim of the present study was to investigate, the cell and tissue reaction to GuttaFlow and AHPlus, both in vitro and in vivo. For the in vitro experiments the materials were incubated with Human Periodontal Ligament Fibroblasts and cell proliferation and cytotoxicity analyses were performed. Additional fluorescence-microscope stainings were carried out in order to visualize cell growth and morphology. For assessment of the tissue reaction to the materials a subcutaneous implantation model in Wistar rats was employed and the inflammatory response to the materials was visualized by means of general and specific histology after 6 weeks. Human gingival fibroblasts proliferation seemed to be dependent upon dental material and cultivation time. After an incubation period of 96 hrs AHPlus proved to be significantly (p<0.002) more cytotoxic than GuttaFlow, as only a small number of fibroblasts survived on AHPlus. In vivo, GuttaFlow was surrounded by a fibrous capsule and no degradation took place, while AHPlus induced a well-vascularized granulation tissue in which the material was phagocyted by macrophages. The results of this study demonstrate that a potential cytotoxic effect of a sealing material may beneficial in order to have antibacterial properties and induce self degradation when accidentally extruded over the apical foramen.

[In vitro trials with single and co-cultures of osteoblasts and endothelial cells : evaluation of new biomaterials for bone reconstruction and regeneration]. / Bewertung von neuartigen Biomaterialien zum Zweck der Knochenrekonstruktion und -regeneration : In-vitro-Versuche mit Mono- und Kokulturen von Osteoblasten und Endothelzellen.

Many different types of bone substitute biomaterials are being developed for different applications in the body. The current dogma is that if osteoblasts and endothelial cells grow and exhibit normal cell functions on these materials in vitro as single cultures or in co-cultures, then the biomaterials are suitable for implantation for bone reconstruction and regeneration. Generally, only in vivo animal studies will prove whether this is the case. However, in vitro studies offer a good pre-screening and selection basis to evaluate the biocompatibility of novel biomaterials prior to animal studies. Multicell type co-culture systems hold a great promise for the future.

Metallic nanoparticles exhibit paradoxical effects on oxidative stress and pro-inflammatory response in endothelial cells in vitro.

Particulate matter is associated with different human diseases affecting organs such as the respiratory and cardiovascular systems. Very small particles (nanoparticles) have been shown to be rapidly internalized into the body. Since the sites of internalization and the location of the detected particles are often far apart, a distribution via the blood stream must have occurred. Thus, endothelial cells, which line the inner surface of blood vessels, must have had direct contact with the particles. In this study we tested the effects of metallic nanoparticles (Co and Ni) on oxidative stress and pro-inflammatory response in human endothelial cells in vitro. Exposure to both nanoparticle types led to a concentration-dependent cytotoxic effect. However, the effects on oxidative stress and pro-inflammatory response differed dramatically. Due to the nanoparticle-induced effects, a comparison between metallic nanoparticle- and metal ion-treatment with the corresponding ions was made. Again, divergent effects of nanoparticles compared with the ions were observed, thus indicating differences in the signaling pathways induced by these compounds. These paradoxical responses to different metallic nanoparticles and ions demonstrate the complexity of nanoparticle-induced effects and suggest the need to design new strategies for nanoparticle toxicology.

Gold nanoparticle interactions with endothelial cells cultured under physiological conditions.

PEGylated gold nanoparticles (AuNPs) have an extended circulation time after intravenous injection in vivo and exhibit favorable properties for biosensing, diagnostic imaging, and cancer treatment. No impact of PEGylated AuNPs on the barrier forming properties of endothelial cells (ECs) has been reported, but recent studies demonstrated that unexpected effects on erythrocytes are observed. Almost all studies to date have been with static-cultured ECs. Herein, ECs maintained under physiological cyclic stretch and flow conditions and used to generate a blood-brain barrier model were exposed to 20 nm PEGylated AuNPs. An evaluation of toxic effects, cell stress, the release profile of pro-inflammatory cytokines, and blood-brain barrier properties showed that even under physiological conditions no obvious effects of PEGylated AuNPs on ECs were observed. These findings suggest that 20 nm-sized, PEGylated AuNPs may be a useful tool for biomedical applications, as they do not affect the normal function of healthy ECs after entering the blood stream.

