Alkaline phosphatase dual-binding sites for collagen dictate cell migration and microvessel assembly in vitro.

Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro- or microvasculature) play a role in the formation of microvessel-like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor-ß1, and interleukin-8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibroblasts and human ECs stimulate collagen synthesis and growth factors production by fibroblasts that ultimately affect the formation and distribution of microvessel-like structures in cell cultures. In this study, areas with activated fibroblasts and high alkaline phosphatase (ALP) activity were also observed in cocultures. Molecular docking assays revealed that ALP has two binding positions for collagen, suggesting its impact in collagen proteins' aggregation, cell migration, and microvessel assembly. These findings indicate that bioinformatics and coculture systems are complementary tools for investigating the participation of proteins, like collagen and ALP in angiogenesis.

Cervical Cancer Screening: Comparison of Conventional Pap Smear Test, Liquid-Based Cytology, and Human Papillomavirus Testing as Stand-alone or Cotesting Strategies.

BACKGROUND: Some countries have implemented stand-alone human papillomavirus (HPV) testing while others consider cotesting for cervical cancer screening. We compared both strategies within a population-based study. METHODS: The MARZY cohort study was conducted in Germany. Randomly selected women from population registries aged ≥30 years (n = 5,275) were invited to screening with Pap smear, liquid-based cytology (LBC, ThinPrep), and HPV testing (Hybrid Capture2, HC2). Screen-positive participants [ASC-US+ or high-risk HC2 (hrHC2)] and a random 5% sample of screen-negatives were referred to colposcopy. Post hoc HPV genotyping was conducted by GP5+/6+ PCR-EIA with reverse line blotting. Sensitivity, specificity (adjusted for verification bias), and potential harms, including number of colposcopies needed to detect 1 precancerous lesion (NNC), were calculated. RESULTS: In 2,627 screened women, cytological sensitivities (Pap, LBC: 47%) were lower than HC2 (95%) and PCR (79%) for CIN2+. Cotesting demonstrated higher sensitivities (HC2 cotesting: 99%; PCR cotesting: 84%), but at the cost of lower specificities (92%-95%) compared with HPV stand-alone (HC2: 95%; PCR: 94%) and cytology (97% or 99%). Cotesting versus HPV stand-alone showed equivalent relative sensitivity [HC2: 1.06, 95% confidence interval (CI), 1.00-1.21; PCR: 1.07, 95% CI, 1.00-1.27]. Relative specificity of Pap cotesting with either HPV test was inferior to stand-alone HPV. LBC cotesting demonstrated equivalent specificity (both tests: 0.99, 95% CI, 0.99-1.00). NNC was highest for Pap cotesting. CONCLUSIONS: Cotesting offers no benefit in detection over stand-alone HPV testing, resulting in more false positive results and colposcopy referrals. IMPACT: HPV stand-alone screening offers a better balance of benefits and harms than cotesting.See related commentary by Wentzensen and Clarke, p. 432.

Short-term hypoxia promotes vascularization in co-culture system consisting of primary human osteoblasts and outgrowth endothelial cells.

Prevascularization of tissue constructs before implantation has been developed as a novel and promising concept for successful implantation. Since hypoxia might induce angiogenesis, we have investigated the effects of hypoxic treatment on vascularization by using co-cultures of primary human osteoblasts (POBs) and outgrowth endothelial cells. Our results show that: (a) repeated short-term hypoxia (2% O2 for 8 hr), not long-term hypoxia (2% O2 for 24 hr), over 1 or 2 weeks, significantly enhances microvessel formation in co-cultures; (b) sustained hypoxia, not short-term or long-term hypoxia, causes cytotoxicity in mono- and co-cultures; (c) the expression of some angiogenic and inflammatory factors such as vascular endothelial growth factor, platelet-derived growth factor subunit B, insulin-like growth factor 1, interleukin-8, and early growth response protein 1 increases significantly in hypoxia-treated POB monoculture and co-cultures after single or multiple 8- or 24-hr hypoxic treatments; (d) long-term (24 hr) hypoxic treatment induces more angiogenic inhibitors compared with short-term hypoxic treatment. Our findings suggest that hypoxia-induced vascularization/angiogenesis is regulated by a complex balance of angiogenic/antiangiogenic factors, and that repeated short-term hypoxia, but not repeated long-term hypoxia, promotes the vascularization and tissue regeneration of bone tissue constructs.

