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Tissue engineering is a method of growing importance regarding clinical application in the genitourinary region. One of the key factors in successfully development of an artificially tissue engineered mucosa equivalent (TEOM) is the optimal choice of the scaffold. Collagen scaffolds are regarded as gold standard in dermal tissue reconstruction. Four distinct collagen scaffolds were evaluated for the ability to support the development of an organotypical tissue architecture. TEOMs were established by seeding cocultures of primary oral epithelial cells and fibroblasts on four distinct collagen membranes. Cell viability was assessed by MTT-assay. The 3D architecture and functionality of the tissue engineered oral mucosa equivalents were evaluated by confocal laser-scanning microscopy and immunostaining. Cell viability was reduced on the TissuFoil E® membrane. A multi-stratified epithelial layer was established on all four materials, however the TEOMs on the Bio-Gide® scaffold showed the best fibroblast differentiation, secretion of tenascin and fibroblast migration into the membrane. The TEOMs generated on Bio-Gide® scaffold exhibited the optimal cellular organization into a cellular 3D network. Thus, the Bio-Gide® scaffold is a suitable matrix for engineering of mucosa substitutes in vitro.

PEG is the gold standard polymer for pharmaceutical applications, however it lacks degradability. Degradation under physiologically relevant pH as present in endolysosomes, cancerous and inflammatory tissues is crucial for many areas. The authors present anionic ring‐opening copolymerization of ethylene oxide with 3,4‐epoxy‐1‐butene (EPB) and subsequent modification to introduce acid‐degradable vinyl ether groups as well as methacrylate (MA) units, enabling radical cross‐linking. Copolymers with different molar ratios of EPB, molecular weights (M n) up to 10 000 g mol−1 and narrow dispersities (Đ, Responsive gels: P(EG‐co‐isoEPB)MA, a PEG copolymer with acidic degradability under physiologically relevant pH is employed for hydrogels. The attached methacrylate units enable radical cross‐linking to form protein‐loaded hydrogels. Both the P(EG‐co‐isoEPB)MA copolymer as well as the respective hydrogels show rapid and tailorable hydrolysis at physiologically relevant pH (pH 5–6), despite excellent storage stability at neutral pH (pH 7.4).

Injectable bone substitutes (IBS) are increasingly being used in the fields of orthopedics and maxillofacial/oral surgery. The rheological properties of IBS allow for proper and less invasive filling of bony defects. Vaterite is the most unstable crystalline polymorph of calcium carbonate and is known to be able to transform into hydroxyapatite upon contact with an organic fluid (e.g., interstitial body fluid). Two different concentrations of hydrogels based on poly(ethylene glycol)-acetal-dimethacrylat (PEG-a-DMA), i.e., 8% (w/v) (VH-A) or 10% (w/v) (VH-B), were combined with vaterite nanoparticles and implanted in subcutaneous pockets of BALB/c mice for 15 and 30 days. Explants were prepared for histochemical staining and immunohistochemical detection methods to determine macrophage polarization, and energy-dispersive X-ray analysis (EDX) to analyze elemental composition was used for the analysis. The histopathological analysis revealed a comparable moderate tissue reaction to the hydrogels mainly involving macrophages. Moreover, the hydrogels underwent a slow cellular infiltration, revealing a different degradation behavior compared to other IBS. The immunohistochemical detection showed that M1 macrophages were mainly found at the material surfaces being involved in the cell-mediated degradation and tissue integration, while M2 macrophages were predominantly found within the reactive connective tissue. Furthermore, the histomorphometrical analysis revealed balanced numbers of pro- and anti-inflammatory macrophages, demonstrating that both hydrogels are favorable materials for bone tissue regeneration. Finally, the EDX analysis showed a stepwise transformation of the vaterite particle into hydroxyapatite. Overall, the results of the present study demonstrate that hydrogels including nano-vaterite particles are biocompatible and suitable for bone tissue regeneration applications.

Fetal calf serum (FCS) is used for in vitro cell culture, as it provides the cells with various growth-promoting compounds. For applications in humans, FCS does not meet the required safety standards and should be replaced by an appropriate substitute. This study analyzed the suitability of using human platelet lysate (hPL) as a substitute for FCS in endothelial cell cultures for in vitro and in vivo tissue engineering applications. The focus was placed on standardized, commercially available hPLs (MultiPL’30, MultiPL’100), which are approved for applications in humans, and compared to laboratory-prepared hPLs (lp-hLP). Human umbilical vein endothelial cells (HUVEC) were cultured with FCS or with different hPLs. Cell morphology, proliferation, viability, apoptosis, and necrosis, as well as the organization of vascular structures, were assessed. No morphological changes were noticed when FCS was replaced by standardized hPLs in concentrations of 1-10%. In contrast, the use of lp-hLPs led to irregular cell shape and increased vacuolization of the cytoplasm. HUVEC proliferation and viability were not compromised by using media supplemented with standardized hPLs or pl-hPLs in concentrations of 1-10%, compared to cells grown in media supplemented with 20% FCS. The apoptosis rate using lp-hPLs was higher compared to the use of standardized hPLs. The necrosis rate tended to be lower when FCS was replaced by hPLs. HUVEC formed more pronounced capillary-like structures when the media were supplemented with hPLs instead of supplementation with FCS. Thus, compared to the use of FCS, the use of hPLs was beneficial for the growth and optimal expression of functional endothelial cell characteristics during in vitro experiments. Commercially available hPLs proved to be particularly suitable, as they led to reproducible results during in vitro experiments, while meeting the safety requirements for in vivo use.

In reconstructive surgery the use of prevascularized soft tissue equivalents is a promising approach for wound coverage of defects after tumor resection or trauma. However, in previous studies to generate soft tissue equivalents on collagen membranes, microcapillaries were restricted to superficial areas. In this study, to understand which factors were involved in the formation of these microcapillaries, the levels of the angiogenic factors vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) in the supernatants of the tissue equivalents were examined at various time points and conditions. Additionally, the influence of these factors on viability, proliferation, migration, and tube formation in monocultures compared to cocultures of fibroblast and endothelial cells was examined. The results showed that VEGF production was decreased in cocultures compared to fibroblast monocultures and the lowest VEGF levels were observed in endothelial cell monocultures. Additionally, the highest levels of IL-8 were observed in cocultures compared to monocultures. Similar results were observed for bFGF with lowest levels seen within the first 24 hr and highest levels in cocultures. VEGF and IL-8 were shown to promote endothelial cell viability, proliferation and migration and angiogenic parameters such as tube density, total tube length, and number of tube branches. Addition of VEGF and IL-8 to cocultures resulted in accelerated and denser formation of capillary-like structures. The results indicate that VEGF, IL-8, and bFGF strongly influence cellular behavior of endothelial cells and this information should be useful in promoting the formation of microcapillary-like structures in complex tissue equivalents.