Monocyte preseeding leads to an increased implant bed vascularization of biphasic calcium phosphate bone substitutes via vessel maturation.

The present study analyzes the influence of the addition of monocytes to a biphasic bone substitute with two granule sizes (400-700 µm and 500-1000 µm). The majority of the added monocytes was detectable as mononuclear cells, while also low amounts of (chimeric) multinucleated giant cells (MNGCs) were found. No increase in the total number of MNGCs was established, but a significantly increased percent vascularization. Altogether, the results show that the added monocytes become involved in the tissue response to a biomaterial without marked changes in the overall reaction. Monocyte addition enables an increased implant bed vascularization especially via induction of vessel maturation and, thus intervenes positively in the healing reaction to a biomaterial. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2928-2935, 2016.

Tissue engineered pre-vascularized buccal mucosa equivalents utilizing a primary triculture of epithelial cells, endothelial cells and fibroblasts.

Artificial generated buccal mucosa equivalents are a promising approach for the reconstruction of urethral defects. Limiting in this approach is a poor blood vessel supply after transplantation, resulting in increased morbidity and necrosis. We generated a pre-vascularized buccal mucosa equivalent in a tri-culture of primary buccal epithelial cells, fibroblasts and microvascular endothelial cells, using a native collagen membrane as a scaffold. A successful pre-vascularization and dense formation of capillary-like structures at superficial areas was demonstrated. The lumen size of pre-formed blood vessels corresponded to the capillary size in vivo (10-30 µm). Comparing native with a highly cross-linked collagen membrane we found a distinct higher formation of capillary-like structures on the native membrane, apparently caused by higher secretion of angiogenic factors such as PDGF, IL-8 and angiopoietin by the cells. These capillary-like structures became functional blood vessels through anastomosis with the host vasculature after implantation in nude mice. This in vitro method should result in an accelerated blood supply to the biomaterial with cells after transplantation and increase the succes rates of the implant material.

Growth of human cells on polyethersulfone (PES) hollow fiber membranes.

A novel material of porous hollow fibers made of polyethersulfone (PES) was examined for its ability to support the growth of human cells. This material was made in the absence of solvents and had pore diameters smaller than 100 microm. Human cell lines of different tissue and cell types (endothelial, epithelial, fibroblast, glial, keratinocyte, osteoblast) were investigated for adherence, growth, spread and survival on PES by confocal laser microscopy after staining of the cells with Calcein-AM. Endothelial cell attachment and growth required pre-coating PES with either fibronectin or gelatin. The other cell types exhibited little difference in growth, spread or survival on coated or uncoated PES. All the cells readily adhered and spread on the outer, inner and cut surfaces of PES. With time confluent monolayers of cells covered the available surface area of PES and in some cases cells grew as multilayers. Many of the cells were able to survive on the PES for up to 7 weeks and in some cases growth was so extensive that the underlying PES was no longer visible. Scanning electron microscope observations of cells on the materials correlated with the confocal morphometric data. Thus, PES is a substrate for the growth of many different types of human cells and may be a useful scaffolding material for tissue engineering.

Selection of HIV-1 genotypes by cultivation in different primary cells.

