In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Fetal ventral mesencephalon of human and rat origin maintained in vitro and transplanted to 6-hydroxydopamine-lesioned rats gives rise to grafts rich in dopaminergic neurons

Free-floating roller tube cultures of human fetal (embryonic age 6-10 weeks post-conception) and rat fetal (embryonic day 13) ventral mesencephalon were prepared. After 7-15 days in vitro, the mesencephalic tissue cultures were transplanted into the striatum of adult rats that had received unilateral injections of 6-hydroxydopamine into the nigrostriatal bundle 3-5 weeks prior to transplantation. Graft survival was assessed in tyrosine hydroxylase (TH)-immunostained serial sections of the grafted brains up to post-transplantation week 4 for the human fetal xenografts and post-transplantation week 11 for the rat fetal allografts. D-amphetamine-induced rotation was monitored up to 10 weeks after transplantation in the allografted animals and compared with that of lesioned-only control animals. All transplanted animals showed large, viable grafts containing TH-immunoreactive (ir) neurons. The density of TH-ir neurons in the human fetal xenografts and in rat fetal allografts was similar. A significant amelioration of the amphetamine-induced rotation was observed in the animals that received cultured tissue allografts. These results promote the feasibility of in vitro maintenance of fetal human and rat nigral tissue prior to transplantation using the free-floating roller tube technique.

Thermoreversible hyaluronan-based hydrogel supports in vitro and ex vivo disc-like differentiation of human mesenchymal stem cells

BACKGROUND CONTEXT The fate of human mesenchymal stem cells (hMSCs) supplied to the degenerating intervertebral disc (IVD) is still not fully understood and can be negatively affected by low oxygen, pH, and glucose concentration of the IVD environment. The hMSC survival and yield upon injection of compromised IVD could be improved by the use of an appropriate carrier and/or by predifferentiation of hMSCs before injection. PURPOSE To optimize hMSC culture conditions in thermoreversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-pNIPAM), to achieve differentiation toward the disc phenotype in vitro, and evaluate whether preconditioning contributes to a better hMSC response ex vivo. STUDY DESIGN In vitro and ex vivo whole-organ culture of hMSCs. METHODS In vitro cultures of hMSCs were conducted in HA-pNIPAM and alginate for 1 week under hypoxia in chondropermissive medium alone and with the supplementation of transforming growth factor β1 or growth and differentiation factor 5 (GDF-5). Ex vivo, hMSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or predifferentiated with GDF-5 for 1 week in HA-pNIPAM and then supplied to the IVDs. Cell viability was evaluated by Live-Dead assay, and DNA, glycosaminoglycan (GAG), and gene expression profiles were used to assess hMSC differentiation toward the disc phenotype. RESULTS The HA-pNIPAM induced hMSC differentiation toward the disc phenotype more effectively than alginate: in vitro, higher GAG/DNA ratio and higher collagen type II, SOX9, cytokeratin-19, cluster of differentiation 24, and forkhead box protein F1 expressions were found for hMSCs cultured in HA-pNIPAM compared with those cultured in alginate, regardless of the addition of growth factors. Ex vivo, direct combination of HA-pNIPAM with the disc environment induced a stronger disc-like differentiation of hMSCs than predifferentiation of hMSCs followed by their delivery to the discs. CONCLUSIONS Hyaluronan-based thermoreversible hydrogel supports hMSC differentiation toward the disc phenotype without the need for growth factor supplementation in vitro and ex vivo. Further in vivo studies are required to confirm the suitability of this hydrogel as an effective stem cell carrier for the treatment of IVD degeneration.

An In Vitro Method to Investigate the Microbicidal Potential of Human Macrophages for Use in Psychosomatic Research

OBJECTIVE: Psychological states relate to changes in circulating immune cells, but associations with immune cells in peripheral tissues such as macrophages have hardly been investigated. Here, we aimed to implement and validate a method for measuring the microbicidal potential of ex vivo isolated human monocyte-derived macrophages (HMDMs) as an indicator of macrophage activation. METHODS: The method was implemented and validated for two blood sampling procedures (short-term cannula insertion versus long-term catheter insertion) in 79 participants (34 women, 45 men) aged between 18 and 75 years. The method principle is based on the reduction of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-dis-ulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) by superoxide anions, the first in a series of pathogen-killing reactive oxygen species produced by phorbol myristate acetate-activated HMDM. Cytochrome c reduction and current generation were measured as reference methods for validation purposes. We further evaluated whether depressive symptom severity (Beck Depression Inventory) and chronic stress (Chronic Stress Screening Scale) were associated with macrophage microbicidal potential. RESULTS: The assay induced superoxide anion responses by HMDM in all participants. Assay results depended on blood sampling procedure (cannula versus catheter insertion). Interassay variability as a measure for assay reliability was 10.92% or less. WST-1 reduction scores correlated strongly with results obtained by reference methods (cytochrome c: r = 0.57, p = .026; current generation: r values ≥ 0.47, p values <.033) and with psychological factors (depressive symptom severity: r = 0.35 [cannula insertion] versus r = -0.54 [catheter insertion]; chronic stress: r = 0.36 [cannula insertion]; p values ≤ .047). CONCLUSIONS: Our findings suggest that the implemented in vitro method investigates microbicidal potential of HMDM in a manner that is valid and sensitive to psychological measures.