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.

We illustrate a reliable and accelerated isolation routine for mucosal epithelial cells, which thereupon can be used for soft tissue engineering. This is highly important in the field of soft tissue engineering because mucosal equivalents are frequently usable in several surgical fields like gynecology, urology, otorhinolaryngology, ophthalmology, maxillofacial surgery, and many others. In this context the isolation of mucosal epithelial cells suitable for tissue engineering is mandatory. The reliable cultivation of mucosal or skin epithelial cells is challenging and there is currently no reproducible method. We demonstrate a solution for this problem by developing an accelerated and nevertheless reliable method.

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-beta 1, 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.

Motivation Deep learning use for quantitative image analysis is exponentially increasing. However, training accurate, widely deployable deep learning algorithms requires a plethora of annotated (ground truth) data. Image collections must contain not only thousands of images to provide sufficient example objects (i.e. cells), but also contain an adequate degree of image heterogeneity. Results We present a new dataset, EVICAN—Expert visual cell annotation, comprising partially annotated grayscale images of 30 different cell lines from multiple microscopes, contrast mechanisms and magnifications that is readily usable as training data for computer vision applications. With 4600 images and ∼26 000 segmented cells, our collection offers an unparalleled heterogeneous training dataset for cell biology deep learning application development. Availability and implementation The dataset is freely available (https://edmond.mpdl.mpg.de/imeji/collection/l45s16atmi6Aa4sI?q=). Using a Mask R-CNN implementation, we demonstrate automated segmentation of cells and nuclei from brightfield images with a mean average precision of 61.6 % at a Jaccard Index above 0.5.

The facile and modular functionalization of gold nanoparticles makes them versatile tools in nanomedicine, for instance, photothermal therapy, contrast agents or as model nanoparticles to probe drug-delivery mechanisms. Since endothelial cells from various locations in the body exhibit unique phenotypes we quantitatively examined the amount of different sized poly(2-hydroxypropylmethacrylamide)-coated gold nanoparticles internalized into primary human dermal endothelial cells or human brain endothelial cells (hCMEC/D3) by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and visualized the nanoparticles using light and electron microscopy. Poly(2-hydroxypropylmethacrylamide)-coated gold nanoparticles exhibited high uptake into brain endothelial cells and were used to examine transport mechanisms across the blood–brain barrier using a well-established in vitro model of the blood–brain barrier. Our results demonstrate that 35 nm-sized gold nanoparticles were internalized best into human brain endothelial cells by a flotillin-dependent endocytotic pathway. The uptake into the cells is not correlated with transport across the blood–brain barrier. We demonstrated that the surface modification of gold nanoparticles impacts the internalization process in different cells. In addition, to evaluating toxicity and uptake potential of nanoparticles into cells, the transport properties across cell barriers are important criteria to classify nanoparticle properties regarding targeted delivery of drugs.

Endothelial cells exhibit distinct properties in morphology and functions in different organs that can be exploited for nanomedicine targeting. In this work, endothelial cells from different organs, i.e. brain, lung, liver, and kidney, were exposed to plain, carboxylated, and amino-modified silica. As expected, different protein coronas were formed on the different nanoparticle types and these changed when foetal bovine serum (FBS) or human serum were used. Uptake efficiencies differed strongly in the different endothelia, confirming that the cells retained some of their organ-specific differences. However, all endothelia showed higher uptake for the amino-modified silica in FBS, but, interestingly, this changed to the carboxylated silica when human serum was used, confirming that differences in the protein corona affect uptake preferences by cells. Thus, uptake rates of fluid phase markers and transferrin were determined in liver and brain endothelium to compare their endocytic activity. Overall, our results showed that endothelial cells of different organs have very different nanoparticle uptake efficiency, likely due to differences in receptor expression, affinity, and activity. A thorough characterization of phenotypic differences in the endothelia lining different organs is key to the development of targeted nanomedicine.

Biomimetic materials have been gaining increasing importance for use as bone biomaterials, because they may provide regenerative alternatives for the use of autologous tissues for bone regeneration. We demonstrate a promising alternative for the use of biomimetic materials based on a biodegradable PEG hydrogel loaded with vaterite nanoparticles as mineral storage. Vaterite, the least stable CaCO3 polymorph, is stable enough to ensure the presence of a potential ion buffer for bone regeneration, but still has sufficient reactivity for the transformation from CaCO3 to hydroxyapatite (HA). A combination of powder X-ray diffraction (PXRD), electron microscopy, and Fourier-transform infrared (FT-IR) and Raman spectroscopy showed the transformation of vaterite nanoparticles incorporated in a PEG-acetal-DMA hydrogel to hydroxycarbonate apatite (HCA) crystals upon incubation in simulated body fluid at human body temperature within several hours. The transformation in the PEG-acetal-DMA hydrogel scaffold in simulated body fluid or phosphate saline buffer proceeded significantly faster than for free vaterite. The vaterite-loaded hydrogels were free of endotoxin and did not exhibit an inflammatory effect on endothelial cells. These compounds may have prospects for future applications in the treatment of bone defects and bone degenerative diseases.

Vaterite, a metastable modification of calcium carbonate, embedded in a flexible microgel packaging with adjustable mechanical properties, functionality, and biocompatibility, provides a powerful scaffolding for bone tissue regeneration, as it is easily convertible to bone-like hydroxyapatite (HA). In this study, the synthesis and physical analysis of a packaging material to encapsulate vaterite particles and osteoblast cells into monodisperse, sub-millimeter-sized microgels, is described whereby a systematic approach is used to tailor the microgel properties. The size and shape of the microgels is controlled via droplet-based microfluidics. Key requirements for the polymer system, such as absence of cytotoxicity as well as biocompatibility and biodegradability, are accomplished with functionalized poly(ethylene glycol) (PEG), which reacts in a cytocompatible thiol-ene Michael addition. On a mesoscopic level, the microgel stiffness and gelation times are adjusted to obtain high cellular viabilities. The co-encapsulation of living cells provides i) an in vitro platform for the study of cellular metabolic processes which can be applied to bone formation and ii) an in vitro foundation for novel tissue-regenerative therapies. Finally, the degradability of the microgels at physiological conditions caused by hydrolysis-sensitive ester groups in the polymer network is examined.