A low-speed centrifugation concept leads to cell accumulation and vascularization of solid platelet-rich fibrin: an experimental study in vivo.

Platelet-rich fibrin (PRF) is generated from the patients' own venous blood by a single centrifugation step without the additional use of anticoagulants. Based on the previously described LSCC (low-speed centrifugation concept), our group showed that modification of the centrifugation setting, that is, reducing the relative centrifugal force (RCF) and mildly increasing the centrifugation time, resulted in modified solid and liquid PRF-matrices with increased number of platelets, leukocytes, and growth factors' concentrations. The aim of this study was to determine whether RCF reduction might also result in different tissue reactions toward the two PRF-based matrices, especially vascularization and cell distribution in vivo. Two centrifugation protocols (PRF-high [719 g] and PRF-medium [222 g]) were compared in a subcutaneous implantation model of SCID mice at 5 and 10 days. Histological and histomorphometrical analyses were performed to quantify lymphocyte, neutrophil, human macrophage, and monocyte populations. CD31 was used to detect newly formed vessels, while all human cells were detected by using human vimentin as a pan-cellular marker. The results demonstrated that PRF-high elicited a dense and stable fibrin structure and prevented cellular penetration of the host tissue. By contrast, PRF-medium was more porous, had a significantly higher in vivo vascularization rate, and included significantly more human cells, especially at day 10, compared to PRF-high. These findings highlight the possibility of modifying the structure and composition of PRF matrices and thus selectively altering their regenerative potential in vivo. Clinical studies now must evaluate the different PRF matrices for bone and soft-tissue regeneration to validate possible benefits using personalized preparation protocols.

Human CD8+ T Cells Damage Noninfected Epithelial Cells during Influenza Virus Infection In Vitro.

During severe influenza A virus (IAV) infections, a large amount of damage to the pulmonary epithelium is the result of the antiviral immune response. Specifically, whilst CD8+ T cells are important for killing IAV-infected cells, during a severe IAV infection, they can damage uninfected epithelial cells. At present, the mechanisms by which this occurs are unclear. Here, we used a novel in vitro coculture model of human NCl-H441 cells and CD8+ T cells to provide a new insight into how CD8+ T cells may affect uninfected epithelial cells during severe IAV infections. Using this model, we show that human IAV-specific CD8+ T cells produce soluble factors that reduce the barrier integrity of noninfected epithelial cells (referred to as "bystander damage"). We show that this bystander damage is the result of a combination of TNF-α and IFN-γ. This bystander damage occurred in the absence of widespread epithelial cell death and was instead associated with decreased expression of epithelial cell ion channels and pumps. Together, these data suggest that ameliorating the function of epithelial cell ion channels and pumps may help reduce immunopathology during severe IAV infections.

Basement Membrane Mimics of Biofunctionalized Nanofibers for a Bipolar-Cultured Human Primary Alveolar-Capillary Barrier Model.