A nonlinear QCT-based finite element model validation study for the human femur tested in two configurations in vitro

Purpose Femoral fracture is a common medical problem in osteoporotic individuals. Bone mineral density (BMD) is the gold standard measure to evaluate fracture risk in vivo. Quantitative computed tomography (QCT)-based homogenized voxel finite element (hvFE) models have been proved to be more accurate predictors of femoral strength than BMD by adding geometrical and material properties. The aim of this study was to evaluate the ability of hvFE models in predicting femoral stiffness, strength and failure location for a large number of pairs of human femora tested in two different loading scenarios. Methods Thirty-six pairs of femora were scanned with QCT and total proximal BMD and BMC were evaluated. For each pair, one femur was positioned in one-legged stance configuration (STANCE) and the other in a sideways configuration (SIDE). Nonlinear hvFE models were generated from QCT images by reproducing the same loading configurations imposed in the experiments. For experiments and models, the structural properties (stiffness and ultimate load), the failure location and the motion of the femoral head were computed and compared. Results In both configurations, hvFE models predicted both stiffness (R2=0.82 for STANCE and R2=0.74 for SIDE) and femoral ultimate load (R2=0.80 for STANCE and R2=0.85 for SIDE) better than BMD and BMC. Moreover, the models predicted qualitatively well the failure location (66% of cases) and the motion of the femoral head. Conclusions The subject specific QCT-based nonlinear hvFE model cannot only predict femoral apparent mechanical properties better than densitometric measures, but can additionally provide useful qualitative information about failure location.

Antitumor effect of SIRT1 inhibition in human HCC tumor models in vitro and in vivo

Sirtuins (SIRT1-7) are a highly conserved family of NAD(+)-dependent enzymes that control the activity of histone and nonhistone regulatory proteins. SIRT1 is purposed to promote longevity and to suppress the initiation of some cancers. Nevertheless, SIRT1 is reported to function as a tumor suppressor as well as an oncogenic protein. Our data show that compared with normal liver or surrounding tumor tissue, SIRT1 is strongly overexpressed in human hepatocellular carcinoma (HCC). In addition, human HCC cell lines (Hep3B, HepG2, HuH7, HLE, HLF, HepKK1, skHep1) were screened for the expression of the sirtuin family members and only SIRT1 was consistently overexpressed compared with normal hepatocytes. To determine its effect on HCC growth, SIRT1 activity was inhibited either with lentiviruses expressing short hairpin RNAs or with the small molecule inhibitor, cambinol. Knockdown or inhibition of SIRT1 activity had a cytostatic effect, characterized by an altered morphology, impaired proliferation, an increased expression of differentiation markers, and cellular senescence. In an orthotopic xenograft model, knockdown of SIRT1 resulted in 50% fewer animals developing tumors and cambinol treatment resulted in an overall lower tumor burden. Taken together, our data show that inhibition of SIRT1 in HCC cells impairs their proliferation in vitro and tumor formation in vivo. These data suggest that SIRT1 expression positively influences the growth of HCC and support further studies aimed to block its activity alone or in combination as a novel treatment strategy.

Comparison of the toxicity of diesel exhaust produced by bio- and fossil diesel combustion in human lung cells in vitro

Alternative fuels are increasingly combusted in diesel- and gasoline engines and the contribution of such exhausts to the overall air pollution is on the rise. Recent findings on the possible adverse effects of biodiesel exhaust are contradictive, at least partly resulting from the various fuel qualities, engine types and different operation conditions that were tested. However, most of the studies are biased by undesired interactions between the exhaust samples and biological culture media. We here report how complete, freshly produced exhausts from fossil diesel (B0), from a blend of 20% rapeseed-methyl ester (RME) and 80% fossil diesel (B20) and from pure rapeseed methyl ester (B100) affect a complex 3D cellular model of the human airway epithelium in vitro by exposing the cells at the air–liquid interface. The induction of pro-apoptotic and necrotic cell death, cellular morphology, oxidative stress, and pro-inflammatory responses were assessed. Compared to B0 exhaust, B20 exhaust decreased oxidative stress and pro-inflammatory responses, whereas B100 exhaust, depending on exposure duration, decreased oxidative stress but increased pro-inflammatory responses. The effects are only very weak and given the compared to fossil diesel higher ecological sustainability of biodiesel, it appears that – at least RME – can be considered a valuable alternative to pure fossil diesel.