Patients diagnosed with osteosarcoma are currently treated with intravenous injections of anticancer agents after tumor resection. However, due to remaining neoplastic cells at the site of tumor removal, cancer recurrence often occurs. Successful bone regeneration combined with the control of residual cancer cells presents a challenge for tissue engineering. Cyclodextrins loaded with chemotherapeutic drugs reversibly release the drugs over time. Hydroxyapatite bone biomaterials coated with doxorubicin-loaded cyclodextrin should release the drug with time after implantation directly at the original tumor site and may be a way to eliminate residual neoplastic cells. In the present study, we have carried out in vitro studies to evaluate such a drug-delivery system and have shown that doxorubicin released from cyclodextrin-coated hydroxyapatite retained biological activity and exhibited longer and higher cytotoxic effects on both cancer (osteosarcoma cells) and healthy cells (primary osteoblasts and endothelial cells) compared to biomaterials without cyclodextrin loaded with doxorubicin. Furthermore, doxorubicin released from biomaterials with cyclodextrin moderately induced the expression of tumor suppressor protein p53 whereas p21 expression was similar to control cells. In addition, hypoxic conditions, which occur after implantation until blood-flow to the area is regenerated, protected endothelial cells and primary osteoblasts from doxorubicin-induced cytotoxicity. This chemo-protective effect was far less prominent for the osteosarcoma cells. These findings indicate that a hydroxyapatite-cyclodextrin-doxorubicin chemotherapeutic strategy may enhance the drug-targeting effect on tumor cells while protecting the more sensitive healthy cells for a period of time after implantation. A successful integration of such a drug delivery system might allow healthy cells to initially survive during the doxorubicin exposure period, while eliminating residual neoplastic cells.

We have previously described a promising alternative to conventional synthetic bone biomaterials using vaterite, a metastable CaCO3 polymorph that increases the local Ca2+ concentration in vitro and leads to an oversaturation of phosphate, the primary bone mineral. This stimulates a natural bone-like mineralisation in a short period of time. In this study, sterile and endotoxin-free vaterite particles were synthesised in a nearly quantitative yield. The 500-1,000 nm vaterite particles did not exhibit any cytotoxic effects as measured by MTS, lactate dehydrogenase, or crystal violet assays on the human osteoblast cell line (MG-63) exposed to concentrations up to 500 μg/ml vaterite up to 72 hr. MG-63, primary human osteoblasts or human umbilical vein endothelial cells in the presence of vaterite up to 500 μg/ml for 7 days exhibited typical growth patterns. Endothelial cells exhibited a normal induction of E-selectin after exposure to LPS and MG-63 cells in osteogenic differentiation medium showed an increased expression of alkaline phosphatase compared with the respective control cells without vaterite. MG-63 cultured on a vaterite-containing degradable poly(ethylene glycol)-hydrogel exhibited strong adhesion and proliferation, similar to cells on cell culture plates. Cells did not attach to gels without vaterite. Our results demonstrate that vaterite particles are biocompatible, do not influence cell gene expression, and that vaterite in hydrogels may be able to serve for adhesion of osteoblasts and as a mineral substrate for natural bone formation by osteoblasts. These characteristics make vaterite particles a highly favourable compound for use in bone regeneration applications.

In addition to macrophages, multinucleated giant cells (MNGCs) are involved in the tissue reaction to a variety of biomaterials. Especially in the case of bone substitute materials it has been assumed that the MNGCs are osteoclasts, based on the chemical and physical similarity of many materials to the calcified matrix and the bony environment in which they are used. However, many studies indicate that these cells belong to the cell line of the foreign body giant cells (FBGCs), which are of "inflammatory origin", although they have been shown to possess both a pro- and also anti-inflammatory phenotype. Moreover, no information is available about their role in the tissue reaction to bone substitute materials. The present study was conducted to analyze the origin of MNGCs in the implant beds of a synthetic and a xenogeneic bone substitute and focused on the application of immunohistochemical methods. Two antibodies against integrin molecules specific for osteoclasts (β-3 integrin) or FBGCs (β-2 integrin) were used to distinguish both giant cell types. The results of the present study indicate that the MNGCs induced by both kinds of bone substitutes are FBGCs, as they express only β-2 integrin in contrast to the osteoclasts outside of the immediate implantation areas, which only demonstrate β-3 integrin expression. These data give new insight into the tissue reaction to both xenogeneic and synthetic bone substitutes. Based on this new knowledge further research concerning the proteomic profile of the FBGCs especially based on the different physicochemical properties of bone substitutes is necessary. This may show that specific characteristics of bone substitutes may exhibit a substantial influence on the regeneration process via the expression of anti-inflammatory molecules by FBGCs. Based on this information it may be possible to formulate and choose bone substitutes that can guide the process of bone tissue regeneration on the molecular level. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1105-1111, 2017.

Spontaneously regressing infantile haemangiomas and aggressive angiosarcomas are vascular tumours with excessive angiogenesis. When analysing haemangiomas and angiosarcomas immunohistochemically with respect to their chaperone profiles we found that angiosarcomas have significantly elevated protein levels of binding immunoglobulin protein (BIP) and PERK with concomitant attenuated IRE1α levels, whereas haemangioma tissue exhibits the same pattern as embryonal skin tissue. We show that BiP is essential for the maintenance of VEGFR2 protein, which is expressed in the endothelium of both tumour types. When studying the effects of BiP, the IRE1α/Xbp1 -, and PERK/ATF4-signalling pathways on the migration and tube-forming potential of endothelial cells, we show that downregulation of BiP, as well as inhibition of the kinase activity of IRE1α, inhibit in vitro angiogenesis. Downregulation of PERK (PKR-like kinase; PKR = protein kinase R) levels promotes Xbp1 splicing in endoplasmic reticulum (ER)-stressed cells, indicating that in angiosarcoma the elevated PERK levels might result in high levels of unspliced Xbp1, which have been reported to promote cell proliferation and increase tumour malignancy. The data presented in this study revealed that in addition to BiP or PERK, the kinase domains of IRE1α and Xbp1 could be potential targets for the development of novel therapeutic approaches for treating angiosarcomas and to control tumour angiogenesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The intestinal microvasculature (iMV) plays multiple pathogenic roles during chronic inflammatory bowel disease (IBD). The iMV acts as a second line of defense and is, among other factors, crucial for the innate immunity in the gut. It is also the therapeutic location in IBD targeting aggravated leukocyte adhesion processes involving ICAM-1 and E-selectin. Specific targeting is stressed via nanoparticulate drug vehicles. Evaluating the iMV in enterocyte barrier models in vitro could shed light on inflammation and barrier-integrity processes during IBD. Therefore, we generated a barrier model by combining the enterocyte cell line Caco-2 with the microvascular endothelial cell line ISO-HAS-1 on opposite sides of a transwell filter-membrane under culture conditions which mimicked the physiological and inflamed conditions of IBD. The IBD model achieved a significant barrier-disruption, demonstrated via transepithelial-electrical resistance (TER), permeability-coefficient (Papp) and increase of sICAM sE-selectin and IL-8. In addition, the impact of a prospective model drug-vehicle (silica nanoparticles, aSNP) on ongoing inflammation was examined. A decrease of sICAM/sE-selectin was observed after aSNP-exposure to the inflamed endothelium. These findings correlated with a decreased secretion of ICAM/E-selectin bearing exosomes/microvesicles, as evaluated via ELISA. Our findings indicate that aSNP treatment of the inflamed endothelium during IBD may hamper exosomal/microvesicular systemic communication.