In vitro reconstruction of an alveolar barrier for modeling normal lung functions and pathological events serve as reproducible, high-throughput pharmaceutical platforms for drug discovery, diagnosis, and regenerative medicine. Despite much effort, the reconstruction of organ-level alveolar barrier functions has failed due to the lack of structural similarity to the natural basement membrane, functionalization with specific ligands for alveolar cell function, the use of primary cells and biodegradability. Here we report a bipolar cultured alveolar-capillary barrier model of human primary cells supported by a basement membrane mimics of fully synthetic bifunctional nanofibers. One-step electrospinning process using a bioresorbable polyester and multifunctional star-shaped polyethylene glycols (sPEG) enables the fabrication of an ultrathin nanofiber mesh with interconnected pores. The nanofiber mesh possessed mechanical stability against cyclic expansion as seen in the lung in vivo. The sPEGs as an additive provide biofunctionality to fibers through the conjugation of peptide to the nanofibers and hydrophilization to prevent unspecific protein adsorption. Biofunctionalized nanofiber meshes facilitated bipolar cultivation of endothelial and epithelial cells with fundamental alveolar functionality and showed higher permeability for molecules compared to microporous films. This nanofiber mesh for a bipolar cultured barrier have the potential to promote growth of an organ-level barrier model for modeling pathological conditions and evaluating drug efficacy, environmental pollutants, and nanotoxicology.

Foreign Body Giant Cell-Related Encapsulation of a Synthetic Material Three Years After Augmentation.

Bone substitute materials of different origin and chemical compositions are frequently used in augmentation procedures to enlarge the local bone amount. However, relatively little data exist on the long-term tissue reactions. The presented case reports for the first time histological and histomorphometrical analyses of a nanocrystaline hydroxyapatite-based bone substitute material implanted in the human sinus cavity after an integration period of 3 years. The extracted biopsy was analyzed histologically and histomorphometrically with focus on the tissue reactions, vascularization, new bone formation, and the induction of a foreign body reaction. A comparably high rate of connective tissue (48.25%) surrounding the remaining bone substitute granules (42.13%) was observed. Accordingly, the amount of bone tissue (9.62%) built the smallest fraction within the biopsy. Further, tartrate-resistant acid phosphatase-positive and -negative multinucleated giant cells (4.35 and 3.93 cells/mm(2), respectively) were detected on the material-tissue interfaces. The implantation bed showed a mild vascularization of 10.03 vessels/mm(2) and 0.78%. The present case report shows that after 3 years, a comparable small amount of bone tissue was observable. Thus, the foreign body response to the bone substitute seems to be folded without further degradation or regeneration.

Recent progress in revealing the biological and medical significance of the non-neuronal cholinergic system.

This special issue of International Immunopharmacology is the proceedings of the Fourth International Symposium on Non-neuronal Acetylcholine that was held on August 28-30, 2014 at the Justus Liebig University of Giessen in Germany. It contains original contributions of meeting participants covering the significant progress in understanding of the biological and medical significance of the non-neuronal cholinergic system extending from exciting insights into molecular mechanisms regulating this system via miRNAs over the discovery of novel cholinergic cellular signaling circuitries to clinical implications in cancer, wound healing, immunity and inflammation, cardiovascular, respiratory and other diseases.

Apoptosis of ileal crypt epithelia after allogeneic bone marrow transplantation without graft-versus-host disease.

Intestinal crypt cell apoptosis may occur after allogeneic bone marrow transplantation without clinically overt graft-versus-host disease. We describe this phenomenon in a case of a 12-year-old girl who had segments of the ileum resected because of a relapse of acute lymphoblastic leukemia. The diagnostic difficulties are discussed.

TRAP-Positive Multinucleated Giant Cells Are Foreign Body Giant Cells Rather Than Osteoclasts: Results From a Split-Mouth Study in Humans.