OBJECTIVES: To determine the representation of particular HIV-1 genotypes during cultivation in different primary cell-culture systems compared with the spectrum of the quasispecies in vivo. METHODS: Primary isolates of HIV-1 were recovered by isolation in cultures of lymphocytes, mixed mononuclear cells (MNC), and monocytes/macrophages. Nucleotide sequence determination of the C2-V3 region of gp120 of HIV was performed on 10-20 independently isolated clones derived by polymerase chain reaction from the culture systems, the uncultured peripheral blood MNC (PBMC) as well as plasma. RESULTS: Several predominant HIV genotypes were found in the uncultured PBMC from each of the patients. The most frequent genotypes in PBMC were also the most frequent types in plasma. In addition, lymphocytes, macrophages or mixed MNC cultures allowed the outgrowth of variants that were underrepresented in uncultured PBMC. We showed that the virus cultivation systems used in this study selected differently for the genetic variants. Whereas some genotypes were present in all three culture systems, although at different frequencies, others were exclusively found in a specific culture system. CONCLUSIONS: These results demonstrate that monocyte/macrophage and mixed MNC culture systems complement the standard lymphocyte culture in terms of the spectrum of genotypically different virus variants obtained in vitro.

Inhibition of HIV type 1 replication by simultaneous infection of peripheral blood lymphocytes with human immunodeficiency virus types 1 and 2.

A productive infection of peripheral blood lymphocytes by HIV-1 was severely inhibited by the simultaneous infection of these cells with HIV-2. A similar reciprocal effect on HIV-2 infection was not observed. The extent of virus replication was determined by virus-specific antigen capture assays of the supernatants of the infections. The inhibitory effect was observed with T cell-tropic, dual-tropic, as well as with primary HIV-1 isolates from different subtypes (A, B, C, E, F, and O). Infection of PBLs with different subtypes of HIV-2 (A and B) as well as with SIV(mac) resulted in the inhibition of HIV-1. However, the inhibitory effect was limited to PBLs; similar results were not observed in a T cell line. The inhibition of HIV-1 replication was independent of HIV-2 concentration; however, the infection by HIV-2 had to take place within 24 hr after PBLs were infected by HIV-1 for inhibition of HIV-1 replication to occur. The inhibition could be reversed by the addition of PHA. Analysis of HIV-1 RNA and DNA demonstrated that the inhibition was not at uptake or reverse transcription and that equal amounts of PBLs were infected by HIV-1 in single infections and coinfections. Immunocytochemical analysis of HIV-1 proteins demonstrated that equal numbers of cells were infected and that equal amounts of intracellular HIV-1 Env and Gag proteins were produced throughout the culture period. Therefore we conclude that HIV-2 can potently inhibit the productive infection of PBLs by HIV-1 and that the mechanism of this inhibition appears to prevent HIV-1 assembly or release from PBLs.

Genetic and biological comparisons of pathogenic and nonpathogenic molecular clones of simian immunodeficiency virus (SIVmac).

Simian immunodeficiency virus (SIV) is a designation for a group of related but unique lentiviruses identified in several primate species. A viral isolate from a rhesus macaque (i.e., SIVmac) causes a fatal AIDS-like disease in experimentally infected macaques, and several infectious molecular clones of this virus have been characterized. This report presents the complete nucleotide sequence of molecularly cloned SIVmac1A11, and comparisons are made with the sequence of molecularly cloned SIVmac239. SIVmac1A11 has delayed replication kinetics in lymphoid cells but replicates as well as uncloned SIVmac in macrophage cultures. Macaques infected with virus from the SIVmac1A11 clone develop antiviral antibodies, but virus does not persist in peripheral blood mononuclear cells and no disease signs are observed. SIVmac239 infects lymphoid cells, shows restricted replication in cultured macrophages, and establishes a persistent infection in animals that leads to a fatal AIDS-like disease. Both viruses are about 98% homologous at the nucleotide sequence level. In SIVmac1A11, the vpr gene as well as the transmembrane domain of env are prematurely truncated, whereas the nef gene of SIVmac239 is prematurely truncated. Sequence differences are also noted in variable region 1 (V1) in the surface domain of the env gene. The potential implications of these and other sequence differences are discussed with respect to the phenotypes of both viruses. This animal model is critically important for investigating the roles of specific viral genes in viral/host interactions that cannot be studied in individuals infected with the human immunodeficiency virus (HIV).

Characterization of rhesus macaque B-lymphoblastoid cell lines infected with simian type D retrovirus.