Gelatin-modified poly(ethylene terephthalate) (PET) surfaces have been previously realized via an intermediate dopamine coating procedure that resulted in surfaces with superior haemocompatibility compared to unfunctionalized PET. The present study addresses the biocompatibility assessment of these coated PET surfaces. In this context, the stability of the gelatin coating upon exposure to physiological conditions and its cell-interactive properties were investigated. The proposed gelatin–dopamine-PET surfaces showed an increased protein coating stability up to 24 days and promoted the attachment and spreading of both endothelial cells (ECs) and smooth muscle cells (SMCs). In parallel, physisorbed gelatin coatings exhibited similar cell-interactive properties, albeit temporarily, as the coating delaminated within 1 week after cell seeding. Furthermore, no or only minimal immunogenic or inflammatory responses were observed during in vitro cytotoxicity and endotoxicity assessment for all gelatin-modified PET surfaces evaluated. Overall, the combined enhanced biocompatibility reported herein together with the previously proven haemocompatibility show the potential of the gelatin–dopamine-PET surfaces to function as vascular graft candidates.

Biodegradable metals in orthopedics are aimed to eliminate secondary the surgical intervention for implant removal, and thus improve patient compliance, reduce surgery costs and the risk of infections. In addition, biodegradable implants could be loaded with different drugs to prevent the growth of pathogens and the development of bone infections, kill remaining cancer cells after tumor resection or stimulate bone regeneration. However, drugs undergo thermal decomposition under the conditions of conventional metal fabrication processes. In this work, we describe the fabrication of a dense drug-eluting biodegradable Fe-Ag nanocomposite containing 10 vol% of Ag and loaded with the antibiotic vancomycin by consolidation at ambient temperature utilizing advanced cold sintering. Fabricated Fe10Ag nanocomposites loaded with 1 wt% VH combined strength of 225 and 123 MPa in compression and bending tests, respectively, and antibacterial activity against Staphylococcus aureus. Finally, the show good cell compatibility with a human osteoblast-like cell line. Our findings show the promise of this simple process to produce drug-eluting biodegradable metals.

Jennifer Y Kasper,1 Maria Iris Hermanns,1 Christian Cavelius,2 Annette Kraegeloh,2 Thomas Jung,3 Rolf Danzebrink,3 Ronald E Unger,1 Charles James Kirkpatrick1 1Institute of Pathology, University Medical Center, Mainz, 2Leibniz Institute for New Materials, 3NanoGate AG, Goettelborn, Saarbrücken, Germany Abstract: The microvascular endothelium of the gut barrier plays a crucial role during inflammation in inflammatory bowel disease. We have modified a commonly used intestinal cell model based on the Caco-2 cells by adding microvascular endothelial cells (ISO-HAS-1). Transwell filters were used with intestinal barrier-forming Caco-2 cells on top and the ISO-HAS-1 on the bottom of the filter. The goal was to determine whether this coculture mimics the in vivo situation more closely, and whether the model is suitable to evaluate interactions of, for example, prospective nanosized drug vehicles or contrast agents with this coculture in a physiological and inflamed state as it would occur in inflammatory bowel disease. We monitored the inflammatory responsiveness of the cells (release of IL-8, soluble intercellular adhesion molecule 1, and soluble E-selectin) after exposure to inflammatory stimuli (lipopolysaccharide, TNF-α, INF-γ, IL1-β) and a nanoparticle (Ba/Gd: coprecipitated BaSO4 and Gd(OH)3), generally used as contrast agents. The barrier integrity of the coculture was evaluated via the determination of transepithelial electrical resistance and the apparent permeability coefficient (Papp) of NaFITC. The behavior of the coculture Caco-1/ISO-HAS-1 was compared to the respective monocultures Caco-2 and ISO-HAS-1. Based on transepithelial electrical resistance, the epithelial barrier integrity of the coculture remained stable during incubation with all stimuli, whereas the Papp decreased after exposure to the cytokine mixture (TNF-α, INF-γ, IL1-β, and Ba/Gd). Both the endothelial and epithelial monocultures showed a high inflammatory response in both the upper and lower transwell-compartments. However, in the coculture, inflammatory mediators were only detected on the epithelial side and not on the endothelial side. Thus in the coculture, based on the Papp, the epithelial barrier appears to prevent a potential inflammatory overreaction in the underlying endothelial cells. In summary, this coculture model exhibits in vivo-like features, which cannot be observed in conventional monocultures, making the former more suitable to study interactions with external stimuli. Keywords: intestinal microvasculature, inflammatory bowel disease, intestinal barrier in vitro, Caco-2, ISO-HAS-1, soluble E-selectin, sICAM-1, nanosized gadolinium contrast agent

A library of polymer-coated gold nanoparticles (AuNPs) differing in size and surface modifications was examined for uptake and induction of cellular stress responses in the endoplasmic reticulum (ER stress) in human brain endothelial cells (hCMEC/D3). ER stress is known to affect the physiology of endothelial cells (ECs) and may lead to inflammation or apoptosis. Thus, even if applied at non-cytotoxic concentrations ER stress caused by nanoparticles should be prevented to reduce the risk of vascular diseases and negative effects on the integrity of barriers (e.g. blood-brain barrier). We exposed hCMEC/D3 to twelve different AuNPs (three sizes: 18, 35, and 65 nm, each with four surface-modifications) for various times and evaluated their effects on cytotoxicity, proinflammatory mediators, barrier functions and factors involved in ER stress. We demonstrated a time-dependent uptake of all AuNPs and no cytotoxicity for up to 72 h of exposure. Exposure to certain AuNPs resulted in a time-dependent increase in the proinflammatory markers IL-8, MCP-1, sVCAM, sICAM. However, none of the AuNPs induced an increase in expression of the chaperones and stress sensor proteins BiP and GRP94, respectively, or the transcription factors ATF4 and ATF6. Furthermore, no upregulation of the UPR stress sensor receptor PERK, no active splicing product of the transcription factor XBP1 and no upregulation of the transcription factor CHOP were detectable. In conclusion, the results of the present study indicate that effects of different-sized gold nanoparticles modified with various polymers were not related to the induction of ER stress in brain microvascular endothelial cells or led to apoptosis.