Impaired genome encapsidation restricts the in vitro propagation of human parvovirus B19.

The lack of a permissive cell culture system hampers the study of human parvovirus B19 (B19V). UT7/Epo is one of the few established cell lines that can be infected with B19V but generates none or few infectious progeny. Recently, hypoxic conditions or the use of primary CD36+ erythroid progenitor cells (CD36+ EPCs) have been shown to improve the infection. These novel approaches were evaluated in infection and transfection experiments. Hypoxic conditions or the use of CD36+ EPCs resulted in a significant acceleration of the infection/transfection and a modest increase in the yield of capsid progeny. However, under all tested conditions, genome encapsidation was impaired seriously. Further analysis of the cell culture virus progeny revealed that differently to the wild-type virus, the VP1 unique region (VP1u) was exposed partially and was unable to become further externalized upon heat treatment. The fivefold axes pore, which is used for VP1u externalization and genome encapsidation, might be constricted by the atypical VP1u conformation explaining the packaging failure. Although CD36+ EPCs and hypoxia facilitate B19V infection, large quantities of infectious progeny cannot be generated due to a failure in genome encapsidation, which arises as a major limiting factor for the in vitro propagation of B19V.

In vitro and in vivo activity of human interleukin-8 in dogs

Interleukin-8 (IL-8), a proinflammatory cytokine produced by human monocytes, fibroblasts, and endothelial and epithelial cells, is effective not only on cells and tissues of human beings but also on those of several animal species. We investigated the importance of recombinant human IL-8 for the activation of canine neutrophils in vitro and its potential for inducing inflammation in vivo. Shape change (10(-9)-10(-7) M IL-8) and chemotaxis (10(-10)-10(-6) M IL-8) assays were used to determine the activation of canine neutrophils in vitro. Chemotaxis was induced by IL-8 at doses > 10(-8) M with a maximum response at 10(-6) M. A rapid shape change of comparable intensity was elicited by 10(-9)-10(-7) M IL-8. Thirty minutes after intradermal injection of 10(-9) moles of IL-8, emigration of neutrophils could be observed and became more intense at 60 minutes and 240 minutes, respectively. Zymosan-activated canine plasma, which served as a positive control, induced a rapid, massive, and more diffuse neutrophil accumulation, whereas the reaction after IL-8 was weaker but still significant. The neutrophil accumulation after IL-8 was preferentially located in perivenular areas of the deep dermis. Recombinant human IL-8 is capable of activating canine neutrophils in vitro and is able to generate significant neutrophil accumulation in dog skin. Its activity is lower than that in human, rabbit, and rat systems.

In vitro-activity of oily calcium hydroxide suspension on microorganisms as well as on human alveolar osteoblasts and periodontal ligament fibroblasts.

BACKGROUND Findings from animal and human studies have indicated that an oily calcium hydroxide suspension (OCHS) may improve early wound healing in the treatment of periodontitis. Calcium hydroxide as the main component is well known for its antimicrobial activity, however at present the effect of OCHS on the influence of periodontal wound healing/regeneration is still very limited. The purpose of this in vitro study was to investigate the effect of OCHS on periodontopathogenic bacteria as well as on the attachment and proliferation of osteoblasts and periodontal ligament fibroblasts. METHODS Human alveolar osteoblasts (HAO) and periodontal ligament (PDL) fibroblasts were cultured on 3 concentrations of OCHS (2.5, 5 and 7.5 mg). Adhesion and proliferation were counted up to 48 h and mineralization was assayed after 1 and 2 weeks. Furthermore potential growth inhibitory activity on microorganisms associated with periodontal disease (e.g. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans) as well as the influence of periodontopathogens and OCHS on the HAO and PDL fibroblasts counts were determined. RESULTS More than a 2-fold increase in adherent HAO cells was observed at 4 h following application of OCHS when compared to the control group (p = 0.007 for 2.5 mg). Proliferation of HAO cells at 48 h was stimulated by moderate concentrations (2.5 mg; 5 mg) of OCHS (each p < 0.001), whereas a high concentration (7.5 mg) of OCHS was inhibitory (p = 0.009). Mineralization was observed only for HAO cells treated with OCHS. OCHS did not exert any positive effect on attachment or proliferation of PDL fibroblasts. Although OCHS did not have an antibacterial effect, it did positively influence attachment and proliferation of HAO cells and PDL fibroblasts in the presence of periodontopathogens. CONCLUSIONS The present data suggests that OCHS promotes osteoblast attachment, proliferation and mineralization in a concentration-dependent manner and results are maintained in the presence of periodontal pathogens.