A simian type D retrovirus designated SRV induces a fatal immunosuppressive disease in rhesus macaques. This syndrome shows many clinical similarities to acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus-infected individuals. To investigate the mechanisms of immune dysfunction in SRV infection, we have focused on the interactions of SRV serotype 1 (SRV-1) with macaque B-lymphoblastoid cell lines (B-LCL). Procedures were optimized for establishing B-LCL by immortalization of macaque B lymphocytes with rhesus Epstein-Barr virus (EBV). These cell lines express B-cell surface markers, secrete immunoglobulins of the IgG or IgM isotypes, and release EBV which transforms monkey B cells. In vitro cultures of B-LCL supported replication of SRV-1. Several B-LCL infected with SRV-1 showed downregulation of major histocompatibility complex (MHC) class II antigen expression whereas levels of MHC class I antigen remained unchanged. Infection of B-LCL with SRV-1 did not alter the level of secreted immunoglobulin. Rhesus EBV was also used to obtain B-LCL from macaques infected with SRV-1; these cell lines were found to release infectious SRV-1. Investigations on the interactions of SRV-1 with B cells will be useful for elucidating mechanisms involved in the immunopathogenesis of primate retroviruses.

Endothelialization of a non-woven silk fibroin net for use in tissue engineering: growth and gene regulation of human endothelial cells.

We have previously shown that a biomaterial consisting of a non-woven fibroin net produced from silk (Bombyx mori) cocoons is an excellent scaffolding material for a wide variety of human cells of different tissue types. Endothelialization must take place for a biomaterial to be successful after implantation. Therefore, primary human endothelial cells and the human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS-1, were examined for adherence and growth patterns on the fibroin nets by confocal laser scanning microscopy after vital staining of the cells and by electron microscopy. Endothelial cells adhered and spread along individual fibers of the nets and did not fill the gaps between individual fibers. Higher attachment and growth coverage was obtained if nets were first coated with gelatin, fibronectin or collagen type I. Proinflammatory markers of endothelial cells on the fibers exhibited a non-activated state and LPS-stimulated cells exhibited activation of these markers. Furthermore, a typical PECAM-1 localization at cell-cell contacts was observed. Scanning electron microscopic examination of fibroin nets after removal of cells did not demonstrate any changes to the fibroin structure. HUVEC and HDMEC on fibroin nets embedded in collagen type I gels formed microvessel-like structures. Thus, silk fibroin nets are a highly endothelial cell-compatible scaffolding material that support the growth, normal and inducible cell functions and angiogenesis potential of human endothelial cells in vitro similar to that observed in vivo.

Tissue response and biomaterial integration: the efficacy of in vitro methods.

Implantation involves tissue trauma, which evokes an inflammatory response, coupled to a wound healing reaction, involving angiogenesis, fibroblast activation and matrix remodelling. Until now the type and extent of such reactions to give optimal integration of various biomaterials are practically unknown. Three principal fields of research can yield useful data to understand these phenomena better: studies on explanted biomaterials, animal models and relevant in vitro techniques. This paper will present examples of the latter field and the application of endothelial cell (EC) culture systems to study the effects of important tissue (e.g. pro-inflammatory cytokines, chemokines) and material (e.g. metal ions, particulate debris) factors on the regulation of the inflammatory and angiogenic response. A central feature is the use of microvascular endothelial cells (MEC), which can be used in both 2-and 3-dimensional (3-D) assays. We have also used genetic manipulation to develop a permanent MEC line from the human lung (HPMEC-ST1), which is being tested for its suitability to study cell-biomaterial interactions. In addition, suitable in vitro techniques are being developed in order to investigate drug delivery systems (DDS). Of particular interest is the targeting of the central nervous system, our approach being to establish a human model of the blood-brain barrier (BBB). A mainstay of our scientific philosophy is that such in vitro methods can make an important contribution to understanding biological reactions at the tissue-biomaterial interface and thus further a causal approach to tissue engineering (TE) and drug delivery applications.