Today, chronic respiratory disease is one of the leading causes of mortality globally. Epithelial dysfunction can play a central role in its pathophysiology. The development of physiologically-representative in vitro model systems using tissue-engineered constructs might improve our understanding of epithelial tissue and disease. This study sought to engineer a bilayered collagen-hyaluronate (CHyA-B) scaffold for the development of a physiologically-representative 3D in vitro tracheobronchial epithelial co-culture model. CHyA-B scaffolds were fabricated by integrating a thin film top-layer into a porous sub-layer with lyophilisation. The film layer firmly connected to the sub-layer with delamination occurring at stresses of 12-15 kPa. Crosslinked scaffolds had a compressive modulus of 1.9 kPa and mean pore diameters of 70 μm and 80 μm, depending on the freezing temperature. Histological analysis showed that the Calu-3 bronchial epithelial cell line attached and grew on CHyA-B with adoption of an epithelial monolayer on the film layer. Immunofluorescence and qRT-PCR studies demonstrated that the CHyA-B scaffolds facilitated Calu-3 cell differentiation, with enhanced mucin expression, increased ciliation and the formation of intercellular tight junctions. Co-culture of Calu-3 cells with Wi38 lung fibroblasts was achieved on the scaffold to create a submucosal tissue analogue of the upper respiratory tract, validating CHyA-B as a platform to support co-culture and cellular organisation reminiscent of in vivo tissue architecture. In summary, this study has demonstrated that CHyA-B is a promising tool for the development of novel 3D tracheobronchial co-culture in vitro models with the potential to unravel new pathways in drug discovery and drug delivery.

The use of beta-tricalcium phosphate (β-TCP) ceramic as a bioresorbable bone substitute is limited to non-load-bearing sites by the material׳s brittleness and low bending strength. In the present work, new biocompatible β-TCP-based composites with improved mechanical properties were developed via reinforcing the ceramic matrix with 30 vol% of a biodegradable iron-magnesium metallic phase. β-TCP-15Fe15Mg and β-TCP-24Fe6Mg (vol%) composites were fabricated using a combination of high energy attrition milling, cold sintering/high pressure consolidation of powders at room temperature and annealing at 400 °C. The materials synthesized had a hierarchical nanocomposite structure with a nanocrystalline β-TCP matrix toughened by a finely dispersed nanoscale metallic phase (largely Mg) alongside micron-scale metallic reinforcements (largely Fe). Both compositions exhibited high strength characteristics; in bending, they were about 3-fold stronger than β-TCP reinforced with 30 vol% PLA polymer. Immersion in Ringer׳s solution for 4 weeks resulted in formation of corrosion products on the specimens׳ surface, a few percent weight loss and about 50% decrease in bending strength. In vitro studies of β-TCP-15Fe15Mg composite with human osteoblast monocultures and human osteoblast-endothelial cell co-cultures indicated that the composition was biocompatible for the growth and survival of both cell types and cells exhibited tissue-specific markers for bone formation and angiogenesis, respectively.

PURPOSE Sinus floor elevation using the lateral approach and bone window repositioning and a xenogeneic bone substitute (Cerabone) has been well documented clinically. The purpose of this histologic and histomorphometric study was to determine the fate of the window, its contributing role in the healing process, and the osseoconductivity and resorption potential of the high-temperature sintered bovine bone used, as well as to correlate the histomorphometric results with sinus depth and lateral wall thickness as determined on cone beam computed tomography (CBCT). MATERIALS AND METHODS Thirty biopsy specimens were harvested from the lateral side of the maxilla of patients operated on for sinus floor elevation and implant placement at two postoperative periods: early, group 1 (mean: 5.73 ± 0.44 months); and late, group 2 (mean: 8.68 ± 1.76 months). Sinus depth and lateral wall thickness were determined on CBCT and correlated to graft maturation. RESULTS The repositioned bone window was microscopically detectable in both study groups and looked well integrated. Bone was found growing out of the repositioned window toward the center of the graft, most often forming a trabecular network independently from the bone matrix, which is in favor of osteogenic potential of the window. Also, newly built bone was found directly attached to the surfaces of the window, indicating bone growth via osseoconduction. Repositioned window sides showed signs of low-grade inflammation. Active osteoclasts were only found to be associated with the newly built bone matrix, hinting at an active bone remodeling process. No signs of biodegradation or remodeling of the window were detected using the tartrate-resistant acid phosphatase (TRAP) technique. The histomorphometric analysis of the tissue distribution showed similar values of newly formed bone in group 1 (22.77% ± 5.89%) and in group 2 (26.15% ± 11.18%) and connective tissue values in both study groups (42.29% ± 8.98% for group 1 vs 46.03% ± 5.84% for group 2). No significant differences were found between group 1 (34.94% ± 7.10%) and group 2 (27.82% ± 11.97%) for xenogeneic bone substitute values. Statistically significant differences were only found between connective tissue values and newly built bone values (P < .01 and P < .001, respectively). Furthermore, a significant difference was found between connective tissue values and that of bone substitute up to 12 months (P < .01). No significant correlation was found between sinus depth and lateral window thickness and histomorphometric results. CONCLUSION The re positioned window technique appears to be a good osteoconductive barrier for bone formation. Its osteogenic potential needs to be confirmed immunochemically. High-temperature sintered bovine bone proved to be an effective slowly resorbing osseoconductive material.

An original fabrication route of high-strength bulk Fe-5Ag and Fe-10Ag nanocomposites with enhanced degradation rate is reported. Near fully dense materials with fine nanostructures and uniform distribution of Ag nanoparticles were obtained employing high energy attrition milling of Fe-Ag2O powder blends followed by cold sintering – high pressure consolidation at ambient temperature that allowed the retention of the nanoscale structure. Annealing in hydrogen flow at 550 °C resulted in enhanced ductility without coarsening the nanostructure. The strength in compression of Fe5Ag and Fe10Ag nanocomposites was several-fold higher than the values reported for similar composites with micrometer grain size. The galvanic action of finely dispersed Ag nanoparticles greatly increased the corrosion rate and degradation kinetics of iron. Following four weeks immersion of Fe-Ag nanocomposites in saline solution, a more than 10% weight loss accompanied by less than 25% decrease in bending strength were measured. The interconnected nanoporosity of cold sintered Fe-Ag nanocomposites was utilized for incorporation of vancomycin that was gradually released upon immersion. In cell culture experiments, the Fe-Ag nanocomposites supported the attachment of osteoblast cells and exhibited no signs of cytotoxicity. The results suggest that the proposed Fe-Ag nanocomposites could be developed into attractive biodegradable load-bearing implant materials with drug delivery capability.