Uptake efficiency of surface modified gold nanoparticles does not correlate with functional changes and cytokine secretion in human dendritic cells in vitro.

Engineering nanoparticles (NPs) for immune modulation require a thorough understanding of their interaction(s) with cells. Gold NPs (AuNPs) were coated with polyethylene glycol (PEG), polyvinyl alcohol (PVA) or a mixture of both with either positive or negative surface charge to investigate uptake and cell response in monocyte-derived dendritic cells (MDDCs). Inductively coupled plasma optical emission spectrometry and transmission electron microscopy were used to confirm the presence of Au inside MDDCs. Cell viability, (pro-)inflammatory responses, MDDC phenotype, activation markers, antigen uptake and processing were analyzed. Cell death was only observed for PVA-NH2 AuNPs at the highest concentration. MDDCs internalize AuNPs, however, surface modification influenced uptake. Though limited uptake was observed for PEG-COOH AuNPs, a significant tumor necrosis factor-alpha release was induced. In contrast, (PEG+PVA)-NH2 and PVA-NH2 AuNPs were internalized to a higher extent and caused interleukin-1beta secretion. None of the AuNPs caused changes in MDDC phenotype, activation or immunological properties.

Retinal differentiation of human bone marrow-derived stem cells by co-culture with retinal pigment epithelium in vitro.

The goal of this study was to assess the in vitro differentiation capacity of human bone marrow-derived stem cells (hBMSCs) along retinal lineages. Mononuclear cells (MNC) were isolated from bone marrow (BM) and mobilized peripheral blood (mPB) using Ficoll-Paque density gradient centrifugation, and were sorted by magnetic-activated cell sorting (MACS) for specific stem cell subsets (CD34(+)CD38(+)/CD34(+)CD38(-)). These cells were then co-cultured on human retinal pigment epithelial cells (hRPE) for 7 days. The expression of stem cell, neural and retina-specific markers was examined by immunostaining, and the gene expression profiles were assessed after FACS separation of the co-cultured hBMSCs by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, in vitro functionality of the differentiated cells was analyzed by quantifying phagocytosis of CY5-labeled photoreceptor outer segments (POS). After 7 days of co-culture, hBMSCs adopted an elongated epithelial-like morphology and expressed RPE-specific markers, such as RPE65 and bestrophin. In addition, these differentiated cells were able to phagocytose OS, one of the main characteristics of native RPE cells. Our data demonstrated that human CD34(+)CD38(-) hBMSC may differentiate towards an RPE-like cell type in vitro and could become a new type of autologous donor cell for regenerative therapy in retinal degenerative diseases.

Inhibition of in vitro metabolism of testosterone in human, dog and horse liver microsomes to investigate species differences.

Testosterone hydroxylation was investigated in human, canine and equine liver microsomes and in human and canine single CYPs. The contribution of the CYP families 1, 2 and 3 was studied using chemical inhibitors. Testosterone metabolites were analyzed by HPLC. The metabolites androstenedione, 6β- and 11β-hydroxytestosterone were found in microsomes of all species, but the pattern of metabolites varied within species. Androstenedione was more prominent in the animal species, and an increase over time was seen in equines. Testosterone hydroxylation was predominantly catalyzed by the CYP3A subfamily in all three species. While CYP2C9 did not metabolise testosterone, the canine ortholog CYP2C21 produced androstenedione. Quercetin significantly inhibited 6β- and 11β-hydroxytestosterone in all species investigated, suggesting that CYP2C8 is involved in testosterone metabolism, whereas sulfaphenazole significantly inhibited the formation of 6β- and 11β-hydroxytestosterone in human microsomes, at 60min in equine microsomes, but not in canine microsomes. A contribution of CYP2B6 in testosterone metabolism was only found in human and equine microsomes. Inhibition of 17β-hydroxysteroid dehydrogenase 2 indicated its involvement in androstenedione formation in humans, increased androstenedione formation was found in equines and no involvement in canines. These findings provide improved understanding of differences in testosterone biotransformation in animal species.