This study compared the material-specific tissue response to the synthetic, hydroxyapatite-based bone substitute material NanoBone (NB) with that of the xenogeneic, bovine-based bone substitute material Bio-Oss (BO). The sinus cavities of 14 human patients were augmented with NB and BO in a split-mouth design. Six months after augmentation, bone biopsies were extracted for histological and histomorphometric investigation prior to dental implant insertion. The following were evaluated: the cellular inflammatory pattern, the induction of multinucleated giant cells, vascularization, the relative amounts of newly formed bone, connective tissue, and the remaining bone substitute material. NB granules were well integrated in the peri-implant tissue and were surrounded by newly formed bone tissue. Multinucleated giant cells were visible on the surfaces of the remaining granules. BO granules were integrated into the newly formed bone tissue, which originated from active osteoblasts on their surface. Histomorphometric analysis showed a significantly higher number of multinucleated giant cells and blood vessels in the NB group compared to the BO group. No statistical differences were observed in regard to connective tissue, remaining bone substitute, and newly formed bone. The results of this study highlight the different cellular reactions to synthetic and xenogeneic bone substitute materials. The significantly higher number of multinucleated giant cells within the NB implantation bed seems to have no effect on its biodegradation. Accordingly, the multinucleated giant cells observed within the NB implantation bed have characteristics more similar to those of foreign body giant cells than to those of osteoclasts.

Synthetic bone substitute material comparable with xenogeneic material for bone tissue regeneration in oral cancer patients: First and preliminary histological, histomorphometrical and clinical results.

BACKGROUND: The present study was first to evaluate the material-specific cellular tissue response of patients with head and neck cancer to a nanocrystalline hydroxyapatite bone substitute NanoBone (NB) in comparison with a deproteinized bovine bone matrix Bio-Oss (BO) after implantation into the sinus cavity. MATERIALS AND METHODS: Eight patients with tumor resection for oral cancer and severely resorbed maxillary bone received materials according to a split mouth design for 6 months. Bone cores were harvested prior to implantation and analyzed histologically and histomorphometrically. Implant survival was followed-up to 2 years after placement. RESULTS: Histologically, NB underwent a higher vascularization and induced significantly more tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated giant cells when compared with BO, which induced mainly mononuclear cells. No significant difference was observed in the extent of new bone formation between both groups. The clinical follow-up showed undisturbed healing of all implants in the BO-group, whereas the loss of one implant was observed in the NB-group. CONCLUSIONS: Within its limits, the present study showed for the first time that both material classes evaluated, despite their induction of different cellular tissue reactions, may be useful as augmentation materials for dental and maxillofacial surgical applications, particularly in patients who previously had oral cancer.

Kinetics and isotherm of fibronectin adsorption to three-dimensional porous chitosan scaffolds explored by ¹²5I-radiolabelling.

In this study, (125)I-radiolabelling was explored to follow the kinetics and isotherm of fibronectin (FN) adsorption to porous polymeric scaffolds, as well as to assess the elution and exchangeability of pre-adsorbed FN following incubation in serum-containing culture medium. Chitosan (CH) porous scaffolds with two different degrees of acetylation (DA 4% and 15%) were incubated in FN solutions with concentrations ranging from 5 to 50 µg/mL. The kinetic and isotherm of FN adsorption to CH were successfully followed using (125)I-FN as a tracer molecule. While on DA 4% the levels of adsorbed FN increased linearly with FN solution concentration, on DA 15% a saturation plateau was attained, and FN adsorbed amounts were significantly lower. These findings were supported by immunofluorescent studies that revealed, for the same FN solution concentration, higher levels of exposed cell-binding domains on DA 4% as compared with DA 15%. Following incubation in serum containing medium, DA 4% also revealed higher ability to exchange pre-adsorbed FN by new FN molecules from serum than DA 15%. In accordance, when assessing the efficacy of passively adsorbed FN to promote endothelial cell (EC) adhesion to CH, ECs were found to adhere at higher levels to DA 4% as compared with DA 15%, 5 µg/mL of FN being already efficient in promoting cell adhesion and cytoskeletal organization on CH with DA 4%. Taken together the results show that protein radiolabelling can be used as an effective tool to study protein adsorption to porous polymeric scaffolds, both from single and complex protein solutions.

Nanocrystalline hydroxyapatite bone substitute leads to sufficient bone tissue formation already after 3 months: histological and histomorphometrical analysis 3 and 6 months following human sinus cavity augmentation.