In vascular tissue engineering, great attention is paid to the immobilization of biomolecules onto synthetic grafts to increase bio- and hemocompatibility-two critical milestones in the field. The surface modification field of poly(ethylene terephthalate) (PET), a well-known vascular-graft material, is matured and oversaturated. Nevertheless, most developed methods are laborious multistep procedures generally accompanied by coating instability or toxicity issues. Herein, a straightforward surface modification procedure is presented engineered to simultaneously promote surface endothelialization and anticoagulation properties via the covalent immobilization of gelatin through a photoactivated azide derivative. A complete physicochemical characterization and biological study including cytotoxicity and endotoxin testing are performed. In addition, biocompatibility toward small (diameter ≤ 6 mm) and/or large caliber (diameter ≥ 6 mm) vessels is assessed by micro- and macrovascular endothelial cell assays. Superior bio- and hemocompatibility properties are seen for the gelatin-covalently modified PET surfaces compared to the conventional surface-modification procedures based on physisorption.

One of the major problems with bone tissue engineering is the development of a rapid vascularization after implantation to supply the growing osteoblast cells with the nutrients to grow and survive as well as to remove waste products. It has been demonstrated that capillary-like structures produced in vitro will anastomose rapidly after implantation and become functioning blood vessels. For this reason, in recent years many studies have examined a variety of human osteoblast and endothelial cell co-culture systems in order to distribute osteoblasts on all parts of the bone scaffold and at the same time provide conditions for the endothelial cells to migrate to form a network of capillary-like structures throughout the osteoblast-colonized scaffold. The movement and proliferation of endothelial cells to form capillary-like structures is known as angiogenesis and is dependent on a variety of pro-angiogenic factors. This review summarizes human 2- and 3-D co-culture models to date, the types and origins of cells used in the co-cultures and the proangiogenic factors that have been identified in the co-culture models.

A major problem in tissue engineering (TE) is graft failure in vivo due to core degradation in in vitro engineered constructs designed to regenerate thick tissues such as bone. The integration of constructs post-implantation relies on the rapid formation of functional vasculature. A recent approach to overcome core degradation focuses on the creation of cell-based, pre-engineered vasculature formed within the TE construct in vitro , prior to implantation in vivo . The primary objective of this study was to investigate whether an amniotic fluid-derived stem cell (AFSC)–human umbilical vein endothelial cell (HUVEC) co-culture could be used to engineer in vitro vasculature in a collagen chondroitin sulphate (CCS) scaffold. The secondary objective was to investigate whether hypoxic conditions (2% O 2 ) could enhance microcapillary-like structure formation by this co-culture. The results of this study demonstrate, for the first time, that the AFSC–HUVEC co-culture was capable of pre-vascularising CCS scaffolds within 7 days and that the AFSCs are capable of behaving as pericytes while interacting with HUVECS to form microcapillary-like structures. However, this microcapillary-like structure formation was reduced in hypoxic conditions. qRT-PCR analysis indicated that an upregulation of VEGFR1 and accompanying decrease of VEGFR2 gene expression may be responsible for the poor response of these microcapillary-like structures to hypoxic conditions. Overall, however, these results demonstrate the potential of this newly developed co-culture system for the formation of pre-engineered vasculature within TE constructs. Statement of Significance This article describes the development of an amniotic fluid-derived stem cell (AFSC)–human umbilical vein endothelial cell (HUVEC) co-culture for use in engineering in vitro vasculature in a collagen chondroitin sulphate (CCS) scaffold. The article also describes the effect of hypoxic conditions on the networks of microcapillary-like structures formed by this co-culture. The AFSC–HUVEC co-culture was capable of pre-vascularising CCS scaffolds within 7 days. However, microcapillary-like structure formation was reduced in hypoxic conditions. Overall, these results demonstrate the potential of this newly developed co-culture system for the formation of pre-engineered vasculature within TE constructs. The proangiogenic nature of this co-culture has the potential to both enhance bone regeneration while also overcoming the problem of inadequate vascularisation of grafts commonly seen in the field of tissue engineering.

We have previously described a promising alternative to conventional synthetic bone biomaterials using vaterite, a metastable CaCO

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations. (C) 2016 Elsevier Inc. All rights reserved.

In this paper, the processing and properties of iron-toughened bioresorbable β-tricalcium phosphate (β-TCP) nanocomposites are reported. β-TCP is chemically similar to bone mineral and thus a good candidate material for bioresorbable bone healing devices; however intrinsic brittleness and low bending strength make it unsuitable for use in load-bearing sites. Near fully dense β-TCP-matrix nanocomposites containing 30vol% Fe, with and without addition of silver, were produced employing high energy attrition milling of powders followed by high pressure consolidation/cold sintering at 2.5GPa. In order to increase pure iron's corrosion rate, 10 to 30vol% silver were added to the metal phase. The degradation behavior of the developed composite materials was studied by immersion in Ringer's and saline solutions for up to 1month. The mechanical properties, before and after immersion, were tested in compression and bending. All the compositions exhibited high mechanical strength, the strength in bending being several fold higher than that of polymer toughened β-TCP-30PLA nanocomposites prepared by the similar procedure of attrition milling and cold sintering, and of pure high-temperature sintered β-TCP. Partial substitution of iron with silver led to an increase in both strength and ductility. Furthermore, the galvanic action of silver particles dispersed in the iron phase significantly accelerated in vitro degradation of β-TCP-30(Fe-Ag) nanocomposites. After 1month immersion, the composites retained about 50% of their initial bending strength. In cell culture experiments, β-TCP-27Fe3Ag nanocomposites exhibited no signs of cytotoxicity towards human osteoblasts suggesting that they can be used as an implant material.

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.