Comparison of dot-blot and Northern blot hybridizations in the determination of genetic relatedness of United States bluetongue virus serotypes.

Dot-blot and Northern blot hybridization methods to determine the genetic relatedness of United States bluetongue virus serotypes 2, 10, 11, 13, and 17 were compared. Both plasmid and insert DNA probes from cloned BTV-17 dsRNA segments 2, 5, 6, and 8 were hybridized to dsRNA from the BTV serotypes and epizootic hemorrhagic disease virus (EHDV). Stringencies of hybridization were kept identical, and experiments differed only in the method in which dsRNA was applied to the membranes (dot-blot or Northern blot). The Northern blot hybridization method yielded more consistent results, and it was visually and unequivocally shown to which dsRNA segment a cDNA probe bound, since the dsRNA segments were separated by PAGE prior to blotting and hybridization. In contrast, the dot-blot hybridization method gave less consistent results. Identical results for plasmid and insert probes were obtained for Northern blot hybridizations but not for dot-blot hybridizations. At least two probes could be used simultaneously in Northern blot hybridizations.

Colony hybridizations using nylon membranes and RNA probes--an improved method for screening bacterial clones containing bluetongue virus genome.

Bluetongue virus (BTV) total genomic and isolated individual segment dsRNAs end-labeled with 32P were successfully used as probes in colony hybridization to detect clones of BTV genomic material. The RNA probes were highly specific for cloned BTV genomic material. DNA probes, however, gave false positive results. DNA from bacterial clones was fixed to nylon and nitrocellulose membranes. The hybridized nylon membranes could be stripped of probe and reprobed at least 6 times without loss of signal strength.

New generation super alloy candidates for medical applications: corrosion behavior, cation release and biological evaluation.

Three super alloy candidates (X1 CrNiMoMnW 24-22-6-3-2 N, NiCr21 MoNbFe 8-3-5 AlTi, CoNiCr 35-20 Mo 10 BTi) for a prolonged contact with skin are evaluated in comparison with two reference austenitic stainless steels 316L and 904L. Several electrochemical parameters were measured and determined (E(oc), E(corr), i(corr), b(a), b(c), E(b), R(p), E(crev) and coulometric analysis) in order to compare the corrosion behavior. The cation release evaluation and in vitro biological characterization also were performed. In terms of corrosion, the results reveal that the 904L steels presented the best behavior followed by the super austenitic steel X1 CrNiMoMnW 24-22-6-3-2 N. For the other two super alloys (NiCr and CoNiCr types alloys) tested in different conditions (annealed, work hardened and work hardened+age hardened) it was found that their behavior to corrosion was weak and close to the other reference stainless steel, 316L. Regarding the extraction a mixture of cations in relatively high concentrations was noted and therefore a cocktail effect was not excluded. The results obtained in the biological assays WST-1 and TNF-alpha were in correlation with the corrosion and extraction evaluation.

In vitro evaluation of biomimetic chitosan-calcium phosphate scaffolds with potential application in bone tissue engineering.

This work reports on the physicochemical properties and in vitro cytotoxicity assessment of chitosan-calcium phosphate (Cs-CP) scaffolds for bone tissue engineering, which were synthesized by a novel biomimetic co-precipitation method. X-ray diffraction (XRD) along with scanning electron microscopy (SEM) analysis confirmed the porous morphology of the scaffolds and the amorphous nature of the inorganic phase with different crystallite sizes and the formation of various forms of calcium phosphate. Compressive mechanical testing revealed that the Young's modulus of the biomaterials is in the range of human trabecular bone. In vitro tests were performed on the biomaterials for up to 14 days to study the behavior of the osteoblast-like human cell line (MG63), primary human osteoblasts (HOS) and human dermal microvascular endothelial cells (HDMEC). The cytotoxicity was evaluated by the MTS assay for cell metabolism and the detection of membrane integrity (lactate dehydrogenase-LDH release). An expression of the vascular endothelial growth factor (VEGF) in the cell supernatants was quantified by ELISA. Cell viability gave values close to untreated controls for MG63 and HOS, while in the case of HDMEC the viability after 2 weeks in the cell culture was between 80-90%. The cytotoxicity induced by the Cs-CP scaffolds on MG63, HOS and HDMEC in vitro was evaluated by the amount of LDH released, which is a sensitive and accurate marker for cellular toxicity. The increased levels of VEGF obtained in the osteoblast culture highlights its important role in the regulation of vascularization and bone remodeling. The biological responses of the Cs-CP scaffolds demonstrate a similar proliferation and differentiation characteristics of the cells comparable to the controls. These results reveal that biomimetic Cs-CP composite scaffolds are promising biomaterials for bone tissue engineering; their in vivo response remains to be tested.