PURPOSE: In this study the de novo bone formation capacity of a nanocrystalline hydroxyapatite bone substitute was assessed 3 and 6 months after its insertion into the human sinus cavity. MATERIALS AND METHODS: Sinus cavity augmentation was performed in a total of 14 patients (n = 7 implantation after 3 months; n = 7 implantation after 6 months) with severely atrophic maxillary bone. The specimens obtained after 3 and 6 months were analyzed histologically and histomorphometrically with special focus on bone metabolism within the residual bone and the augmented region. RESULTS: This study revealed that bone tissue formation started from the bone-biomaterial-interface and was directed into the most cranial parts of the augmented region. There was no statistically significant difference in new bone formation after 3 and 6 months (24.89 ± 10.22% vs 31.29 ± 2.29%), respectively. CONCLUSIONS: Within the limits of the present study and according to previously published data, implant insertion in regions augmented with this bone substitute material could be considered already after 3 months. Further clinical studies with bone substitute materials are necessary to validate these findings.

Wild-type JAK2 secondary acute erythroleukemia developing after JAK2-V617F-mutated primary myelofibrosis.

A 54-year-old female patient developed acute erythroleukemia after an 8-year course of primary myelofibrosis. The latter harbors the JAK2-V617F mutation and was treated with hydroxyurea and anagrelide. A bone marrow trephine biopsy disclosed 2 morphologically distinct areas of chronic primary myelofibrosis and acute erythroleukemia. Microdissection and a separate molecular pathological analysis was performed. Although the activating JAK2-V617F mutation was not maintained in blasts of acute erythroleukemia, it was detectable in the chronic phase of primary myelofibrosis, indicating that this mutation did not play a role in the leukemic transformation of erythroid cells.

Contribution of outgrowth endothelial cells from human peripheral blood on in vivo vascularization of bone tissue engineered constructs based on starch polycaprolactone scaffolds.

In the present study we assessed the potential of human outgrowth endothelial cells (OEC), a subpopulation within endothelial progenitor cell cultures, to support the vascularization of a complex tissue engineered construct for bone. OEC cultured on starch polycaprolactone fiber meshes (SPCL) in monoculture retained their endothelial functionality and responded to angiogenic stimulation by VEGF (vascular endothelial growth factor) in fibrin gel-assays in vitro. Co-culture of OEC with human primary osteoblasts (pOB) on SPCL, induced an angiogenic activation of OEC towards microvessel-like structures achieved without additional supplementation with angiogenic growth factors. Effects of co-cultures with pOB on the vascularization process by OEC in vivo were tested by subcutaneous implantation of Matrigel plugs containing both, OEC and pOB, and resulted in OEC-derived blood vessels integrated into the host tissue and anastomosed to the vascular supply. In addition, morphometric analysis of the vascularization process by OEC indicated a better performance of OEC in the co-cultures with primary osteoblasts compared to monocultures of OEC. The contribution of OEC to vascular structures and the beneficial effect of the co-culture with primary human osteoblasts on the vascularization in vivo was additionally proven by subcutaneous implantation of pre-cellularized and pre-cultured SPCL constructs. OEC contributed to the vascular structures, by generating autogenic vessels or by incorporation into chimeric vessels consisting of both, human and mouse endothelial cells. The current data highlight the vasculogenic potential of OEC for bone tissue engineering applications and indicate a beneficial influence of constructs including both osteoblasts and endothelial cells for vascularization strategies.

Dynamic processes involved in the pre-vascularization of silk fibroin constructs for bone regeneration using outgrowth endothelial cells.