Limitations of current treatments for intervertebral disc (IVD) degeneration have promoted interest in the development of tissue-engineering approaches. Injectable hydrogels loaded with cells can be used as a substitute material for the inner IVD part, the nucleus pulposus (NP), and provide an opportunity for minimally invasive treatment of IVD degeneration. The NP is populated by chondrocyte-like cells; therefore, chondrocytes and mesenchymal stem cells (MSCs), stimulated to differentiate along the chondrogenic lineage, could be used to promote NP regeneration. In this study, the in vitro and in vivo response of human bone marrow-derived MSCs and nasal chondrocytes (NCs) to modified gellan gum-based hydrogels was investigated. Both ionic- (iGG-MA) and photo-crosslinked (phGG-MA) methacrylated gellan gum hydrogels show no cytotoxicity in extraction assays with MSCs and NCs. Furthermore, the materials do not induce pro-inflammatory responses in endothelial cells. Moreover, MSCs and NCs can be encapsulated into the hydrogels and remain viable for at least 2 weeks, although apoptosis is observed in phGG-MA. Importantly, encapsulated MSCs and NCs show signs of in vivo chondrogenesis in a subcutaneous implantation of iGG-MA. Altogether, the data endorse the potential use of modified gellan gum-based hydrogel as a suitable material in NP tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

Previous studies have shown that plastic compression (PC) of collagen gels allows a rapid and controlled fabrication of matrix- and cell-rich constructs in vitro that closely mimic the structure and characteristics of tissues in vivo. Microvascular endothelial cells, the major cell type making up the blood vessels in the body, were added to the PC collagen to determine whether cells attach, survive, grow, and express endothelial cell characteristics when seeded alone or in coculture with other cells. Endothelial cells seeded on the PC collagen containing human foreskin fibroblasts (HFF) or human osteoblasts (HOS) formed vessel-like structures over 3 weeks in culture without the addition of exogenous growth factors in the medium. In contrast, on the PC scaffolds without HFF or HOS, human dermal microvascular endothelial cells (HDMEC) exhibited a typical cobblestone morphology for 21 days under the same conditions. We propose that the coculture of primary endothelial cells with PC collagen constructs, containing a stromal cell population, is a valuable technique for in vitro modeling of proangiogenic responses toward such biomimetic constructs in vivo. A major observation in the cocultures was the absence of gel contraction, even after 3 weeks of fibroblast culture. This collagen form could, for example, be of great value in tissue engineering of the skin, as contractures are both aesthetically and functionally disabling.

Objective Placental growth and villous maturation are critical parameters of placental function at the end of pregnancy. A failure in these processes leads to the development of placental dysfunction, as well as fetal and neonatal mortality and morbidity. The aim of the study was to determine the relevant diagnostic markers associated with pathological placental development. Study design Forty tissue samples from normal placentas of different gestational age and 68 pathological term placentas with defective villous maturation (GDM, idiopathic IUFD, preeclamsia, HELLP syndrome) comprised the comparative immunohistochemical study (CD15, CD45 and CD34). Positive immunohistochemical reactions were quantitatively assessed in the chorionic plate and vessels of the villi of different histological type. Results Physiologically immature placentas of the first and second trimester and pathologically immature term placentas were characterized by marked endothelial CD15-immunostaining. A significant loss of CD15-positive endothelium of the placentas was associated with a physiological and accelerated villous maturity. A spatio-temporal correlation was shown for CD15+ endothelial cells (ECs) and the number of CD45+ stromal cells (SCs). A negative temporal correlation was shown for CD15+ ECs and CD15+ myelomonocytes in the fetal blood. CD34 expression in the ECs was stable during the pregnancy. Conclusion A correlation between a transient CD15-positive endothelial phenotype and a physiological and pathological fetoplacental immaturity was demonstrated. Physiological and accelerated placental maturation was accompanied by a significant disappearance of CD15-positive endothelium. We propose that “immature” CD15+ endothelium is an important diagnostic marker of the physiological and pathological fetoplacental immaturity.

The repair and regeneration of large bone defects, including the formation of functional vasculature, represents a highly challenging task for tissue engineering and regenerative medicine. Recent studies have shown that vascularization and ossification can be stimulated by mild heat stress (MHS), which would offer the option to enhance the bone regeneration process by relatively simple means. However, the mechanisms of MHS-enhanced angiogenesis and osteogenesis, as well as potential risks for the treated cells are unclear. We have investigated the direct effect of MHS on angiogenesis and osteogenesis in a co-culture system of human outgrowth endothelial cells (OECs) and primary osteoblasts (pOBs), and assessed cytotoxic effects, as well as the levels of various heat shock proteins (HSPs) synthesized under these conditions. Enhanced formation of microvessel-like structures was observed in co-cultures exposed to MHS (41°C, 1 h), twice per week, over a time period of 7-14 days. As shown by real-time polymerase chain reaction (PCR), the expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and tumor necrosis factor-alpha was up-regulated in MHS-treated co-cultures 24 h post-treatment. At the protein level, significantly elevated VEGF and Ang-1 concentrations were observed in MHS-treated co-cultures and pOB mono-cultures compared with controls, indicating paracrine effects associated with MHS-induced angiogenesis. MHS-stimulated co-cultures and OEC mono-cultures released higher levels of Ang-2 than untreated cultures. On the other hand MHS treatment of co-cultures did not result in a clear effect regarding osteogenesis. Nevertheless, real-time PCR demonstrated that MHS increased the expression of mitogen-activated protein kinase, interleukin-6, and bone morphogenetic protein 2, known as HSP-related molecules in angiogenic and osteogenic regulation pathways. In agreement with these observations, the expression of some selected HSPs also increased at both the mRNA and protein levels in MHS-treated co-cultures.

Endotoxin, also known as lipopolysaccharide (LPS) produced by bacteria can be present in any liquid or on any biomaterial even if the material is sterile. Endotoxin in mammals can cause fever, inflammation, cell and tissue damage and irreversible septic shock and death. In the body, endothelial cells making up the blood vasculature and endothelial cells in vitro rapidly react to minute amounts of endotoxin resulting in a rapid induction of the cell adhesion molecule E-selectin. In this study we have used immunofluorescent staining to evaluate the expression of E-selectin on human microvascular endothelial cells from the skin (HDMEC) and human umbilical vein endothelial cells (HUVEC) exposed to various concentrations of LPS. In addition, the sensitivity of detection was compared with the most widely used assay for the presence of endotoxin, the Limulus Amebocyte Lysate assay (LAL). The detection of E-selectin on endothelial cells in the presence of LPS for 4 h was found to be at least as sensitive in detecting the same concentration using the LAL assay. A cell adhesion molecule-enzyme immunosorbent assay was also developed and used to quantify LPS using the endothelial cell model. A comparison of LAL and the immunofluorescent staining method was carried out with solutions, nanoparticles, biomaterial extracts and endothelial cells grown directly on biomaterials. Under all conditions, the endothelial/E-selectin model system was positive for the test samples that were positive by LAL. Thus, we propose the use of this highly sensitive, rapid, reproducible assay for the routine testing of endotoxin in all steps in the manufacturing process of materials destined for use in humans. This can give a rapid feedback and localization of bacterial contamination sources with the LAL being reserved for the testing of the final product.