Improving cytocompatibility of Co28Cr6Mo by TiO2 coating: gene expression study in human endothelial cells.

Cobalt-based materials are widely used for coronary stents, as well as bone and joint implants. However, their use is associated with high corrosion incidence. Titanium alloys, by contrast, are more biocompatible owing to the formation of a relatively inactive titanium oxide (TiO2) layer on their surface. This study was aimed at improving Co28Cr6Mo alloy cytocompatibility via sol-gel TiO2 coating to reduce metal corrosion and metal ion release. Owing to their role in inflammation and tissue remodelling around an implant, endothelial cells present a suitable in vitro model for testing the biological response to metallic materials. Primary human endothelial cells seeded on Co28Cr6Mo showed a stress phenotype with numerous F-actin fibres absent on TiO2-coated material. To investigate this effect at the gene expression level, cDNA microarray analysis of in total 1301 genes was performed. Compared with control cells, 247 genes were expressed differentially in the cells grown on Co28Cr6Mo, among them genes involved in proliferation, oxidative stress response and inflammation. TiO2 coating reduced the effects of Co28Cr6Mo on gene expression in endothelial cells, with only 34 genes being differentially expressed. Quantitative real-time polymerase chain reaction and protein analysis confirmed microarray data for selected genes. The effect of TiO2 coating can be, in part, attributed to the reduced release of Co(2+), because addition of CoCl2 resulted in similar cellular responses. TiO2 coating of cobalt-based materials, therefore, could be used in the production of cobalt-based devices for cardiovascular and skeletal applications to reduce the adverse effects of metal corrosion products and to improve the response of endothelial and other cell types.

Ni-Cr based dental alloys; Ni release, corrosion and biological evaluation.

In the last years the dental alloy market has undergone dramatic changes for reasons of economy and biocompatibility. Nickel based alloys have become widely used substitute for the much more expensive precious metal alloys. In Europe the prevalence of nickel allergy is 10-15% for female adults and 1-3% for male adults. Despite the restrictions imposed by the EU for the protection of the general population in contact dermatitis, the use of Ni-Cr dental alloys is on the increase. Some questions have to be faced regarding the safety risk of nickel contained in dental alloys. We have collected based on many EU markets, 8 Ni-Cr dental alloys. Microstructure characterization, corrosion resistance (generalized, crevice and pitting) in saliva and the quantities of cations released in particular nickel and CrVI have been evaluated. We have applied non parametric classification tests (Kendall rank correlation) for all chemical results. Also cytotoxicity tests and an evaluation specific to TNF-alpha have been conducted. According to the obtained results, it was found that their behavior to corrosion was weak but that nickel release was high. The quantities of nickel released are higher than the limits imposed in the EU concerning contact with the skin or piercing. Surprisingly the biological tests did not show any cytotoxic effect on Hela and L929 cells or any change in TNF-alpha expression in monocytic cells. The alloys did not show any proinflammatory response in endothelial cells as demonstrated by the absence of ICAM-1 induction. We note therefore that there is really no direct relationship between the in vitro biological evaluation tests and the physico-chemical characterization of these dental alloys. Clinical and epidemiological studies are required to clarify these aspects.