For successful bone regeneration tissue engineered bone constructs combining both aspects, namely a high osteogenic potential and a rapid connection to the vascular network are needed. In this study we assessed the formation of pre-vascular structures by human outgrowth endothelial cells (OEC) from progenitors in the peripheral blood and the osteogenic differentiation of primary human osteoblasts (pOB) on micrometric silk fibroin scaffolds. The rational was to gain more insight into the dynamic processes involved in the differentiation and functionality of both cell types depending on culture time in vitro. Vascular tube formation by OEC was assessed quantitatively at one and 4 weeks of culture. In parallel, we assessed the temporal changes in cell ratios by flow cytometry and in the marker profiles of endothelial and osteogenic markers by quantitative real-time PCR. In terms of OEC, we observed an increase in tube length, tube area, number of nodes and number of vascular meshes within a culture period of 4 weeks, but a decrease in endothelial markers in real-time PCR. At the same time early osteogenic markers were downregulated, while marker expression associated with progressing mineralized matrix was upregulated in later stages of the culture. In addition, deposition of matrix components, such as collagen type I, known as a pro-angiogenic substrate for endothelial cells, appeared to increase with time indicated by immunohistochemistry. In summary, the study suggests a progressing maturation of the tissue construct with culture time which seems to be not effected by culture conditions mainly designed for outgrowth endothelial cells.

Functionality of endothelial cells on silk fibroin nets: comparative study of micro- and nanometric fibre size.

Biomimetic material design, such as mimicking nanostructured components of the extracellular matrix, is an actual challenge for biomaterial research with a high impact on tissue engineering and regenerative medicine. Thus, understanding the cellular response at the cell biological and molecular level and the consequences of various chemically or physically modified biomaterials is highly important. In the present study we assessed the response of human umbilical vein endothelial cells (HUVEC) and outgrowth endothelial cells (OEC) from endothelial progenitor cells to different variants of nanofibrous silk fibroin nets in comparison to microfibrous silk fibroin scaffolds with regard to cellular morphology, proliferation, formation of intercellular contacts as well as integrin-dependent adhesion. Endothelial cells (ECs) grown on nanometric nets formed a differentiated and interconnected endothelial monolayer with no significant changes in the expression of intercellular contact molecules or proliferation rates compared with cells grown on micrometric nets. Nevertheless, quantitative real-time PCR revealed a higher expression level of integrin-beta1 in ECs grown on nanofibrous fibroin nets compared to the microfibrous samples. In addition, single nano-fibres were recognised by the integrin-receptor mechanism supporting the formation of focal adhesion at the interface of ECs and nanometric nets. These findings indicate that the nanometric silk fibroin scaffolds did not interfere with the formation of a differentiated and interconnected EC layer. On the contrary, nanofibre variation of the fibroin net architecture induced changes in ECs at the molecular level in terms of the increased expression of adhesion molecules such as integrin-beta1.

Efficacy and safety of imatinib in adult patients with c-kit-positive acute myeloid leukemia.

This phase 2 pilot study was conducted to determine the efficacy and safety of imatinib mesylate in patients with c-kit-positive acute myeloid leukemia (AML) refractory to or not eligible for chemotherapy. Twenty-one patients were enrolled and received imatinib 600 mg orally once daily. Five responses were seen primarily in patients, starting with relatively low blast counts in bone marrow (BM) and peripheral blood (PB): 2 patients who were considered refractory on chemotherapy on the basis of persistence of blasts in PB and BM met the criteria for complete hematologic remission, 1 patient had no evidence of leukemia, and 2 patients achieved a minor response. Treatment with imatinib demonstrated a good safety profile and was well tolerated. Western blot analysis and immunohistochemistry demonstrated c-Kit activation in primary AML cells. Further, imatinib treatment of primary AML cells inhibited c-Kit tyrosine-phosphorylation. Genomic DNA-sequencing of c-KIT showed no mutations in exons 2, 8, 10, 11, 12, and 17. Although some of the responses derived from relatively small reductions in leukemic blasts and may be attributable, in part, to prior chemotherapy, these cases suggest that imatinib has interesting clinical activity in a subset of patients with c-kit-positive AML. Further clinical trials are warranted to explore the clinical potential of imatinib in AML and to identify the underlying molecular mechanism.