Inflammation is an important step in the early phase of tissue regeneration around an implanted metallic orthopaedic device. However, prolonged inflammation, which can be induced by metallic corrosion products, can lead to aseptic loosening and implant failure. Cells in peri-implant tissue as well as metal corrosion can induce reactive oxygen species (ROS) formation, thus contributing to an oxidative microenvironment around an implant. Understanding cellular reactions to implant-induced oxidative stress and inflammatory activation is important to help prevent an adverse response to metallic materials. In an earlier study we have shown that endothelial cells grown on Ti6Al4V alloy are subjected to oxidative stress. Since endothelial cells play a critical role in inflammation, in this study we examined the role of oxidative stress in their response to pro-inflammatory activation. Therefore, we stimulated endothelial cells in contact with Ti6Al4V with tumour necrosis factor-α (TNF-α) and monitored the expression of inflammation-associated molecules, such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8). The induction of these proteins was lower in endothelial cells on Ti6Al4V compared to control tissue culture conditions. There was, however, a discrepancy in pro-inflammatory activation at protein compared to mRNA level in the cells on Ti6Al4V. To examine the role of oxidative stress in this response we utilized different ROS scavengers and showed that ROS depletion improved cellular response to TNF-α on Ti6Al4V. These results could contribute to developing strategies to improve tissue response to metal implants.

Drug and gene delivery via nanoparticles across biological barriers such as the alveolar-capillary barrier of the lung constitutes an interesting and increasingly relevant field in nanomedicine. Nevertheless, potential hazardous effects of nanoparticles (NPs) as well as their cellular and systemic fate should be thoroughly examined. Hence, this study was designed to evaluate the effects of amorphous silica NPs (Sicastar) and (poly)organosiloxane NPs (AmOrSil) on the viability and the inflammatory response as well as on the cellular uptake mechanisms and fate in cells of the alveolar barrier. For this purpose, the alveolar epithelial cell line (NCI H441) and microvascular endothelial cell line (ISO-HAS-1) were used in an experimental set up resembling the alveolar-capillary barrier of the lung. In terms of IL-8 and sICAM Sicastar resulted in harmful effects at higher concentrations (60μg/ml) in conventional monocultures but not in the coculture, whereas AmOrSil showed no significant effects. Immunofluorescence counterstaining of endosomal structures in NP-incubated cells showed no evidence for a clathrin- or caveolae-mediated uptake mechanism. However, NPs were enclosed in flotillin-1 and -2 marked vesicles in both cell types. Flotillins appear to play a role in cellular uptake or trafficking mechanisms of NPs and are discussed as indicators for clathrin- or caveolae-independent uptake mechanisms. In addition, we examined the transport of NPs across this in vitro model of the alveolar-capillary barrier forming a tight barrier with a transepithelial electrical resistance of 560±8Ωcm2. H441 in coculture with endothelial cells took up much less NPs compared to monocultures. Moreover, coculturing prevented the transport of NP from the epithelial compartment to the endothelial layer on the bottom of the filter insert. This supports the relevance of coculture models, which favour a differentiated and polarised epithelial layer as in vitro test systems for nanoparticle uptake.

Stem cells are used to generate differentiated somatic cells including neuronal cells. Synthesis and release of acetylcholine, a neurotransmitter and widely expressed signaling molecule, were investigated in the murine embryonic stem cell line CGR8 during early differentiation, i.e. in the presence of leukemia inhibitory factor (LIF) to maintain pluripotency and in the absence of LIF to induce early differentiation. CGR8 cells express choline acetyltransferase (ChAT) as demonstrated by measurement of enzyme activity and substantial inhibition by bromoacetylcholine. Pluripotent CGR8 cells showed a ChAT activity of 250 pmol acetylcholine/mg/h, contained 1.1 pmol acetylcholine/10⁶ cells and released about 12.00 pmol acetylcholine/1 x 10⁶ cells/6 h. Removal of LIF induced early differentiation as evidenced by reduced transcription factors Oct-4 and Nanog and a substantial slowing of the proliferation rate. Under this condition acetylcholine synthesis increased to 1640 pmol/mg/h; related to the pluripotent state the content of acetylcholine increased 10-fold and the release to about 32 pmol acetylcholine/1 x 10⁶ cells/6 h. Enzyme kinetic analysis showed a significant increase of the K(m) for the precursor acetyl-CoA and of V(max) without a change of the K(m) for the precursor choline. In conclusion, early differentiation of the stem cell line CGR8 is associated with a substantial increase in ChAT activity and acetylcholine release.

Bone tissue engineering takes part in the complex process of bone healing by combining cells, chemical/physical signals, and scaffolds with the scaffolds providing an artificial extracellular matrix network. The role of the support template for cell activity is crucial to guide the healing process. This in vitro study compared three different poly(D,L-lactic acid) scaffolds obtained by varying the pore size generated by applying different salt leaching processes. The influence of pore dimensions on the extracellular matrix produced by human osteosarcoma-derived osteoblasts (MG63 cell line) seeded on these different materials was analyzed. This study is targeted on the intermediate stage of the bone healing process, where a collagen network is beginning to develop by the growing osteoblasts representing the template for the ultimate stage of bone formation. Imaging analyses assessed by confocal laser microscopy were combined with gene expression measurements of the most common genes involved in the bone healing process. Furthermore, in vitro evaluations were carried out to investigate cell morphology, proliferation, and viability. It was found that the different pore size matrixes can affect extracellular matrix development and that cell organization, collagen I assembly, and mineralization are strictly correlated.

We previously demonstrated that the co-cultivation of endothelial cells with neural cells resulted in an improved integrity of the in vitro blood-brain barrier (BBB), and that this model could be useful to evaluate the transport properties of potential central nervous system disease drugs through the microvascular brain endothelial. In this study we have used real-time PCR, fluorescent microscopy, protein arrays and enzyme-linked immunosorbent assays to determine which neural- and endothelial cell-derived factors are produced in the co-culture and improve the integrity of the BBB. In addition, a further improvement of the BBB integrity was achieved by adjusting serum concentrations and growth factors or by the addition of brain pericytes. Under specific conditions expression of angiogenic, angiostatic and neurotrophic factors such as endostatin, pigment epithelium derived factor (PEDF/serpins-F1), tissue inhibitor of metalloproteinases (TIMP-1), and vascular endothelial cell growth factor (VEGF) closely mimicked the in vivo situation. Freeze-fracture analysis of these cultures demonstrated the quality and organization of the endothelial tight junction structures and their association to the two different lipidic leaflets of the membrane. Finally, a multi-cell culture model of the BBB with a transendothelial electrical resistance up to 371 (±15) Ω×cm2 was developed, which may be useful for preliminary screening of drug transport across the BBB and to evaluate cellular crosstalk of cells involved in the neurovascular unit.